CN117368289B - Method for coupling protein aptamer on surface of carbon electrode, carbon electrode and electrochemical immunosensor - Google Patents
Method for coupling protein aptamer on surface of carbon electrode, carbon electrode and electrochemical immunosensor Download PDFInfo
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 229910052799 carbon Inorganic materials 0.000 title claims abstract description 64
- 238000010168 coupling process Methods 0.000 title claims abstract description 30
- 230000008878 coupling Effects 0.000 title claims abstract description 27
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 27
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000012460 protein solution Substances 0.000 claims abstract description 12
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 claims abstract description 10
- MZMNEDXVUJLQAF-UHFFFAOYSA-N 1-o-tert-butyl 2-o-methyl 4-hydroxypyrrolidine-1,2-dicarboxylate Chemical compound COC(=O)C1CC(O)CN1C(=O)OC(C)(C)C MZMNEDXVUJLQAF-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 37
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 14
- 238000004140 cleaning Methods 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 10
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 7
- 229920000053 polysorbate 80 Polymers 0.000 claims description 7
- 235000011164 potassium chloride Nutrition 0.000 claims description 7
- 239000001103 potassium chloride Substances 0.000 claims description 7
- 238000002791 soaking Methods 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- QXYRRCOJHNZVDJ-UHFFFAOYSA-N 4-pyren-1-ylbutanoic acid Chemical compound C1=C2C(CCCC(=O)O)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 QXYRRCOJHNZVDJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000002041 carbon nanotube Substances 0.000 claims description 2
- 229910021393 carbon nanotube Inorganic materials 0.000 claims description 2
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 2
- 229910021389 graphene Inorganic materials 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims 2
- 150000001721 carbon Chemical class 0.000 claims 1
- 239000003575 carbonaceous material Substances 0.000 abstract description 28
- 230000009145 protein modification Effects 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 230000021523 carboxylation Effects 0.000 abstract description 5
- 238000006473 carboxylation reaction Methods 0.000 abstract description 5
- 239000000203 mixture Substances 0.000 abstract description 4
- 238000011534 incubation Methods 0.000 abstract description 3
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 34
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 31
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 31
- 102000004169 proteins and genes Human genes 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 238000009826 distribution Methods 0.000 description 10
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 150000007942 carboxylates Chemical group 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 238000010976 amide bond formation reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000018732 detection of tumor cell Effects 0.000 description 1
- 238000003487 electrochemical reaction Methods 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 108091008104 nucleic acid aptamers Proteins 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000003115 supporting electrolyte Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
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Abstract
The invention relates to a method for coupling protein aptamer on the surface of a carbon electrode, the carbon electrode and an electrochemical immunosensor, wherein the coupling method is based on a mode of carboxylation of N-hydroxysuccinimide ester of 1-pyrene butyric acid on the surface of a carbon material, and the effect of natural incubation coupling modification of protein on the surface of a conventional carbon material is effectively improved by optimizing the composition of a protein solution and combining direct current treatment. The carbon electrode obtained by the embodiment of the invention can be used in an electrochemical immunosensor.
Description
Technical Field
The invention belongs to the technical field of electrochemical immunosensors, and particularly relates to a method for coupling protein aptamer on the surface of a carbon electrode, the carbon electrode and an electrochemical immunosensor.
Background
The carbon material is often manufactured into an electrode required in an electrochemical reaction because of its characteristics of stable chemical properties and good conductivity, and its advantages of low cost and mass-production in industrialization. In biochemical detection, due to the characteristic that the carbon material is easy to functionalize and has a wide voltage window, the carbon electrode is often subjected to chemical modification on the surface of the material, and some immune active substances (such as antibody proteins, enzymes and the like) are coupled to the carbon electrode by means of chemical modification.
At present, the surface of a carbon material is subjected to carboxylation treatment to ensure that the surface of the carbon material has free carboxylate groups, wherein a plurality of carboxylation methods are available, and comprise: and (3) etching carbon elements by surface plasma to form carboxylate radicals, and adding substances (such as PEG marked with carboxyl) of the linking protein on the surface of the carbon material. The protein is coupled to the surface of the carbon material by amide bond formation with free carboxylate groups.
For example, patent application CN109507256a discloses a method for preparing a label-free electrochemiluminescence aptamer sensor for detecting carcinoembryonic antigen, and patent application CN103257167a discloses an electrode modification method for highly sensitive detection of tumor cells by using a nucleic acid aptamer. However, the above method only focuses on whether the protein is coupled to the surface of the carbon material, and does not focus on whether the number of coupling modifications of the protein on the carbon material is sufficient and the problem of aggregation of the protein on the surface of the carbon material.
Disclosure of Invention
Based on the method, the invention provides a method for coupling protein aptamer on the surface of a carbon electrode, so as to solve the problems of insufficient coupling modification quantity of the protein on a carbon material and agglomeration of the protein on the surface of the carbon material, thereby further pushing the technology to the commercial market.
In one aspect, the invention provides a method for coupling protein aptamer on the surface of a carbon electrode, comprising the following steps:
(1) Soaking a carbon electrode in a methanol solution of N-hydroxysuccinimide ester of 1-pyrene butyric acid, taking out, cleaning the surface by using a PBS solution, and naturally drying at room temperature;
(2) Dissolving a protein aptamer to be coupled in a mixed solution consisting of PBS, potassium chloride and Tween-80, regulating the pH value of the solution to be lower than the isoelectric point of the protein aptamer, and uniformly vortex-oscillating to obtain a protein solution;
(3) Taking the carbon electrode treated in the step (1) as a negative electrode, taking an untreated pure carbon electrode as a positive electrode, putting the pure carbon electrode into the protein solution obtained in the step (2) together, and applying direct current between the two electrodes for treatment;
(4) And (3) cleaning the carbon electrode treated in the step (3) by using a PBS solution, cleaning by using deionized water, and drying at room temperature.
The method is based on a mode of carboxylation of N-hydroxysuccinimide ester of 1-pyrene butyric acid on the surface of a carbon material, and the effect of coupling modification of protein on the surface of the carbon material is optimized by optimizing the composition of a protein solution and combining direct current treatment, and utilizing electrode migration to enable a protein aptamer and carboxylate to form an amide bond, so that the protein aptamer is coupled on the surface of the electrode of the carbon material. Wherein PBS in the protein solution is a protein buffer solution for adjusting pH; potassium chloride is used as a supporting electrolyte for coupling protein and carboxylate, tween-80 is used as a surfactant for preventing protein from precipitating and polymerizing in solution, and in addition, the pH of the protein solution is lower than the isoelectric point of the coupled aptamer, so that the aptamer can be positively charged, and electrode migration is convenient to realize.
The carbon electrode of the present invention may be a conventional electrode of various carbon materials; preferably, the carbon nanotube material electrode, graphene material electrode, glassy carbon electrode, or screen-printed carbon electrode.
Preferably, in the step (1), the concentration of the methanol solution of the N-hydroxysuccinimide ester of 1-pyrene butyrate is 5-10mmol/L, such as 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L, 10mmol/L and the like, the soaking time is more than or equal to 30min, and the concentration of the PBS solution is 0.01M.
Preferably, in the step (2), the mixed solution is prepared from 0.01M PBS, 0.1M potassium chloride and 0.01wt% Tween-80 according to a volume ratio of 1:1:1, and mixing.
Further preferably, in step (2), the concentration of the protein aptamer in the protein solution is 100 μg/mL.
Preferably, in the step (3), the direct current is supplied for more than or equal to 10min, such as 10min, 15min, 20min, 30min and the like. The voltage ranges from 0.1V to 1.0V, such as 0.1V,0.2V,0.3V,0.4V,0.5V,0.6V,0.7V,0.8V,0.9V,1V, etc.
Preferably, in step (4), the PBS solution has a concentration of 0.01M to 0.1M, such as 0.01M,0.02M,0.03M,0.04M,0.05M, 0.07M,0.08M,0.09M,0.1M, etc.
The invention also provides a carbon electrode prepared by the method for coupling the protein aptamer on the surface of the carbon electrode.
In still another aspect of the present invention, an electrochemical immunosensor prepared from the carbon electrode is provided.
Compared with the prior art, the invention has the following beneficial effects:
according to the method, based on the carboxylation mode of using the N-hydroxysuccinimide ester of 1-pyrene butyric acid on the surface of the carbon material, the effect of coupling modification of the protein on the surface of the carbon material is optimized by optimizing the composition of the protein solution and combining direct current treatment, so that the coupling quantity of the protein on the surface of the carbon material is more abundant, and the distribution of the modified protein on the surface of the carbon material is more uniform. The carbon electrode obtained by the method for coupling protein aptamer on the surface of the carbon electrode can be used in the application fields of electrochemical immunosensors and the like.
Drawings
FIG. 1 is a schematic diagram showing the coupling and distribution of the protein modification on the surface of a carbon electrode according to example 1 of the present invention;
FIG. 2 is a schematic diagram showing the coupling and distribution of the protein modification on the surface of the carbon electrode in comparative example 1;
FIG. 3 is a schematic diagram showing the coupling and distribution of the protein modification on the surface of the carbon electrode according to comparative example 2.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention.
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents, materials and equipment used in the examples are all commercially available sources unless otherwise specified; the test methods are conventional in the art unless otherwise specified.
Example 1
A carbon electrode, the surface of which is coupled with carcinoembryonic antigen CEA antibody, which is coupled with carcinoembryonic antigen CEA antibody by the following method, comprising the steps of:
(1) Soaking the screen-printed carbon electrode in 8mmol/L methanol solution of N-hydroxysuccinimide ester of 1-pyrene butyrate for more than 30min, taking out, cleaning the surface with 0.01M PBS solution, and naturally drying at room temperature;
(2) Under the condition of avoiding light, the carcinoembryonic antigen CEA antibody marked with FITC fluorescein is dissolved in a solution prepared from 0.01M PBS, 0.1M potassium chloride and 0.01 percent Tween-80 according to the volume ratio of 1:1:1, preparing a CEA antibody solution of 100 mug/mL in the mixed solution, and regulating the pH of the solution to 6.5 (the isoelectric point of the CEA antibody used in the experiment is 7.5, purchased from Beijing Yiqiao Shenzhou science and technology Co., ltd., batch number: CEA PAb:11077-RP 01), and carrying out vortex vibration uniformly;
(3) Immersing the carbon electrode treated in the step (1) in the CEA antibody solution treated in the step (2) to serve as a working electrode, inserting an untreated pure carbon electrode into the solution system, switching on a power supply between the two electrodes, switching on a power supply negative electrode, switching on a power supply positive electrode and switching on a direct current for 10min, and setting the voltage to be 0.5V;
(4) And (3) cleaning the carbon electrode treated in the step (3) by using a PBS solution with the concentration of 0.01M, cleaning by using deionized water, and drying at room temperature in a dark place.
The surface of the carbon electrode treated by the steps is placed under a forward fluorescence microscope to observe the modified coupling and distribution condition of fluorescent protein, the excitation wavelength is set to 488nm, the emission wavelength is set to 525nm, the observation result is shown in figure 1, and the surface of the carbon electrode is coupled to a carcinoembryonic antigen CEA antibody which is uniformly and abundantly.
Comparative example 1
A carbon electrode, the surface of which is coupled with carcinoembryonic antigen CEA antibody, which is coupled with carcinoembryonic antigen CEA antibody by the following method, comprising the steps of:
(1) Soaking the screen-printed carbon electrode in 8mmol/L methanol solution of N-hydroxysuccinimide ester of 1-pyrene butyrate for more than 30min, taking out, cleaning the surface with 0.01M PBS solution, and naturally drying at room temperature;
(2) Under the condition of avoiding light, dissolving CEA antibody of carcinoembryonic antigen marked with FITC fluorescein in 0.01M PBS solution to prepare 100 mug/mL CEA antibody solution, and uniformly vortex and shake the test solution with the pH of 7.5 (the experiment uses CEA antibody isoelectric point of 7.5, purchased from Beijing Yiqiao Shenzhou science and technology Co., ltd., batch number: CEA PAb:11077-RP 01);
(3) Immersing the carbon electrode treated in the step (1) in the CEA antibody solution treated in the step (2) to serve as a working electrode, inserting an untreated pure carbon electrode into the solution system, switching on a power supply between the two electrodes, switching on a power supply negative electrode, switching on a power supply positive electrode and switching on a direct current for 10min, and setting the voltage to be 0.5V;
(4) And (3) cleaning the carbon electrode treated in the step (3) by using a PBS solution with the concentration of 0.01M, cleaning by using deionized water, and drying at room temperature in a dark place.
The surface of the carbon electrode treated by the steps is placed under a forward fluorescence microscope to observe the modification coupling and distribution condition of fluorescent protein, the excitation wavelength is set to 488nm, the emission wavelength is set to 525nm, the observation result is shown in figure 2, and the CEA antibody is seriously agglomerated on the surface of the carbon electrode and is unevenly distributed.
Comparative example 2
A carbon electrode, the surface of which is coupled with carcinoembryonic antigen CEA antibody, which is coupled with carcinoembryonic antigen CEA antibody by the following method, comprising the steps of:
(1) Soaking the screen-printed carbon electrode in 8mmol/L methanol solution of N-hydroxysuccinimide ester of 1-pyrene butyrate for more than 30min, taking out, cleaning the surface with 0.01M PBS solution, and naturally drying at room temperature;
(2) Under the condition of avoiding light, the carcinoembryonic antigen CEA antibody marked with FITC fluorescein is dissolved in 0.01MPBS, 0.1M potassium chloride and 0.01 percent Tween-80 according to the volume ratio of 1:1:1, preparing a CEA antibody solution of 100 mug/mL in the mixed solution, and regulating the pH of the solution to 6.5 (the isoelectric point of the CEA antibody used in the experiment is 7.5, purchased from Beijing Yiqiao Shenzhou science and technology Co., ltd., batch number: CEA PAb:11077-RP 01), and carrying out vortex vibration uniformly;
(3) Immersing the carbon electrode treated in the step (1) in the CEA antibody solution treated in the step (2) for 10min;
(4) And (3) cleaning the carbon electrode treated in the step (3) by using a PBS solution with the concentration of 0.01M, cleaning by using deionized water, and drying at room temperature in a dark place.
The surface of the carbon electrode treated by the steps is placed under a forward fluorescence microscope to observe the modified coupling and distribution condition of fluorescent protein, the excitation wavelength is set to 488nm, the emission wavelength is set to 525nm, and the observation result is shown in figure 3, so that the CEA antibody of carcinoembryonic antigen is improved in the uniformity of the distribution of the carbon electrode, but the quantity is small.
As can be seen by comparing the effect patterns of CEA antibody modification under a fluorescence microscope, the CEA antibody protein modified on the surface of a carbon material by the method of the invention in example 1 has the greatest quantity and even distribution; in the embodiment 2, a conventional PBS solution is used as an antibody protein buffer solution, and the pH is not regulated, so that the CEA antibody is modified on the surface of a carbon material electrode to form aggregation, and the distribution is uneven; the CEA antibody was coupled to carboxylate groups on the surface of the carbon material using conventional natural incubation methods in example 3, so that the number of CEA antibody modifications on the surface of the carbon material electrode was very small. Benefit (benefit)The fluorescence microscope device is used for self-contained analysis software, and the average fluorescence intensity values of the graphs in the figures 1-3 are respectively read as follows: 39.39 FU/. Mu.m 2 ，18.47FU/μm 2 ，2.27FU/μm 2 From this, it can be seen that the coupling modification effect of the protein on the surface of the carbon electrode is greatly improved, and the average fluorescence intensity value is improved by approximately 20 times after the coupling method is used for treatment.
In conclusion, the coupling method of the invention effectively improves the effect of natural incubation coupling modification of protein on the surface of the conventional carbon material by optimizing the composition of protein solution and combining direct current treatment, greatly improves the average fluorescence intensity value of fluorescent microscope analysis, solves the problem of insufficient quantity of coupling modification of protein on the carbon material, and ensures that the protein on the surface of the carbon material is uniformly distributed, thereby being beneficial to further pushing the technology to the commercial market application.
While the above examples illustrate the conjugation methods of the present invention in terms of binding CEA antibodies and antigens, it will be apparent to those skilled in the art that they can also be applied to other conventional protein aptamers.
The carbon electrode obtained by the embodiment of the invention can be used for preparing an electrochemical immunosensor.
If the carbon electrode obtained in example 1 can be subjected to site blocking, CEA antigen can be detected as an immunosensor.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (4)
1. A method for coupling protein aptamer on the surface of a carbon electrode, which is characterized by comprising the following steps:
(1) Soaking a carbon electrode in a methanol solution of N-hydroxysuccinimide ester of 1-pyrene butyric acid, taking out, cleaning the surface by using a PBS solution, and naturally drying at room temperature;
(2) Dissolving the coupled protein aptamer in a mixed solution consisting of PBS, potassium chloride and Tween-80, regulating the pH value of the solution to be lower than the isoelectric point of the protein aptamer, and uniformly vortex-oscillating to obtain a protein solution;
(3) Taking the carbon electrode treated in the step (1) as a negative electrode, taking an untreated pure carbon electrode as a positive electrode, putting the pure carbon electrode into the protein solution obtained in the step (2) together, and applying direct current between the two electrodes for treatment;
(4) Washing the carbon electrode treated in the step (3) by using PBS solution, washing by using deionized water, and drying at room temperature to obtain a modified carbon electrode;
in the step (1), the concentration of the methanol solution of the N-hydroxysuccinimide ester of 1-pyrene butyrate is 5-10mmol/L, the soaking time is more than or equal to 30min, and the concentration of the PBS solution is 0.01M;
in the step (2), the mixed solution is prepared from 0.01M PBS, 0.1M potassium chloride and 0.01wt% Tween-80 according to the volume ratio of 1:1:1, mixing to obtain the product;
the concentration of protein aptamer in the protein solution is 100 μg/mL;
in the step (3), the direct current is conducted for more than or equal to 10min, and the voltage range is 0.1V-1.0V;
in the step (4), the concentration of the PBS solution is 0.01M-0.1M.
2. The method of coupling protein aptamers on a carbon electrode surface of claim 1, wherein the carbon electrode is a carbon nanotube material electrode, a graphene material electrode, a glassy carbon electrode, or a screen-printed carbon electrode.
3. A carbon electrode prepared by the method of any one of claims 1-2 by coupling protein aptamer to the surface of the carbon electrode.
4. An electrochemical immunosensor prepared from the carbon electrode of claim 3.
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