CN109097029B - Synthesis of a Silicon Nanoparticle/Au Nanocluster Ratio Fluorescent Probe and Its Application to Rifampicin Ratiometric Fluorescence Detection - Google Patents
Synthesis of a Silicon Nanoparticle/Au Nanocluster Ratio Fluorescent Probe and Its Application to Rifampicin Ratiometric Fluorescence Detection Download PDFInfo
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Abstract
本发明提供了一种硅纳米粒子/金纳米簇比率荧光探针的合成方法：首先采用水热还原法制备氨基保护的硅纳米粒子；然后采用搅拌还原法制备羧基保护的金纳米簇；最后将硅纳米粒子和金纳米簇进行自组装，合成比率荧光探针。本发明室温下自组装合成硅纳米粒子/金纳米簇比率荧光探针，过程简单、易操作、条件温和、耗时短。本发明提供的硅纳米粒子/金纳米簇比率荧光探针能够准确采用比率荧光方法检测利福平，将所述硅纳米粒子/金纳米簇比率荧光探针用于检测人血清中的痕量利福平，结果表明其回收率为97.0%～103.5%，且相对标准差(RSDs)均低于4%。
The invention provides a method for synthesizing a silicon nanoparticle/gold nanocluster ratio fluorescent probe: firstly, the amino-protected silicon nanoparticle is prepared by a hydrothermal reduction method; then the carboxyl-protected gold nanocluster is prepared by a stirring reduction method; Silicon nanoparticles and gold nanoclusters self-assemble to synthesize ratiometric fluorescent probes. The present invention self-assembles and synthesizes the silicon nanoparticle/gold nanocluster ratio fluorescent probe at room temperature, and has the advantages of simple process, easy operation, mild conditions and short time-consuming. The silicon nanoparticle/gold nanocluster ratio fluorescent probe provided by the present invention can accurately detect rifampicin by the ratio fluorescence method, and the silicon nanoparticle/gold nanocluster ratio fluorescent probe is used to detect trace amounts of rifampicin in human serum. Fuping, the results showed that the recoveries ranged from 97.0% to 103.5%, and the relative standard deviations (RSDs) were all lower than 4%.
Description
Technical Field
The invention relates to the technical field of fluorescent probes, in particular to synthesis of a silicon nanoparticle/gold nanocluster ratiometric fluorescent probe and application thereof to rifampicin ratiometric fluorescent detection.
Background
Rifampin (RIF), also known as rifamycin, vefluanid, mesona, rifampin or amitriptyline. The chemical name is 3- [ [ (4-methyl-1-piperazinyl) imino ] methyl ] -rifamycin. RIF is a broad-spectrum antibiotic drug belonging to rifamycin family, and has strong antibacterial effect on tubercle bacillus. RIF binds strongly to the beta subunit of DNA-dependent RNA polymerase, inhibiting synthesis of bacterial RNA, preventing the enzyme from linking to DNA, thereby blocking the RNA transcription process and stopping DNA and protein synthesis. The antibiotic has obvious bactericidal effect on mycobacterium tuberculosis and partial nontuberculous mycobacteria (including mycobacterium leprae, etc.) inside and outside host cells. RIF has good antibacterial effect on aerobic gram-positive bacteria, including Staphylococcus enzyme-producing strain and methicillin-resistant strain, Streptococcus pneumoniae, other Streptococcus, enterococcus, Listeria, Bacillus anthracis, Clostridium perfringens, diphtheria bacillus, anaerobic coccus, etc. However, when used as an effective drug for tuberculosis, RIF can cause damage to liver and kidney functions of a human body after being taken for a long time, and mental retardation and various skin syndromes can be caused by taking RIF excessively. Patients with primary liver disease, alcoholics, or patients taking other hepatotoxic drugs may cause death. The transport of RIF in humans is mainly dependent on serum proteins in blood, so it is essential to develop a method capable of detecting RIF in human serum with high selectivity and high sensitivity.
The existing methods for detecting the RIF trace mainly comprise a high performance liquid chromatography method, an electrochemical method, a nuclear magnetic resonance spectroscopy method and a spectrophotometry method. However, most of these methods require expensive equipment or much time for preliminary preparation. The existing fluorescence detection technology has the advantages of good selectivity, high sensitivity, easy operation, economy, practicability and the like, and is widely used in the fields of trace detection, fluorescence labeling and the like. The traditional fluorescent probe mainly comprises organic dye molecules, has the defects of low fluorescence brightness, poor light stability and light bleaching resistance and the like, and limits the further development and application of the fluorescence detection technology. The nanometer fluorescent probe which appears in recent years comprises fluorescent silicon nano particles (SiNPs), gold nano clusters (AuNCs) and the like, has the advantages of high fluorescence quantum yield, good light stability and photobleaching resistance, good biocompatibility, simplicity in synthesis and modification and the like, and becomes a novel fluorescent probe for replacing the traditional fluorescent probe. At present, the nano fluorescent probe is usually based on single fluorescence intensity change in the detection process, and the method is easily interfered by a detection instrument and a test environment. The novel nanometer ratiometric fluorescent probe with the ratiometric fluorescent characteristic detects a target object by adopting a fluorescence intensity ratio based on two different emission wavelengths, and can effectively eliminate the external interference factor by utilizing the self-calibration effect of ratiometric fluorescence. Therefore, the ratiometric fluorescent probe can be used for more accurately detecting the trace amount of RIF in human serum.
Disclosure of Invention
The invention aims to provide synthesis of a silicon nano particle/gold nano cluster (SiNPs/AuNCs) ratiometric fluorescent probe and application of the probe to RIF ratiometric fluorescence detection. The ratiometric fluorescent probe consists of two parts, namely amino-protected silicon nano particles (SiNPs) and carboxyl-protected gold nano clusters (AuNCs). The ratiometric fluorescent probe is directly self-assembled by SiNPs and AuNCs with different emission wavelengths, and has the advantages of simple process, easy operation, mild condition and short time consumption; the prepared SiNPs/AuNCs ratiometric fluorescent probe can be applied to trace detection of RIF by a ratiometric fluorescence method.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a synthetic method of a SiNPs/AuNCs ratiometric fluorescent probe, which comprises the following steps.
(1) Preparation of amino-protected SiNPs by hydrothermal method
(a) Mixing a water-soluble silicon source, a reducing agent and water to obtain a primary mixture;
(b) stirring the primary mixture in the step (a) at room temperature for 10-20 min to obtain a reaction precursor;
(c) and (c) putting the reaction precursor in the step (b) into a reaction kettle with a Teflon lining, wherein the volume of the reaction kettle is 20mL, and carrying out hydrothermal reaction at 220-240 ℃ for 1-3 h to obtain the SiNPs protected by amino groups.
(2) Preparation of carboxyl-protected AuNCs by stirring reduction method
(a) Mixing chloroauric acid (HAuCl)4·3H2O), mercaptoundecanoic acid (MUA) and water to obtain a primary mixture;
(b) adjusting the pH value of the primary mixture in the step (a) to 10-12 by using a pH regulator to obtain a reaction precursor;
(c) and (c) stirring the reaction precursor in the step (b) at room temperature, and reacting for 4-7 h to obtain the carboxyl protected AuNCs.
(3) Self-assembling the SiNPs and AuNCs in the steps (1) and (2) to synthesize the SiNPs/AuNCs ratiometric fluorescent probe
(a) Mixing SiNPs and AuNCs in a buffer solution to obtain a primary mixture;
(b) and (c) uniformly mixing the primary mixture in the step (a) at room temperature, and incubating for 0.5-2 h to obtain the SiNPs/AuNCs ratiometric fluorescent probe.
Preferably, the water-soluble silicon source used in step (1) comprises 3-Aminopropyltriethoxysilane (APTES) or 3-Aminopropyltrimethoxysilane (APTMS).
Preferably, the reducing agent used in step (1) comprises Ascorbic Acid (AA) or sodium citrate.
Preferably, in the step (1), the molar ratio of the reducing agent to the water-soluble silicon source is 1 (50-150), wherein the substance concentration of the reducing agent is 100 mM.
Preferably, the pH regulator used for adjusting the pH of the primary mixture in step (2) includes sodium hydroxide solution or potassium hydroxide solution.
Preferably, the concentration of the pH regulator is 1.0-1.2M.
Preferably, HAuCl is used in the step (2)4·3H2The molar ratio of O to MUA is 1 (4-8), and HAuCl in the final primary mixture4·3H2The concentration of the substance amount of O was 500. mu.M.
Preferably, the buffer solution in the step (3) comprises HEPES, Tris-HCl and PBS buffer solution, and the pH value is in the range of 7.0-8.0.
Preferably, the mass ratio of the SiNPs to AuNCs in the step (3) is 200:1, wherein the preferred mass concentration of the SiNPs is 4 mg/mL; the mass concentration of AuNCs is 20 mug/mL.
The SiNPs/AuNCs ratiometric fluorescent probe prepared by the preparation method provided by the invention comprises SiNPs and AuNCs, and is simple in process, easy to operate, mild in condition and short in time consumption. The preferable SiNPs/AuNCs ratiometric fluorescent probe synthesized based on the step (3) has the excitation wavelength of 300-310 nm, the emission wavelength of 440-445 nm and 610-615 nm, and the ratiometric fluorescent characteristic (as shown in FIG. 3).
The invention provides an application of a SiNPs/AuNCs ratio fluorescence probe in ratio fluorescence detection of RIF. AuNCs are protected and modified by MUA, and carboxyl functional groups contained in the MUA chelate RIF, so that the strongest fluorescence with the emission wavelength of about 610-615 nm in the SiNPs/AuNCs ratiometric fluorescent probe is caused (the fluorescent probe is protected by MUA)I 615) Quenching; and the most intense fluorescence with emission wavelength of about 440-445 nm (I 440) The intensity remains unchanged (as shown in fig. 4). Indicating that the SiNPs/AuNCs ratiometric fluorescent probes can adopt the ratiometric fluorescence method, i.e. the fluorescence intensity ratio (I 615/I 440) Can realize ratiometric fluorescence detection of RIF. Common anions, cations and 20 natural amino acids have no influence on the detection of RIF by the SiNPs/AuNCs ratiometric fluorescent probe, which indicates that the ratiometric fluorescent probe can realize specific identification and detection on RIF. The compound nano ratiometric fluorescent probe is used for detecting trace RIF in human serum, and the result shows that the recovery rate is 97.0-103.5%, and the Relative Standard Deviations (RSDs) are all lower than 4%.
Drawings
FIG. 1 is a bar graph showing the effect of molar ratio of AA to APTES solution on the fluorescence intensity of SiNPs in example 1;
FIG. 2 shows HAuCl in example 24·3H2Bar graph of the effect of O to MUA molar ratio on the fluorescence intensity of AuNCs;
FIG. 3 is an emission fluorescence spectrum of the ratiometric fluorescent probe SiNPs/AuNCs prepared in example 3;
FIG. 4 shows the fluorescence intensity ratio of the SiNPs/AuNCs ratiometric fluorescent probes prepared in example 3 (ratio of fluorescence intensity to fluorescence intensity of the SiNPs/AuNCs ratiometric fluorescent probes: (I 615/I 440) Fluorescence spectra as a function of RIF concentration;
FIG. 5 shows the fluorescence intensity ratio of the SiNPs/AuNCs ratiometric fluorescent probes after addition of 20 μ M RIF described in example 4 (ratio of fluorescence intensity to fluorescence intensity of the SiNPs/AuNCs ratiometric fluorescent probes after addition of 20 μ M RIF (R))I 615/I 440) Graph of the change with incubation time;
FIG. 6 shows the ratio of SiNPs/AuNCs fluorescence prepared in example 5Ratio of fluorescence intensity of the optical probe (1I 615/I 440) A graph of the relationship as a function of RIF concentration;
FIG. 7 is a bar graph showing the selectivity of the fluorescence response of SiNPs/AuNCs ratiometric fluorescent probes prepared in example 6 to RIF in positive and negative ions;
FIG. 8 is a bar graph of the selectivity analysis of the fluorescence response of SiNPs/AuNCs ratiometric fluorescent probes prepared in example 7 to RIF in 20 natural amino acids.
Detailed Description
The invention provides an application of a SiNPs/AuNCs ratio fluorescence probe in ratio fluorescence detection of RIF, which comprises the following steps:
(1) preparing SiNPs protected by amino groups by a hydrothermal reduction method;
(2) preparing carboxyl-protected AuNCs by adopting a stirring reduction method;
(3) and (3) carrying out self-assembly on the SiNPs and AuNCs in the steps (1) and (2) to synthesize the SiNPs/AuNCs ratiometric fluorescent probe.
In the step (1), a water-soluble silicon source, a reducing agent and water are mixed to obtain a primary mixture. In the invention, the molar ratio of the reducing agent to the water-soluble silicon source is preferably 1 (50-150); more preferably 1 (75-150); most preferably 1 (75-100), wherein the mass concentration of the reducing agent is 100 mM. In the present invention, the water-soluble silicon source comprises 3-Aminopropyltriethoxysilane (APTES) or 3-Aminopropyltrimethoxysilane (APTMS). In the present invention, the reducing agent includes Ascorbic Acid (AA) or sodium citrate. In the present invention, the water is preferably ultrapure water.
The method for mixing the water-soluble silicon source, the reducing agent and the water is not particularly limited, and the technical scheme of mixing materials, which is well known to those skilled in the art, can be adopted. In the present invention, it is preferable that the water-soluble silicon source and water are mixed first, and the obtained mixture is mixed with the reducing agent to ensure that the materials are fully mixed.
After a primary mixture is obtained, stirring the primary mixture at room temperature for 10-20 min to obtain a reaction precursor; and (3) putting the reaction precursor into a reaction kettle with a Teflon lining of which the volume is 20mL, and carrying out hydrothermal reaction for 1-3 h at 220-240 ℃ to obtain the SiNPs protected by amino groups. In the invention, the hydrothermal temperature is preferably 220-240 ℃, more preferably 225-240 ℃, and most preferably 235-240 ℃. The reaction time is preferably 1 to 3 hours, more preferably 2 to 3 hours, and most preferably 2.5 to 3 hours.
In the step (2) of the present invention, HAuCl is added4·3H2O, MUA and water to obtain a primary mixture. In the present invention, the HAuCl is4·3H2The mol ratio of O to MUA is preferably 1 (4-8); more preferably 1 (4-7); most preferably 1 (5-6), HAuCl in the final primary mixture4·3H2The concentration of the substance amount of O was 500. mu.M. In the present invention, the water is preferably ultrapure water.
The invention is directed to the HAuCl4·3H2O, MUA and water, and the method of mixing the materials is not particularly limited and may be any known method of mixing materials. In the present invention, HAuCl is preferably first used in the present invention4·3H2And mixing the O and the water, mixing the obtained mixture with MUA, and stirring for 10-15 min to ensure that the materials are fully mixed.
After the primary mixture is obtained, the pH value of the primary mixture is adjusted to 10-12, and a reaction precursor is obtained. In the present invention, the pH adjuster includes a sodium hydroxide solution or a potassium hydroxide solution. In the invention, the concentration of the pH regulator is preferably 1.0-1.2M; more preferably 1.0-1.1M; most preferably 1.0 to 1.05M.
And stirring the reaction precursor at room temperature, and reacting for 4-7 h to obtain the carboxyl-protected AuNCs. In the invention, the reaction time is preferably 4-7 h, more preferably 5-7 h, and most preferably 5-6 h.
In the present invention, the apparatus used for carrying out the hydrothermal reaction and the agitation reduction reaction is not particularly limited, and apparatuses for carrying out the hydrothermal reaction and the agitation reduction reaction, which are well known to those skilled in the art, may be used. Specifically, the hydrothermal reaction is carried out by adopting a reaction kettle with a Teflon lining, and a DF-101S heat collection type constant temperature stirrer is used as a stirring reduction reaction device.
After the reaction is completed, the present invention preferably performs post-treatment on the obtained reaction material to obtain solid phase SiNPs and AuNCs. In the present invention, the post-treatment preferably comprises the steps of: and (3) dialyzing and drying the obtained hydrothermal reaction material and the stirring reduction reaction material in sequence to respectively obtain solid-phase SiNPs and AuNCs. The invention preferably adopts a dialysis bag with 3500 Da molecular weight for the dialysis; the dialysis time is preferably 20-28 h, more preferably 22-26 h, and most preferably 22-24 h. In the present invention, the drying is preferably vacuum drying; the temperature of the vacuum drying is preferably 35-45 ℃, and more preferably 40 ℃; the vacuum drying time is preferably 10-24 hours, and more preferably 15-20 hours; the vacuum degree of the vacuum drying is preferably-0.1 MPa.
In the step (3), the solid phase SiNPs, AuNCs and buffer solution are mixed to obtain a primary mixture. In the invention, the mass ratio of the SiNPs to AuNCs is most preferably 200:1, wherein the mass concentration of the SiNPs is 4mg/mL, and the mass concentration of the AuNCs is 20 mu g/mL. The method for mixing the SiNPs, AuNCs and the buffer solution is not particularly limited, and the technical scheme of material mixing, which is well known to a person skilled in the art, is adopted. In the invention, the solid phase SiNPs and the buffer solution are preferably mixed firstly; and then mixed with AuNCs.
And uniformly mixing the primary mixture at room temperature, and incubating for 0.5-2 h to obtain the self-assembled SiNPs/AuNCs ratiometric fluorescent probe. In the invention, the buffer solution comprises HEPES, Tris-HCl and PBS buffer solution, and the pH value range is preferably 7.0-8.0; more preferably 7.0 to 7.5. Uniformly mixing and standing for 0.5-1 h preferably; more preferably 0.5 to 0.6 hour.
The invention provides a SiNPs/AuNCs ratiometric fluorescent probe prepared by the preparation method in the technical scheme, which comprises two fluorescent materials of SiNPs and AuNCs. The two fluorescent nanomaterials form ratiometric fluorescent probes with two different emission wavelengths by a self-assembly technique.
The invention provides the SiNPs/AuNCs ratio fluorescence in the technical schemeUse of a probe for ratiometric fluorescence detection of RIF. In the embodiment of the invention, the SiNPs/AuNCs ratiometric fluorescent probe (the SiNPs concentration is 4 mg/mL; the AuNCs concentration is 20 mu g/mL) is added into a HEPES buffer solution (the pH value is 7.4), then RIFs with different concentrations are respectively added, after incubation for 18 min, to-be-detected liquids with RIFs concentrations of 0, 0.1, 0.5, 1, 2, 3, 5, 10 and 20 mu M are obtained, and fluorescence test is carried out at room temperature (the excitation wavelength is 300 nm). At a ratio of fluorescence intensities at 615nm and 440 nm of (I 615/I 440) As an ordinate, the amount of substance concentration (C) of RIF, as an abscissa, a linear curve of the SiNPs/AuNCs ratio fluorescence probe for RIF detection was established (as shown in FIG. 6). The line curve is specificallyI 615/I 440= -0.034036C + 3.9377. Fluorescence intensity ratio at 440 nm and 615 nm: (I 615/I 440) The linear response of the substance quantity concentration (C) to the RIF is 0.1-5 mu M (R)2=0.99952), detection limit is 0.04 μ M.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The amounts of APTES for the different substances were mixed with 12 mL of water to give APTES solutions. Adding 30 μ L of AA with mass concentration of 100mM respectively to make molar ratio of AA to APTES 1:50, 1:75, 1:100 and 1:150 respectively, and stirring for 10 min; and (3) putting the mixture into a 20mL reaction kettle with a Teflon lining, and carrying out hydrothermal reaction at 240 ℃ for 3h to obtain the SiNPs fluorescent probe.
FIG. 1 is a bar graph of the effect of AA and APTES solution volume ratio on the fluorescence intensity of SiNPs. As can be seen from FIG. 1, the fluorescence intensity of the SiNPs fluorescent probe is highest when the molar ratio of AA to APTES is 1: 100.
Example 2
Different volume concentrations of 10 mM HAuCl4·3H2O was mixed with 10 mL of water to give HAuCl4·3H2O solution, adding different amounts of MUA and 100. mu.L of NaOH with a mass concentration of 1M to HAuCl4·3H2And the molar ratio of O to MUA is 1:4, 1:5, 1:6, 1:7 and 1:8 respectively, and stirring is carried out for 5 hours to obtain the AuNCs fluorescent probe.
FIG. 2 shows HAuCl4·3H2Bar graph of the effect of volume ratio of O to MUA solutions on AuNCs fluorescence intensity, as shown in FIG. 1, when HAuCl4·3H2The AuNCs fluorescence intensity was highest when the molar ratio of O to MUA solution was 1: 6.
Example 3
Adding the solid-phase SiNPs into a HEPES buffer solution (pH value is 7.4) to enable the mass concentration of the solid-phase SiNPs to be 4mg/mL, adding the solid-phase AuNCs to enable the mass concentration of the solid-phase AuNCs to be 20 mu g/mL, uniformly mixing, and incubating for 0.5 h to obtain the SiNPs/AuNCs ratiometric fluorescent probe.
FIG. 3 is a graph showing the fluorescence emission spectrum of the SiNPs/AuNCs ratiometric fluorescent probe. As can be seen from FIG. 3, the peak positions of the emission spectra of the SiNPs/AuNCs ratiometric fluorescent probes were respectively 440 nm and 615nm under an excitation spectrum having a wavelength of 300 nm.
Example 4
20 μ M RIF was added to HEPES buffer (pH 7.4) solution of SiNPs/AuNCs ratiometric fluorescent probes (wherein the concentration of SiNPs was 4 mg/mL; the concentration of AuNCs was 20 μ g/mL). The influence of different incubation times on the fluorescence intensity of the SiNPs/AuNCs ratio fluorescent probe is obtained.
FIG. 5 shows the fluorescence intensity ratio of the SiNPs/AuNCs ratiometric fluorescent probe (I 615/I 440) Graph of the change with incubation time after addition of 20 μ M RIF. As can be seen from FIG. 5, the fluorescence intensity ratio of the SiNPs/AuNCs ratiometric fluorescent probes (ratio of fluorescence intensity to fluorescence intensity of the SiNPs/AuNCs: (ratio of fluorescence intensity to fluorescence intensity of the SiNPs/AuNCs) increases with time (ratio of fluorescence intensity to fluorescence intensity of the SiNPs/AuNCI 615/I 440) The gradual decrease eventually stabilized after 18 min of incubation.
Example 5
HEPES buffer solution (pH 7.4) was added with the SiNPs/AuNCs ratiometric fluorescent probe (wherein the concentration of SiNPs is 4mg/mL, and the concentration of AuNCs is 4 mg/mL)20 mug/mL), then adding RIF with different concentrations respectively, obtaining liquid to be tested with RIF concentrations of 0, 0.1, 0.5, 1, 2, 3, 5, 10 and 20 muM respectively after incubation for 18 min, and carrying out fluorescence test (excitation wavelength is 300 nm) at room temperature. At a ratio of fluorescence intensities at 615nm and 440 nm of (I 615/I 440) As an ordinate, the amount of substance concentration (C) of RIF, as an abscissa, a linear curve of the SiNPs/AuNCs ratio fluorescence probe for RIF detection was established (as shown in FIG. 7). The line curve is specificallyI 615/I 440= -0.034036C + 3.9377. Fluorescence intensity ratio at 440 nm and 615 nm: (I 615/I 440) The linear response of the substance quantity concentration (C) to the RIF is 0.1-5 mu M (R)2=0.99952), detection limit is 0.04 μ M.
Example 6
20 μ M of different common ions (Na)+、K+、Ca2+、Mg2+、Zn2+、Cl-、NO3 -、SO4 2-、CO3 2-、PO4 3-) Respectively adding the mixture into SiNPs/AuNCs ratio fluorescent probe solution (wherein the concentration of the SiNPs is 4mg/mL, and the concentration of the AuNCs is 20 mu g/mL), and incubating for 18 min to obtain the influence of different ions on the RIF selectivity.
FIG. 7 is a bar graph showing the selectivity of the fluorescence response of SiNPs/AuNCs ratiometric fluorescent probes prepared in example 6 to RIF in positive and negative ions. As can be seen from FIG. 7, the selectivity of various common anions and cations on RIF is not significantly affected.
Example 7
Respectively adding 20 common natural amino acids (glycine Gly, alanine Ala, valine Val, leucine Leu, isoleucine Ile, phenylalanine Phe, proline Pro, tryptophan Trp, serine Ser, tyrosine Tyr, cysteine Cys, methionine Met, asparagine Asn, glutamine Gln, threonine Thr, aspartic acid Asp, glutamic acid Glu, lysine Lys, arginine Arg and histidine His) into the SiNPs/AuNCs ratio fluorescence probe solution (wherein the concentration of the SiNPs is 4mg/mL, the concentration of the AuNCs is 20 mu g/mL), wherein the concentration of the amino acids is 20 mu M, and incubating for 18 min to obtain the influence of different amino acids on the RIF selectivity.
FIG. 8 is a bar graph of the selectivity analysis of the fluorescence response of SiNPs/AuNCs ratiometric fluorescent probes prepared in example 7 to RIF in 20 natural amino acids. As can be seen from fig. 8, the 20 natural amino acids had no significant effect on the RIF fluorescence response.
Example 8
RIF (1.00, 2.00 and 4.00 mu M) with different concentrations are respectively added into human serum of a SiNPs/AuNCs ratiometric fluorescent probe (wherein the concentration of the SiNPs is 4mg/mL, and the concentration of the AuNCs is 20 mu g/mL) for object detection, and the RIF concentration in the sample I is 0.97 mu M, the recovery rate is 97 percent, and the relative standard deviation is 3.4 percent. The RIF concentration in the second detection sample is 2.07 mu M, the recovery rate is 103.5 percent, and the relative standard deviation is 2.6 percent. The RIF concentration in the third detection sample is 4.11 mu M, the recovery rate is 102.8 percent, and the relative standard deviation is 2.1 percent. And the Relative Standard Deviations (RSDs) of the three are all lower than 4 percent. Therefore, the SiNPs/AuNCs ratiometric fluorescent probe can be applied to material object detection (as shown in Table 1).
TABLE 1 use of SiO2Trace data of RIF in human serum measured by NPs/AuNCs ratiometric fluorescent probe
It should also be noted that the particular embodiments of the present invention are provided for illustrative purposes only and do not limit the scope of the present invention in any way, and that modifications and variations may be made by persons skilled in the art in light of the above teachings, but all such modifications and variations are intended to fall within the scope of the invention as defined by the appended claims.
Claims (7)
1. A preparation method of a silicon nanoparticle/gold nanocluster ratiometric fluorescent probe is characterized by comprising the following steps of:
(1) preparing amino-protected silicon nanoparticles by a hydrothermal reduction method: (a) mixing a water-soluble silicon source, a reducing agent and water to obtain a primary mixture; (b) stirring the primary mixture in the step (a) at room temperature for 10-20 min to obtain a reaction precursor; (c) putting the reaction precursor in the step (b) into a reaction kettle with a Teflon lining, the volume of which is 20mL, and carrying out hydrothermal reaction for 1-3 h at 220-240 ℃ to obtain silicon nano particles protected by amino;
(2) preparing the carboxyl-protected gold nanoclusters by adopting a stirring reduction method: (a) adding HAuCl chloroauric acid4·3H2Mixing O, MUA (mercapto undecanoic acid) and water to obtain a primary mixture; (b) adjusting the pH value of the primary mixture in the step (a) to 10-12 by using a pH regulator to obtain a reaction precursor; (c) stirring the reaction precursor in the step (b) at room temperature, and reacting for 4-7 h to obtain a carboxyl-protected gold nanocluster;
(3) self-assembling the silicon nano particles and the gold nano clusters in the steps (1) and (2) to synthesize the silicon nano particle/gold nano cluster ratiometric fluorescent probe: (a) mixing the silicon nano particles and the gold nano clusters in a buffer solution to obtain a primary mixture; (b) uniformly mixing the primary mixture in the step (a) at room temperature, and incubating for 0.5-2 h to obtain a silicon nanoparticle/gold nanocluster ratiometric fluorescent probe;
the molar ratio of the reducing agent to the water-soluble silicon source in the step (1) is 1 (50-150), wherein the mass concentration of the reducing agent is 100 mM; HAuCl in the step (2)4·3H2The molar ratio of O to MUA is 1 (4-8), and HAuCl in the final primary mixture4·3H2The concentration of the substance of O is 500. mu.M; the mass ratio of the silicon nano particles to the gold nano clusters in the step (3) is 200:1, wherein the mass concentration of the silicon nano particles is 4 mg/mL; the mass concentration of the gold nanoclusters is 20 mug/mL.
2. The method according to claim 1, wherein the water-soluble silicon source in step (1) comprises 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane APTMS; the reducing agent comprises ascorbic acid AA or sodium citrate.
3. The method according to claim 1, wherein the pH adjusting agent used for adjusting the pH of the initial mixture in the step (2) comprises sodium hydroxide solution or potassium hydroxide solution; the concentration of the pH regulator is 1.0-1.2M.
4. The method according to claim 1, wherein the buffer solution in the step (3) comprises HEPES, Tris-HCl and PBS buffer solution; the pH value of the buffer solution is 7.0-8.0.
5. The silicon nanoparticle/gold nanocluster ratiometric fluorescent probe prepared by the preparation method of any one of claims 1 to 4, which comprises silicon nanoparticles and gold nanoclusters chelated on the surfaces of the silicon nanoparticles.
6. The silicon nanoparticle/gold nanocluster ratiometric fluorescent probe of claim 5, wherein the silicon nanoparticle/gold nanocluster ratiometric fluorescent probe has ratiometric fluorescent characteristics with the strongest excitation wavelength of 300-310 nm, the strongest emission wavelengths of 440-445 nm and 610-615 nm.
7. Use of the silicon nanoparticle/gold nanocluster ratiometric fluorescent probe of claim 5 or 6 for ratiometric fluorescent detection of rifampicin.
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