WO2018119003A1 - Virus composite biosensor - Google Patents

Abstract

Provided here are, inter alia, virus-enabled biosensors, electrochemical cells, and method of use thereof.

Description

VIRUS COMPOSITE BIOSENSOR CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No.62/436,373 filed

December 19, 2016, the disclosure of which is incorporated by reference herein in its entirety. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under grant number CHE-1306928 awarded by The National Science Foundation, and grant number 1R33CA206955-01 awarded by the National Instituted of Health. The Government has certain rights in this invention.

BACKGROUND

[0003] Biosensor technologies that enable the rapid measurement of disease biomarkers in unprocessed biological samples, including blood, urine, saliva, lacrimal fluid, nipple aspirate fluid, and cerebrospinal fluids, remain elusive and highly sought. The ultimate goal is devices that can be used with minimal training by physicians and patients to provide actionable information at the point- of-care (PoC) (see, e.g., Gubala, V. et al. (2012) Anal. Chem.84:487-515; Soper, S. A. et al. (2006) Biosens. Bioelectron.21:1932-1942; Luo, X. L. and Davis, J. J. (2013) Chem. Soc. Rev.42:5944- 5962). In addition to simplicity, analysis speed and sensitivity are critically important metrics for PoC biosensors but the technology must also provide for sensor-to-sensor reproducibility, manufacturability, and low cost.

[0004] A new approach to point of care detection of protein disease markers involves the use of virus particles, rather than antibodies, within a bioaffinity capture layer. Relative to antibodies, virus particles have several advantages that make them attractive for emerging PoC sensor technologies: First, virus particles can be engineered to bind virtually any protein– even toxic proteins for which antibody development is difficult (see, e.g., Beekwilder, J. et al. (1999) Gene 228:23-31; Pacheco, S. et al. (2015) Amb Express, 5). Second, virus particles are less thermally and chemically labile than antibodies, dramatically simplifying the large-scale production, storage and transport of biosensors that rely on virus–based bioaffinity layers (see, e.g., Hayhurst, A. and Georgiou, G. (2001) Curr. Opin. Chem. Biol.5:683-689). Finally, virus particles that are capable of antibody-like affinities can be produced in quantity at lower costs (see, e.g., Weiss, G. A. and Penner, R. M. (2008) Anal. Chem. 80:3082-3089).

[0005] The use of whole virus particles as a bioaffinity matrix for biosensors dates to 2003, when it was demonstrated that engineered M13 phage could be immobilized by physisorption onto the gold transducer of an acoustic wave sensor (see, e.g., Petrenko, V. A. and Vodyanoy, V. J. (2003) J. Microbiol. Meth.53:253-262) and, somewhat later (see, e.g., Nanduri, V. et al. (2007) Biosens. Bioelectron.22:986-992), to a gold quartz crystal microbalance electrode, enabling the detection in both cases of β-galactosidase (see, e.g., Petrenko, V. A. and Vodyanoy, V. J. (2003) J. Microbiol. Meth.53:253-262; Nanduri, V. et al. (2007) Biosens. Bioelectron.22:986-992). Subsequently, in 2007 Cosnier et al. (see, e.g., Ionescu, R. E. et al. (2007) Anal. Chem.79:8662-8668) demonstrated biosensors based upon the virus T7 capable of detecting human antibodies to the West Nile virus.

[0006] There are provided herein inter alia solutions to these and other problems in the art.

BRIEF SUMMARY

[0007] Provided herein are electrochemical cells that comprise a potentiostat electronically connected to a first electrode and a second electrode, where the first electrode and the second electrode are coated with a viral composition that comprises: (i) a whole viral particle comprising a charged protein coat, the charged protein coat comprising a plurality of charged coat proteins; (ii) a first polymer electrostatically bound to the plurality of charged coat proteins; and (iii) a covalent linker linking the first polymer to a recognition moiety. The electrochemical cells comprise a cell layer forming a liquid-holding cell capable of holding a liquid test sample, such as a biological test sample.

[0008] Provided herein are methods of detecting biomolecules (e.g., cancer cell markers) in a liquid test sample, the method comprising: (i) contacting the first electrode and the second electrode of the electrochemical cell with a test sample, (ii) measuring the resistance of the test sample; and (iii) comparing the resistance to a control in order to detect the biomolecules.

[0009] These and other embodiments and aspects of the disclosure are described in more detail herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG.1. Biosensor and flow cell schematic diagrams. (FIG.1A) An assembled flow cell attached to a gold-electrode device comprises two gold contacts connected to a potentiostat for EIS measurements. (FIG.1B) A gold-electrode device and detailed structure of a single

poly(methylmethacrylate) (PMMA) flow cell; (FIG.1C) a single device with a box showing the two planar gold electrodes used for sensing. The two gold electrodes have a length (L) of 2 mm, width (w) of 0.85 mm, and are separated by a 50 µm gap. (FIG.1D) Dimensions of the first PMMA flow cell layer which creates a cell holding 6 µL of solution over the gold electrodes; (FIG.1E) Top view representation of assembled flow cell. Solution is added to a reservoir with a 75 µL capacity, flows from the inlet port (right), through the cell, and exits through the outlet port (left).

[0011] FIG.2. Electrodeposition and SEM characterization of virus-PEDOT bioaffinity coatings. (FIG.2A) Electrodeposition of a virus-PEDOT film by cyclic voltammetry. Film prepared by two cycles in aqueous EDOT solution (2.5 mM EDOT, 12.5 mM LiClO₄) followed by eight cycles in a virus-EDOT solution (2.5 mM EDOT, 12.5 mM LiClO4, 8 nM HSA phage). Virus-EDOT solution was replenished every two cycles. All scan rates were 20 mV/s. Optical image of: (FIG.2B) bare gold electrodes and (FIG.2C) gold electrodes after electrodeposition of virus-PEDOT film. (FIG. 2D, FIG.2F, FIG.2E, FIG.2G) Scanning electron microscopy images of uncoated films. (FIG.2D) PEDOT film prepared by ten consecutive cycles of deposition in aqueous EDOT solution (2.5 mM EDOT, 12.5 mM LiClO4). (FIG.2E) PEDOT edge showing film height of approximately 220 nm. (FIG.2F) Virus-PEDOT film prepared as described in (FIG.2A) showing dense incorporation of phage bundles on the surface. (FIG.2G) Virus-PEDOT edge showing primer layer of PEDOT with thickness of approximately 160 nm and PEDOT-coated phage on top.

[0012] FIG.3. Atomic force microscopy of virus-PEDOT bioaffinity films and AFM line scans shown at the bottom. (FIG.3A) PEDOT-only film prepared by ten cycles of deposition in EDOT solution (2.5 mM EDOT, 12.5 mM LiClO4). Topography of the middle (left) and the edge (right) of films imaged by atomic force microscopy. The film-edge height shown in line scans includes the gold electrode layer (60 nm). (FIG.3B) Virus-PEDOT film prepared by two cycles of deposition in EDOT solution followed by eight cycles in virus-EDOT solution (2.5 mM EDOT, 12.5 mM LiClO4, 8 nM HSA phage); virus-EDOT solution replenished every two cycles. The rms roughness for PEDOT and virus-PEDOT films is≈10 nm and≈150 nm, respectively. [0013] FIG.4. Using EIS to electrically transduction HSA binding. The EIS response of virus- PEDOT biosensors upon exposure to 500 nM BSA and 500 nM HSA is compared. No redox species are added to the solution in these measurements. Errors bars represent the standard deviation, ±1σ, of five consecutive EIS measurements on a single electrode. (FIG.4A, FIG.4B) Nyquist plots for virus-PEDOT films in solutions of run buffer and 500 nM BSA or HSA. Plots of (FIG.4C) ΔZre and (FIG.4D) ΔZ_im versus frequency, where ΔZ is defined as Z_analyte– Z_buffer. Corresponding (FIG.4E) ΔZ_re and (FIG.4F) ΔZ_im signal-to-noise ratio, defined as ΔZ/σ, as a function of frequency.

[0014] FIG.5. Signal-to-noise (S/N) of the HSA detection using ΔZre and ΔZim. Calibration plots of (FIG.5A) ΔZre and (FIG.5B) ΔZim versus frequency for virus-PEDOT films in varying concentrations of HSA in run buffer. Each HSA concentration was measured using a different biosensor. Errors bars are defined as the standard deviation, ±1σ, of five consecutive impedance measurements on a single electrode. (FIG.5C) S/N, defined as ΔZre/σ, versus frequency for ΔZre, and (FIG.5D) S/N, defined as ΔZ_im/σ, versus frequency for ΔZ_im.

[0015] FIG.6. Sensor-to-sensor reproducibility of HSA detection. Calibration plot of (FIG.6A) ΔZre, and (FIG.6B) ΔZim versus frequency for multiple virus-PEDOT films exposed to varying concentrations of HSA. ΔZ values for n = 3 independent virus-PEDOT electrodes were averaged to obtain each curve, errors bars indicate, ±1σ. Corresponding coefficient of variation, defined as the relative standard deviation for n=3 virus-PEDOT electrodes of (FIG.6C) ΔZre and (FIG.6D) ΔZim versus frequency plots for each HSA concentration. ΔZre shows regions of COV values < 20 % while, ΔZ_im COV values are too high for reliable measurements. At each frequency, ΔZ_re versus [HSA] was fitted to the Hill equation and the square of the regression coefficient, R², versus frequency plot (FIG.6E). R² = 1 represents the best fit of the Hill equation to the data. The highlighted interval in (FIG.6A, FIG.6C, FIG.6E) indicates the frequency range where ΔZ_re signal is largest, COV is at a minimum, and the peak for goodness of fit occur, respectively. (FIG.6F) Calibration plot of ΔZre, measured at 340 Hz, versus concentration. Each data point represents a different virus-PEDOT electrode with error bars defined as the standard deviation, ±1σ, of five consecutive impedance measurements. Impedance data for HSA exposures to virus-PEDOT films containing HSA phage are fitted to the hill equation. Three controls to confirm specific binding to HSA are shown: BSA exposure to virus-PEDOT films containing HSA binding phage, HSA exposure to virus-PEDOT films containing a control phage having no affinity for HSA, and HSA exposure to pure PEDOT films containing no phage.

[0016] FIG.7. Process flow for lithographic preparation of gold electrodes. FIG.7 shows a process flow of lithographically patterned gold films. Schematic diagram of gold film electrodes prepared by photolithography:(1) Positive photoresist is spin coated onto a glass substrate, (2) the photoresist is patterned by a photomask and developed, (3) slides are coated with thermally- evaporated gold, (4) and lift off is performed.

[0017] FIG.8. Enzyme linked immunosorbent assay. FIG.8 shows an enzyme-linked

immunosorbent assay for HSA phage binding. Ligand binding is compared to the negative control Stop-4 phage, which shows significantly less binding activity. A phage-based ELISA for HSA phage binding: HSA-phage binding to HSA compared to two negative controls, HSA-phage binding to BSA and Stop-4 phage binding to HSA.

[0018] FIG.9. Effect of pH and blocking agents on non-specific binding. FIG.9 shows a plot of the change in Zre at 340 Hz when exposed to 500 nM BSA under various conditions. Preventing non-specific binding is a critical challenge for non-faradaic impedance based biosensors; in this study pH and blocking agents were explored. Non-specific binding was characterized by exposing a PEDOT film to BSA protein in various buffers. Initial studies of PEDOT films in PBS buffer at pH 8 shows significant non-specific binding to BSA. This response from non-specific binding is reduced as the pH of the buffer is decreased, suggesting that non-specific binding is caused by electrostatic interactions between the positively-charged PEDOT and BSA protein. Non-specific was also reduced by addition of a blocking agent, casein. At pH 8, PEDOT films that were blocked with casein in PBS show less response from BSA than PEDOT films that were not blocked. Bar plot of ∆Zre for PEDOT films to determine optimal buffer conditions for reduced non-specific binding: (FIG.9A- FIG.9C) PEDOT films, at varying pH’s, were equilibrated in PBS and then exposed to 500 nM BSA in PBS. (FIG.9D) A PEDOT film was blocked with casein in PBS for 15 minutes, equilibrated in PCT, and exposed to 500 nM BSA in PCT at pH 8. Lowering the buffer pH or implementing a casein blocking agent significantly reduced non-specific binding.

[0019] FIG.10. An equivalent circuit for virus-PEDOT films. FIG.10 shows an equivalent circuit corresponding to virus-PEDOT films on two gold electrodes. R1, C2 , R2, and C3 represent the two virus-PEDOT films where most of the change induced by HSA binding is in the two resistors. There is little change in the R2, the solution resistance, and C1, the geometrical capacitance between the two films. A constant phase element, Q, represents deviation from a perfect capacitor caused by surface roughness or composition differences between the phage and PEDOT the films. (FIG.10A) Diagram of an equivalent circuit used to model virus-PEDOT films on two planar-gold electrodes. Circuit elements represent: capacitance between the two electrodes (C1), the solution resistance of PBS buffer (R₂), the impedance imposed by one virus-PEDOT film (R_1,C_2, Q₁), and the impedance imposed by the second virus-PEDOT film (R₃, C₃, Q₂). (FIG.10B) Plot of impedance versus frequency for Zre and Zim. The simulated impedance (solid line) data produced by the parameters in Table 4 is plotted on top of the raw impedance data (open circle) of a virus-PEDOT film in PCT buffer. (FIG.10C) Plot of Z_re versus frequency for the range of frequencies, 50 Hz to 10 kHz, where a response to HSA binding is observed. Both the raw and simulated data show an increase in impedance from virus-PEDOT films in PCT buffer to 500 nM HSA in PCT buffer.

[0020] FIG.11._.HSA sensing in synthetic urine. FIG.11 includes analysis of signal-to-noise and R2 values for the Hill equation for virus-PEDOT films in synthetic urine.∆Zre increases

monotonically with HSA concentration from 100 nM to 5 µM in synthetic urine. Peak∆Zre response, signal-to-nose, and R2 > 0.95 are observed at 136 Hz. (FIG.11A) Calibration plot of ΔZ_re versus frequency for virus-PEDOT films in varying concentrations of HSA in synthetic urine. Each HSA concentration was measured using a different biosensor. Errors bars are defined as the standard deviation, ±1σ, of five consecutive impedance measurements on a single electrode. (FIG.11B) S/N, defined as ΔZre/σ, versus frequency for ΔZre. (FIG.11C) At each frequency, ΔZre versus (HAS) was fitted to the Hill equation and the square of the regression coefficient, R², versus frequency plot. R² = 1 represents the best fit of the Hill equation to the data.

[0021] FIG.12. Real-time HSA biosensing. Plot of∆Zre verus time, of a single virus-PEDOT, using a control virus that did not bind HSA and HSA virus, electrode when exposed to three concentrations of HSA. A freshly electrodeposited virus-PEDOT film was first immersed in run buffer (PBS-casein-tween) until reaching an equilibration signal. The time scan was then paused and five EIS spectra were acquired in rapid succession. Immediately following this, the virus-PEDOT film was exposed to 100 nM HSA in run buffer and the time scan was restarted within 5 seconds of exposure. This procedure was repeated for exposures to 500 nM and 5000 nM HSA. [0022] FIG.13. Virus-PEDOT sensors in synthetic urine. Calibration plot of (FIG.13A) ΔZre versus frequency for multiple virus-PEDOT films exposed to varying concentrations of HSA. ΔZ values for n = 3 independent virus-PEDOT electrodes were averaged to obtain each curve, errors bars indicate, ±1σ. (FIG.13B) Corresponding coefficient of variation (COV), defined as the relative standard deviation for n=3 virus-PEDOT electrodes of ΔZre versus frequency plots for each HSA concentration. (FIG.13C) Calibration plot of ΔZ_re, measured at 136 Hz, versus concentration. Each data point represents an independent virus-PEDOT electrode with error bars defined as the standard deviation, ±1σ, of five consecutive impedance measurements. Impedance data for HSA exposures to virus-PEDOT films containing HSA phage are fitted to the hill equation. Three controls to confirm specific binding to HSA are shown: BSA exposure to virus-PEDOT films containing HSA phage, HSA exposure to virus-PEDOT films containing a control phage that did not bind HSA, and HSA exposure to pure PEDOT films containing no phage.

[0023] FIG.14. Schematic depicting photolithography device fabrication and flow cell.

[0024] Figures and drawings can be seen in color in Ogata et al,“Virus-Enabled Biosensor for Human Serum Albumin,” Analytical Chemistry, 89(2):1373-1381 (2017), the disclosure of which is incorporated by reference herein in its entirety.

DETAILED DESCRIPTION

[0025] Definitions

[0026] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al.,

Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0027] “Electrochemical impedence spectroscopy” or“EIS” refers to a method of measuring the electrical impedence of a substance as a function of the frequency of an applied electrical current in an electrochemical cell. [0028] The terms“gap” or“space” refer to a distance between electrodes that allows for the passage or flow of a voltage or current between the electrodes that can be measured by, for example, electrochemical impedance spectroscopy (EIS).

[0029] “Electrically conductive polymers” refer to organic polymer that conduct electricity.

Examples of electrically conductive polymers include polyfluorenes, polyphenylenes, polypyrenes, polyazulenes, polynaphthalenes, polypyrroles, polycarbazoles, polyindoles, polyazepines, polyanilines, polythiophenes, poly(3,4-ethylenedioxythiophene), poly(p-phenylene sulfide), polyacetylenes, poly(p-phenylene vinylene) and the like. Electrically conductive polymers can be modified with functional groups (e.g., hydroxy, sulfo) to impart desired properties to the polymer (e.g., water solubility).

[0030] “Acrylic polymer” or“acrylates” or“polyacrylic acid” refers to polymers comprised of acrylate monomers, e.g., homopolymers of acrylic acid crosslinked with allyl ether pentaerythritol, allether of sucrose, or allyl ether of propylene. Exemplary acrylic monomers include acrylic acid, methacrylate (methacrylic acid), methyl acrylate, ethyl acrylate, butyl acrylate, 2-chloroethyl vinyl ether, 2-ethylhexyl acrylate, hydroxyethyl methacrylate, methyl methacrylate, ethyl methacrylate, butyl methacrylate, and the like. Acrylic polymers are commercially available in varying molecular weights, such as from about 2,000 daltons to about 1,500,000 daltons.

[0031] “Acrylic copolymer” refers to polymers comprised of at least two different acrylate monomers. Exemplary acrylic monomers include acrylic acid, methacrylate (methacrylic acid), methyl acrylate, ethyl acrylate, butyl acrylate, 2-chloroethyl vinyl ether, 2-ethylhexyl acrylate, hydroxyethyl methacrylate, methyl methacrylate, ethyl methacrylate, butyl methacrylate, and the like. Exemplary acrylic copolymers include copolymers of methacrylic acid and ethyl acrylate, and copolymer of methacrylic acid and methyl methacrylate. Acrylic copolymers are commercially available.

[0032] The term“electrochemical cell” refers to a device having two electrodes connected by an electron conductor and spatially separated by an ionic conductor and that converts chemical energy into electrical energy or vice versa when a chemical reaction is occurring in the cell. In

embodiments, the electrochemical cell comprises a potentiostat electronically connected to a first electrode and a second electrode, wherein the first electrode and the second electrode are coated with a viral composition.

[0033] The term“potentiostat” refers to a device to control or maintain the potential difference between electrodes at a constant level in an electrochemical cell.

[0034] The term“liquid-holding cell” refers to a compartment, a cavity, a hollow or a unit in a device receiving an approximately-determined volume of a sample liquid and containing electrodes immersed in an electrolyte.

[0035] The term“biosensor” refers to a device for detecting and measuring very small quantities or changes in a biochemical or chemical substance, in which a microelectronic component registers reactions related to the substance and translates them into data, or a device that detects, records, and transmits information regarding a physiological change or process, or a device that uses biological materials, such as enzymes, to monitor the presence of various chemicals in a substance. In embodiments, the biosensor is a point of care (PoC) biosensor that exploits electrodeposited bioaffinity layers that contain a composite of virus particles, as described herein.

[0036] The term“biomolecule” refers to a molecule that is made or naturally occurs in a living organism, such as amino acids, sugars, nucleic acids, proteins, polysaccharides, DNA and RNA. In embodiments, the biomolecules are hormones, cytokines, proteins, nucleic acids, lipids,

carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands. In embodiments, the biomolecules are cancer cell markers. In embodiments, the biomolecule is human serum albumin.

[0037] "Biological sample" or "sample" refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, white blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, cerebral spinal fluid, lacrimal fluid, nipple aspirate fluid, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. [0038] A "solid support" as provided herein refers to any appropriate material that can be modified to contain discrete individual sites for the attachment or association of an electronically conductive polymer as provided herein including embodiments thereof and is amenable to the methods provided herein including embodiments thereof. Examples of solid supports include without limitation, glass and modified or functionalized glass (e.g., carboxymethyldextran functionalized glass), plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene, TEFLON® (The Chemours Co.), etc.), polysaccharides, nylon or nitrocellulose, composite materials, ceramics, and plastic resins, silica or silica-based materials including silicon and modified silicon (e.g., patterned silicon), carbon, metals, quartz (e.g., patterned quartz), inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers (e.g., electronically conductive polymers such as poly-3,4- ethylenedioxythiophene, PEDOT). In general, the solid support allows optical detection and do not appreciably fluoresce. The solid support may be planar (e.g., flat planar substrates such as glass, polystyrene and other plastics and acrylics). Although it will be appreciated by a person of ordinary skill in the art that other configurations of solid supports may be used as well; for example, three dimensional configurations can be used. The solid support may be modified to contain discrete, individual sites (also referred to herein as "wells") for polymer binding. These sites generally include physically altered sites, i.e. physical configurations such as wells or small depressions in the substrate that can retain the polymers. The wells may be formed using a variety of techniques well known in the art, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques. It will be appreciated by a person of ordinary skill in the art that the technique used will depend on the composition and shape of the solid support. In embodiments, physical alterations are made in a surface of the solid support to produce wells. In embodiments, the solid support is a microtiter plate.

[0039] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts. Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH₂O- is equivalent to -OCH₂-. [0040] The term“alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched non-cyclic carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-). An alkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. An alkyl moiety may be fully saturated.

[0041] The term“alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited

to, -CH₂CH₂CH₂CH₂-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term“alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0042] The term“heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched non-cyclic chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited

to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, - S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH ₃)-CH₃, -O-CH₃, -O-CH₂-CH₃, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and–CH2-O-Si(CH3)3. A heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P).

[0043] Similarly, the term“heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH₂-CH₂-S-CH₂-CH₂- and -CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents

both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such

as -C(O)R', -C(O)NR', -NR'R'', -OR', -SR', and/or -SO₂R'. Where“heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R'' or the like, it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R'' or the like.

[0044] The terms“cycloalkyl” and“heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, non-aromatic cyclic versions of“alkyl” and“heteroalkyl,” respectively, wherein the carbons making up the ring or rings do not necessarily need to be bonded to a hydrogen due to all carbon valencies participating in bonds with non-hydrogen atoms.

Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, 3- hydroxy-cyclobut-3-enyl-1,2, dione, 1H-1,2,4-triazolyl-5(4H)-one, 4H-1,2,4-triazolyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A“cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. A heterocycloalkyl moiety may include one ring heteroatom (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include two optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include three optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include four optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A

heterocycloalkyl moiety may include five optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include up to 8 optionally different ring heteroatoms (e.g., O, N, S, Si, or P).

[0045] The terms“halo” or“halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C1- C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2- trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0046] The term“acyl” means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0047] The term“aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term“heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term“heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,

3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An“arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. Non-limiting examples of aryl and heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl,

quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. The examples above may be substituted or unsubstituted and divalent radicals of each heteroaryl example above are non-limiting examples of heteroarylene. A heteroaryl moiety may include one ring heteroatom (e.g., O, N, or S). A heteroaryl moiety may include two optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include three optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include four optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include five optionally different ring heteroatoms (e.g., O, N, or S). An aryl moiety may have a single ring. An aryl moiety may have two optionally different rings. An aryl moiety may have three optionally different rings. An aryl moiety may have four optionally different rings. A heteroaryl moiety may have one ring. A heteroaryl moiety may have two optionally different rings. A heteroaryl moiety may have three optionally different rings. A heteroaryl moiety may have four optionally different rings. A heteroaryl moiety may have five optionally different rings.

[0048] A fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring

heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring

heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl- cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.

[0049] For brevity, the term“aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g.,

phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

[0050] The term“oxo,” as used herein, means an oxygen that is double bonded to a carbon atom.

[0051] The term“alkylsulfonyl,” as used herein, means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g.,“C₁-C₄ alkylsulfonyl”).

[0052] Each of the above terms (e.g.,“alkyl,”“heteroalkyl,”,“cycloalkyl”,“heterocycloalkyl”, “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0053] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''',

-OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2 R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', −NR'NR''R''',−ONR'R'',−NR'C=(O)NR''NR'''R'''', -CN, -NO2, in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such radical. R, R', R'', R''', and R'''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R'', R''', and R'''' group when more than one of these groups is present. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1- pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl

(e.g., -C(O)CH₃, -C(O)CF₃, -C(O)CH₂OCH₃, and the like).

[0054] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO₂R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)₂R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)₂R', -S(O)₂NR'R'', -NR SO2R',−NR'NR''R''',−ONR'R'',−NR'C=(O)NR''NR'''R'''', -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1- C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R'', R''', and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R'', R''', and R'''' groups when more than one of these groups is present.

[0055] Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0056] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR')_q-U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the

formula -A-(CH₂)_r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O) -, -S(O)₂-, -S(O)2NR'-, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X'- (C''R''R''')d-, where s and d are independently integers of from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituents R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or

unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0057] As used herein, the terms“heteroatom” or“ring heteroatom” are meant to include, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0058] A“substituent group,” as used herein, means a group selected from the following moieties: (A) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, −NHNH₂,−ONH₂,−NHC=(O)NHNH₂,−NHC=(O) NH₂, -NHSO₂H, -NHC= (O)H, -NHC(O)- OH, -NHOH, -OCF₃, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl; and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: (i) oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -SH, - SO₃H, -SO₄H, -SO₂NH₂,−NHNH₂,−ONH₂,−NHC=(O)NHNH₂,−NHC=(O) NH₂, -NHSO₂H, - NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF₃, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: (a) oxo,

halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2,−NHNH2, −ONH2,−NHC=(O)NHNH2,−NHC=(O) NH2, -NHSO2H, -NHC= (O)H, -NHC(O)- OH, -NHOH, -OCF₃, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -SH, - SO3H, -SO4H, -SO2NH2,−NHNH2,−ONH2,−NHC=(O)NHNH2,−NHC=(O) NH2, -NHSO2H, - NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0059] A“size-limited substituent” or“ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.

[0060] A“lower substituent” or“ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₇ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. [0061] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

[0062] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈ cycloalkylene, each substituted or unsubstituted

heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.

[0063] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C₁-C₈ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C₆- C₁₀ arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In some embodiments, the compound is a chemical species set forth in the Examples section, figures, or tables below.

[0064] As used herein, the term“isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

[0065] Unless otherwise stated, structures depicted herein are also meant to include all

stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope disclosed herein.

[0066] The symbol“-“ or“ ” denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

[0067] The terms "a" or "an," as used in herein means one or more. In addition, the phrase "substituted with a[n]," as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is "substituted with an unsubstituted C1-C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl," the group may contain one or more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as“R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different.

[0068] Descriptions of compounds of the present invention are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.

[0069] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The term

"polynucleotide" refers to a linear sequence of nucleotides. The term“nucleotide” typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides,

deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA.

Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0070] Nucleic acids, including nucleic acids with a phosphothioate backbone can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amio acid on a protein or polypeptide through a covalent, non-covalent or other interaction.

[0071] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate), phosphorodithioate, phosphonocarboxylic acids,

phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA)), including those described in U.S. Patent Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0072] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism. An "inhibitory nucleic acid" is a nucleic acid (e.g. DNA, RNA, polymer of nucleotide analogs) that is capable of binding to a target nucleic acid (e.g. an mRNA translatable into a protein) and reducing transcription of the target nucleic acid (e.g. mRNA from DNA) or reducing the translation of the target nucleic acid (e.g.mRNA) or altering transcript splicing (e.g. single stranded morpholino oligo).

[0073] A "labeled nucleic acid or oligonucleotide" is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the nucleic acid may be detected by detecting the presence of the detectable label bound to the nucleic acid. Alternatively, a method using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin. In embodiments, the phosphorothioate nucleic acid or phosphorothioate polymer backbone includes a detectable label, as disclosed herein and generally known in the art.

[0074] The term "probe" or "primer", as used herein, is defined to be one or more nucleic acid fragments whose specific hybridization to a sample can be detected. A probe or primer can be of any length depending on the particular technique it will be used for. For example, PCR primers are generally between 10 and 40 nucleotides in length, while nucleic acid probes for, e.g., a Southern blot, can be more than a hundred nucleotides in length. The probe may be unlabeled or labeled as described below so that its binding to the target or sample can be detected. The probe can be produced from a source of nucleic acids from one or more particular (preselected) portions of a chromosome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The length and complexity of the nucleic acid fixed onto the target element is not critical to the invention. One of skill can adjust these factors to provide optimum hybridization and signal production for a given

hybridization procedure, and to provide the required resolution among different genes or genomic locations.

[0075] The probe may also be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. In some embodiments, the probe may be a member of an array of nucleic acids as described, for instance, in WO 96/17958. Techniques capable of producing high density arrays can also be used for this purpose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol.8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Patent No.5,143,854).

[0076] The words "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.

[0077] The term "isolated", when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0078] The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In some embodiments, the nucleic acid or protein is at least 50% pure, optionally at least 65% pure, optionally at least 75% pure, optionally at least 85% pure, optionally at least 95% pure, and optionally at least 99% pure.

[0079] The term "isolated" may also refer to a cell or sample cells. An isolated cell or sample cells are a single cell type that is substantially free of many of the components which normally

accompany the cells when they are in their native state or when they are initially removed from their native state. In certain embodiments, an isolated cell sample retains those components from its natural state that are required to maintain the cell in a desired state. In some embodiments, an isolated (e.g. purified, separated) cell or isolated cells, are cells that are substantially the only cell type in a sample. A purified cell sample may contain at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of one type of cell. An isolated cell sample may be obtained through the use of a cell marker or a combination of cell markers, either of which is unique to one cell type in an unpurified cell sample. In some embodiments, the cells are isolated through the use of a cell sorter. In some embodiments, antibodies against cell proteins are used to isolate cells.

[0080] As used herein, the term "conjugate" refers to the association between atoms or molecules. The association can be direct or indirect. For example, a conjugate between a polymer and a ligand or recognition moiety provided herein can be direct, e.g., by covalent bond, or indirect, e.g., by non- covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, conjugates are formed using conjugate chemistry including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol.198, American Chemical Society, Washington, D.C., 1982. In embodiments, the polymer is non-covalently attached to the ligand through a non-covalent chemical reaction between a component of the polymer and a component of the ligand. In other embodiments, the polymer is covalently bound to the ligand or recognition moiety using a covalent linker, wherein the covalent linker is attached to the polymer at one end and to the ligand or recognition moiety at the other end. The linker attachment to the polymer or to the ligand or recognition moiety may be accomplished using one or more reactive moieties, e.g., bioconjugate techniques, a covalent reactive moiety, as described herein (e.g., alkyne, azide, maleimide or thiol reactive moiety).

[0081] Useful reactive moieties or functional groups (chemical reactive functional groups) used for conjugate chemistries (click chemistries) herein include, for example: (a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N- hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.; (c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom; (d) dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups; (e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition; (f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold; (h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated or oxidized; (i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, and the like; (j) epoxides, which can react with, for example, amines and hydroxyl compounds; (k)

phosphoramidites and other standard functional groups useful in nucleic acid synthesis; (l) metal silicon oxide bonding; (m) metal bonding to reactive phosphorus groups (e.g. phosphines) to form, for example, phosphate diester bonds; and (n) sulfones, for example, vinyl sulfone.

[0082] Chemical synthesis of compositions by joining modular units using conjugate (click) chemistry may also be sued to attach the covalent linker to the polymer and/or to the ligand or recognition moiety, which is well known in the art and described, for example, in H. C. Kolb, M. G. Finn and K. B. Sharpless ((2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions". Angewandte Chemie International Edition 40 (11): 2004–2021); R. A. Evans ((2007). "The Rise of Azide–Alkyne 1,3-Dipolar 'Click' Cycloaddition and its Application to Polymer Science and Surface Modification". Australian Journal of Chemistry 60 (6): 384–395; W.C. Guida et al. Med. Res. Rev. p 31996; Spiteri, Christian and Moses, John E. ((2010). "Copper-Catalyzed Azide–Alkyne Cycloaddition: Regioselective Synthesis of 1,4,5-Trisubstituted 1,2,3-Triazoles". Angewandte Chemie International Edition 49 (1): 31–33); Hoyle, Charles E. and Bowman,

Christopher N. ((2010). "Thiol–Ene Click Chemistry". Angewandte Chemie International Edition 49 (9): 1540–1573); Blackman, Melissa L. and Royzen, Maksim and Fox, Joseph M. ((2008).

"Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder

Reactivity". Journal of the American Chemical Society 130 (41): 13518–13519); Devaraj, Neal K. and Weissleder, Ralph and Hilderbrand, Scott A. ((2008). "Tetrazine Based Cycloadditions:

Application to Pretargeted Live Cell Labeling". Bioconjugate Chemistry 19 (12): 2297–2299);

Stöckmann, Henning; Neves, Andre; Stairs, Shaun; Brindle, Kevin; Leeper, Finian ((2011).

"Exploring isonitrile-based click chemistry for ligation with biomolecules". Organic & Biomolecular Chemistry), all of which are hereby incorporated by reference in their entirety and for all purposes.

[0083] The reactive functional groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the proteins described herein. By way of example, the polymer or ligand/recognition moiety can include a vinyl sulfone or other reactive moiety (e.g., maleimide). Optionally, the polymer or ligand can include a reactive moiety having the formula S- S-R. R can be, for example, a protecting group. Optionally, R is hexanol. As used herein, the term hexanol includes compounds with the formula C₆H₁₃OH and includes, 1-hexanol, 2-hexanol, 3- hexanol, 2-methyl-1-pentanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 2-methyl-2-pentanol, 3- methyl-2-pentanol, 4-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-3-pentanol, 2,2-dimethyl-1- butanol, 2,3-dimethyl-1-butanol, 3,3-dimethyl-1-butanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-2- butanol, and 2-ethyl-1-butanol. Optionally, R is 1-hexanol.

[0084] As used herein, the term "about" means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term "about" means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about means the specified value.

[0085] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The terms apply to macrocyclic peptides, peptides that have been modified with non- peptide functionality, peptidomimetics, polyamides, and macrolactams. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0086] The term "peptidyl" and "peptidyl moiety" means a monovalent peptide.

[0087] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O- phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms "non-naturally occurring amino acid" and "unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0088] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0089] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0090] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0091] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0092] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0093] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0094] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0095] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then the to be "substantially identical." This definition also refers to the complement of a test sequence.

Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0096] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0097] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.

48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0098] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res.25:3389-3402, and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the

BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA

89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0099] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0100] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0101] A polypeptide, or a cell is“recombinant” when it is artificial or engineered, or derived from or contains an artificial or engineered protein or nucleic acid (e.g. non-natural or not wild type). For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example a variant of a naturally occurring gene, is recombinant.

[0102] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0103] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0104] A "control" sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). One of skill in the art will understand which controls are most appropriate in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

[0105] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0106] A "labeled protein or polypeptide" is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the labeled protein or polypeptide may be detected by detecting the presence of the label bound to the labeled protein or polypeptide. Alternatively, methods using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.

[0107] A "cell" as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.

[0108] The term“cell surface marker” as used herein, refers to a protein or a group of proteins expressed on the surface of cells that serve as markers of specific cell types.

[0109] The term "polymer" or "polymers" as provided herein refers to synthetic or natural molecules, or macromolecules, composed of multiple repeated subunits (monomers). Synthetic polymers (e.g., synthetic plastics such as polystyrene) and natural biopolymers (e.g., DNA, proteins) may be distinguished. Polymers, both natural and synthetic, are created via polymerization of many small molecules, known as monomers. In embodiments, polymers have a large molecular mass relative to small molecule compounds and, therefore, produce unique physical properties (e.g., toughness, viscoelasticity, tendency to form glasses and semicrystalline structures). In

embodiments, the polymers are charged (charged polymers). The charged polymers provided herein may include a positive charge or a negative charge. Thus, in embodiments, the charged polymer is an anionic polymer. In embodiments, the charged polymer is a cationic polymer. Non-limiting examples of polymers useful for the compositions and methods provided herein include gum arabic, gum acacia, gum tragacanth, locust bean gum, guar gum, hydroxypropyl guar, xanthan gum, talc, cellulose gum, sclerotium gum, carageenan gum, karaya gum, cellulose gum, rosin, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxymethylcellulose ,

hydroxypropylmethylcellulose, methylhydroxyethylcellulose, cetyl hydroxyethylcellulose, carboxymethylcellulose, corn starch, hydroxypropyl starch phosphate, distarch phosphate, distarch dimethylene urea, aluminum starch octenyl succinate, maltodextrin, dextran, poly(acrylamide), PEG- 150 distearate, PEG-150/decyl alcohol/SMDI copolymer, PEG-150/stearyl alcohol/SMDI copolymer, PEG-180/Laureth-50/TMMG copolymer, Polyether 1, acrylic acid/acrylamidomethyl propane sulfonic acid copolymer, acrylate/C10-30 alkyl acrylate cross polymer, acrylate/beheneth- 25 methacrylate copolymer, acrylate/steareth-20 methacrylate copolymer, acrylate/steareth-20 copolymer, acrylate/VA cross polymer, acrylic acid/acrylonitrogen copolymer, ammonium acryloyldimethyltaurate/beheneth-25 methacrylate copolymer, ammonium

acryloyldimethyltaurate/VP copolymer, sodium acrylate copolymer, PVM/MA decadiene cross polymer, alginic acid, propylene glycol alginate, dimethicone, silica dimethyl silylate, a

dimethylacrylamide/acrylic acid/polystyrene ethyl methacrylate copolymer, PLGA polymer, polylactide, polyethylene glycol, carbomer, trolamine, derivatives thereof, and mixtures thereof. In embodiments, the polyethylene glycol is PEG3380. PEG3380 refers, in the customary sense, to CAS Registry No.71767-64-1. In embodiments, the carbomer is CARBOPOL® 980. The term “carbomer” refers to cross linked polyacrylate polymers as known in the art and, for example, to CARBOPOL® 980 or CARBOPOL® 980 polymer, which are defined by CAS Registry Nos.9063- 87-0, 9003-01-4, or 600-07-7, respectively. The polyacrylate polymer may be, but is not limited to, poly-2-methylbutanoic acid, poly-prop-2-enoic acid, polyacrylic acid.

[0110] The term“polymerizable monomer” is used in accordance with its meaning in the art of polymer chemistry and refers to a compound that may covalently bind chemically to other monomer molecules (such as other polymerizable monomers that are the same or different) to form a polymer.

[0111] In embodiments, the polymer is a block polymer. The term“block copolymer” is used in accordance with its ordinary meaning and refers to two or more portions (e.g., blocks) of

polymerized monomers linked by a covalent bond. In embodiments, a block copolymer is a repeating pattern of polymers. In embodiments, the block copolymer includes two or more monomers in a periodic (e.g., repeating pattern) sequence. For example, a diblock copolymer has the formula:–B-B-B-B-B-B–A-A-A-A-A–, where‘B’ is a first subunit and‘A’ is a second subunit covalently bound together. A triblock copolymer therefore is a copolymer with three distinct blocks, two of which may be the same (e.g.,–A-A-A-A-A–B-B-B-B-B-B–A-A-A-A-A–) or all three are different (e.g.,–A-A-A-A-A–B-B-B-B-B-B–C-C-C-C-C–) where‘A’ is a first subunit,‘B’ is a second subunit, and‘C’ is a third subunit, covalently bound together. In embodiments, the block polymer is a Lysine₁₄ block polymer. A Lysine₁₄ block polymer (“K₁₄”) as provided herein refers to a polymer derived from lysine homopolymer subunits (monomers), which are linked by covalent bonds.

[0112] The terms“virus” or“virus particle” are used according to its plain ordinary meaning within Virology and refers to a virion including the viral genome (e.g. DNA, RNA, single strand, double strand), viral capsid and associated proteins, and in the case of enveloped viruses (e.g.

herpesvirus), an envelope including lipids and optionally components of host cell membranes, and/or viral proteins.

[0113] The term“recognition moiety” or“ligand” (also referred to herein as a ligand domain) refers to a composition (e.g., atom, molecule, ion, molecular ion, compound, particle, protein, peptide, nucleic acid, oligosaccharide, polysaccharide, or small molecule) capable of binding (e.g. specifically binding) to a second complementary ligand-binding composition (e.g., analyte, polymer, protein, marker, small molecule, ligand, polysaccharide, aptamer, or other binder) to form a complex. A recognition moiety as provided herein may without limitation bind to biomolecules (e.g., hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors or their ligands)); whole cells or lysates thereof (e.g., prokaryotic (e.g., pathogenic bacteria), eukaryotic cells (e.g., mammalian tumor cells); viruses (e.g., retroviruses, herpesviruses, adenoviruses, lentiviruses and spores); chemicals (e.g., solvents, polymers, organic materials, small molecules); therapeutic molecules (e.g., therapeutic drugs, abused drugs, antibiotics); environmental pollutants (e.g., pesticides, insecticides, toxins). In embodiments, the recognition moiety is a cell surface marker binding moiety (i.e., a composition that recognizes and binds to a cell surface marker). In embodiments, the recognition moiety is a polypeptide. In embodiments, the recognition moiety is an antibody or a fragment thereof.

[0114] As used herein, the terms "specific binding" or "specifically binds" refer to two molecules forming a complex that is relatively stable under physiologic conditions.

[0115] Methods for determining whether a ligand binds to a protein and/or the affinity for a ligand to a protein are known in the art. For example, the binding of a ligand to a protein can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), isothermal titration calorimetry (ITC), or enzyme-linked

immunosorbent assays (ELISA).

[0116] Immunoassays which can be used to analyze immunospecific binding and cross-reactivity of the ligand include, but are not limited to, competitive and non- competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, and fluorescent immunoassays. Such assays are routine and well known in the art.

[0117] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or functional fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0118] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one“light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms“variable heavy chain,”“V_H,” or“VH” refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv , dsFv or Fab; while the terms“variable light chain,”“VL” or“VL” refer to the variable region of an immunoglobulin light chain, including of an Fv, scFv , dsFv or Fab.

[0119] Examples of antibody functional fragments include, but are not limited to, complete antibody molecules, antibody fragments, such as Fv, single chain Fv (scFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab)2' and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen (see, e.g., FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th ed.2001). As appreciated by one of skill in the art, various antibody fragments can be obtained by a variety of methods, for example, digestion of an intact antibody with an enzyme, such as pepsin; or de novo synthesis. Antibody fragments are often synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., (1990) Nature 348:552). The term "antibody" also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J. Immunol.148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al.( 1993), PNAS. USA 90:6444, Gruber et al. (1994) J Immunol.152:5368, Zhu et al. (1997) Protein Sci.6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res.53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

[0120] The term "diagnosis" refers to a relative probability that a disease (e.g. cancer, urinary tract infection, infection, or other disease) is present in the subject. Similarly, the term "prognosis" refers to a relative probability that a certain future outcome may occur in the subject with respect to a disease state. For example, in the context of the present invention, prognosis can refer to the likelihood that an individual will develop a disease (e.g. cancer, urinary tract infection, infection, or other disease), or the likely severity of the disease (e.g., duration of disease). The terms are not intended to be absolute, as will be appreciated by any one of skill in the field of medical diagnostics.

[0121] As used herein, a "diagnostically effective amount" of a composition described herein is an amount sufficient to produce a clinically useful characterization or measurement of a disease state, such as an infection or cancer, (e.g. in an individual, patient, human, mammal, clinical sample, tissue, biopsy). A clinically useful characterization or measurement of a disease state, such as an infection or cancer, (e.g. in an individual, patient, human, mammal, clinical sample, tissue, biopsy) is one containing sufficient detail to enable an experienced clinician to assess the degree and/or extent of disease for purposes of diagnosis, monitoring the efficacy of a therapeutic intervention, and the like.

[0122] The terms "disease" or "condition" refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. In embodiments, the disease is cancer (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma).

[0123] The compounds disclosed herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I), or carbon- 14 (¹⁴C). All isotopic variations of the compounds disclosed herein, whether radioactive or not, are encompassed within the scope disclosed herein.

[0124] “Subject,”“patient,”“subject in need thereof” and the like refer to a living organism suffering from or prone to a disease or condition that can be treated by administration of a compound or pharmaceutical composition, as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In embodiments, a subject is human.

[0125] As defined herein, the term“inhibition,”“inhibit,”“inhibiting” and the like in reference to a protein-inhibitor interaction means negatively affecting (e.g. decreasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the inhibitor. In embodiments inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g. an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein from an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation).

[0126] The terms“inhibitor,”“repressor” or“antagonist” or“downregulator” interchangeably refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein. The antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of the antagonist. An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even where there is no identified agonist. In embodiments, inhibitors are molecules that decrease, block, prevent, delay activation, inactivate, desensitize, or down-regulate, e.g., a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity. An“antagonist” is a molecule that opposes the action(s) of an agonist.

[0127] The term "associated" or "associated with" in the context of a substance or substance activity or function associated with a disease (e.g., diabetes, cancer (e.g. prostate cancer, renal cancer, metastatic cancer, melanoma, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma)) means that the disease (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. In embodiments, the disease or disorder includes cardiovascular disorders (e.g., stroke, coronary artery disease, heart disease), metabolic diseases (e.g., diabetes), renal disorders, and liver or kidney failures.

[0128] Compositions

[0129] In one aspect, provided herein is a viral composition. The viral composition includes (a) a whole viral particle comprising a charged protein coat that has a charged coat protein; (b) a first polymer electrostatically bound to the charged coat protein; and (c) a covalent linker linking the first polymer to a recognition moiety. In embodiments, the viral composition is a conjugated wrapped phage.

[0130] The term“conjugated wrapped phage” and the like as used herein means a bacteriophage (also referred to herein as a phage) which is in contact with a charged polymer which at least partially encircles or enfolds the phage charged protein coat as disclosed herein. In embodiments, the polymer in this context binds the phage non-covalently via e.g., electrostatic attraction between the charged protein coat of the phage and charges on the polymer. The charged protein coat includes a plurality of charged coat proteins in contact with the charged polymer. The plurality of charged coat proteins has the opposite charge of the charged polymer. In embodiments, a phage described herein is in contact with a charged polymer (e.g., a first polymer) that fully encircles or enfolds the phage charged protein coat as disclosed herein. In embodiments, the charged polymer (e.g., a first polymer) encircles 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times the charged protein coat.

[0131] The term“whole viral particle” as used herein, refers to a complete viral particle that includes the genetic material made from either DNA or RNA and a protein coat, also called the capsid, which surrounds and protects the genetic material. In embodiments, where appropriate, the whole viral particle includes; an envelope of lipids that surrounds the protein coat (e.g. when the viral particle is outside a cell). A protein coat or a capsid is the protein shell of a virus. A charged protein coat, as used herein, refers to a protein coat having either a net positive or a net negative electric charge. In embodiments, a charged protein coat has a net negative electric charge.

[0132] In embodiments, the whole viral particle is a whole bacteriophage (or phage) that includes the genetic material made from either DNA or RNA and a protein coat, also called the capsid, which surrounds and protects the genetic material. A protein coat or a capsid is the protein shell of the phage. A charged protein coat, as used herein, refers to a protein coat having either a net positive or a net negative electric charge. In embodiments, a charged protein coat has a net negative electric charge.

[0133] A coat protein, as used herein, refers to a protein within the capsid (or protein coat). A charged coat protein refers to a coat protein having either a net positive or a net negative electric charge. In embodiments, a charged coat protein has a net negative electric charge.

[0134] The term“covalent linker,”“linker,”“spacer” are used herein interchangeably and refer to a divalent chemical moiety attached at each end to the remainder of the compound. In embodiments, the covalent linker is -L¹-L²-L³-L⁴-L⁵-L⁶-.

[0135] In embodiments, L¹ is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or

unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl or -L^1A-L^1B-L^1C-L^1D-L^1E-L^1F-L^1G-L^1H-L^1I-L^1J-.

[0136] In embodiments, L^1A is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or

unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0137] In embodiments, L^1B is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or

unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0138] In embodiments, L^1C is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0139] In embodiments, L^1D is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0140] In embodiments, L^1E is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0141] In embodiments, L^1F is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0142] In embodiments, L^1G is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0143] In embodiments, L^1H is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0144] In embodiments, L^1I is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0145] In embodiments, L^1J is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0146] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^1A, L^1B, L^1C, L^1D, L^1E, L^1F, L^1G, L^1H, L^1I, and L^1J is not a bond

[0147] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^1A, L^1B, L^1C, L^1D, L^1E, L^1F, L^1G, L^1H, L^1I, and L^1J is a cleavable linker.

[0148] In embodiments, L² is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl or -L^2A-L^2B-L^2C-L^2D-L^2E-L^2F-L^2G-L^2H-L^2I-L^2J-.

[0149] In embodiments, L^2A is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0150] In embodiments, L^2B is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0151] In embodiments, L^2C is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0152] In embodiments, L^2D is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0153] In embodiments, L^2E is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0154] In embodiments, L^2F is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0155] In embodiments, L^2G is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0156] In embodiments, L^2H is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0157] In embodiments, L^2I is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0158] In embodiments, L^2J is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0159] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^2A, L^2B, L^2C, L^2D, L^2E, L^2F, L^2G, L^2H, L^2I, and L^2J is not a bond.

[0160] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^2A, L^2B, L^2C, L^2D, L^2E, L^2F, L^2G, L^2H, L^2I, and L^2J is a cleavable linker.

[0161] In embodiments, L³ is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl or -L^3A-L^3B-L^3C-L^3D-L^3E-L^3F-L^3G-L^3H-L^3I-L^3J-.

[0162] In embodiments, L^3A is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0163] In embodiments, L^3B is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0164] In embodiments, L^3C is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0165] In embodiments, L^3D is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0166] In embodiments, L^3E is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0167] In embodiments, L^3F is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0168] In embodiments, L^3G is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0169] In embodiments, L^3H is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0170] In embodiments, L^3I is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0171] In embodiments, L^3J is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0172] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^3A, L^3B, L^3C, L^3D, L^3E, L^3F, L^3G, L^3H, L^3I, and L^3J is not a bond.

[0173] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^3A, L^3B, L^3C, L^3D, L^3E, L^3F, L^3G, L^3H, L^3I, and L^3J is a cleavable linker.

[0174] In embodiments, L⁴ is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl or -L^4A-L^4B-L^4C-L^4D-L^4E-L^4F-L^4G-L^4H-L^4I-L^4J-.

[0175] In embodiments, L^4A is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0176] In embodiments, L^4B is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0177] In embodiments, L^4C is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0178] In embodiments, L^4D is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0179] In embodiments, L^4E is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0180] In embodiments, L^4F is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0181] In embodiments, L^4G is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0182] In embodiments, L^4H is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0183] In embodiments, L^4I is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0184] In embodiments, L^4J is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0185] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^4A, L^4B, L^4C, L^4D, L^4E, L^4F, L^4G, L^4H, L^4I, and L^4J is not a bond.

[0186] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^4A, L^4B, L^4C, L^4D, L^4E, L^4F, L^4G, L^4H, L^4I, and L^4J is a cleavable linker.

[0187] In embodiments, L⁵ is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl or -L^5A-L^5B-L^5C-L^5D-L^5E-L^5F-L^5G-L^5H-L^5I-L^5J-.

[0188] In embodiments, L^5A is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0189] In embodiments, L^5B is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0190] In embodiments, L^5C is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0191] In embodiments, L^5D is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0192] In embodiments, L^5E is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0193] In embodiments, L^5F is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0194] In embodiments, L^5G is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0195] In embodiments, L^5H is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0196] In embodiments, L^5I is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0197] In embodiments, L^5J is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0198] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^5A, L^5B, L^5C, L^5D, L^5E, L^5F, L^5G, L^5H, L^5I, and L^5J is not a bond.

[0199] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^5A, L^5B, L^5C, L^5D, L^5E, L^5F, L^5G, L^5H, L^5I, and L^5J is a cleavable linker.

[0200] In embodiments, L⁶ is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl or -L^6A-L^6B-L^6C-L^6D-L^6E-L^6F-L^6G-L^6H-L^6I-L^6J-.

[0201] In embodiments, L^6A is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0202] In embodiments, L^6B is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0203] In embodiments, L^6C is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0204] In embodiments, L^6D is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0205] In embodiments, L^6E is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0206] In embodiments, L^6F is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0207] In embodiments, L^6G is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0208] In embodiments, L^6H is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0209] In embodiments, L^6I is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0210] In embodiments, L^6J is a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, -S-, -S(O)₂NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0211] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^6A, L^6B, L^6C, L^6D, L^6E, L^6F, L^6G, L^6H, L^6I, and L^6J is not a bond.

[0212] In embodiments, at least one (e.g.1, 2, 3 or 4) of L^6A, L^6B, L^6C, L^6D, L^6E, L^6F, L^6G, L^6H, L^6I, and L^6J is a cleavable linker.

[0213] In embodiments, L¹ is substituted or unsubstituted heteroalkyl, L² is substituted or unsubstituted heteroaryl, L³ is substituted or unsubstituted heteroalkyl, L⁴ is substituted or unsubstituted heterocycloalkyl, L⁵ is a substituted or unsubstituted heteroalkyl, and L⁶ is a bond. [0214] In embodiments, L⁴ is

, where the carbon at the 3 position is covalently attached to L⁵.

[0215] In embodiments, L⁵ is–S-CH₂-CH(NH₂)-C(O)- or–S-CH₂-CH(C(O)OH)-NH-, wherein the sulfur of L⁵ is attached to L⁴.

[0216] In embodiments, L³ comprises a polyethylene glycol linker. In embodiments, polyethylene glycol linker comprises 2 to 150 oxyethylene units (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 oxyethylene units).

[0217] In some embodiments, each substituted group described for L¹, L², L³, L⁴, L⁵, L⁶, L^1A, L^1B, L^1C, L^1D, L^1E, L^1F, L^1G, L^1H, L^1I, L^1J, L^2A, L^2B, L^2C, L^2D, L^2E, L^2F, L^2G, L^2H, L^2I, L^2J, L^3A, L^3B, L^3C, L^3D, L^3E, L^3F, L^3G, L^3H, L^3I, L^3J, L^4A, L^4B, L^4C, L^4D, L^4E, L^4F, L^4G, L^4H, L^4I, L^4J, L^5A, L^5B, L^5C, L^5D, L^5E, L^5F, L^5G, L^5H, L^5I, L^5J, L^6A, L^6B, L^6C, L^6D, L^6E, L^6F, L^6G, L^6H, L^6I, and L^6J is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described for L¹, L², L³, L⁴, L⁵, L⁶, L^1A, L^1B, L^1C, L^1D, L^1E, L^1F, L^1G, L^1H, L^1I, L^1J, L^2A, L^2B, L^2C, L^2D, L^2E, L^2F, L^2G, L^2H, L^2I, L^2J, L^3A, L^3B, L^3C, L^3D, L^3E, L^3F, L^3G, L^3H, L^3I, L^3J, L^4A, L^4B, L^4C, L^4D, L^4E, L^4F, L^4G, L^4H, L^4I, L^4J, L^5A, L^5B, L^5C, L^5D, L^5E, L^5F, L^5G, L^5H, L^5I, L^5J, L^6A, L^6B, L^6C, L^6D, L^6E, L^6F, L^6G, L^6H, L^6I, and L^6J is substituted with at least one substituent group.

[0218] In some embodiments, each substituted group described for L¹, L², L³, L⁴, L⁵, L⁶, L^1A, L^1B, L^1C, L^1D, L^1E, L^1F, L^1G, L^1H, L^1I, L^1J, L^2A, L^2B, L^2C, L^2D, L^2E, L^2F, L^2G, L^2H, L^2I, L^2J, L^3A, L^3B, L^3C, L^3D, L^3E, L^3F, L^3G, L^3H, L^3I, L^3J, L^4A, L^4B, L^4C, L^4D, L^4E, L^4F, L^4G, L^4H, L^4I, L^4J, L^5A, L^5B, L^5C, L^5D, L^5E, L^5F, L^5G, L^5H, L^5I, L^5J, L^6A, L^6B, L^6C, L^6D, L^6E, L^6F, L^6G, L^6H, L^6I, and L^6J is substituted with at least one lower substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described for L¹, L², L³, L⁴, L⁵, L⁶, L^1A, L^1B, L^1C, L^1D, L^1E, L^1F, L^1G, L^1H, L^1I, L^1J, L^2A, L^2B, L^2C, L^2D, L^2E, L^2F, L^2G, L^2H, L^2I, L^2J, L^3A, L^3B, L^3C, L^3D, L^3E, L^3F, L^3G, L^3H, L^3I, L^3J, L^4A, L^4B, L^4C, L^4D, L^4E, L^4F, L^4G, L^4H, L^4I, L^4J, L^5A, L^5B, L^5C, L^5D, L^5E, L^5F, L^5G, L^5H, L^5I, L^5J, L^6A, L^6B, L^6C, L^6D, L^6E, L^6F, L^6G, L^6H, L^6I, and L^6J is substituted with at least one lower substituent group.

[0219] In some embodiments, each substituted group described for L¹, L², L³, L⁴, L⁵, L⁶, L^1A, L^1B, L^1C, L^1D, L^1E, L^1F, L^1G, L^1H, L^1I, L^1J, L^2A, L^2B, L^2C, L^2D, L^2E, L^2F, L^2G, L^2H, L^2I, L^2J, L^3A, L^3B, L^3C, L^3D, L^3E, L^3F, L^3G, L^3H, L^3I, L^3J, L^4A, L^4B, L^4C, L^4D, L^4E, L^4F, L^4G, L^4H, L^4I, L^4J, L^5A, L^5B, L^5C, L^5D, L^5E, L^5F, L^5G, L^5H, L^5I, L^5J, L^6A, L^6B, L^6C, L^6D, L^6E, L^6F, L^6G, L^6H, L^6I, and L^6J is substituted with at least one size-limited substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described for L¹, L², L³, L⁴, L⁵, L⁶, L^1A, L^1B, L^1C, L^1D, L^1E, L^1F, L^1G, L^1H, L^1I, L^1J, L^2A, L^2B, L^2C, L^2D, L^2E, L^2F, L^2G, L^2H, L^2I, L^2J, L^3A, L^3B, L^3C, L^3D, L^3E, L^3F, L^3G, L^3H, L^3I, L^3J, L^4A, L^4B, L^4C, L^4D, L^4E, L^4F, L^4G, L^4H, L^4I, L^4J, L^5A, L^5B, L^5C, L^5D, L^5E, L^5F, L^5G, L^5H, L^5I, L^5J, L^6A, L^6B, L^6C, L^6D, L^6E, L^6F, L^6G, L^6H, L^6I, and L^6J is substituted with at least one size-limited substituent group.

[0220] In embodiments, each of the plurality of charged coat proteins is a negatively charged coat protein. In embodiments, each of the plurality of charged coat proteins is a positively charged coat protein. In embodiments, each of the plurality of charged coat proteins includes one or more negatively charged amino acid residues. In embodiments, each of the plurality of charged coat proteins includes one or more Glu or one or more Asp residues. In embodiments, each of the plurality of charged coat proteins includes one or more Glu and one or more Asp residues. In embodiments, the one or more Glu or one or more Asp residues form part of the N-terminus of the charged coat protein. In embodiments, the charged coat protein is P8. [0221] In embodiments, a plurality of charged coat proteins includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 or more of charged coat proteins. In embodiments, the charged proteins of the plurality of charged coat proteins are the same. In embodiments, the charged coat proteins plurality of charged coat proteins are not the same.

[0222] The term "P8" or "P8 protein" as provided herein includes any of the recombinant or naturally-occurring forms of the viral coat protein P8 or variants or homologs thereof that maintain P8 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to P8). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring P8 polypeptide. In embodiments, P8 is the protein as identified by the NCBI sequence reference GI:402239556, homolog or functional fragment thereof.

[0223] In embodiments, the whole viral particle is a whole bacteriophage viral particle. In embodiments, the whole viral particle is an M13 filamentous phage.

[0224] In embodiments, the first polymer is a cationic polymer. In embodiments, the first polymer is an anionic polymer. Where the charged coat protein is electrostatically bound to a first polymer, the charged coat protein and the first polymer are connected through an ionic bond.

[0225] In embodiments, the first polymer is or includes a polypeptide. In embodiments, the polypeptide has a net positive charge. In embodiments, the polypeptide encompasses a polymer of lysine (aka oligolysine, e.g., 3 to 20 lysine, 4 to 19 lysine, 5 to 18 lysine, 6 to 17 lysine, 7 to 17 lysine, 8 to 16 lysine, 9 to 16 lysine, 10 to 15 lysine, 11 to 14 lysine, 12 to 14 lysine, 13 to 14 lysine, 14 lysine). In embodiments, the polypeptide includes a polymer of lysine. In embodiments, the polymer of lysine is K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, K14, K15, K16, K17, K18, K19, or K20. The term“AAx” refers in the usual and customary sense to a polymer of amino acid“AA” having“x” repeating amino acid units. Thus,“K2” refers to a lysine-lysine polymer or polymer portion (e.g. KK),“K₃” a lysine-lysine-lysine polymer or polymer portion (e.g. KKK), and so forth. In embodiments, the polymer of lysine includes 2 lysine residues (K2), 3 lysine residues (K3), 4 lysine residues (K4), 5 lysine residues (K5), 6 lysine residues (K6), 7 lysine residues (K7), 8 lysine residues (K₈), 9 lysine residues (K₉), 10 lysine residues (K₁₀),11 lysine residues (K₁₁), 12 lysine residues (K₁₂), 13 lysine residues (K₁₃), 14 lysine residues (K₁₄), 15 lysine residues (K₁₅), 16 lysine residues (K16), 17 lysine residues (K17), 18 lysine residues (K18), 19 lysine residues (K19) or 20 lysine residues (K20). In embodiments, the polymer of lysine includes14 lysine residues (K14).

[0226] In embodiments, the recognition moiety or ligand is a composition (e.g., atom, molecule, ion, molecular ion, compound, particle, protein, peptide, nucleic acid, oligosaccharide,

polysaccharide, small molecule) capable of binding (e.g. specifically binding) to another

complementary composition (e.g., analyte, polymer, protein, marker, small molecule, ligand, polysaccharide, aptamer, or other binder) to form a complex. In embodiments, a recognition moiety as provided herein may without limitation bind to biomolecules (e.g., hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors or their ligands)); whole cells or lysates thereof (e.g., prokaryotic (e.g., pathogenic bacteria), eukaryotic cells (e.g., mammalian tumor cells); viruses (e.g., retroviruses, herpesviruses, adenoviruses, lentiviruses and spores); chemicals (e.g., solvents, polymers, organic materials, small molecules); therapeutic molecules (e.g., therapeutic drugs, abused drugs, antibiotics); environmental pollutants (e.g., pesticides, insecticides, toxins).

[0227] In embodiments, the recognition moiety or ligand is a cell surface marker binding moiety. In embodiments, the recognition moiety is a cancer cell surface marker binding moiety. In embodiments, the recognition moiety is a prostate-specific membrane antigen (PMSA) binding moiety.

[0228] The term“cancer cell marker” as used herein, refers to a protein or a polypeptide derived from a cancer cell or tumor that can be used to identify the cancer cell. A number of cancer cell markers have been established. The recognition moiety of the viral composition described herein can be designed to bind any cancer cell marker that is located on the surface of the cancer cell (i.e., any cancer cell surface marker) known in the art. Exemplary cancer cell markers that can be recognized and bound by the recognition moiety of the viral composition described herein include, but are not limited to:

[0229] (a) where the tumor cell is a breast cancer cell, the antigen may be one of EpCAM

(epithelial cell adhesion molecule), Her2/neu (Human Epidermal growth factor Receptor 2), MUC-1, EGFR (epidermal growth factor receptor), TAG-12 (turnor associated glycoprotein 12), IGFl R (insulin-like growth factor 1 receptor), TACSTD2 (tumor associated calcium signal transducer 2), CD318, CD340, CD104, or N-cadherin;

[0230] (b) where the tumor cell is a prostate cancer cell, the antigen may be one of EpCAM, MUC-1, EGFR, PSMA (prostate specific membrane antigen), PSA (prostate specific antigen), TACSTD2, PSCA (prostate stem cell antigen), PCSA (prostate cell surface antigen), CD318, CD104, or N-cadherin;

[0231] (c) where the tumor cell is a colorectal cancer cell, the antigen may be one of EpCAM, CD66c, CD66e, CEA (carcinoembryonic antigen), TACSTD2, CK20 (cytokeratin 20), CD104, MUC-1, CD318, or N-cadherin;

[0232] (d) where the tumor cell is a lung cancer cell the antigen may be one or CK18, CK19, CEA, EGFR, TACSTD2, CD318, CD104, or EpCAM;

[0233] (e) where the tumor cell is a pancreatic cancer cell the antigen may be one of HSP70, mHSP70, MUC-1, TACSTD2, CEA, CD104, CD318, N-cadherin, or EpCAM1;

[0234] (f) where the tumor cell is an ovarian cancer cell the antigen may be one of MUC-1, TACSTD2, CD318, CD104, N-cadherin, or EpCAM;

[0235] (g) where the tumor cell is a bladder cancer cell, the antigen may be one of CD34, CD146, CD62, CD105, CD106, VEGF receptor (vascular endothelial growth factor receptor), MUC-1, TACSTD2, EpCAM, CD318, EGFR, 6B5 or Folate binding receptor;

[0236] (h) where the tumor cell is a cancer stem cell, the antigen may be one of CD133, CD135, CD 117, or CD34; and

[0237] (i) where the tumor cell is a melanoma cancer cell, the antigen may be one of the melanocyte differentiation antigens, oncofetal antigens, tumor specific antigens, SEREX antigens or a combination thereof. Examples of melanocyte differentiation antigens, include but are not limited to tyrosinase, gp75, gpl00, MART 1 or TRP-2. Examples of oncofetal antigens include antigens in the MAGE family (MAGE-Al, MAGE-A4), BAGE family, GAGE family or NY–ESO1. Examples of tumor-specific antigens include CDK4 and 13-catenin. Examples of SEREX antigens include D-1 and SSX-2.

[0238] In embodiments, the recognition moiety is a polypeptide. In embodiments, the recognition moiety is a peptide having the sequence of CALCEFLG. In embodiments, the recognition moiety is a peptide having the sequence of SECVEVFQNSCDW. In embodiments, the recognition moiety is a polypeptide that is selected through a phage display library and this polypeptide selectively recognizes and binds to a cell surface marker. In embodiments, the recognition moiety encompasses an antibody, a variant or a fragment thereof, where the antibody (a variant or a fragment thereof) specifically recognizes and binds to a surface marker on a cell (e.g., a cancer cell).

[0239] In embodiments, cell is a target cell. The term“target cell” and the like refer, in the usual and customary sense, to a cell which can indicate a pathological condition or the potential for a pathological condition, e.g., a disease. In embodiments, the target cell expresses a surface marker for a disease, as disclosed herein. In embodiments, the target cell is a non-pathological cell, e.g., a normal cell, the identification of which is desired, e.g., within a biological sample.

[0240] In embodiments, the linker is attached to one end of the main backbone of the recognition moiety (e.g., -COOH group at the C-terminus of a peptide recognition moiety). In embodiments, the linker is attached to a side chain of the recognition moiety (e.g., a -COOH group of a side chain of a peptide recognition moiety).

[0241] In embodiments, provided herein are articles of manufacture or kits containing any compositions described herein (e.g. viral particle, phage, phage wrappers or linkers, ligands or recognition moieties, first polymer) and instructions for their use in the methods described herein.

[0242] In embodiments, the recognition moiety of each viral composition used in the method described herein is the same. In embodiments, the recognition moiety of each viral composition used in the method described herein is not the same. In embodiments, the recognition moiety of each viral composition is a same cell surface marker binding moiety. In embodiments, the recognition moiety of each viral composition is not a same cell surface marker binding moiety. In embodiments, the recognition moieties of the 2 or more viral compositions are not the same cell surface marker binding moieties, but they recognize and bind to the same cell surface marker. For example, they recognize and bind to different sites of the same cell surface marker. In embodiments, the recognition moieties of the 2 or more viral compositions recognize and bind to 2 or more different cell surface markers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more cell surface markers) of a cell (e.g., cancer cell).

[0243] In embodiments, the molar ratio of different types of recognition moieties used in the compositions/methods described herein is optimized. In embodiments, the optimization leads to a synergistic binding between the recognition moieties and the cell surface marker. In embodiments, the synergistic binding between the recognition moieties and the cell surface marker results in higher sensitivity and/or higher specificity of the method described herein.

[0244] In embodiments, methods described herein utilize two types of viral compositions that each includes one unique recognition moiety. In embodiments, the molar ratio of these two types of viral compositions used in the methods described herein is optimized. In embodiments, the ratio of two recognition moieties or two types of viral compositions is, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1.

[0245] In embodiments, the one or more viral compositions used in the methods described herein are immobilized to a solid support.

[0246] Methods for detecting a cell-viral composition complex are known in the art. In

embodiments, the detecting includes an antibody based reaction. In embodiments, the binding of a viral composition to a cell (i.e., the binding of a recognition moiety to a cell surface marker) can be detected and/or quantified using a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), isothermal titration calorimetry (ITC), or enzyme-linked

immunosorbent assays (ELISA).

[0247] Immunoassays which can be used to analyze immunospecific binding and cross-reactivity of the recognition moiety include, but are not limited to, competitive and non- competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, and fluorescent immunoassays. Such assays are routine and well known in the art.

[0248] In another aspect, there is provided a method for generating a viral composition described herein. The method includes (a) synthesizing a covalent linker comprising -L¹-L²-L³-L⁴-L⁵-L⁶-, where L¹, L², L³, L⁴, L⁵ and L⁶ are independently a bond, -O-, -C(O)O-, -C(O)-, -C(O)NH-, -NH-, - S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; (b) contacting the linker with a recognition moiety thereby forming an intermediate linker-recognition moiety conjugate; and (c) contacting the intermediate linker-recognition moiety conjugate with an intermediate wrapped phage thereby forming the viral composition, and the intermediate wrapped phage includes a whole viral particle having a charged protein coat that encompasses a charged coat protein and a first polymer electrostatically bound to the charged coat protein.

[0249] Polymer viral compositions and methods for making them are also described in US Publication No.2017/0097353, the disclosure of which is incorporate by reference herein.

[0250] Biosensors/Electrochemical Cells

[0251] In embodiments, the disclosure provides an electrochemical cell comprising a potentiostat electronically connected a first electrode and a second electrode, wherein the first electrode and the second electrode are coated with a viral composition, the viral composition comprising: (i) a whole viral particle comprising a charged protein coat, the charged protein coat comprising a plurality of charged coat proteins; (ii) a first polymer electrostatically bound to the plurality of charged coat proteins; and (iii) a covalent linker linking the first polymer to a recognition moiety.

[0252] With reference to FIGS.1A and 1C, the first and second electrodes are connected to the potentiostat for measurements by electrochemical impedance spectroscopy (EIS). The electrodes have contacts for connection to the potentiostat. In embodiments, the electrodes and contacts are metal. In embodiments, the electrodes and contacts are graphite (carbon). In embodiments, the metal electrodes are gold, platinum, silver, palladium, rhodium, lead, copper, or zinc. In

embodiments, the metal electrodes are gold. The first and second electrodes have a length (L) and a width (w), and are separated by a gap. The skilled artisan will appreciate that the length and width of the electrodes and the gap between them will be of an appropriate size for the intended use based on the teachings herein. In embodiments, the length is from about 0.1 mm to about 5 mm; or from about 0.1 mm to about 4 mm. In embodiments, the length is from about 0.5 mm to about 3.5 mm; or about 1 mm to about 3 mm, or about 1.5 mm to about 2.5 mm; or about 2 mm. In embodiments, the width is from about 0.1 mm to about 2 mm; or from about 0.1 mm to about 1.5 mm; or from about 0.5 mm to about 1.55 mm; or from about 0.7 mm to about 1 mm; or from about 0.8 mm to about 0.9 mm; or about 0.85 mm.

[0253] In aspects, the gap between the electrodes is about 500 microns or less. In embodiments, the gap between the electrodes is about 475 microns or less. In embodiments, the gap between the electrodes is about 450 microns or less. In embodiments, the gap between the electrodes is about 425 microns or less. In embodiments, the gap between the electrodes is about 400 microns or less. In embodiments, the gap between the electrodes is about 375 microns or less. In embodiments, the gap between the electrodes is about 350 microns or less. In embodiments, the gap between the electrodes is about 325 microns or less. In embodiments, the gap between the electrodes is about 300 microns or less. In embodiments, the gap between the electrodes is about 275 microns or less. In embodiments, the gap between the electrodes is about 250 microns or less. In embodiments, the gap between the electrodes is about 225 microns or less. In embodiments, the gap between the electrodes is about 200 microns or less. In embodiments, the gap between the electrodes is from about 1 micron to about 200 microns. In embodiments, the gap between the electrodes is from about 1 micron to about 100 microns. In embodiments, the gap between the electrodes is from about 10 microns to about 90 microns.

[0254] In aspects, the gap between the electrodes is from about 20 microns to about 80 microns. In embodiments, the gap between the electrodes is from about 20.5 microns to about 79.5 microns. In embodiments, the gap between the electrodes is from about 21 microns to about 79 microns. In embodiments, the gap between the electrodes is from about 21.5 microns to about 78.5 microns. In embodiments, the gap between the electrodes is from about 22 microns to about 78 microns. In embodiments, the gap between the electrodes is from about 22.5 microns to about 77.5 microns. In embodiments, the gap between the electrodes is from about 23 microns to about 77 microns. In embodiments, the gap between the electrodes is from about 23.5 microns to about 76.5 microns. In embodiments, the gap between the electrodes is from about 24 microns to about 76 microns. In embodiments, the gap between the electrodes is from about 24.5 microns to about 75.5 microns. In embodiments, the gap between the electrodes is from about 25 microns to about 75 microns. In embodiments, the gap between the electrodes is from about 25.5 microns to about 74.5 microns. In embodiments, the gap between the electrodes is from about 26 microns to about 74 microns. In embodiments, the gap between the electrodes is from about 26.5 microns to about 73.5 microns. In embodiments, the gap between the electrodes is from about 27 microns to about 73 microns. In embodiments, the gap between the electrodes is from about 27.5 microns to about 72.5 microns. In embodiments, the gap between the electrodes is from about 28 microns to about 72 microns. In embodiments, the gap between the electrodes is from about 28.5 microns to about 71.5 microns. In embodiments, the gap between the electrodes is from about 29 microns to about 71 microns. In embodiments, the gap between the electrodes is from about 29.5 microns to about 70.5 microns. In embodiments, the gap between the electrodes is from about 30 microns to about 70 microns. In embodiments, the gap between the electrodes is from about 30.5 microns to about 69.5 microns. In embodiments, the gap between the electrodes is from about 31 microns to about 69 microns. In embodiments, the gap between the electrodes is from about 31.5 microns to about 68.5 microns. In embodiments, the gap between the electrodes is from about 32 microns to about 68 microns. In embodiments, the gap between the electrodes is from about 32.5 microns to about 67.5 microns. In embodiments, the gap between the electrodes is from about 33 microns to about 67 microns. In embodiments, the gap between the electrodes is from about 33.5 microns to about 66.5 microns. In embodiments, the gap between the electrodes is from about 34 microns to about 66 microns. In embodiments, the gap between the electrodes is from about 34.5 microns to about 65.5 microns. In embodiments, the gap between the electrodes is from about 35 microns to about 65 microns. In embodiments, the gap between the electrodes is from about 35.5 microns to about 64.5 microns. In embodiments, the gap between the electrodes is from about 36 microns to about 64 microns. In embodiments, the gap between the electrodes is from about 36.5 microns to about 63.5 microns. In embodiments, the gap between the electrodes is from about 37 microns to about 63 microns. In embodiments, the gap between the electrodes is from about 37.5 microns to about 62.5 microns. In embodiments, the gap between the electrodes is from about 38 microns to about 62 microns. In embodiments, the gap between the electrodes is from about 38.5 microns to about 61.5 microns. In embodiments, the gap between the electrodes is from about 39 microns to about 61 microns. In embodiments, the gap between the electrodes is from about 39.5 microns to about 60.5 microns.

[0255] In aspects, the gap between the electrodes is from about 40 microns to about 60 microns. In embodiments, the gap between the electrodes is from about 40.5 microns to about 59.5 microns. In embodiments, the gap between the electrodes is from about 41 microns to about 59 microns. In embodiments, the gap between the electrodes is from about 41.5 microns to about 58.5 microns. In embodiments, the gap between the electrodes is from about 42 microns to about 58 microns. In embodiments, the gap between the electrodes is from about 42.5 microns to about 57.5 microns. In embodiments, the gap between the electrodes is from about 43 microns to about 57 microns. In embodiments, the gap between the electrodes is from about 43.5 microns to about 56.5 microns. In embodiments, the gap between the electrodes is from about 44 microns to about 56 microns. In embodiments, the gap between the electrodes is from about 44.5 microns to about 55.5 microns.

[0256] In aspects, the gap between the electrodes is from about 45 microns to about 55 microns. In embodiments, the gap between the electrodes is from about 46.5 microns to about 54.5 microns. In embodiments, the gap between the electrodes is from about 46 microns to about 54 microns. In embodiments, the gap between the electrodes is from about 46.1 microns to about 53.9 microns. In embodiments, the gap between the electrodes is from about 46.2 microns to about 53.8 microns. In embodiments, the gap between the electrodes is from about 46.3 microns to about 53.7 microns. In embodiments, the gap between the electrodes is from about 46.4 microns to about 53.6 microns. In embodiments, the gap between the electrodes is from about 46.5 microns to about 53.5 microns. In embodiments, the gap between the electrodes is from about 46.6 microns to about 53.4 microns. In embodiments, the gap between the electrodes is from about 46.7 microns to about 53.3 microns. In embodiments, the gap between the electrodes is from about 46.8 microns to about 53.2 microns. In embodiments, the gap between the electrodes is from about 46.9 microns to about 53.1 microns.

[0257] In aspects, the gap between the electrodes is from about 47 microns to about 53 microns. In embodiments, the gap between the electrodes is from about 47.1 microns to about 52.9 microns. In embodiments, the gap between the electrodes is from about 47.2 microns to about 52.8 microns. In embodiments, the gap between the electrodes is from about 47.3 microns to about 52.7 microns. In embodiments, the gap between the electrodes is from about 47.4 microns to about 52.6 microns. In embodiments, the gap between the electrodes is from about 47.5 microns to about 52.5 microns. In embodiments, the gap between the electrodes is from about 47.6 microns to about 52.4 microns. In embodiments, the gap between the electrodes is from about 47.7 microns to about 52.3 microns. In embodiments, the gap between the electrodes is from about 47.8 microns to about 52.2 microns. In embodiments, the gap between the electrodes is from about 47.9 microns to about 52.1 microns.

[0258] In aspects, the gap between the electrodes is from about 48 microns to about 52 microns. In embodiments, the gap between the electrodes is from about 48.1 microns to about 51.9 microns. In embodiments, the gap between the electrodes is from about 48.2 microns to about 51.8 microns. In embodiments, the gap between the electrodes is from about 48.3 microns to about 51.7 microns. In embodiments, the gap between the electrodes is from about 48.4 microns to about 51.6 microns. In embodiments, the gap between the electrodes is from about 48.5 microns to about 51.5 microns. In embodiments, the gap between the electrodes is from about 48.6 microns to about 51.4 microns. In embodiments, the gap between the electrodes is from about 48.7 microns to about 51.3 microns. In embodiments, the gap between the electrodes is from about 48.8 microns to about 51.2 microns. In embodiments, the gap between the electrodes is from about 48.9 microns to about 51.1 microns.

[0259] In aspects, the gap between the electrodes is from about 49 microns to about 51 microns. In embodiments, the gap between the electrodes is from about 49.1 microns to about 50.9 microns. In embodiments, the gap between the electrodes is from about 49.2 microns to about 50.8 microns. In embodiments, the gap between the electrodes is from about 49.2 microns to about 50.8 microns. In embodiments, the gap between the electrodes is from about 49.3 microns to about 50.7 microns. In embodiments, the gap between the electrodes is from about 49.4 microns to about 50.6 microns. In embodiments, the gap between the electrodes is from about 49.5 microns to about 50.5 microns. In embodiments, the gap between the electrodes is from about 49.6 microns to about 50.4 microns. In embodiments, the gap between the electrodes is from about 49.7 microns to about 50.3 microns. In embodiments, the gap between the electrodes is from about 49.8 microns to about 50.2 microns. In embodiments, the gap between the electrodes is from about 49.9 microns to about 50.1 microns. In embodiments, the gap between the electrodes is about 50 microns.

[0260] In embodiments, the gap between the electrodes is about 40 microns. In embodiments, the gap between the electrodes is about 40.5 microns. In embodiments, the gap between the electrodes is about 41 microns. In embodiments, the gap between the electrodes is about 42.5 microns. In embodiments, the gap between the electrodes is about 42 microns. In embodiments, the gap between the electrodes is about 42.5 microns. In embodiments, the gap between the electrodes is about 43 microns. In embodiments, the gap between the electrodes is about 43.5 microns. In embodiments, the gap between the electrodes is about 44 microns. In embodiments, the gap between the electrodes is about 44.5 microns. In embodiments, the gap between the electrodes is about 45 microns. In embodiments, the gap between the electrodes is about 45.5 microns. In embodiments, the gap between the electrodes is about 46 microns. In embodiments, the gap between the electrodes is about 46.5 microns. In embodiments, the gap between the electrodes is about 47 microns. In embodiments, the gap between the electrodes is about 47.5 microns.

[0261] In embodiments, the gap between the electrodes is about 48 microns. In embodiments, the gap between the electrodes is about 48.1 microns. In embodiments, the gap between the electrodes is about 48.2 microns. In embodiments, the gap between the electrodes is about 48.3 microns. In embodiments, the gap between the electrodes is about 48.4 microns. In embodiments, the gap between the electrodes is about 48.5 microns. In embodiments, the gap between the electrodes is about 48.6 microns. In embodiments, the gap between the electrodes is about 48.7 microns. In embodiments, the gap between the electrodes is about 48.8 microns. In embodiments, the gap between the electrodes is about 48.9 microns.

[0262] In embodiments, the gap between the electrodes is about 49 microns. In embodiments, the gap between the electrodes is about 49.1 microns. In embodiments, the gap between the electrodes is about 49.2 microns. In embodiments, the gap between the electrodes is about 49.3 microns. In embodiments, the gap between the electrodes is about 49.4 microns. In embodiments, the gap between the electrodes is about 49.5 microns. In embodiments, the gap between the electrodes is about 49.6 microns. In embodiments, the gap between the electrodes is about 49.7 microns. In embodiments, the gap between the electrodes is about 49.8 microns. In embodiments, the gap between the electrodes is about 49.9 microns.

[0263] In embodiments, the gap between the electrodes is about 50 microns. In embodiments, the gap between the electrodes is about 50.1 microns. In embodiments, the gap between the electrodes is about 50.2 microns. In embodiments, the gap between the electrodes is about 50.3 microns. In embodiments, the gap between the electrodes is about 50.4 microns. In embodiments, the gap between the electrodes is about 50.5 microns. In embodiments, the gap between the electrodes is about 50.6 microns. In embodiments, the gap between the electrodes is about 50.7 microns. In embodiments, the gap between the electrodes is about 50.8 microns. In embodiments, the gap between the electrodes is about 50.9 microns. In embodiments, the gap between the electrodes is about 51 microns. In embodiments, the gap between the electrodes is about 51.1 microns. In embodiments, the gap between the electrodes is about 51.2 microns. In embodiments, the gap between the electrodes is about 51.3 microns. In embodiments, the gap between the electrodes is about 51.4 microns. In embodiments, the gap between the electrodes is about 51.5 microns. In embodiments, the gap between the electrodes is about 51.6 microns. In embodiments, the gap between the electrodes is about 51.7 microns. In embodiments, the gap between the electrodes is about 51.8 microns. In embodiments, the gap between the electrodes is about 51.9 microns.

[0264] In embodiments, the gap between the electrodes is about 52 microns. In embodiments, the gap between the electrodes is about 52.5 microns. In embodiments, the gap between the electrodes is about 53 microns. In embodiments, the gap between the electrodes is about 53.5 microns. In embodiments, the gap between the electrodes is about 54 microns. In embodiments, the gap between the electrodes is about 54.5 microns. In embodiments, the gap between the electrodes is about 55 microns. In embodiments, the gap between the electrodes is about 55.5 microns. In embodiments, the gap between the electrodes is about 56 microns. In embodiments, the gap between the electrodes is about 56.5 microns. In embodiments, the gap between the electrodes is about 57 microns. In embodiments, the gap between the electrodes is about 57.5 microns. In embodiments, the gap between the electrodes is about 58 microns. In embodiments, the gap between the electrodes is about 58.5 microns. In embodiments, the gap between the electrodes is about 59 microns. In embodiments, the gap between the electrodes is about 59.5 microns. In embodiments, the gap between the electrodes is about 60 microns.

[0265] It has been unexpectedly discovered that the gap between the electrodes, as described herein, allows for the production of sine waves that provide the optimal signal for the detection of biomolecules and analytes. In aspects, the sine wave between the electrodes described herein is between about 1 millivolts (mv) and about 30 millivolts (mv). In embodiments, the sine wave between the electrodes is between about 5 mv and about 25 mv. In embodiments, the sine wave between the electrodes is between about 6 mv and about 24 mv. In embodiments, the sine wave between the electrodes is between about 7 mv and about 23 mv. In embodiments, the sine wave between the electrodes is between about 8 mv and about 22 mv. In embodiments, the sine wave between the electrodes is between about 8.5 mv and about 21.5 mv. In embodiments, the sine wave between the electrodes is between about 9 mv and about 21 mv. In embodiments, the sine wave between the electrodes is between about 9.1 mv and about 20.9 mv. In embodiments, the sine wave between the electrodes is between about 9.2 mv and about 20.8 mv. In embodiments, the sine wave between the electrodes is between about 9.3 mv and about 20.7 mv. In embodiments, the sine wave between the electrodes is between about 9.4 mv and about 20.6 mv. In embodiments, the sine wave between the electrodes is between about 9.5 mv and about 20.5 mv. In embodiments, the sine wave between the electrodes is between about 9.6 mv and about 20.4 mv. In embodiments, the sine wave between the electrodes is between about 9.7 mv and about 20.3 mv. In embodiments, the sine wave between the electrodes is between about 9.8 mv and about 20.2 mv. In embodiments, the sine wave between the electrodes is between about 9.9 mv and about 20.1 mv. In embodiments, the sine wave between the electrodes is between about 10 mv and about 20 mv. In embodiments, the sine wave (current) is measured by electrochemical impedance spectroscopy.

[0266] The cell layer of the electrochemical cell comprises a non-conducting materal. In embodiments, the non-conducting material is an acrylic polymer, an acrylic copolymer, or a combination thereof. In embodiments, the non-conducting material is poly(methyl methacrylate). With reference to FIG.1B, the cell layer can comprise multiple layers which can comprise the same non-conducting material or different non-conducting material. In embodiments, the cell layer comprises multiple layers of the same non-conducting material

[0267] With reference to FIGS.1 and 14, the electrochemical cell comprises an inlet port and an outlet port, where the sample enters the inlet port, enters into a liquid holding cell over over the first and second electrodes, and then exits through the outlet port into a reservoir. The skilled artisan will appreciate that the liquid holding cell will have a volume appropriate for the intended use based on the teachings herein. In embodiments, the liquid holding cell has a volume from about 0.1 microliters to about 25 microliters; or from about 0.1 microliters to about 20 microliters; or from about 0.1 microliters to about 15 microliters; or from about 0.1 microliters to about 10 microliters; or from about 1 microliter to about 10 microliters; or from about 3 microliters to about 9 microliters; or from about 4 microliters to about 8 microliters; or from about 5 microliters to about 7 microlitesr; or from about 5.5 microliters to about 6.5 microliters. In embodiments, the liquid holding cell has a volume of about 3 microliters; or about 4 microliters; or about 5 microliters; or about 6 microliters; or about 7 microliters; or about 8 microliters; or about 9 microliters; or about 10 microliters. he skilled artisan will appreciate that the reservoir will have a volume appropriate for the intended use based on the teachings herein. In embodiments, the reservoir will have a volume from about 1 microliter to about 250 microliters; or from about 1 microliters to about 200 microliters; or from about 5 microliters to about 150 microliters; or from about 10 microliters to about 140 microliters; or from about 15 microliters to about 135 microliters; or from about 20 microliters to about 130 microliters; or from about 25 microliters to about 125 micrliters; or from about 30 microliters to about 120 microliters; or from about 35 microliters to about 115 microliters; or from about 40 microliters to about 110 microliters; or from about 45 microliters to about 105 microliters; or from about 50 microliters to about 100 microliters; or from about 55 microliters to about 95 microliters; or from about 60 microliters to about 90 microliters; or from about 65 microliters to about 85 microliters; or from about 70 microliters to about 80 microliters. In embodiments, the reservoir will have a volume of about 70 microliters, about 75 microliters, or about 80 microliters.

[0268] Methods

[0269] In embodiments, the disclosure provides methods of detecting a biomolecule in a liquid sample by (i) adding a liquid sample to the inlet of the electrochemical cell, (ii) measuring the current of the liquid sample by electrochemical impedence spectroscopy, thereby detecting the biomolecule in the sample. In embodiments, the liquid sample is a biological sample. In

embodiments, the biological sample is blood. In embodiments, the biological sample is urine. In embodiments, the biological sample is saliva. In embodiments, the biological sample is

cerebrospinal fluid. In embodiments, the biological sample is lacrimal fluid. In embodiments, the biological sample is nipple aspirate fluid. Any biomolecules can be detected by the methods described herein, and the skilled artisan can select a recognition moiety appropriate for the biomolecule that is to be detected. In embodiments, the biomolecule is a cancer cell marker.

[0270] In embodiments, the disclosure provides methods of detecting a biomolecule in a liquid sample by (i) adding a liquid sample to the inlet of the electrochemical cell, (ii) measuring the current of the liquid sample by electrochemical impedence spectroscopy; and (iii) comparing the current to a control to detect the presence of the biomolecule in the liquid sample. In embodiments, the liquid sample is a biological sample. In embodiments, the biological sample is blood. In embodiments, the biological sample is urine. In embodiments, the biological sample is saliva. In embodiments, the biological sample is cerebrospinal fluid. In embodiments, the biological sample is lacrimal fluid. In embodiments, the biological sample is nipple aspirate fluid. Any biomolecules can be detected by the methods described herein, and the skilled artisan can select a recognition moiety appropriate for the biomolecule that is to be detected. In embodiments, the biomolecule is a cancer cell marker.

[0271] In embodiments, the disclosure provides methods of detecting a biomolecule in a liquid sample by (i) adding a liquid sample to the inlet of the electrochemical cell, (ii) measuring the current of the liquid sample, thereby detecting the biomolecule in the sample. In embodiments, the liquid sample is a biological sample. In embodiments, the biological sample is blood. In embodiments, the biological sample is urine. In embodiments, the biological sample is saliva. In embodiments, the biological sample is cerebrospinal fluid. In embodiments, the biological sample is lacrimal fluid. In embodiments, the biological sample is nipple aspirate fluid. Any biomolecules can be detected by the methods described herein, and the skilled artisan can select a recognition moiety appropriate for the biomolecule that is to be detected. In embodiments, the biomolecule is a cancer cell marker.

[0272] In embodiments, the disclosure provides methods of detecting a biomolecule in a liquid sample by (i) adding a liquid sample to the inlet of the electrochemical cell, (ii) measuring the current of the liquid sample; and (iii) comparing the current to a control to detect the presence of the biomolecule in the liquid sample. In embodiments, the liquid sample is a biological sample. In embodiments, the biological sample is blood. In embodiments, the biological sample is urine. In embodiments, the biological sample is saliva. In embodiments, the biological sample is

cerebrospinal fluid. In embodiments, the biological sample is lacrimal fluid. In embodiments, the biological sample is nipple aspirate fluid. Any biomolecules can be detected by the methods described herein, and the skilled artisan can select a recognition moiety appropriate for the biomolecule that is to be detected. In embodiments, the biomolecule is a cancer cell marker.

EXAMPLES

[0273] Described herein is a point of care (PoC) biosensor that exploits electrodeposited bioaffinity layers that consist of a composite of virus particles with an electrically conductive polymer, poly(3,4-ethylenedioxythiophene) or PEDOT. Exemplary receptors in the biosensors are M13 virus particles. Peptides are“displayed” as fusions to the N-terminus of a subset of this virus’ major P8 coat proteins that compose the virus capsid. From libraries of more than 10¹⁰ unique sequences, the displayed peptide on the phage surface is selected based upon its target binding affinity and specificity.

[0274] Virus-PEDOT films provide a simple and reproducible method for immobilizing virus on an electrode that involves entraining it in a film of the conductive polymer PEDOT. Applicants have demonstrated two types of biosensors based upon this bioaffinity matrix: virus-PEDOT nanowires prepared using the lithographically patterned nanowire electrodeposition (LPNE) process (see, e.g., Arter, J. A. et al. (2012) Anal. Chem.84:2776-2783; Arter, J. A. et al. (2010) Nano. Lett. 10:4858-4862), and virus-PEDOT films on planar gold electrodes (see, e.g., Donavan, K. C. et al. (2011) Anal. Chem.83:2420-2424: Mohan, K. et al. (2013) J. Am. Chem. Soc.135:7761-7767). Biosensors based upon virus-PEDOT nanowires transduce target binding using the through- nanowire resistance (see, e.g., Arter, J. A. et al. (2012) Anal. Chem.84:2776-2783; Arter, J. A. et al. (2010) Nano. Lett.10:4858-4862) whereas films of virus-PEDOT use electrical impedance spectroscopy (EIS) without added redox species to transduce the binding of a target molecule to the virus-PEDOT composite (see, e.g., Donavan, K. C. et al. (2011) Anal. Chem.83:2420-2424: Mohan, K. et al. (2013) J. Am. Chem. Soc.135:7761-7767). Applicants have demonstrated that virus- PEDOT biosensors can detect prostate specific membrane antigen (PSMA), a 90 kDa glycoprotein that is a promising prostate cancer marker, with a limit-of-detection in synthetic urine of 0.50 nM concentration in synthetic urine (see, e.g., Mohan, K. et al. (2013) J. Am. Chem. Soc.135:7761- 7767). These experiments establish the current baseline capabilities for this technology in terms of its sensitivity and limit-of-detection. But conventional laboratory research grade gold electrodes, three-electrode potentiostats, and stand-alone reference and counter electrodes were employed in this prior work. Applicants have translated this sensing modality into manufacturable and minaturizable biosensor architectures that have excellent sensitivity, signal-to-noise, and sensor-to-sensor reproducibility.

[0275] Provide herein, the exemplary analyte of interest was human serum albumin (HSA, MW = 66.5 kDa) in urine. The skilled artisan will recognize that other analytes can be measured. HSA is a well-established urinary biomarker that can indicate a wide range of adverse health conditions such as stroke, coronary artery disease, heart disease, renal disease, and liver or kidney failure, especially for those with diabetes (see, e.g., Meigs, J. B. et al. (2002) Diabetes Care 25:977-983). In healthy adults, HSA is excreted in urine at a concentration below 20 mg/mL (or 300 nM) (see, e.g., Wu, H. Y. et al. (2014) JAMA. Intern. Med.174:1108-1115; Chavers, B. M. et al. (1994) Diabetes 43:441- 446; Gross, J. L. et al. (2005) Diabetes Care 28:164-176). HSA levels of 20 mg/L to 200 mg/L (300 nM to 3 µM) indicate microalbuminuria, a moderate increase in albumin related to a risk of kidney disease (see, e.g., Viberti, G. C. et al. (1982) Lancet 1:1430-1432; Watts, G. F. et al. (1986) Clin. Chem.32:1544-1548), and patients with HSA concentrations above 200 mg/L are diagnosed with macroalbuminuria (see, e.g., Wu, H. Y. et al. (2014) JAMA. Intern. Med.174:1108-1115; Jones, C. A. et al. (2002) Am. J. Kidney. Dis.39:445-459). Current dipstick tests are only sensitive to macroalbuminuria, a diagnosis that usually occurs when kidney disease has irreversibly progressed to kidney failure (see, e.g., Viberti, G. C. et al. (1982) Lancet 1:1430-1432). Early detection of microalbuminuria through routine screening is imperative for a successful treatment plan that can be implemented in the beginning stages of kidney disease. This need for an accessible urinalysis test for HSA that is able to detect HSA over the range from < 20 mg/L to > 200 mg/L corresponding to < 300 nM to >3 µM.

[0276] Abbreviations: HSA: human serum albumin, BSA: bovine serum albumin, QSM: quartz crystal microbalance, EIS: electrochemical impedance spectroscopy, DPV: differential pulse voltammetry, PCS: poly(chloromethyl)styrene, PEDOT: poly(3,4 thiophene)

[0277] Example 1 - Virus-poly(3,4 ethylenedioxythiophene) composite biosensor for human serum albumin.

[0278] The label-free detection of human serum albumin (HSA) in aqueous buffer is demonstrated using a simple, monolithic, two-electrode electrochemical biosensor. In this device, both millimeter- scale electrodes were coated with a thin layer of a composite containing M13 virus particles and the electronically conductive polymer poly(3,4 ethylenedioxy thiophene) (PEDOT). These virus particles, engineered to selectively bind HSA, served as receptors in this biosensor. The resistance component of the electrical impedance, Zre, measured between these two electrodes provided electrical transduction of HSA binding to the virus-PEDOT film. The analysis of sample volumes as small as 50 µL was made possible using a microfluidic cell. Upon exposure to HSA, virus-PEDOT films showed a prompt increase in Zre within 5 seconds and a stable Zre signal within 15 minutes. HSA concentrations in the range from 100 nM to 5 µM were detectable. Sensor-to-sensor reproducibility of the HSA measurement was characterized by a coefficient of variance (COV) ranging from 2-8% across this entire concentration range. In addition, virus-PEDOT sensors successfully detected HSA in synthetic urine solutions.

[0279] Materials. All chemicals and solvents were purchased from Sigma Aldrich and used as received, unless noted. Nichromix solutions (Godax Laboratories) were prepared in sulfuric acid (Macron Fine Chemicals) by package directions. Positive photoresist (Shipley S-1827) and developer MF-219 (Microchem Corporation), gold pellets (5 N purity, Kurt J. Lesker Co.), and chromium powder (3 N purity, American Elements) were used for the photolithography of gold films. Devices were O2 plasma cleaned using a basic plasma cleaner (PDC-32G, Harrick Plasma). Flow cells were designed and manufactured by Wainamics Inc., Fremont CA. Milli-Q UV water (ρ >18 MΩ cm) was used as the solvent for all aqueous solutions. Phosphate-buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 pH 8) was filtered through a 0.22 µM pore size membrane (Corning). The wash buffer was 0.1% Tween 20 (Fisher Scientific) in PBS.2 mg/mL of Casein in PBS was used as blocking solution. Human serum albumin (HSA) of purity >97% based on SDS-PAGE was used as received. Bovine serum albumin (BSA, Calbiochem Omnipur) was used as received. The buffer for all blank and analyte solutions used for EIS measurement contained 2 mg/mL casein and 0.1% Tween 20 (henceforth“tween”) in PBS buffer. Synthetic urine (Ricca Chemical Co.) solutions composed of 18.01 g/mol water, 60.05 g/mol urea, 58.44 g/mol sodium chloride, magnesium sulfate heptahydrate, and 147.02 g/mol calcium chloride dihydrate.

[0280] Phage Library Design and the Selection of HSA Binders. The procedures associated with design of the phage library and the selection of HSA binders from this library are described herein.

[0281] Device Fabrication. Gold-film electrodes on glass substrate were fabricated by

photolithography (FIG.7). 1 in. by 1 in. glass slides were soaked in nichromix solution overnight, rinsed with Millie Q-UV water, and dried with pure air. Each slide was spincoated with positive photoresist and baked in a 90 °C oven for 30 min. The slides were then patterned using a contact photomask, 365 nm UV light source, and alignment stage (Newport, 83210i-line, 4 s), developed (MF-319), and rinsed with Millie Q-UV water. A 2 nm thick layer of chromium followed by a 60 nm thick layer of gold were thermally evaporated onto the slides. The slides were then soaked in acetone and sonicated for 10 min to lift off the photoresist layer and subsequently rinsed with Millie Q-UV water. Each 1 in by 1 in slide contained three pairs of gold electrodes, and were cut into three separate devices.

[0282] Synthesis of Phage-PEDOT Films. Gold-film electrodes and flow cells were cleaned by O2 plasma for 10 min prior to electroplating. The flow cell was then mounted on the gold-film electrodes. A flame-cleaned platinum foil counter electrode-wrapped around a mercurous sulfate electrode (MSE) - was used during electropolymerization. Film growth occurred by cycling between 0.2 V and 0.8 using a PalmSens3 controlled by a PS-Trace software (PalmSens BV, Houten, Netherlands) at a scan rate of 20 mV/s in plating solution. Gold-film electrodes were first exposed to EDOT plating solution (12.5 mM LiClO4, 2.5 mM EDOT) for 2 cycles of electropolymerization. Electrodes were then exposed to phage-EDOT plating solution (8 nM M13 bacteriophage, 12.5 mM LiClO₄, 2.5 mM EDOT) and electropolymerized for 2 cycles. Electropolymerization of phage- EDOT was repeated with new phage-EDOT plating solution three times for a total of 8 cycles.

[0283] Electrochemical Impedance Spectroscopy. Various concentrations of HSA in run buffer (casein, tween, PBS) were prepared immediately prior to exposure of phage electrodes. Newly plated phage-PEDOT films were exposed to blocking solution (casein, PBS) for 15 min followed by rinsing with wash buffer (PBS, tween). The electrode was then rinsed three times with run buffer and allowed to equilibrate while monitoring the impedance signal over time. Equilibration was determined by having less than 1 Ω of change over a 2 min period. Once equilibration was reached, five consecutive EIS measurements were taken using a PalmSens3 controlled by a PS-Trace software (PalmSens BV, Houten, Netherlands). The amplitude of the applied voltage was 10 mV, and 50 data points were acquired spanning a frequency range of 5 Hz to 40 kHz. Phage electrodes were then exposed to HSA solutions in run buffer, monitored for equilibration, and five consecutive EIS measurements were collected. Independent electrodes were used for EIS measurements of HSA solutions and BSA solutions for a positive and negative response, respectively.

[0284] AFM and SEM Analysis. Scanning electron microscopy (SEM) was performed on uncoated films using a FEI Magellan 400L XHR SEM operating at 2 keV. Atomic force microscopy (AFM) images of PEDOT-only and phage-PEDOT films were acquired using an Asylum MFP-3D- SA atomic force microscopy (Asylum Research, Santa Barbara, CA) equipped with Olympus AC160TSAFM tips (Olympus) in laboratory ambient air. AC Mode AFM images were obtained over a 20 µm range at 512 x 512 pixels. Images and amplitude traces were analyzed using the Asylum image processing software. [0285] Results and Discussion

[0286] Electrodeposition and Characterization of virus-PEDOT Films. Applicants describe a biosensor that contained a pair of gold electrodes lithographically patterned onto a microscope slide, mated to a flow cell (FIG.1A). The flow cell, in three parts, was assembled on the sensor electrode before electrodeposition of the virus-PEDOT bioaffinity layer (FIG.1B). Each of the two gold electrodes is 1 mm x 3 mm, and the two electrodes were separated by 50 µm (FIG.1C). These electrodes spanned the 3 mm width of the flow channel from edge to edge, and were centered along its 2.3 mm length (FIG.1D). Plating and sample solutions were introduced into the 75 µL reservoir at top (FIG.1E), and the 6 µL volume of the flow channel quickly filled by capillary action. Both gold electrodes were modified with identical virus-PEDOT films that served as bioaffinity layers. The virus incorporated into the PEDOT film was engineered to selectively bind HSA with an affinity in the 10-100 nM range, as estimated using enzyme-linked immunosorbent assay (ELISA, FIG.8). (see, e.g., Sidhu, S. S. et al. (2000) Methods Enzymol.328:333-363)

[0287] The sensor operated without counter and reference electrodes, but these additional electrodes were used for the electrodeposition of virus-PEDOT films. Both virus-PEDOT films were electrodeposited simultaneously using an aqueous plating solution containing 8 nM virus, 2.5 mM EDOT, and 12.5 mM NaClO₄. Ten voltammetric scans from +0.80 V to +0.20 V vs. MSE (FIG.2A) were used for the preparation of each pair of films and the plating solution within the flow cell, which was quiescent during the deposition process, was replaced every two cycles. Electrodeposited virus-PEDOT films were a uniform dark blue in color (FIG.2C).

[0288] Scanning electron micrographs of pure PEDOT films prepared from the same plating solution without the addition of virus (FIG.2D) show a textured surface, dotted with 50-500 nm diameter protrusions. The apparent film thickness measured in the SEM is in the 200-300 nm range (FIG.2E). Virus-PEDOT films prepared from plating solution containing added 8 nM virus showed a surface with much greater roughness; the filamentous texture observed is characteristic of the virus-PEDOT composite (FIG.2F). At the film edge, SEM images acquired at high angular incidence of the electron beam show that a thin PEDOT layer of≈150-180 nm is present on the gold surface and virus particles protrude from this PEDOT base layer like a shag carpet (FIG.2G).

Several SEM images were obtained at random spots on the PEDOT-phage films and were visually identical, showing complete and uniform coverage over the gold electrodes. [0289] Refreshing the plating solution every two scans dramatically increased the amount of virus present in the resulting virus-PEDOT films, as qualitatively assessed from these SEM images (FIG. 2D, FIG.2F and FIG.8). Such difference in phage loading suggests that phage particles diffuse slowly to the surface of the electrode and are depleted within two cycles of deposition. An increase in phage loading enables high density of receptors on the phage-PEDOT sensors.

[0290] Atomic force microscopy (AFM) images of PEDOT only films prepared using this procedure (FIG.3A) allow for determination of the RMS surface roughness which is 15.596 nm. At the edges of the gold electrode (FIG.3A, right), the film thickness could be determined, and was in the range from 350– 450 nm– significantly thicker than measured in the high vacuum environment of the SEM. We attribute the difference to the removal of water associated with film swelling in the SEM whereas films imaged by AFM were not subjected to high vacuum and likely retain a greater water content as a result. AFM images of virus-PEDOT films (FIG.3B) show a much more pronounced topography and a greater RMS roughness of 101.150 nm. The apparent film thickness for these films (FIG.3B, right) was in the range from 550 to 650 nm, but this value included the PEDOT film and the protruding phage layer, which can not be distinguished in these images.

[0291] Detection of HSA in Buffer. Applicants begin by comparing the impedance response of virus-PEDOT biosensors in BSA and HSA in order to ascertain the degree to which HSA can be selectively detected. BSA and HSA are identical in size (66.5 kDa) and have 76% sequence homology so this comparison provides a challenging test for HSA selectivity (see, e.g., Huang, B. X. et al. (2004) J. Am. Soc. Mass Spectrom.15:1237-1247). Nyquist plots (Z_im vs. Z_re) for virus- PEDOT films immersed in run buffer (FIG.4A) shows behavior characteristic of a series RC circuit, as expected. An equivalent circuit for the virus-PEDOT film quantitatively accounting for these observations is presented in the Supporting Information. The addition of 500 nM of BSA to the buffer causes a slight, 1-4Ω, shift in this curve to higher Zre, but almost no change in Zim. For a different virus-PEDOT film (FIG.4B) a larger shift in Zre of 8-10Ω, is observed upon exposure to 500 nM HSA in buffer and a much smaller shift is seen in Z_im. In all subsequent discussion, we refer to the shifts in Zre and Zim relative to buffer as∆Zre and∆Zim. Plots of∆Zre and∆Zim versus frequency (FIG.4C and FIG.4D) show that∆Zre is superior to∆Zim for detecting HSA at 500 nM across the entire frequency spectrum from 5 Hz to 10 kHz. The error bars shown in these two plots represent the standard deviation of the mean for multiple impedance measurements (n = 5) on a single biosensor. The measurement-to-measurement dispersion in impedance seen both for∆Zre and ∆Zim is simply noise, and a signal-to-noise ratio (S/N) can therefore be calculated at each frequency point as:∆Z_re/σ_re and∆Z_im/σ_im (FIG.4E and FIG.4F) where σ_re is the standard deviation of∆Z_re across these five EIS measurements at each frequency. These plots show that for∆Zre (FIG.4E) a S/N of 50 is obtained at 500-600 Hz for HSA whereas BSA at the same 500 nM concentration and frequency is detected with a S/N of 1-3. For∆Zim, on the other hand (FIG.4E), both HSA and BSA produce a comparable S/N ranging from 10-50 across the frequency spectrum. The tentative conclusion is that measurement of∆Zre is superior to∆Zim for the detection of HSA under these conditions. Since∆Zre represents the change in resistance of the virus-PEDOT layer, and∆Zim, is the change in the quantity (ωC)^-1 where ω is the angular frequency and C is the capacitance the resistance of the virus-PEDOT bioaffinity layer, we conclude that the resistance of the virus-PEDOT layer is preferentially perturbed by HSA binding, relative to the capacitance.

[0292] The extension of these EIS measurements to a range of HSA concentrations from 50 nM to 2 µM (FIG.5) confirms that∆Zre increases monotonically with HSA concentration from 50 nM to 5 µM. The peak S/N of 20-140 for this response occurs in the range from 200-500 Hz. Below 100 Hz,∆Zim shows an even larger increase with increasing HSA concentration– as high as 50Ω - but noise is also more prominent and the resulting S/N is lower than seen for∆Zre. A more serious problem is that no single frequency provides reliable HSA quantitation across this HSA

concentration range using∆Z_im. In addition to sensitivity and signal-to-noise, the speed of biosensor response is also critically important. Real time measurements of∆Z_re at f = 340 Hz (FIG.6) show that increasing the HSA concentration in the flow cell causes a prompt increase in∆Z_re on the 5 s time scale followed by a slower increase in∆Z_re over the next 200 s or so. Using the slower of these two time-scales, a single measurement comprising exposure to buffer and then to sample can be accomplished in≈8-10 min.

[0293] The sensor-to-sensor reproducibility for HSA detection can be assessed by making repetitive measurements of HSA at a particular concentration using different sensors. The impedance versus frequency data shown in FIG.12 (for∆Z_re) show error bars representing coefficient-of-variation (COV) for measurements at three different biosensors at each concentration. For∆Z_re in the frequency range from 200-500 Hz, no overlap of these error bars occurs between the seven concentration plots, suggesting that the biosensor cleanly resolves these seven concentrations. COV values are minimized for all concentrations in the 200-500 Hz window (FIG.6D) and equal to 2-35 across this frequency range, and for HSA concentrations from 50 nM to 2 µM. For∆Z_im on the other hand, error bars overlap across the frequency spectrum (FIG.6B) and larger COV values apply (FIG.6E) demonstrating again that∆Zim is a less effective discriminator of HSA than∆Zre. [0294] While FIG.6A shows that∆Z_re progresses to higher values as the HSA concentration increases, one can ask whether this progression conforms to the Hill Equation, which is expected to model the sensor response (see, e.g., Kurganov, B. I. et al. (2001) Anal. Chim. Acta.427:11-19):

where∆Z_re,lim is the limiting∆Z_re seen at high HSA concentrations,∆Z_re,0 is the minimum value of ∆Z_re seen at low C_HSA, and K_D is the dissociation constant, which corresponds to the value of C_HSA at which∆Z_re = (∆Z_re,0−∆Z_re,lim)/2. The Hill coefficient, h, equals 1.0 when no cooperativity is present, it has a positive value when positive cooperativity is operating (that is, KD,app increases with HSA loading) and it has a negative value when negative cooperativity is indicated (see, e.g., Kurganov, B. I. et al. (2001) Anal. Chim. Acta.427:11-19). The R² value computed for the best 4- parameter fit of Eq. (1) to the data of FIG.6A is plotted in FIG.6C. R² when > 0.9 is seen for all frequencies below 800 Hz, and R² > 0.95 is observed in the frequency range 200-500 Hz (yellow). The calibration of phage-PEDOT biosensors will ultimately be determined at a single frequency. Based on the maximum signal-to-noise ratio, minimum COV, and maximum R² fits to the Hill equation, the frequency of choice is 340 Hz. Using f = 340 Hz, for example, the∆Z_re versus C_HSA calibration curve shown in FIG.6F is obtained. A summary of the Hill equation fit parameters for ∆Z_re is summarized in Table 1. The Hill coefficient of h = 1.0 ± 0.2 indicates that there is no cooperativity in phage-HSA binding, consistent with previous studies (see, e.g., Mohan, K. et al. (2013) J. Am. Chem. Soc.135:7761-7767).

[0295] Table 1. Parameters for the Best Fit of the Hill Equation (Eq.1) to HSA Calibration Curves Acquired in PBS Buffer and Synthetic Urine

[0296] Nonspecific adsorption is well-controlled by casein blocking of these virus-PEDOT films. Blue data points in FIG.6F are BSA while green and black data points represent measurements of HSA conducted using a Stop 4 phage which has no measurable affinity to HSA (green), and a pure PEDOT film containing no phage (black). Phage-PEDOT films do show slight non-specific binding to BSA. Therefore, a conservative limit of detection for phage-PEDOT films is 100 nM HSA.

Corresponding COV values for 100 nM - 5 µM HSA are within 2% - 8%. Nonspecific binding is attributed to electrostatic interactions between proteins and the positively charged PEDOT backbone. The isoelectric point of HSA and BSA is ~5, rendering the proteins negatively charged in solutions with pH values above 5 such as buffer solutions (pH=8) used in phage-HSA sensing. FIG.9 demonstrates that lowering the pH of buffer solution suppresses the negative charge on BSA and results in a decrease of non-specific binding with the inherently positive PEDOT-only film. The addition of casein blocking solution similarly inhibits non-specific adsorption compared to sensing in pH 5 buffer. While casein is an effective blocking agent for phage-PEDOT films, it can also block binding sights and suppress sensor response. Such effects are apparent in the observed KD of phage- PEDOT biosensors, which is slightly higher than K_D = 10 nM - 100 nM obtained by ELISA immunosorbent assays.

[0297] Detection of HSA in Synthetic Urine. To validate phage-PEDOT sensor capabilities for urine analysis, EIS measurements for HSA detection were repeated in synthetic, buffered urine. Casein blocking was not implemented in synthetic urine sensing experiments. Negative controls in synthetic urine show little non-specific binding in agreement with previous studies that show synthetic urine improves specificity (see, e.g., Mohan, K. et al. (2013) J. Am. Chem. Soc.135:7761- 7767: the entire contents of these publications are incorporated herein by reference in their entirety for all purposes). Urea disrupts protein interactions and mimics the blocking activity of casein which mitigates the non-specific binding of HSA and BSA. Phage-PEDOT sensors in synthetic urine demonstrated a concentration dependent response to HSA similar to buffer (FIG.13A). Sensor-to- sensor reproducibility in synthetic urine is maintained at <10 % COV centered around 100 Hz (FIG. 13B). Optimal frequency point for calibrating virus-PEDOT films in synthetic urine was 136 Hz (FIG.13C) based on the maximum signal-to-noise ratio, minimum COV, and maximum R² fits to the Hill equation (FIG.11). Although the impedance response in synthetic urine and buffer follow a similar trend, assessment of the Hill equation reveals significant differences in the fit parameters (Table 2). In synthetic urine sensitivity decreases by an order of magnitude and the Hill coefficient indicates negative cooperativity binding of virus-PEDOT films to HSA. We attribute this to interactions between urea and the PEDOT backbone that induce a reduction-like reaction. Amines participate in a nucleophilic attack on the delocalized positive charge on PEDOT, displacing charge carriers, and decreasing the conductivity of the PEDOT film. The two amine groups on urea enable such interactions with PEDOT to increase the film resistance and ultimately reduce efficiency in impedance sensing towards analytes (see, e.g., Hojati-Talemi, P. et al. (2013) Chem. Mater.

25:1837-1841).

[0298] Phage Library Design. The HSA binding phage was selected from a mega random peptide library (MRPL) created by pooling 24, individually constructed, peptide libraries (Table 2).

Individual peptide libraries contain five to 18 amino acids codons encoding the 20 naturally occurring amino acids or none or two cysteines. Each library has a theoretical peptide diversity of 10⁶ to 10²³, respectively. Among the library members, X₈ is the only designed linear library, and the other libraries are structurally constrained by disulfide bonds. The library of peptides is fused to the N-terminus of P8, which is localized in the oxidizing environment of the periplasm prior to assembly in the phage (see, e.g., Dottavio, D. (1996) CHAPTER 7 - Phagemid-Displayed Peptide Libraries. In: Phage Display of Peptides and Proteins. Burlington: Academic Press, 113-125; du Plessis, D. H. and Jordaan, F. (1996) CHAPTER 9 - Phage Libraries Displaying Random Peptides Derived from a Target Sequence. In: Phage Display of Peptides and Proteins. Burlington: Academic Press, 141-150).

[0299] Table 2. The MRPL components representing theoretical and actual peptide diversity.

[0300] A highly efficient site-directed mutagenesis method, Kunkel mutagenesis was used to clone libraries in the phagemid (see, e.g., Kunkel, T. A. et al. (1987) Methods in Enzymology 154:367- 382). The pooled libraries were electroporated, into SS320 cells, a strain of E. coli optimized for efficient electroporation (see, e.g., Sidhu, S. S. et al. (2000) Method Enzymol.328:333-363). The P8 phagemid, carries an antibiotic resistance marker, bacterial and phage origins of replication, and a phage packaging signal. Virions extruded from co-infected cells contain single-stranded

recombinant phagemid DNA, and display a mixture of recombinant and wild-type P8 proteins (see, e.g., Smith, G. P. (1985) Science 228:1315-1317). Phage-displayed peptides have a physical linkage between phenotype (the expressed peptide) and its encoding phagemid DNA. This linkage provides easy access to the DNA sequence of any MRPL phage-displayed peptide that binds a target of interest.

[0301] The cells were grown in 2YT media supplemented with carbenicilin (50 μg/ml) and tetracyclin (5 μg/ml), and infected with KO7 helper phage (10¹⁰ phage/mL) before growth in 2YT/carbenicilin media supplemented with kanamycin (20 μg/ml). The culture was shaken at 250 rpm for 16-18 h at 37 °C. To isolate the phage from the cells, the culture was centrifuged for 10 min at 10 krpm at 4 °C. The supernatant was decanted into separate tubes, and the phage was precipitated by addition of 1/5th volume of PEG-NaCl (2.5 M NaCl, 20% PEG-8000). The solution was placed on ice for 1 h. Next, the phage was recovered by centrifugation for 10 min at 10 krpm. The phage pellet was resuspended in phosphate-buffered saline (PBS, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.2) with addition of 0.05% Tween-20. After additional centrifugation for 10 min at 15 krpm, the phage precipitation step was repeated as described above. Phage were isolated by PEG-NaCl precipitation, and their concentration was determined by UV absorbance at 268 nm.

[0302] Selection of HSA Binders. In each of five rounds of selection, 15 wells of a 96-well microtiter plate (Nunc) were coated with 10 μg/mL HSA in 100 μL of PBS (pH 8.0), and incubated overnight at 4 °C on a shaker. After removal of the coating solution, 400 μL of a solution of 0.2% w/v casein in PBS was used to block the wells for 30 min on a shaker at room temperature. In successive rounds, the blocking reagent was switched to BSA, ovalbumin, or nonfat milk (NFM). The plate was then washed three times with 300 μL per well 0.05% Tween-20 in PBS. Phage were added to the wells in a buffer containing 0.2% w/v BSA, 0.05% Tween-20 in PBS. After 90 min incubation on a shaker at room temperature, the wells were washed with 0.05% Tween-20 in PBS. The numbers of washes increased with each round from 3, 5, 5, 7, and 9 times respectively for the five rounds. The bound phage was eluted by adding 100 μL of 0.1 M HCl and shaking vigorously at room temperature for 5 min. The phage were neutralized with 33 μL of 1 M Tris-HCl, pH 8.0. Before incubation for 45 min at 37 °C, 2 mL of the eluted phage was used to infect 20 mL of log phase E. coli XL-1 Blue cells. Helper phage KO7 was added at≈6X10¹² phage/mL, and after 45 min of incubation, the culture was transferred to 200 mL of 2YT supplemented with 50 μg/mL carbenicillin and 20 μg/mL kanamycin and shaken overnight at 37 °C for 16-18 h.

[0303] After 5 rounds of selection, spot assays were performed on around 200 selectants targeting HSA (10 μg/mL). The assay was performed based on a sandwich ELISA format in 96-well microtiter plates. Four potential HSA binders were obtained from the selections and spot assay. The four peptides were further examined for specificity to HSA. In the specificity assay, the selectants were screened against seven different proteins including kinases, membrane proteins, and high pI proteins. Furthermore, to increase accuracy, the binders were also screened against E. coli and mammalian cancer (fibroblasts, kidney, and prostate) cell lysates. From the selected variants, only two binders demonstrated high affinity and specificity to the HSA protein. [0304] The apparent Kd of both binders was calculated by dose–dependent ELISA. The phagemid containing the genes encoding P8 fused to either peptide 1 or 2 were transformed in CaCl2 competent E. coli XL-1 Blue cells. Similar steps were followed for the phage growth and precipitation as mentioned in the previous section. The phage concentration was determined by UV absorbance at 268 nm. For the ELISA, 96-well microtiter plates (Nunc) were coated with 10 μg/mL of HSA in PBS (pH 8.0), and incubated on a shaker at 4 °C overnight. The wells were blocked with 0.2% w/v solution of BSA in PBS at room temperature on a shaker for 30 min and washed three times with wash buffer (0.05% v/v Tween-20 in PBS). The phage were then serially diluted along with a negative control (Stop4 phage) in phage dilution buffer (0.2% w/v BSA, 0.05% v/v Tween-20 in PBS). The plates were incubated with the samples at room temperature on a shaker for 1 h and then washed five times with wash buffer. Anti-M13/HRP conjugate (GE Healthcare) was diluted 1:5000 in the phage dilution buffer, added to the wells, and incubated for 30 min on a shaker at room temperature. The wells were washed four times with wash buffer and once with PBS. 100 μL of a solution of 2 mg/mL o-phenylenediamine dihydrochloride, 0.02% w/v H2O2, in citric acid buffer (50 mM citric acid, 50 mM Na2HPO4, pH 5.0) was added to each well. After 10 min incubation, the absorbance at 450 nm was measured using a microtiter plate reader (Bio-Tek). The data was further analyzed by Prism (GraphPad) software, which estimated the apparent K_D for the binders. Since, the apparent KD of binder 2 (0.08 nM) was higher than binder 1 (45 nM), future experiments were conducted with binder 2. For incorporation of phage in the virus-PEDOT films, the phage pellet obtained after the above protocol was re-suspended in aqueous LiClO₄ (12 mM) solution.

[0305] The mega random peptide library (MRPL) from which HSA binders were selected was constructed from 24 individual peptide libraries. Each library was designed to be structured, yet as unbiased as possible, and thus provide potential binders for a wide range different targets. The individual libraries were designed to be structurally different using degenerate codons and varied placement of cysteine-based disulfide bonds. Each amino acid residue in the peptide was randomly assigned using NNS as the codon designation; where N is any nucleotide, and S is C or G. Thus, each degenerate position is designed to encode all 20 natural amino acids, termed“hard

randomization.” The codon choice prevents the occurrence of the non-suppressible stop codons TGA and TAA that could result in non-displayed peptides. Due to the degeneracy of the genetic code, some amino acids will be represented twice (A, G, P, T and V; in one-letter amino acid code) or three times (R, L and S). In addition, a TAG stop codon in each position could be encoded. In an amber suppressor strain of E. coli such as XL1 Blue used here, the TAG will be read as mixture of glutamine and a stop codon minimizing the impact of terminating translation to <50%.

[0306] The constrained libraries have random residues flanked by cysteines, which spontaneously form disulfide bonds creating constrained loops of two to ten amino acids. In addition, the libraries are constructed to limit the number of possible loop conformations to improve the overall free energy of binding compared with the unconstrained library. The design can increase binding affinity by limiting the entropic cost upon peptide binding to the target. Also, the X8 linear library is included to provide conformations missing from the constrained libraries. Therefore, engineering and using multiple primary libraries with unconstrained and constrained peptides forming large or small loops, MRPL has the diversity to yield productive results when applied to selections against a variety of targets. The theoretical diversity of the MRPL far exceeds the capability of any known system to accommodate full expression and maintenance of 10²⁴ individual library members.

[0307] The twenty-four libraries were mixed and subjected to thermodynamic or equilibrium- based selections for binding to HSA. The target protein, human serum albumin (HSA) was purchased from Sigma Aldrich as lyophilized powder, and was dissolved in PBS (pH 8.0) for rounds of selection. After bio panning targeted to HSA, a total of 200 selectants were screened from the different rounds of selections. Furthermore, the binders were tested for specificity to HSA against various proteins aurora kinase A (AKA), bovine serum albumin (BSA), non-fat milk, ovalbumin, hen egg white lysozyme (HEWL), and cav(1-104) (see, e.g., Majumdar, S. et al. (2011) J. Am.

Chem. Soc.133: 9855-9862) by phage-based ELISA. To increase stringency for specificity, cell lysates of E. coli and human cancerous cell lines (LnCAP, 3T3, 293T, and PC3) were also included in the specificity assay.

Table 3. Sequence of the binders selected after affinity maturation and specificity assay.

[0308] Two binders of 20 residues each were selected with high affinity and specificity to HSA (Table 3). Binder 1 and 2 emerged from round 5, with binder 2 being cysteine rich. Binder 2 had four cysteines, compared to binder 1, which had two cysteines (Table 3). This probably suggest that intramolecular disulfide bond formation within the peptide might have advantage in binding HSA to acquire binding energy. The apparent K_D of both the binders through phage-based ELISA was calculated in sub-nanomolar range. Binder 2, which exhibited the strongest relative affinity and specificity to HSA in the ELISA, was chosen for further bio-sensing studies.

[0309] Applicants describe herein a monolithic, two-electrode electrochemical biosensor for the label-free detection of HSA in PBS buffer and synthetic urine. This biosensor relies upon phage- PEDOT bioaffinity layer that are electrodeposited on both electrodes. An EIS measurement of the shift in Zre at an optimum frequency of≈300 Hz is then used to transduce the binding of HSA. HSA concentrations in a physiologically relevant range of 100 nM to 5 µM were detected using this biosensor. The resulting calibration curves are well-described by the Hill equation for receptor- ligand binding. These single-use biosensors exhibit a sensor-to-sensor reproducibility characterized by a coefficient-of-variation of 2-8% across the entire concentration range. It is also demonstrated that phage-PEDOT biosensors are capable of HSA quantitation in synthetic urine. This simple biosensor architecture is readily manufacturable, is compatible with small sample volumes (≈ 50 µL), and affords rapid analysis times (< 15 min). All of these attributes provide motivation for the further development of this and related biosensing technologies.

[0310] Advantages of the biorecognition element. Viruses are superior to antibodies due their robust nature, low production costs, and tunability while exhibiting binding affinities comparable to antibodies. Large batches of virus can be grown at minimal expenses, making it a cost effective biorecognition element for biosensors.

[0311] Advantages of the biosensor device design. Previous studies using virus-based biosensors depended on reference, counter, and working electrode. The requirement of a individual reference and counter electrode adds to the cost of the overall biosensor set up. The current biosensor design in composed of a glass slide and thin gold films and does not require a separate reference and counter electrode for sensing measurements. The fabrication of the current devices is simple and can be easily transformed into other inexpensive materials such as carbon electrodes. The addition of a flowcell, which is made from an inexpensive plastic, creates an overall durable sensor that is much less fragile and sensitive to handling compared to previous virus-based sensors. Due to the low costs of each device, these biosensors are designed for one time use in order to progress towards cheap, disposable, commercial biosensors.

[0312] Advantages of the virus-biosensor performance. In this study, we describe the label-free detection of human serum albumin (HSA) in aqueous buffer using a simple, monolithic, two- electrode electrochemical biosensor. In this device, both millimeter-scale electrodes are coated with a thin layer of the virus-PEDOT composite. The resistance component of the electrical impedance measured between these two electrodes, Z_re, provides electrical transduction of HSA binding to the virus-PEDOT composite. The analysis of sample volumes as small as 50 µL is made possible using a microfluidic cell. Upon exposure to HSA, virus-PEDOT films show a prompt increase in Zre within 5 s and a stable Z_re signal within 5 min. HSA concentrations in the range from 100 nM to 5 µM are detectable using this biosensor. Sensor-to-sensor reproducibility of the HSA measurement is characterized by a coefficient of variance (COV) ranging from 2-8% across this concentration range.

[0313] An additional characteristic of effective biosensors is high sensitivity in terms of signal response per concentration unit (Z_re / nM). Although phage-PEDOT films can differentiate between 100 nM, 300 nM, 500 nM, 1 µM, 2 µM, and 5 µM HSA, the sensitivity is not adequate for calibrating with 1 nM resolution. Sensitivity can be increased by either minimizing noise or enhancing the signal response. I will focus on the latter for phage-PEDOT sensors. The avenues to be explored for improving sensor signal is assessment of blocking solutions, introduction of a redox probe, and transition to a field-effect- transistor device. The blocking step is crucial in standard enzyme-linked immunosorbant assays and is dependent on the blocking agent, blocking

concentration, and blocking time. Casein protein is a common blocking agent of approximately 30 kDa in size. However, casein can also block active binding sites on phage-PEDOT films suppressing the overall signal response. It is worth exploring other blocking agents such as BSA, pepticase, and tween to investigate the effect of blocking non-specific binding versus masking active binding. Optimizing the blocking step in HSA detection could increase the sensor response by several ohms. A more significant enhancement in signal can be accomplished by adding a redox species and measuring the corresponding faradaic impedance. For example, the redox species Fe^III(CN)₆ ^3- / Fe^II(CN)6^4- can be added to buffer solutions to measure the reversible electron transfer between ferrocene and ferricinium ions. Phage-PEDOT films will sense how proteins bound to the electrode surface perturb the redox charge transfer. In comparison to non-faradaic impedance, faradaic currents are more sensitive and can amplify the binding response by 100 fold. Additionally, fundamental information about the electrochemistry can be elucidated in faradaic impedance measurements and nyquist plots are easier to fit to electrical circuit models. Another approach to improving HSA biosensors will be to apply phage-PEDOT films in field-effect-transistor (FET) based sensors. FET technology is a rapidly growing field producing sensors with fast response times, high sensitivity, label-free sensing, and tunable transduction for optimization of sensitivity and selectivity. FETs monitor charge carriers from a source terminal through a transistor of high input impedance and out of a drain terminal. A gate potential is applied to influence the conductivity of the channel between the source and drain. Charge carrier mobility is influenced by the gate voltage which can be in the form of a liquid electrolyte, termed liquid ion gated FET (LIG-FET). Channel conductivity is additionally perturbed when changes on the surface of the transistor occur, such as binding to a biomolecule. One challenge of LIG-FETs is the ability to incorporate a functionalized semiconductor film that can withstand liquid environments. Phage-PEDOT films provide an ideal bioaffinity transistor for FET with simple fabrication. Phage-PEDOT films are stable in liquid analytes, as demonstrated in this report, and will be configured onto an LIG-FET system using PBS buffer or synthetic urine as the liquid gate.

[0314] Table 4

[0315] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are incorporated by reference herein in their entirety for any purpose.

[0316] While various embodiments and aspects are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art. It should be understood that various alternatives to the embodiments described herein may be employed.

Claims

What is claimed is: 1. An electrochemical cell comprising a potentiostat electronically connected a first electrode and a second electrode, wherein the first electrode and the second electrode are coated with a viral composition, the viral composition comprising:

(i) a whole viral particle comprising a charged protein coat, the charged

protein coat comprising a plurality of charged coat proteins;

(ii) a first polymer electrostatically bound to the plurality of charged coat

proteins; and

(iii) a covalent linker linking the first polymer to a recognition moiety.

2. The electrochemical cell of claim 1, wherein the first electrode and the second electrode are separated by a space of about 40 micrometers to about 60 micrometers.

3. The electrochemical cell of claim 1, wherein the first electrode and the second electrode are separated by a space of about 45 micrometers to about 55 micrometers.

4. The electrochemical cell of claim 1, wherein the first electrode and the second electrode are separated by a space of about 49 micrometers to about 51 micrometers. 5. The electrochemical cell of claim 1, wherein the first electrode and the second electrode are separated by a space of about 49.5 micrometers to about 50.

5 micrometers.

6. The electrochemical cell of claim 1, wherein the first electrode and the second electrode are separated by a space of about 50 micrometers.

7. The electrochemical cell of claim 1, wherein the covalent linker is -L¹-L²-L³- L⁴-L⁵-L⁶-, wherein L¹, L², L³, L⁴, L⁵ and L⁶ are independently a bond, -O-, -C(O)O-, -C(O)-, - C(O)NH-, -NH-, -S-, -S(O)2NH-, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.

8. The electrochemical cell of claim 7, wherein L¹ is substituted or unsubstituted heteroalkyl; L² is substituted or unsubstituted heteroaryl; L³ is substituted or unsubstituted heteroalkyl; L⁴ is substituted or unsubstituted heterocycloalkyl; L⁵ is a substituted or unsubstituted heteroalkyl; and L⁶ is a bond.

9. The electrochemical cell of claim 8, L⁴ is:

wherein the carbon at the 3 position is covalently attached to L⁵.

10. The electrochemical cell of claim 9, wherein L⁵ is–S-CH₂-CH(NH₂)-C(O)- or –S-CH2-CH(C(O)OH)-NH-, wherein the sulfur of L⁵ is attached to L⁴.

11. The electrochemical cell of claim 7, wherein L³ comprises a polyethylene glycol linker.

12. The electrochemical cell of claim 11, wherein the polyethylene glycol linker comprises 2 to 150 oxyethylene units.

13. The electrochemical cell of claim 1, wherein the virus particle is M13 filamentous virus particle.

14. The electrochemical cell of claim 8, wherein the M13 filamentous virus particle is engineered to selectively bind a human serum albumin.

15. The electrochemical cell of claim 1, wherein the first polymer comprises a polypeptide.

16. The electrochemical cell of claim 15, wherein the polypeptide has a net positive charge.

17. The electrochemical cell of claim 15, wherein the polypeptide comprises a

18. The electrochemical cell of claim 17, wherein the polymer of lysine is K₂, K₃, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13, K14, K15, K16, K17, K18, K19, or K20.

19. The electrochemical cell of claim 17, wherein the polymer of lysine is K14.

20. The electrochemical cell of claim 1, wherein the first polymer is a net positively charged polymer.

21. The electrochemical cell of claim 1, wherein the first polymer is a net negatively charged polymer.

22. The electrochemical cell of claim 1, wherein the recognition moiety is a cell surface marker binding moiety.

23. The electrochemical cell of claim 22, wherein the cell surface marker is a cancer cell surface marker.

24. The electrochemical cell of claim 1, wherein the recognition moiety is a polypeptide.

25. The electrochemical cell of claim 24, wherein the polypeptide is an antibody or a fragment thereof.

26. The electrochemical cell of one of claims 1 to 25, further comprising a cell layer forming a liquid-holding cell capable of holding liquid; wherein the liquid-holding cell comprises a bottom portion comprising the first electrode and the second electrode.

27. The electrochemical cell of claim 26, wherein the liquid-holding cell is a flow cell comprising an inlet port and an outlet port within the cell layer.

28. The electrochemical cell of one of claims 1 to 27, wherein the first electrode and the second electrode comprise a metal or carbon.

29. The electrochemical cell of one of claims 1 to 27, wherein the first electrode

30. The electrochemical cell of one of claims 1 to 27, wherein the first electrode and the second electrode are adjacent to a solid support.

31. The electrochemical cell of claim 30, wherein the solid support comprises a non-conducting material.

32. The electrochemical cell of claim 30, wherein the solid support comprises glass.

33. The electrochemical cell of one of claims 26 to 32, wherein the cell layer comprises a non-conducting material.

34. The electrochemical cell of one of claims 26 to 32, wherein the cell layer comprises an acrylic polymer or an acrylic copolymer.

35. The electrochemical cell of one of claims 26 to 32, wherein the cell layer comprises poly(methylmethacrylate).

36. A biosensor comprising the electrochemical cell of any one of claims 1 to 35.

37. The biosensor of claim 36, further comprising a biological sample..

38. The biosensor of calim 37, wherein the biological sample is blood, urine, saliva, lacrimal fluid, nipple aspirate fluid, or cerebrospinal fluid.

39. A method of detecting a biomolecule in a sample, the method comprising: (i) contacting the first electrode and the second electrode of the electrochemical cell of any one of claims 1 to 35 with the sample;

(ii) measuring the current of the sample, thereby detecting the biomolecule in the sample.

40. The method of claim 39, wherein the current is measured by electrochemical impedance spectroscopy.

41. The method of claim 39 or 40, further comprising comparing the current to a control.

42. The method of any one of claims 39 to 41, wherein the sample is a biological sample.

43. The method of claim 42, wherein the biological sample is blood, urine, saliva, lacrimal fluid, nipple aspirate fluid, or cerebrospinal fluid.

44. The method of claim 42, wherein the biological sample is urine.

45. The method of any one of claims 39 to 44, wherein the biomolecule is a cancer cell marker.

46. The method of any one of claims 39 to 45, wherein the biomolecule is human serum albumin.

47. A diagnostic kit comprising the electrochemical cell of any one of claims 1 to 35 and instructions for use.

48. A diagnostic kit comprising the biosensor of claim 36.