CN114577879A - Protein detection system based on electrophoresis and molecular imprinting principles and application thereof - Google Patents
Protein detection system based on electrophoresis and molecular imprinting principles and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of protein detection, and particularly relates to a protein detection system based on electrophoresis and molecular imprinting principles and application thereof. The electrochemical molecularly imprinted sensor for target enrichment of protein based on the electrophoresis principle is constructed for the first time and used for detecting protein, and the principle is that a detection object with negative charges is rapidly enriched and combined to a cavity of a molecularly imprinted film under the action of an electric field to cause the change of the electrochemical property of the surface of an electrode. The protein detection system reduces the lowest detection concentration to 7 × 10‑8g/ml, the incubation time of the traditional electrochemical molecular imprinting sensor is shortened from 15min to 5min, the detection time is greatly shortened, the interference of an interfering substance on the sensor can be selectively avoided by the protein detection system, and the protein detection system can be provided for quickly and accurately detecting the proteinA new strategy is provided.
Description
Technical Field
The invention belongs to the field of protein detection, and particularly relates to a protein detection system based on electrophoresis and molecular imprinting principles and application thereof.
Background
Proteins are important components for maintaining homeostasis in the body and play a very critical role in vital activities. The rapid and efficient detection of the protein is significant in prevention, diagnosis and treatment of diseases, screening of drug treatment and monitoring of life processes.
Common protein detection methods mainly include immunoassay, enzyme-linked immunosorbent assay, resonance light scattering and mass spectrometry. The detection mode generally has a plurality of problems of high detection limit, long detection time, high price, operation by professional personnel and the like, and greatly limits the development of protein detection. The electrochemical biosensor has the advantages of strong specificity, low cost, high accuracy, simple operation, high analysis speed and the like in the detection process, and is widely concerned and researched. Sensors such as immune and aptamer sensors cannot be widely used for the moment due to the defects of poor reagent stability, difficult production, high cost and the like. Researchers are more inclined to develop inexpensive, highly sensitive and reusable sensors. Therefore, more and more research focuses on the molecular imprinting sensor because of its advantages of low cost, high specificity, easy operation, rapid analysis, etc.
Molecularly imprinted polymers provide specific recognition sites for template molecules that are removed from the polymer, providing complementary binding sites that can subsequently recognize the template molecules. In recent years, molecularly imprinted polymers have been increasingly selected for the construction of recognition elements and for the electrochemical detection of proteins. The use of molecular imprinting provides a simple and cheap recognition receptor for universal protein detection, and o-phenylenediamine is widely used for the synthesis of protein molecular imprinting in recent years.
However, the conventional electrochemical molecular imprinting sensor has problems of high detection limit, long detection time and the like in detecting proteins. The process of binding the template molecule and the recognition element takes a lot of time in a detection loop, and when the detection object and the recognition element coexist in the buffer solution, the detection object can be continuously bound with the recognition element along with the increase of time, but part of the detection object can not be identified and detected, so that the existing sensor can not realize lower detection limit in a certain detection system.
In order to solve the problems, the patent develops a novel sensing model of an electrochemical molecularly imprinted sensor for electrophoresis mediated detection object targeted enrichment to a sensing interface, and the principle is that a detection object with negative charges is rapidly enriched and combined to a cavity of a molecularly imprinted film under the action of an electric field to cause the change of the electrochemical properties of the surface of an electrode.
The most common method for detecting protein by electrophoresis is polyacrylamide gel electrophoresis, and the detection principle is that the protein is subjected to motion separation in the gel under the action of an electric field force, and development is carried out by utilizing an antigen-antibody combination mode to realize semi-quantitative detection. It has been proposed that electric field enhancement can be used for detection of DNA fragments, but detection of proteins has not yet appeared, so that electrophoresis of proteins for electrochemical detection of proteins would have great research significance.
The inventor designs a novel protein detection method and a novel sensing device based on molecular imprinting technology and electrophoresis principle to realize universal rapid detection of trace proteinThe detection method comprises the step of constructing the electrochemical molecularly imprinted sensor for targeted protein enrichment based on the electrophoresis principle for the first time for detecting bovine serum albumin. The sensor has the lowest concentration of 7x10 in the initial detection-6The g/ml is reduced to 7X10-8g/ml, and shortens the incubation time of the traditional electrochemical molecular imprinting sensor from 15 minutes to 5 minutes, thereby greatly shortening the detection time. And the interference of interferents to the sensor can be selectively avoided, and a new strategy is provided for quickly and accurately detecting the protein.
The invention patent with publication number CN112763553A discloses an electrochemical detection method for protein based on molecular imprinting technology, which has high selectivity and high sensitivity, and can rapidly detect target protein, but the invention patent does not adopt electrophoresis technology, and does not have the functions of broadening detection limit and improving anti-interference performance.
Disclosure of Invention
One of the objectives of the present invention is to provide a protein detection system based on electrophoresis and molecular imprinting principles, which rapidly enriches and binds a negatively charged detection object to a cavity of a molecular imprinting film under the action of an electric field, thereby causing the change of the electrochemical properties of the electrode surface, and has the advantages of widening the detection limit, shortening the detection time, improving the anti-interference capability, and the like.
In order to realize the purpose, the invention adopts the following technical scheme:
a protein detection system based on electrophoresis and molecular imprinting principles, which consists of an electrochemical molecular imprinting sensor and an electrophoresis device;
the electrophoresis device is an electrolytic cup with a Pt disk at the bottom;
the electrochemical molecular imprinting sensor is suspended in the electrolytic cup through a fixing table;
the Pt disk and the electrochemical molecular imprinting sensor are respectively connected with two poles of a power supply, so that electric field acting force mediated target enrichment of an object to be detected to a sensing interface is realized.
Further, the electrochemical molecular imprinting sensor is parallel to the Pt disk.
Furthermore, the electrochemical molecular imprinting sensor comprises a working electrode, a reference electrode and a counter electrode, wherein the working electrode is an MIP/GCE electrode, the counter electrode is a platinum wire electrode, and the reference electrode is an Ag/AgCl electrode.
Further, the preparation method of the MIP/GCE electrode comprises the following steps:
(1) polishing with alumina and bare GCE as substrate electrode;
(2) electropolymerizing the polished GCE electrode obtained in the step (1) by adopting o-phenylenediamine to obtain a molecular imprinting membrane electrode;
(3) and (3) eluting the molecularly imprinted membrane electrode in the step (2) by using an eluent, and washing away template molecules to obtain the MIP/GCE electrode.
Further, the step (1) is specifically as follows: polishing the naked GCE by using 0.3 mu m of alumina powder, and ultrasonically cleaning the polished naked GCE in ultrapure water for 15 seconds; polishing the naked GCE by using 0.05-micrometer aluminum oxide powder, and ultrasonically cleaning the polished naked GCE in ultrapure water, pure ethanol and ultrapure water for 15 seconds in sequence; in the presence of 5mM [ Fe (CN)6]3-/4-The response of naked GCE was recorded by CV scanning in PBS solution, with voltage cycling from-0.2V to +0.6V at a rate of 100mV/s until a stable cyclic voltammogram was obtained; the electrode was rinsed with ultrapure water and dried at room temperature.
The step (2) is specifically as follows: containing o-phenylenediamine-PD (1.0g/L, 9.3mmol/L) and bovine serum albumin BSA (1.4x 10)-2g/L) in PBS (pH 6.0) for 20min, and then electropolymerizing by cyclic voltammetry to form a molecularly imprinted Membrane (MIP); the synthesis of non-molecularly imprinted membranes is similar to that of molecularly imprinted membranes, but no bovine serum albumin is added during electropolymerization.
Further, the o-PD concentration is 0.5-2.5 g/L, preferably 1.0 g/L; BSA concentration of 0.7X10-2～3.5x10- 2g/L, preferably 1.4x10-2g/L; the number of electropolymerization circles is 5-15 circles; the eluent is prepared by mixing ethanol-water in a volume ratio of 2: 1, the elution time of the eluent containing 1M NaOH is 5-25 min, preferably 15 min.
Furthermore, the scanning rate in the step (2) is 50mV/s, and the potential range is as follows: -0.1-1.0 v, and the number of electropolymerization turns is 15.
Furthermore, the molecularly imprinted membrane electrode needs to be soaked for 15min by eluent at the temperature of 50 ℃ in water bath.
The second purpose of the invention is to provide a method for detecting protein by the protein detection system, which is convenient and rapid and can realize universal rapid detection of trace protein.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting protein by using the protein detection system specifically comprises the following steps:
(1) adding a substance to be tested into the electrolytic cup, and incubating;
(2) electrochemically measuring the incubated electrode of step (1) by using a DPV method.
The electrochemical device CHI660 used differential pulse voltammetry to test the current response of the surface of the electrode and the probe after the molecular imprinting and the protein combination, according to K3Fe(CN)6And K4Fe(CN)6The electrochemical signal is generated by oxidation-reduction on the electrode, the probe is used for researching the response of BSA to the prepared MIP, and the probe can diffuse into and out of the MIP matrix through a print cavity formed in the MIP.
The step (2) is specifically as follows: using DPV method in a medium containing 5mM [ Fe (CN)6]3-/4-And 1M KCl in PBS (pH 6.0), and electrochemically measuring the incubated electrode at a scanning rate of 100mV/s, a voltage range of-0.4-0.8V, a pulse amplitude of 50mV, and a pulse width of 50 ms.
Further, the analyte is negatively charged.
Further, in the step (1), the height between the surface of the electrode and the liquid level is 0-25 mm, the incubation time is 3-15 min, and the voltage range is 0-200 mV.
Furthermore, in the step (1), the height of the electrode surface from the liquid level is 5mm, the incubation time is 5min, and the voltage is 100 mV.
Incubation for 5min was to ensure that the protein molecules bound to the MIP cavity; the 100mV voltage is chosen because it can introduce the analyte into the molecular imprinting and bring the interferent away from the surface of the molecular imprinting membrane.
Further, in the step (2), the electric field operation time was 5 min.
The invention also aims to provide a protein detection method based on electrophoresis and molecular imprinting principles, which is convenient and rapid and can realize universal rapid detection of trace protein.
In order to achieve the purpose, the invention adopts the following technical scheme:
a protein detection method based on electrophoresis and molecular imprinting principles specifically comprises the following steps:
(1) designing a molecularly imprinted membrane according to the property of charges carried by a substance to be detected, the size of molecules and the like, and preparing an electrochemical molecularly imprinted sensor;
(2) one end of the two poles of a power supply is connected with the molecularly imprinted sensor in the step (1), the other end of the power supply is connected with an electrolytic cup containing a Pt disk, the electrochemical molecularly imprinted sensor is parallel to the Pt disk, and the distance between the electrochemical molecularly imprinted sensor and the upper liquid level is 5 mm;
(3) adding the substance to be tested into an electrolytic cup for incubation, wherein the incubation time is 5min, and the voltage is 100 mV;
(4) electrochemically measuring the incubated electrode of step (1) by using a DPV method.
The invention has the advantages that:
(1) the incubation time of the protein detection system in the detection process is shortened to 1/3: by applying an electric field, the charged substance to be detected is subjected to directional electric field force to rapidly move, and the substance to be detected is enriched to the molecular imprinting position on the surface of the electrode and rapidly combined with the molecular imprinting to achieve rapid detection. The complete enrichment effect can be achieved in the same system by selecting the electric field running time of 5min, so that signal amplification is realized.
(2) Compared with the traditional electrochemical molecular imprinting sensor, the detection limit of the protein detection system disclosed by the invention for detecting the protein is expanded by 100 times: the electric field is utilized to enable the substance to be detected in the detection system to be subjected to the electric field force, move and be enriched on the surface of the electrode, so that the response signal is enlarged compared with the ordinary detection, and the signal intensity which is one hundred times of the concentration of the substance to be detected in the ordinary detection can be obtained under a certain concentration of the substance to be detected. When the voltage is selected, the molecular size and the amount of charges of the substance to be detected, the size of interfering substances and molecular motion channels in a system and the properties of the molecularly imprinted membrane are comprehensively considered, and 100mV is selected, so that the substance to be detected can be introduced into the molecularly imprinted membrane, and the interfering substances can be carried on the surface of the molecularly imprinted membrane.
(3) When the protein concentration is 7X10-6When the protein is in g/ml, the current response of the protein detection system is improved by 33.3 muA compared with the current response of the traditional electrochemical molecular imprinting sensor.
(4) This patent provides new tactics in order to reduce interfering substance to identification element's interference, and the electrified nature difference according to determinand and other impurity leads them respectively to the both ends of electric field to the interference killing feature is better. And because the electric conduction property of the o-phenylenediamine is that the electric conduction effect of the o-phenylenediamine is far lower than that of a Pt disk forming an electric field cathode after the molecular imprinting is formed on the surface of the electrode, the adsorption effect of a cathode field on positively charged interferents is stronger in the work.
Drawings
FIG. 1 is a graph showing the performance analysis of the electrophoresis group and the normal group 1;
FIG. 2 is a surface appearance analysis diagram of the molecularly imprinted membrane;
FIG. 3 is a graph showing the results of stability analysis of the protein detection system of this patent;
FIG. 4 is a graph showing the results of an anti-interference assay of the protein detection system of this patent;
FIG. 5 is a schematic diagram showing a protein detection system of the present invention, wherein 1 is a power supply, 2 is a fixing base, 3 is an electrochemical molecular imprinting sensor, 4 is an electrolytic cup, 5 is a Pt disk, and the electrolytic cup 4 and the Pt disk 5 constitute an electrophoresis apparatus.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art can make insubstantial modifications and adaptations to the embodiments described above without departing from the scope of the present invention.
In the examples of the present invention, bovine serum albumin (BSA, 96%), o-phenylenediamine (o-PD, 99%), and potassium ferricyanide (K)3[Fe(CN)6]Not less than 99 percent) and potassium ferricyanide trihydrate (K)4[Fe(CN)6·3H2O]99%) from alatin (china, shanghai); potassium dihydrogen phosphate (KH)2PO4And not less than 99%) produced by Jiangsu Qiangsheng functional chemical corporation (China, Jiangsu); anhydrous disodium hydrogen phosphate (Na)2HPO499%) from chengdu dragon chemical limited (china, chengdu); sodium hydroxide (NaOH, 98%) ordered bio-biotechnology limited (china, shanghai); potassium chloride (KCl) and absolute ethanol (CH)3CH2OH) from chongqing chuandong chemical limited (china, chongqing); fetal bovine serum was purchased from bio-engineering, inc (china, shanghai); alanine (Ala) was supplied by shanghai kangda chemical company, ltd (china, shanghai); lysine (Lys) was purchased from shanghai baisai biotechnology member limited (china, shanghai); glutamic acid (Glu) is produced by shanghai tatatake technologies ltd (china, shanghai); glycine was ordered from the health materials factory of Tianjin, China; trypsin was supplied by Biosharp; all solutions were prepared with ultra pure water (specific resistance 18.2 M.OMEGA.cm).
All electrochemical experiments were performed on the CHI 660E electrochemical workstation (chenhua instruments ltd, china, shanghai), all at room temperature; the electrochemical measurement adopts a traditional three-electrode system, MIP/GCE is used as a working electrode (the diameter is 4mm), a platinum wire electrode is used as a counter electrode, and an Ag/AgCl electrode is used as a reference electrode; the Atomic Force Microscope (AFM) (Packo, Inc., Korea) is used for representing different modified electrodes, the UTP1306S type series economic direct current stable power supply is from Ulideke technologies, Inc., the voltage continuous adjustable range is 0-32V, and the maximum output current is 0-6.0A.
Example 1 electrophoresis set
1) Before electropolymerization, polishing bare GCE by using 0.3 mu m alumina powder, and ultrasonically cleaning in ultrapure water for 15 seconds; the bare GCE was then polished using 0.05 μm alumina powder,ultrasonic cleaning in ultrapure water, pure ethanol and ultrapure water for 15 seconds; followed by a reaction in the presence of 5mM [ Fe (CN)6]3-/4-Recording the response of naked GCE by CV scanning in PBS solution; the voltage was cycled from-0.2V to +0.6V at a rate of 100mV/s until a stable cyclic voltammogram was obtained; finally, the electrode was rinsed with ultrapure water and dried at room temperature.
2) Preparation of MIP and NIP modified electrodes:
containing o-phenylenediamine-PD (1.0g/L, 9.3mmol/L) and bovine serum albumin BSA (1.4x 10)-2g/L) of PBS (pH 6.0) for 20min, then electropolymerization is carried out by using cyclic voltammetry within a potential range of-0.1-1.0 v, wherein the scanning rate is 50mV/s, and a molecularly imprinted Membrane (MIP) is formed after scanning for 15 cycles. The synthesis of non-molecularly imprinted membranes (NIP) was similar to the synthesis of molecularly imprinted membranes, but no bovine serum albumin was added during electropolymerization. Then washing the electropolymerized electrode with water, then soaking the electrode in an eluent prepared from ethanol-water (volume ratio is 2: 1) and containing 1M NaOH for 15 minutes at the temperature of 50 ℃ in a water bath, finally washing with ultrapure water and drying.
3) Testing the current response of the surface of the electrode and the probe after the molecular imprinting is combined with the protein by using a differential pulse voltammetry method through an electrochemical device CHI 660; according to K3Fe(CN)6And K4Fe(CN)6Redox at the electrode to generate an electrochemical signal; the response of BSA to MIPs was studied by probes, which could diffuse into and out of the MIP matrix through the imprinted cavities formed in the MIPs. The MIP/GCE was immersed in different concentrations of BSA solution and the working electrode was placed on a stationary stage to keep the electrophoresis parallel to the bottom Pt disk. This arrangement ensures that the working electrode is 5mm from the upper level. Then, the dc power supply was turned on to provide 100mv dc to the electrophoresis apparatus, and after the addition of the analyte to be detected, incubation was carried out for 5 minutes to ensure that the protein molecules were bound to the MIP cavity.
4) After washing, the DPV method was used in a medium containing 5mM [ Fe (CN)6]3-/4-And 1M KCl in PBS (pH 6.0), under the conditions of scan rate of 100mV/s, voltage range of-0.4-0.8V, pulse amplitude of 50mV, and pulse width of 50msAnd electrochemically determining the incubated electrode. And after the electrophoresis device runs for 5 minutes, the direct current power supply is closed, the working electrode is taken out, and the working electrode is washed by ultrapure water and then is subjected to DPV scanning to obtain detection data.
FIG. 5 is a schematic diagram showing a protein detection system of the present invention, which is composed of a power supply 1, a fixing base 2, an electrochemical molecular imprinting sensor 3, an electrolytic cup 4 and a Pt disk 5, wherein the electrolytic cup 4 and the Pt disk 5 constitute an electrophoresis apparatus. The electrochemical molecular imprinting sensor 3 is suspended in the electrolytic cup 4 through the fixed platform 2, and the Pt disk 5 at the bottom of the electrolytic cup 4 and the electrochemical molecular imprinting sensor 3 are respectively connected with two poles of a power supply.
Comparative example 1 general group 1
The procedure was identical to that of the electrophoresis group of example 1, except that the electrophoresis apparatus was not applied in the incubation and detection of the ordinary group 1; other experimental conditions were consistent.
Comparative example 2 general group 2
The steps are the same as those of the electrophoresis group in the embodiment 1, and the difference is that the electrophoresis device is not applied in the incubation and detection of the common group 2, and the incubation time is 15min after the substance to be detected is added in the step 3); other experimental conditions were consistent.
Example 2 electrophoretic group and general group 1
Controls were performed with and without the electrophoretic sensor and incubated in the same system for 5 minutes at a protein concentration of 7X10-6g/ml. The excellent performance of the electrophoresis device is analyzed, the real sample bovine serum is taken as a sample, and the current response of the sensor with electrophoresis and the sensor without electrophoresis are respectively measured. As a result, the sensor effect of electrophoresis was improved by 33.3 μ A as shown in FIG. 1.
EXAMPLE 3 method feasibility examination
The detection of a real sample is carried out by fetal calf serum, and the feasibility of the method in the actual sample determination is examined. Diluting fetal calf serum with 0.1mol/L PBS 10000 times, performing differential pulse method, and recovering by standard addition method to obtain measurement data, as shown in Table 1, with the addition concentration of 7 × 10-8、7×10-7And 7X10-6The recoveries of BSA in g/ml were 105.13%, 93.98% and 116.93%, respectively, and the relative standard deviations were 5.75% and 5.04%, respectivelyAnd 5.66%. The results demonstrate that the method can be used to detect BSA in complex biological samples, thereby providing a reliable and direct protein detection means for clinical laboratories.
TABLE 1
| BSA concentration (g/ml) | Recovery rate | Relative Standard Deviation (SD) |
| 7×10-8 | 105.13％ | 5.75％ |
| 7×10-7 | 93.98％ | 5.04％ |
| 7×10-6 | 116.93％ | 5.66％ |
Example 4 characterization of AFM
The surface appearance of the molecularly imprinted thin film formed by electropolymerization is analyzed by an atomic force microscope, the result of AFM is shown in fig. 2, in fig. 2A, the roughness of the bare glassy carbon electrode is 1.496, and the roughness of the electrode surface is significantly changed after the electrode is electropolymerized and modified by o-phenylenediamine in the process of molecular imprinting. Before the bovine serum albumin template molecules are removed, the roughness of the molecular imprinting is 2.750, as shown in fig. 2B; after removal of the template molecules, the MIP roughness is 3.217, as in fig. 2C; the NIP roughness was 3.374 in the template-free molecular polymerization, as shown in fig. 2D. The NIP is relatively flat and compact, and the surface roughness of the NIP, the MIP and the eluted MIP is different from that of the NIP, the MIP and the eluted MIP, which indicates that the electro-deposition of o-phenylenediamine and the removal of the template molecule bovine serum albumin are successful.
Example 5 comparison of bovine serum detection ranges of the Normal group 2 and the electrophoresis group
Detecting the target substance under the optimal experimental conditions. The incubation detection range of the electrophoresis group is 7 multiplied by 10-8～7×10-6g/ml; the detection range of a conventional electrochemical MIP sensor is 7x10-6～7×10-4g/ml. The results show that the detection range is increased by about 100 times after the electrophoresis device is added due to the enrichment effect of the electrophoresis device on BSA.
TABLE 2
| Without addition of electrophoresis | Error bar | Additive electrophoresis | Error bar |
| 7.00E-06 | 1.27E-06 | 7.00E-08 | -7.33333E-07 |
| 1.40E-05 | -2.09E-06 | 3.50E-07 | 3.03E-06 |
| 7.00E-05 | -2.61E-06 | 7.00E-07 | -1.17E-06 |
| 1.40E-04 | 1.32E-06 | 3.50E-06 | -5.87E-06 |
| 7.00E-04 | 2.10E-06 | 7.00E-06 | 1.52E-06 |
Example 6 stability testing
Influence of the electrophoretic device on the sensor: as shown in FIG. 3, the same electrode was tested repeatedly under the action of electric field, the electrode was placed in phosphate buffer and run for five minutes, and the test was repeated 5 times, and after DPV test, the electrode was found to have substantially no change in the current response difference, which indicates that the sensor was not disturbed by the electric field under the electrophoresis of 100mV and 5min, and the protein detection system of the present invention has a certain stability.
EXAMPLE 7 tamper resistance testing
The anti-interference test is carried out by selecting various amino acids and trypsin, and the test result shows that the protein detection system disclosed by the invention has excellent anti-interference performance on interferents such as amino acids and the like. The system can specially separate certain proteins under the action of electrophoresis, and reduces the interference on detection, as shown in fig. 4, the system has higher interference resistance on trypsin, mainly because the isoelectric point pI of the trypsin is 10, and the trypsin is positively charged in phosphate buffer with pH 6. The sensor of the added electrophoresis device has a much lower interference effect than a normal incubation in a corresponding interference-free test. In addition, due to the non-conducting property of the o-phenylenediamine, the conducting effect of the o-phenylenediamine is far lower than that of a Pt disk forming an electric field cathode after molecular imprinting is formed on the surface of the electrode, and therefore the adsorption effect of a cathode field on positively charged interferents is stronger.
Claims (10)
1. A protein detection system based on electrophoresis and molecular imprinting principles is characterized by comprising an electrochemical molecular imprinting sensor and an electrophoresis device;
the electrophoresis device is an electrolytic cup with a Pt disk at the bottom;
the electrochemical molecular imprinting sensor is suspended in the electrolytic cup through a fixing table;
the Pt disk and the electrochemical molecular imprinting sensor are respectively connected with two poles of a power supply, so that electric field acting force mediated target enrichment of an object to be detected to a sensing interface is realized.
2. The protein detection system of claim 1, wherein the electrochemical molecular imprinting sensor is parallel to the Pt disk.
3. The protein detection system of claim 1, wherein the electrochemical molecular imprinting sensor comprises a working electrode, a reference electrode, and a counter electrode, wherein the working electrode is a MIP/GCE electrode, the counter electrode is a platinum wire electrode, and the reference electrode is a Ag/AgCl electrode.
4. The protein detection system of claim 3, wherein the MIP/GCE electrode preparation method comprises the steps of:
(1) polishing with alumina and bare GCE as substrate electrode;
(2) electropolymerizing the polished GCE electrode obtained in the step (1) by adopting o-phenylenediamine to obtain a molecular imprinting membrane electrode;
(3) and (3) eluting the molecularly imprinted membrane electrode in the step (2) by using an eluent, and washing away template molecules to obtain the MIP/GCE electrode.
5. The method according to claim 4, wherein the scanning rate in the step (2) is 50mV/s, and the potential range is: -0.1-1.0 v, and the number of electropolymerization turns is 15.
6. The preparation method according to claim 4, wherein in the step (3), the molecularly imprinted membrane electrode is soaked for 15min at 50 ℃ in a water bath with an eluent.
7. A method for detecting a protein using the protein detection system of claim 1, comprising the steps of:
(1) adding a substance to be tested into the electrolytic cup, and incubating;
(2) electrochemically measuring the incubated electrode of step (1) by using DPV method.
8. The method for detecting a protein according to claim 8, wherein the analyte is negatively charged.
9. The method for detecting a protein according to claim 8, wherein the height of the electrode surface from the liquid surface in step (1) is 5mm, the incubation time is 5min, and the voltage is 100 mV.
10. The method for detecting proteins according to claim 8, wherein the electric field running time in step (2) is 5 min.
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