CN103048462A - Multi-parameter electrochemical immunosensor based on electrode array and preparation method thereof - Google Patents

Abstract

The invention discloses a multi-parameter lung cancer electrochemical immunosensor based on an electrode array and a preparation method thereof, and relates to biosensor technology and bioelectronic technology. The sensor integrates an electrode array, a counter electrode, an electrochemical reference electrode, leads and an interface on a substrate. The sensor adopts micro-electro-mechanical system (MEMS) process to prepare the basic electrode array. The counter electrode and reference electrode are modified with silver and silver chloride (Ag|AgCl), and the electronic mediator and porous shielding film are fixed on the detection electrode. Nanomaterials, antibodies, etc. form a multi-parameter detection system. The sensor has the characteristics of good stability and convenient use, and can simultaneously detect multiple lung cancer markers, which is beneficial to clinical application.

Description

Multi-parameter electrochemical immunosensor based on electrode array and its preparation method

technical field

The invention relates to biosensors and bioelectronics, in particular to a multi-parameter electrochemical immunosensor based on an electrode array and a preparation method thereof.

Background technique

At present, lung cancer is the cancer with the highest incidence rate in the world, with 1.2 million new patients every year; the total number of lung cancer patients in my country ranks first in the world. In 2005, the data of our country's assessment of lung cancer showed that there were 500,000 new patients. Tumor markers are chemical substances that reflect the presence of tumors. They either do not exist in normal adult tissues but are only found in embryonic tissues, or their content in tumor tissues greatly exceeds that in normal tissues. Their existence or quantitative changes can indicate the nature of tumors, so as to understand the histogenesis and cell differentiation of tumors , cell function, to help tumor diagnosis, classification, prognosis judgment and treatment guidance. It is clinically found that tumor markers may exist in pleural effusion before tumor cells, and pleural effusion is mainly obtained during surgery. Therefore, detection of tumor markers in pleural effusion during surgery has important guiding significance for the diagnosis of lung cancer and postoperative intervention . There are many markers for lung cancer, and cancer, especially lung cancer, cannot be detected by relying on one alone. A joint detection of multiple parameters is required to make a diagnosis. Among them, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE) and cytokeratin fragment (CYFRA21-1) are common lung cancer markers. The positive rate of CEA for the diagnosis of lung cancer is 40%-50%, especially in lung adenocarcinoma, the positive rate and specificity are high; NSE is closely related to lung small cell carcinoma and is a marker of lung small cell carcinoma; CYFRA21-1 The positive rate in lung squamous cell carcinoma can be as high as 85.1%. The rapid on-site detection of these three tumor markers can provide an important reference for the early diagnosis of lung cancer. However, at present, large-scale instruments are used to detect these markers, and samples need to be sent to specialized laboratories, so rapid on-site detection cannot be achieved.

The immunoelectrochemical method combines immunoassay and electrochemical detection, and the specific combination of antigen and antibody can improve the specificity of detection. The use of electrochemical method has the advantages of simple operation and high sensitivity. Electrode arrays prepared by MEMS technology can form multi-parameter joint detection biosensors and rapidly detect multiple tumor markers at the same time. The sensor can detect the contents of multiple tumor markers sensitively, rapidly and reliably, thereby providing an accurate reference value for clinical tumor diagnosis, staging, observation of postoperative curative effect and prognosis.

Contents of the invention

The object of the present invention is to provide a multi-parameter electrochemical immunosensor for lung cancer based on electrode arrays and a preparation method thereof for the deficiencies of the above-mentioned prior art.

The present invention provides a multi-parameter electrochemical immunosensor based on an electrode array, which includes: a detection electrode array 1, an electrochemical reference electrode 2, a counter electrode 3, a lead wire 4, a socket 6 and a substrate; wherein the detection The electrode array 1 includes a plurality of detection electrodes, and the detection electrodes are immunoelectrochemical electrodes for detecting various disease markers.

The present invention also proposes a preparation method of the electrode array-based multi-parameter electrochemical immunosensor for lung cancer, the method comprising:

Step 1, using the plastic substrate as the substrate, etching the mask pattern of the detection electrode array, reference electrode and counter electrode on the protective film, and sputtering the transition layer and electrode layer thereon;

Step 2, spaced silver and silver chloride mixed paste on the reference electrode and the counter electrode to form an integrated silver and silver chloride reference electrode and counter electrode;

Step 3. Fix the electron mediator on the detection electrode in the detection electrode array to form an electron mediator film, use nano-gold particles as additives, and then use the perfluorinated sulfonic acid resin to form a film of the perfluorinated sulfonic acid resin. The gold hybrid material nafion-AuNPs film is fixed on the electron mediator film to form a porous shielding film mixed with nanomaterials;

Step 4. Immobilizing different cancer marker antibodies on the three detection electrodes.

The electrode array-based multi-parameter electrochemical immunosensor for lung cancer and the preparation method thereof provided by the present invention can realize multi-parameter simultaneous detection of lung cancer markers. The sensor has stable performance, fast detection speed and is easy to use.

Description of drawings

Fig. 1 is the structural diagram of the multi-parameter lung cancer electrochemical immunosensor based on the electrode array of the present invention;

Fig. 2 a is a schematic diagram of engraving the substrate protective film into the required electrode mask pattern in the present invention;

Figure 2b is a schematic diagram of sputtering a conductive film on the electrode mask pattern in the present invention;

Figure 2c is a schematic diagram of using stainless steel as a mask to form a reference electrode and a counter electrode;

Figure 2d is a schematic diagram of immobilizing electron mediators, porous shielding films and nanomaterials on the detection electrodes;

Figure 2e is a schematic diagram of immobilizing glutaraldehyde and antibodies on the detection electrode;

Fig. 2f is a schematic diagram of the structure of pasting an insulating film on the prepared electrode array-based multi-parameter electrochemical immunosensor for lung cancer.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

As shown in Figure 1, the present invention proposes a multi-parameter lung cancer electrochemical immunosensor based on an electrode array, which includes: a detection electrode array 1, an electrochemical reference electrode 2, a counter electrode 3, a lead wire 4, a socket 6 and a lining Bottom 5.

Wherein the detection electrode 1 is an immunoelectrochemical electrode, which is prepared by MEMS technology and used to detect disease markers; it is cleaned by ultrasonic or plasma methods, and electron mediators, porous shielding films, nanomaterials, antibodies, etc. are respectively fixed on the surface of the electrodes; Each detection electrode has a matching counter electrode 3 and an electrochemical reference electrode 2, the counter electrode is used to provide a return circuit, and the electrochemical reference electrode is used to provide a reference potential and keep the potential stable; the substrate The material used is an organic or inorganic insulating material with good biocompatibility, preferably one of silicon dioxide, silicon nitride, silicon oxynitride, SU8, polyimide or parylene; the electrodes 1～ The material of 3 is a metal or metal compound conductive film with good biocompatibility, preferably gold, platinum, titanium nitride or indium tin oxide.

With reference to accompanying drawing 2, the present invention also proposes a kind of preparation method of multi-parameter lung cancer electrochemical immunosensor based on electrode array, and its specific preparation process is described in detail as follows:

1) The protective film on the plastic substrate (130mm×100mm) as the substrate is engraved into the required electrodes, that is, the mask pattern 7 of the detection electrode array, the reference electrode and the counter electrode, as shown in Figure 2a; then sputter on it Sputter the transition layer, and then sputter the electrode layer (100nm), wherein, the transition layer is a metal layer such as chromium with a thickness between 10nm and 30nm, and the electrode layer can be a metal or metal oxide with a thickness of about 100nm, such as gold , platinum, titanium nitride or indium tin oxide, as shown in Figure 2b; after the mask 7 is removed, the plastic substrate is put into a plasma etching machine, and _O2 is introduced for cleaning; wherein the plasma etching The working air pressure of the etching machine is 3.0Pa, the flow rate of O ₂ is 40ml/min, and the flow rate of N ₂ is 120ml/min.

2) A stainless steel mask plate 81 is used to cover the etched detection electrode, and a mixed slurry of silver and silver chloride is screen-printed on the reference electrode and the counter electrode to form an integrated silver and silver chloride Ag|AgCl21 reference electrode. The ratio electrode and the counter electrode are shown in Figure 2c.

3) Use a stainless steel mask plate 81 to cover the reference electrode and the counter electrode, and use chitosan (CHIT) as an intermediate medium to fix the electron mediator (such as potassium ferricyanide) on the detection electrode to form an electron mediator film 11 ; Nano-gold particles are used as additives, and then the perfluorinated sulfonic acid resin nano-gold hybrid material nafion-AuNPs film is fixed on the electronic mediator film by the film-forming effect of perfluorinated sulfonic acid resin (nafion) to form a mixture of nanomaterials The porous shielding film 12, as shown in Figure 2d, adopts a three-dimensional structure to represent the electron mediator film 11 and the porous shielding film 12 successively formed on the detection electrode;

4) A stainless steel mask plate 83 is used to cover the reference electrode, the counter electrode and two of the detection electrodes, and only the remaining detection electrode is exposed, and the positively charged polyethyleneimine (PEI) is adsorbed by electrostatic adsorption. Form a PEI film on the surface of the nafion-AuNPs film of the negatively charged detection electrode, and then connect the PEI film and carcinoembryonic antigen (CEA) monoclonal antibody 14 through glutaraldehyde 13 to form a carcinoembryonic antigen (CEA) detection electrode, For the detection of carcinoembryonic antigen (CEA), as shown in Figure 2e.

5) Replace the stainless steel mask, repeat the previous step, and fix neuron-specific enolase (NSE) monoclonal antibody and cytokeratin fragment (CYFRA21-1) monoclonal antibody on the other two detection electrodes respectively, and complete the other steps. Modification of two detection electrodes for detecting neuron-specific enolase (NSE) and cytokeratin fragment (CYFRA21-1), respectively.

6) Place the prepared immunosensor in BSA solution to block the residual glutaraldehyde active groups on the immunosensor to avoid non-specific adsorption; then stick an insulating film 15 on the surface of the sensor ( FIG. 2 f ).

The invention also proposes a method for using the electrode array-based multi-parameter electrochemical immunosensor for lung cancer.

1) Preparation of reagents and samples.

Take 100ul of concentrated washing solution and dilute it with deionized water according to 1:100 for later use; pretreat the samples to be tested (blood, pleural effusion, sputum, saliva, semen, urine, ascites), etc., take the supernatant, Place at room temperature to equilibrate for 10-15min.

2) Operation steps.

Place the sensor electrode in the test solution for immunoreaction for 10 minutes, take it out; wash it repeatedly with diluted washing solution for 3 times; place the sensor in PBS solution, and connect it to the CHI electrochemical workstation through the socket (6) for detection , to obtain the concentration value of the analyte.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. multiparameter electrochemical immunosensor based on electrod-array, it comprises: detecting electrode array (1), galvanochemistry contrast electrode (2), to electrode (3), lead-in wire (4), socket (6) and substrate (5); Wherein, described detecting electrode array (1) comprises a plurality of detecting electrodes, and described detecting electrode is immune electrochemical electrode, for detection of the various diseases mark.

2. the multiparameter electrochemical immunosensor based on electrod-array according to claim 1, it is characterized in that: each electrode surface in the described detecting electrode array (1) all is fixed with electron mediator, porous screened film, nano material and antibody; Described detecting electrode adopts the preparation of MEMS technique, and adopts ultrasonic or plasma method is cleaned.

3. the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 1, it is characterized in that: each detecting electrode all has supporting to electrode (3) and galvanochemistry contrast electrode (2), wherein saidly be used for providing current return to electrode, described galvanochemistry contrast electrode is used for providing reference potential and keeps current potential stable.

4. the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 1, it is characterized in that: the employed material of described substrate is the organic or inorganic insulating material of good biocompatibility; Detecting electrode in the described detecting electrode array, galvanochemistry contrast electrode and electrode is selected metal or the metallic compound conductive film of good biocompatibility.

5. the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 4 is characterized in that: described substrate select silicon dioxide, silicon nitride, nitrogen-oxygen-silicon, SU8, polyimide or Parylene one of them; Described electrode select gold, platinum, titanium nitride or indium tin oxide one of them.

6. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array as claimed in claim 1, the method comprises:

Step 1, with plastic substrate as substrate, etching detecting electrode array, reference electrode and to the mask pattern of electrode on its diaphragm, and thereon sputter transition bed and electrode layer;

Step 2, the mixed slurry of interval silk-screen silver and silver chloride at contrast electrode and on to electrode form integrated silver and silver chloride contrast electrode and to electrode;

Step 3, electron mediator is fixed on forms the electron mediator film on the detecting electrode in the detecting electrode array, adopt nanogold particle as adjuvant, then the filming function by the perfluorinated sulfonic acid resin is with the perfluorinated sulfonic acid resin | and nm of gold composite material nafion-AuNPs film is fixed in the porous screened film that formation on the electron mediator film is mixed with nano material;

Step 4, on three detecting electrodes, fix respectively different disease marker antibody.

7. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 6, it is characterized in that: described electron mediator is the potassium ferricyanide.

8. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 6, it is characterized in that: in the described step 1, behind the complete transition bed of sputter and electrode layer, described plastic substrate is put into plasma etching machine, pass into O ₂And N ₂Clean, the operating air pressure of described plasma etching machine is 3.0Pa, the O that passes into ₂Flow is 40ml/ minute, N ₂Flow 120ml/ minute.

9. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 6, it is characterized in that, the disease marker antibody of fixing at detecting electrode in the described step 4 is cancer markers antibody, comprise carcinomebryonic antigen, neuronspecific enolase and cytokeratin fragment, its fixation procedure is as follows:

Step 41: adopt the stainless steel mask plate, cover contrast electrode, electrode is reached wherein two detecting electrodes, only expose a remaining detecting electrode, the way of utilizing Electrostatic Absorption forms the PEI film with the perfluorinated sulfonic acid resin surface that the polyethyleneimine PEI of positive electricity is adsorbed onto electronegative detecting electrode, connect described PEI film and Cea Monoclonal Antibodies by glutaraldehyde again, form a carcinomebryonic antigen detecting electrode, with for detection of carcinomebryonic antigen;

Step 42: change the stainless steel mask plate, repeat previous step, respectively fixedly neuronspecific enolase monoclonal antibody and neuronspecific enolase monoclonal antibody on other two detecting electrodes are respectively applied to detect neuronspecific enolase and neuronspecific enolase.

10. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 9, it is characterized in that, described on the detecting electrode respectively fixedly behind carcinomebryonic antigen, neuronspecific enolase and the cytokeratin fragment, the method also comprises:

Step 5, the described electrochemical immunosensor that will prepare place BSA solution to seal residual glutaraldehyde reactive group on it, and at its surface label upper nonconductive Film.

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