CN114577879B - Protein detection system based on electrophoresis and molecular imprinting principle and application thereof - Google Patents

Protein detection system based on electrophoresis and molecular imprinting principle and application thereof Download PDF

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CN114577879B
CN114577879B CN202210220237.5A CN202210220237A CN114577879B CN 114577879 B CN114577879 B CN 114577879B CN 202210220237 A CN202210220237 A CN 202210220237A CN 114577879 B CN114577879 B CN 114577879B
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CN114577879A (en
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臧广超
李钰莎
兰华林
鲁清
张玉婵
王钰耀
兰远胜
武晓婷
明小卿
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CHONGQING MEDICAL EQUIPMENT QUALITY INSPECTION CENTER
Chongqing Medical University
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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 a molecular imprinting principle and application thereof. The electrochemical molecular imprinting sensor for protein detection based on the electrophoresis principle is constructed for the first time, and the principle is that a negatively charged detection object is rapidly enriched and combined to a cavity of a molecular imprinting film under the acting force of an electric field, so that the electrochemical property of the surface of an electrode is changed. The protein detection system reduces the minimum detection concentration to 7X10 ‑8 g/ml, the time for incubating the traditional electrochemical molecularly imprinted sensor is shortened from 15min to 5min, the detection time is greatly shortened, and the protein detection system can also selectively avoid the interference of an interfering object on the sensor, thereby providing a new strategy for rapidly and accurately detecting the protein.

Description

Protein detection system based on electrophoresis and molecular imprinting principle and application thereof
Technical Field
The invention belongs to the field of protein detection, and particularly relates to a protein detection system based on electrophoresis and a molecular imprinting principle and application thereof.
Background
Proteins are important components for maintaining homeostasis in the body and play a critical role in life activities. The realization of rapid and efficient detection of protein has great significance in the prevention, diagnosis and treatment of diseases, screening of drug treatment and monitoring of life process.
Common protein detection modes mainly include immunoassay, enzyme-linked immunosorbent assay, resonance light scattering and mass spectrometry. The detection mode has the advantages of high detection limit, long detection time, high price, and great limitation of the development of protein detection due to the fact that a professional is required to operate the detection mode. Electrochemical biosensors are widely focused and studied because of the advantages of strong specificity, low cost, high accuracy, simple operation, high analysis speed and the like in the detection process. Sensors such as immunity and aptamer have been temporarily unable to be widely used due to the disadvantages of poor reagent stability, difficult production, high cost, etc. Researchers are more inclined to develop inexpensive, highly sensitive and reusable sensors. Therefore, more and more researches focus on the molecular imprinting sensor, because the molecular imprinting sensor has the advantages of low cost, high specificity, simple operation, rapid analysis and the like.
Molecularly imprinted polymers provide specific recognition sites for template molecules, which are removed from the polymer, providing complementary binding sites that are capable of subsequently recognizing the template molecule. In recent years, molecularly imprinted polymers have been increasingly selected for the construction of recognition elements and for application in electrochemical detection of proteins. The use of molecular imprinting provides a simple and low-cost recognition receptor for detecting proteins in a general formula, and o-phenylenediamine is widely used for synthesizing the molecular imprinting of the proteins in recent years.
However, the conventional electrochemical molecularly imprinted sensor has the problems of high detection limit, long detection time and the like in detecting protein. The process of combining the template molecule and the recognition element can consume a great deal of time in the detection link, and when the detection object and the recognition element coexist in the buffer solution, the detection object can be continuously combined with the recognition element along with the increase of time, but still part of the detection object can not be recognized 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 the electrophoresis-mediated targeted enrichment of the detection objects to a sensing interface, and the principle is that the negatively charged detection objects are rapidly enriched and combined to a cavity of a molecularly imprinted film under the action force of an electric field, so that the electrochemical properties of the electrode surface are changed.
The protein can automatically move towards the direction of an electric field force under the action of the electric field force, and the most common electrophoresis method for detecting the protein is polyacrylamide gel electrophoresis, and the detection principle is that the protein is subjected to motion separation in gel under the action of the electric field force, and development is performed by utilizing an antigen-antibody combination mode to realize semi-quantitative detection. There have been studies to suggest that electric field enhancement can be used for detection of DNA fragments, but detection of proteins has not yet emerged, so that electrophoresis of proteins for electrochemical detection of proteins would have great research significance.
The inventor is based on molecular imprinting technologyThe electrophoresis principle designs a novel protein detection method and a sensing device to realize the universal rapid detection of trace proteins, and an electrochemical molecularly imprinted sensor for detecting bovine serum albumin based on the electrophoresis principle and targeted enrichment of proteins is constructed for the first time. The sensor detects the lowest concentration from 7×10 -6 The g/ml is reduced to 7X10 -8 g/ml, and the time for incubating the traditional electrochemical molecularly imprinted sensor is shortened from 15 minutes to 5 minutes, so that the detection time is greatly shortened. And the interference of an interfering object on the sensor can be selectively avoided, and a new strategy is provided for rapidly and accurately detecting the protein.
The invention patent with publication number of CN112763553A discloses an electrochemical detection method for proteins based on a molecular imprinting technology, which has high selectivity and high sensitivity and can rapidly detect target proteins, but the patent does not adopt an electrophoresis technology, and does not have the effects of widening detection limit and improving anti-interference performance.
Disclosure of Invention
The invention aims to provide a protein detection system based on electrophoresis and molecular imprinting principles, which can rapidly enrich and combine negatively charged detection objects into a cavity of a molecular imprinting film under the action of an electric field to cause the change of electrochemical properties of the surface of an electrode and has the advantages of widening detection limit, shortening detection time, improving anti-interference capability and the like.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the protein detection system based on electrophoresis and molecular imprinting principle comprises an electrochemical molecular imprinting sensor and an electrophoresis device;
the electrophoresis device is an electrolysis cup with a Pt disk at the bottom;
the electrochemical molecular imprinting sensor is suspended in the electrolytic cup through a fixed table;
the Pt disc and the electrochemical molecular imprinting sensor are respectively connected with two poles of a power supply, so that the electric field acting force mediated targeted enrichment of the object to be detected to a sensing interface is realized.
Further, the electrochemical molecularly imprinted sensor is parallel to the Pt disk.
Further, the electrochemical molecularly imprinted 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 an Ag/AgCl electrode.
Further, the MIP/GCE electrode preparation method comprises the following steps:
(1) Taking bare GCE as a substrate electrode, and polishing with aluminum oxide;
(2) Adopting o-phenylenediamine to electropolymerize the polished GCE electrode in the step (1) and molecular imprinting the membrane electrode;
(3) And (3) eluting the molecularly imprinted membrane electrode in the step (2) by using an eluent, and washing template molecules to obtain the MIP/GCE electrode.
Further, the step (1) specifically comprises: polishing bare GCE with 0.3 μm alumina powder, and ultrasonically cleaning in ultrapure water for 15 seconds; then polishing the bare GCE with 0.05 μm alumina powder, and ultrasonically cleaning in ultrapure water, pure ethanol and ultrapure water for 15 seconds; containing 5mM of [ Fe (CN) 6 ] 3-/4- The response of bare 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) comprises the following steps: in the presence of o-phenylenediamine omicron-PD (1.0 g/L,9.3 mmol/L) and bovine serum albumin BSA (1.4x10) -2 g/L) of PBS (ph=6.0) was purged with nitrogen for 20min, and then electropolymerized by cyclic voltammetry to form a molecularly imprinted Membrane (MIP); the synthesis of the non-molecularly imprinted membrane is similar to the synthesis of the molecularly imprinted membrane, but no bovine serum albumin is added during electropolymerization.
Further, the o-PD concentration is 0.5 to 2.5g/L, preferably 1.0g/L; BSA concentration of 0.7x10 -2 ～3.5x10 - 2 g/L, preferably 1.4x10 -2 g/L; the electropolymerization turns are 5-15 turns; the eluent is ethanol-water in a volume ratio of 2:1, the elution time of the eluent containing 1M NaOH is 5-25 min, preferably 15min.
Further, the scanning rate in the step (2) is 50mV/s, and the potential range is: 0.1-1.0 v, the number of electropolymerization turns is 15.
Further, the molecularly imprinted membrane electrode is soaked in eluent for 15min at the temperature of 50 ℃ by heating in water bath.
The second object of the present invention is to provide a method for detecting protein by using the protein detection system, which is convenient and rapid, and can realize the general rapid detection of trace protein.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for detecting protein by the protein detection system, which specifically comprises the following steps:
(1) Adding an object to be detected into the electrolytic cup for incubation;
(2) Electrochemical measurement of the incubated electrode of step (1) was performed using the DPV method.
Current response of electrode surface and probe after molecular imprinting binding protein was tested by electrochemical device CHI660 using differential pulse voltammetry according to K 3 Fe(CN) 6 And K 4 Fe(CN) 6 The electrochemical signal is generated by oxidation-reduction on the electrode, and the probe is used for researching the response of BSA to the prepared MIP, and can diffuse into and out of the MIP matrix through a imprinting cavity formed in the MIP.
The step (2) comprises the following steps: DPV method is adopted to contain 5mM [ Fe (CN) 6 ] 3-/4- And 1M KCl in PBS (pH=6.0), at a scan rate of 100mV/s, a voltage in the range of-0.4 to 0.8V, a pulse amplitude of 50mV, a pulse width of 50ms, and electrochemically measuring the electrode after incubation.
Further, the analyte is negatively charged.
Further, in the step (1), the height of the electrode surface from the liquid level is 0-25 mm, the incubation time is 3-15 min, and the voltage range is 0-200 mV.
Further, 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 100mV.
Incubation for 5min was to ensure binding of protein molecules to the MIP cavity; the 100mV voltage was chosen because it was able to both introduce the analyte into the molecular blot and carry the interferents off the surface of the molecularly imprinted membrane.
Further, in the step (2), the electric field operation time is 5min.
The invention further aims to provide a protein detection method based on electrophoresis and molecular imprinting principles, which is convenient and rapid and can realize the universal rapid detection of trace proteins.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a protein detection method based on electrophoresis and molecular imprinting principle specifically comprises the following steps:
(1) Designing a molecular imprinting membrane according to the charged property, molecular size and the like of a substance to be detected, and preparing an electrochemical molecular imprinting sensor;
(2) One end of the two poles of the power supply is connected with the molecular imprinting sensor in the step (1), one end of the power supply is connected with an electrolysis cup containing a Pt disk, the electrochemical molecular imprinting sensor is parallel to the Pt disk, and the distance between the electrochemical molecular imprinting sensor and the upper liquid level is 5mm;
(3) Adding an object to be detected into an electrolytic cup for incubation, wherein the incubation time is 5min, and the voltage is 100mV;
(4) Electrochemical measurement of the incubated electrode of step (1) was performed using the 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 of that of a conventional electrochemical molecularly imprinted sensor: by applying an electric field, the charged substance to be detected is subjected to directional electric field force to rapidly move, is enriched to the molecular imprinting position on the surface of the electrode and is rapidly combined with the molecular imprinting, so that rapid detection is achieved. The complete enrichment effect can be achieved in the same system by selecting the electric field operation 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 widened by 100 times: the utilization of the electric field enables the substances to be detected in the detection system to be subjected to the electric field force and to be moved and enriched on the surface of the electrode, so that compared with the common detection, the response signal is enlarged, and the signal intensity of the concentration of the substances to be detected in the common detection can be obtained at a certain concentration of the substances to be detected. When the voltage is selected, the size of the molecules of the substance to be detected, the charged amount, the size of the interference substance and the molecular motion path in the system and the properties of the molecular imprinting membrane are comprehensively considered, and 100mV is selected, so that the substance to be detected can be introduced into the molecular imprinting and the interference substance can be brought away from the surface of the molecular imprinting membrane.
(3) When the protein concentration is 7×10 -6 At g/ml, the current response of the protein detection system disclosed by the invention is improved by 33.3 mu A compared with that of a traditional electrochemical molecularly imprinted sensor.
(4) This patent provides new tactics in order to reduce the interference effect of interfering substance to identification element, according to the charged nature difference of measured object and other impurity with their both ends that guide the electric field respectively to the anti-interference effect is better. And because of the non-conductive property of the o-phenylenediamine, the conductive 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, so that the adsorption effect of the cathode field on the positively charged interferents is stronger in the work.
Drawings
FIG. 1 is a graph of performance analysis of electrophoresis group and normal group 1;
FIG. 2 is a surface topography of a molecularly imprinted membrane;
FIG. 3 is a graph showing the results of stability analysis of the protein detection system of the present patent;
FIG. 4 is a graph showing the results of an anti-interference analysis of the protein detection system of the present patent;
fig. 5 is a model diagram of a protein detection system of the present patent, wherein 1 is a power supply, 2 is a fixed table, 3 is an electrochemical molecular imprinting sensor, 4 is an electrolytic cup, 5 is a Pt plate, and the electrolytic cup 4 and the Pt plate 5 constitute an electrophoresis device.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that many modifications and variations of the embodiments are possible in light of the above teaching, while still remaining within the scope of the present invention.
In the examples of the present invention, bovine serum albumin (BSA, 96%), o-phenylenediamine (o-PD, 99%), potassium ferricyanide (K) 3 [Fe(CN) 6 ]Greater than or equal to 99 percent) and potassium ferricyanide trihydrate (K) 4 [Fe(CN) 6 ·3H 2 O]99%) from aledine (china, shanghai); monopotassium phosphate (KH) 2 PO 4 More than or equal to 99%) produced by Jiangsu Qiangsheng functional chemical industry Co., ltd (China, jiangsu); anhydrous disodium hydrogen phosphate (Na) 2 HPO 4 99%) by chengdouke chemical industry limited (chengdou, china); sodium hydroxide (NaOH, 98%) order biotechnology limited (china, shanghai); potassium chloride (KCl) and absolute ethanol (CH) 3 CH 2 OH) from Chongqing Chuan Dong chemical Co., ltd (Chongqing, china); fetal bovine serum was purchased from bioengineering, inc. (China, shanghai); alanine (Ala) is supplied by Shanghai Kangda chemical company, inc. (China, shanghai); lysine (Lys) was purchased from Shanghai hundred Siemens biotechnology Co., ltd (China, shanghai); glutamic acid (Glu) was produced by shanghai taitan technologies limited (china, shanghai); glycine was ordered from the sanitary materials factory in the Tianjin market (Tianjin, china); trypsin is supplied by Biosharp corporation; all solutions were prepared with ultrapure water (specific resistance 18.2 M.OMEGA.cm).
All electrochemical experiments were performed on the CHI 660E electrochemical workstation (morning glory instruments limited, china, shanghai) and all electrochemical experiments were performed at room temperature; the electrochemical measurement uses a traditional three-electrode system, wherein MIP/GCE is used as a working electrode (the diameter is 4 mm), a platinum wire electrode is used as a counter electrode, and an Ag/AgCl electrode is used as a reference electrode; different modified electrodes were characterized by Atomic Force Microscope (AFM) (pak corporation, korea), UTP1306S series of economical dc stabilized power supplies were from the company eudragit technologies, inc, with voltage continuously adjustable range of 0-32V and maximum output current of 0-6.0A.
Example 1 electrophoresis set
1) Prior to electropolymerization, the reaction mixture is first allowed to reactPolishing the bare GCE with 0.3 μm alumina powder, and ultrasonically cleaning in ultrapure water for 15 seconds; then polishing the bare GCE with 0.05 μm alumina powder, and ultrasonically cleaning in ultrapure water, pure ethanol and ultrapure water for 15 seconds; then in the presence of 5mM [ Fe (CN) 6 ] 3-/4- The response of bare GCE was recorded by CV scan 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:
in the presence of o-phenylenediamine omicron-PD (1.0 g/L,9.3 mmol/L) and bovine serum albumin BSA (1.4x10) -2 g/L) of PBS (ph=6.0) was purged with nitrogen for 20min and then electropolymerized using cyclic voltammetry at a scan rate of 50mV/s at a potential range of-0.1 to 1.0v, after 15 cycles of scanning, a molecularly imprinted Membrane (MIP) was formed. The synthesis of non-molecularly imprinted membranes (NIP) was similar to the molecularly imprinted membrane synthesis, but no bovine serum albumin was added during electropolymerization. Washing the electropolymerized electrode with water, then washing with ethanol-water (volume ratio of 2:1) eluent containing 1M NaOH, heating in water bath at 50deg.C for 15min, washing with ultrapure water, and drying.
3) Testing the current response of the surface of the electrode and the probe after molecular imprinting binding protein by using a differential pulse voltammetry through an electrochemical device CHI 660; according to K 3 Fe(CN) 6 And K 4 Fe(CN) 6 Redox at the electrode to produce an electrochemical signal; by studying the response of BSA to MIPs with probes, probes can diffuse into and out of the MIP matrix through the blotting cavity formed in the MIPs. The MIP/GCE was immersed in BSA solutions of different concentrations and the working electrode was placed on a stationary stage to keep the electrophoresis parallel to the bottom Pt tray. This arrangement ensures that the working electrode is 5mm from the upper level. Then, the DC power supply is turned on to provide 100 millivolts DC voltage for the electrophoresis device, and after the substance to be detected is added, the electrophoresis device is incubated for 5 minutes to ensure that protein molecules are bound to the MIP cavity.
4) After washing, the DPV method was used to obtain a solution containing 5mM [ Fe (CN) 6 ] 3-/4- And 1M KCl in PBS (ph=6.0)In the solution, the electrode after incubation is electrochemically measured under the conditions of 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. After the electrophoresis device runs for 5 minutes, the direct current power supply is turned off, the working electrode is taken out, and DPV scanning is carried out after the working electrode is washed by ultrapure water to obtain detection data.
FIG. 5A model diagram of the protein detection system of the present patent, which is composed of a power supply 1, a fixed table 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 form an electrophoresis device. The electrochemical molecular imprinting sensor 3 is suspended in the electrolytic cup 4 through the fixing table 2, and the Pt tray 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 example 1, except that no electrophoresis device was applied during incubation and detection in general group 1; other experimental conditions were consistent.
Comparative example 2 general group 2
The procedure was identical to that of example 1, except that no electrophoresis apparatus was applied during incubation and detection in general group 2, and that the incubation time after addition of the test substance in step 3) was 15min; other experimental conditions were consistent.
Example 2 electrophoresis set and general set 1
Control of the electrophoresis-applied and electrophoresis-not-applied sensors, incubation in the same system for 5min, protein concentration 7×10 -6 g/ml. The excellent performance of the electrophoresis device was analyzed, and the current responses of the sensor with and without electrophoresis were measured using the real sample bovine serum as a sample. As a result, as shown in FIG. 1, the effect of the sensor to which electrophoresis was applied was improved by 33.3. Mu.A.
Example 3 method feasibility study
The feasibility of the method in the measurement of the actual sample is examined by detecting the actual sample by the fetal bovine serum. Diluting fetal bovine serum 10000 times with 0.1mol/L PBS, and performing differential pulse method to obtain measurement data by standard recovery method, wherein the concentration of the additive is 7×10 as shown in Table 1 -8 、7×10 -7 And 7X10 -6 The recovery rates of BSA in g/ml were 105.13%, 93.98% and 116.93%, respectively, and the relative standard deviations were 5.75%, 5.04% and 5.66%, respectively. The results demonstrate that the method can be used to detect BSA in complex biological samples, thereby providing a reliable, direct means of protein detection for clinical laboratories.
TABLE 1
BSA concentration (g/ml) Recovery rate Relative standard deviation
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 film formed by electropolymerization is analyzed by an atomic force microscope, the result of AFM is shown in figure 2, in figure 2A, the roughness of the bare glassy carbon electrode is observed to be 1.496, and in the molecularly imprinted process, after the electrode is modified by o-phenylenediamine electropolymerization, the roughness of the electrode surface is obviously changed. The roughness of the molecular imprinting was 2.750 before removal of the bovine serum albumin template molecules, as shown in fig. 2B; after removal of the template molecule, the roughness of the MIP is 3.217, as in fig. 2C; the roughness of the NIP was 3.374 upon template-free molecular polymerization, as shown in fig. 2D. Where the NIP appears to be relatively flat and compact, as can be seen by atomic force microscopy, there is a difference in surface roughness of the NIP, MIP and eluted MIP, indicating that both electrodeposition of o-phenylenediamine and removal of the template molecule bovine serum albumin was successful.
Example 5 comparison of bovine serum ranges for group 2 and electrophoresis
The target substance is detected under optimal experimental conditions. The incubation detection range of the electrophoresis group is 7×10 -8 ～7×10 -6 g/ml; the detection range of the conventional electrochemical MIP sensor is 7 multiplied by 10 -6 ～7×10 -4 g/ml. The result shows that the detection range is increased by about 100 times after the electrophoresis device is added due to the enrichment of BSA by the electrophoresis device.
TABLE 2
Electrophoresis is not added Error bar Addition of 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 test
Effect of electrophoresis device on sensor: as shown in FIG. 3, the same electrode is repeatedly measured under the action of an electric field, the electrode is placed in a phosphate buffer solution to run for five minutes and is repeated for 5 times, the electrode is found to have basically no change of a current response difference value after DPV detection, and the result can indicate that the sensor is not interfered by the electric field under the electrophoresis action of 100mV and 5min, and the protein detection system disclosed by the invention has certain stability.
Example 7 tamper resistance test
A plurality of amino acids and trypsin are selected for anti-interference test, and test results show that the protein detection system disclosed by the invention has excellent anti-interference performance on interference objects such as amino acids. The system can specifically separate certain proteins under the action of electrophoresis, reduces interference to detection, and as shown in fig. 4, the system shows higher interference resistance to trypsin, mainly because the isoelectric point pi=10 of trypsin is positively charged in phosphate buffer with ph=6. The sensor added to the electrophoresis device has a much lower interference effect than normal incubation in the corresponding anti-interference test. In addition, due to the non-conductive property of o-phenylenediamine, the conducting effect of the o-phenylenediamine after molecular imprinting is formed on the surface of the electrode is far lower than that of a Pt disk forming an electric field cathode, so that the adsorption effect of the cathode field on the positively charged interferents is stronger.

Claims (9)

1. The protein detection system based on electrophoresis and molecular imprinting principle is characterized by comprising an electrochemical molecular imprinting sensor of BSA and an electrophoresis device;
the electrophoresis device is an electrolysis cup with a Pt disk at the bottom;
the electrochemical molecular imprinting sensor of the BSA is suspended in an electrolytic cup through a fixed table;
the Pt disc and the electrochemical molecular imprinting sensor of the BSA are respectively connected with two poles of a power supply, so that the targeted enrichment of the object to be detected to a sensing interface is realized under the mediation of the action force of an electric field;
the protein is bovine serum albumin; the electrochemical molecularly imprinted sensor of the BSA comprises a working electrode, a reference electrode and a counter electrode, wherein the working electrode is a MIP/GCE electrode of the BSA, the counter electrode is a platinum wire electrode, and the reference electrode is an Ag/AgCl electrode.
2. The protein detection system of claim 1, wherein the electrochemical molecularly imprinted sensor of the BSA is parallel to the Pt disk.
3. The protein detection system according to claim 1, wherein the preparation method of the MIP/GCE electrode of BSA comprises the steps of:
(1) Taking bare GCE as a substrate electrode, and polishing with aluminum oxide;
(2) Adopting o-phenylenediamine and BSA molecular templates to carry out electropolymerization on the GCE electrode polished in the step (1), and obtaining a molecular imprinting membrane electrode;
(3) And (3) eluting the molecularly imprinted membrane electrode in the step (2) by using an eluent, and washing template molecules to obtain the MIP/GCE electrode of BSA.
4. A method according to claim 3, wherein the scanning rate in step (2) is 50mV/s and the potential range is: 0.1-1.0 v, the number of electropolymerization turns is 15.
5. The method according to claim 3, wherein in the step (3), the molecularly imprinted membrane electrode is immersed in the eluent at 50 ℃ in a water bath for 15min.
6. A method for detecting BSA using the protein detection system of claim 1, comprising the steps of:
(1) Adding an object to be detected into the electrolytic cup, and incubating the electrode;
(2) And (3) electrochemically determining the electrode after incubation in the step (1) by adopting a DPV method.
7. The method of claim 6, wherein the analyte is negatively charged.
8. The method for detecting BSA according to claim 6, wherein the electrode surface in step (1) is 5mm from the liquid surface, the incubation time is 5min, and the voltage is 100mV.
9. The method for detecting BSA according to claim 6, wherein in step (2), the electric field operation time is 5min.
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