Electrochemical sensor for detecting p-tau-181 based on electrophoresis release

Abstract

The invention discloses an electrochemical sensor for detecting p-tau-181 protein based on electrophoresis release as well as a preparation method and a detection method of the electrochemical sensor, and verifies application of the sensor in detection of the p-tau-181 protein. According to the sensor, a biological recognition technology and an electrochemical sensing technology are connected through an electrophoresis technology, a signal amplification effect is generated through the synergistic effect of the biological recognition technology and the electrochemical sensing technology, high-sensitivity and high-reproducibility detection of p-tau-181 is achieved, and the sensor belongs to the technical field of electrochemical biosensors.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing, and particularly relates to an electrochemical sensor for detecting p-tau-181 protein based on electrophoresis release, and a preparation and detection method thereof. Background Art

[0002] Early diagnosis of Alzheimer's Disease (AD) relies on the detection of biomarkers with high sensitivity and specificity. As one of the core pathological biomarkers of AD, phosphorylated tau protein (p-tau-181) is closely related to neuronal damage and cognitive function decline due to its abnormal accumulation. However, traditional detection methods (such as ELISA, chemiluminescence, mass spectrometry) have problems such as complex operation, strong instrument dependence, and insufficient sensitivity, and it is difficult to achieve accurate detection especially in low-abundance samples (the content of p-tau-181 and other phosphorylated tau proteins in human whole blood is at the pg / ml level).

[0003] In recent years, electrochemical sensors have shown significant advantages in the field of protein detection due to their high sensitivity, low cost, and convenience. However, the detection of p-tau-181 still faces great challenges, such as high background interference caused by cross-reactions of non-phosphorylated tau and other phosphorylated isomers. Summary of the Invention

[0004] To solve the above problems, the present invention provides an electrochemical sensor for detecting p-tau-181 protein based on electrophoresis release, which realizes low detection limit, high sensitivity, and high specificity detection of p-tau-181, is applicable to the diagnosis of the asymptomatic stage of Alzheimer's disease, and is of great significance for the early intervention of people at risk of Alzheimer's disease.

[0005] The present application adopts the following technical solutions:

[0006] In the first aspect, the present application provides an electrochemical sensor for detecting p-tau-181 protein based on electrophoresis release, which includes an upper electrode slot 2, an insulating gasket 3, a coating film 4, and a lower electrode slot 5 that are sequentially overlapped;

[0007] The upper electrode slot is divided into a conductive area and a detection area. An exhaust hole 1 is provided at the edge of the detection area, and a conductive paste is coated to form an electrode penetrating the conductive area and the detection area;

[0008] The insulating gasket is a circular insulating sheet made of plastic or rubber material, and insulating hydrophobic glue is coated on the upper and lower sides of the circle for connecting the coating film and the electrode slot;

[0009] The coating film is an insulating film with a uniform pore structure, and p-tau-181 antibody is coated in the pores of the porous film;

[0010] The lower groove of the electrode is divided into a conductive area and a detection area. An injection hole 6 is provided at the edge of the detection area. The injection hole is provided with a leak-proof plug, and the Ag / AgCl paste is coated to form an electrode that penetrates the conductive area and the detection area.

[0011] Further, the detection area is a circular area with a diameter of 3 - 15 mm. Detection electrodes are formed by coating electrode materials on the top of the upper groove and the bottom of the lower groove in the detection area.

[0012] Further, the shape of the insulating gasket is an annular ring with an inner diameter of 3 - 15 mm and an outer diameter of 5 - 20 mm, and the thickness is 0.1 - 3 mm.

[0013] Further, the upper electrode groove and the lower electrode groove are rectangles with a length of 30 - 60 mm, a width of 5 - 20 mm, and a height of 0.1 - 3 mm, and the material is insulating plastic.

[0014] Further, the dimensions of the upper electrode groove, the lower electrode groove, and the insulating gasket are used not only to specify the volume of the test solution but also to adjust the voltage range and test time during the detection process.

[0015] Further, the electrode material is one or more of Au, Pt, Ag, Ag / AgCl, and carbon electrodes.

[0016] In a second aspect, the present application provides a preparation method of the above-mentioned electrochemical sensor based on electrophoresis release, which includes:

[0017] Loading a polymer with positively enriched surface into the pores of the porous membrane to obtain an adsorption membrane;

[0018] Electrostatically adsorbing an antibody / aptamer into the pores of the adsorption membrane, and then using BSA to block the remaining active sites in the pores to obtain a coated membrane;

[0019] Gluing and fixing the insulating gasket to the detection areas of the upper electrode groove and the lower electrode groove respectively, and gluing and fixing the coated membrane in the center of the insulating gaskets of the upper electrode groove and the lower electrode groove to obtain an electrochemical sensor.

[0020] Further, according to the required detection linear range, the pore density of the porous membrane is selected to be 10000 - 2×10 11 per cm 2 , and the pore diameter of the porous membrane is selected to be 0.01 - 5 μm.

[0021] Further, the above-mentioned preparation method of loading a polymer with positively enriched surface into the pores of the porous membrane includes:

[0022] Immerse the PET nuclear pore membrane in a mixed solution of 1 - 25 mg / ml of EDC and 1 - 25 mg / ml of NHS, adjust the pH to 4 - 7, activate at 4°C for 2 - 4 h, and wash with PBS and ultrapure water after the reaction;

[0023] Immerse the activated nuclear pore membrane in a 1 - 10 mg / ml PEI solution, react at 37°C for 2 - 18 h, and wash with PBS solution and ultrapure water after the reaction to obtain an adsorption membrane.

[0024] Furthermore, the preparation method of the above coating membrane includes:

[0025] Immerse the adsorption membrane in a 1 - 100 μg / ml antibody solution, react on a shaker at 37°C for 1 - 4 h, and wash with PBS and ultrapure water after the reaction to obtain an antibody adsorption membrane;

[0026] Immerse the washed antibody adsorption membrane in a 1% BSA solution, react on a shaker at 37°C for 0.5 - 2 h, and wash with PBS and ultrapure water after the reaction to obtain a coating membrane.

[0027] In a third aspect, the present application provides a detection method for the above electrochemical sensor based on electrophoresis release, which includes:

[0028] Step 1, slowly inject the test solution into the sensor through the lower leak-proof plug, so that the test solution completely discharges the air in the sensor through the exhaust hole above the sensor;

[0029] Step 2, connect the sensor to an electrochemical workstation, where the working electrode is connected to the upper slot electrode of the sensor, and the counter electrode and reference electrode are connected to the lower slot of the sensor. Use chronopotentiometry, set the test voltage to 0.5 - 3 V, and the time to 30 - 600 s;

[0030] Step 3, use chronopotentiometry, set the test voltage to -0.5 - -1 V, and the time to 10 - 300 s;

[0031] Step 4, use cyclic voltammetry, with an initial potential of 0 V, a final potential of 0 V, an upper limit voltage of 50 - 500 mV, a lower limit voltage of -50 - -500 mV, a scan rate of 1 - 100 mV / s, and the number of cycles of 3 - 20 times;

[0032] Step 5, use chronopotentiometry, set the test voltage to 1 - 3 V, and the time to 15 - 600 s. After the current stabilizes, record the current value as the response value;

[0033] Take the current value stabilized in Step 2 as the baseline value, and plot the concentration-baseline current curve. Considering the complex protein components in plasma during actual detection, to correct for endogenous interference, take the current value stabilized in Step 5 as the response value, calculate the current change rate, and substitute it into the quantification curve to obtain the concentration of the analyte in the test solution.

[0034] Further, a method for differentiating false positives is provided. If the results obtained from the two curves are inconsistent, it indicates that there is significant endogenous interference in the sample. After excluding interfering factors such as elevated rheumatoid factor, further testing is required.

[0035] Further, the test solution is a PBS solution or serum containing the analyte, with a pH of 6 - 8.

[0036] Further, sucrose can be added to the analyte solution to increase the solution viscosity in order to adjust the test parameters.

[0037] Further, the purpose of regulating the voltage and test time is to control the electrophoresis rate of the analyte in the sensor, enabling the analyte to be purposefully enriched and moved, thereby improving the capture efficiency and response signal.

[0038] Further, Step 2 enriches the analyte onto the electrode in the upper slot of the electrode.

[0039] Further, Step 3 allows the uncaught analyte to pass through the coating membrane and enriches it at the junction of the coating membrane and the lower slot of the electrode.

[0040] Further, Step 4 allows the uncaught analyte to repeatedly pass through the pores of the coating membrane, enabling the analyte to be captured as much as possible.

[0041] Further, Step 5 causes the antigen-antibody in the pores to desorb from the pores by overcoming the electrostatic interaction under a strong electric field and enriches it onto the electrode in the upper slot of the electrode.

[0042] Further, the electric field strength is calculated by v = μ / E, where v is the electrophoresis migration rate, μ is the electrophoresis mobility, and E is the applied electric field strength.

[0043] Further, the electrophoresis mobility is calculated by, where Z is the net charge number, e is the electric charge amount, η is the viscosity coefficient, and r is the radius of gyration.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] 1. The present invention combines immunoassay with electrochemical analysis technology to quantitatively detect the content of p-tau-181 in serum samples. The detection principle is as follows: A substance with a large number of positive charges is modified in the pores of the porous membrane. Relying on the electrostatic adsorption between molecules, the substance specifically binding to the analyte is temporarily fixed inside the pores. After assembling the sensor, the air inside the sensor is emptied by injecting liquid from below to form a complete electrolytic cell circuit. Subsequently, by changing the electric field strength and direction, relying on electrophoresis, the analyte is incubated inside the sensor. Finally, a unidirectional strong electric field is applied to cause the probe specifically bound in the pores to desorb from the electrostatic interaction with the analyte. By detecting the current change brought about by the desorption in the pores, the specific detection of the analyte is achieved.

[0046] 2. The present invention uses two quantification curves drawn under different detection states, one is for rapid incubation and the other is the quantification curve after stable incubation. The patient group with Alzheimer's disease is an aging population and is more likely to be interfered by rheumatoid factors and heterophilic antibodies. If there is no obvious interference in the sample, the two curves should show consistent detection results. When the two curves show different detection results, the above-mentioned endogenous interference should be preferentially investigated, providing a means for differentiating false positives.

[0047] 3. The present invention proposes a combined strategy of electrophoresis release - electrochemical sensing, innovatively combining electrophoresis with bioelectrochemical sensing technology. Utilizing the low isoelectric point characteristic of p-tau-181, p-tau-181 is driven to migrate and accumulate on the sensor surface at a specific pH by regulating the electric field, achieving highly selective capture and highly sensitive quantification of the target protein. This technology not only overcomes the dependence on complex sample pretreatment in traditional methods but also can effectively distinguish p-tau-181 from other tau isoforms through the characteristic of charge difference, providing a new tool for the early diagnosis of AD and the study of pathological mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a schematic diagram of the electrochemical biosensor device for the present invention.

[0049] Figure 2 It is a test mechanism diagram of the electrochemical biosensor of the present invention.

[0050] Figure 3 It is the concentration - baseline current quantification curve of the electrochemical sensor detecting p-tau-181 in Example 2.

[0051] Figure 4 It is the concentration - current change rate quantification curve of the electrochemical sensor detecting p-tau-181 in Example 2.

[0052] Figure 5 It is the specificity of the electrochemical sensor in Example 3. Detailed implementation manners

[0053] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments.

[0054] Example 1 Preparation of a sensor for detecting p-tau-181

[0055] Immerse a 15*15 mm PET nuclear pore membrane in a mixed solution of 15 mg / ml EDC and 3 mg / ml NHS, adjust the pH to 6, activate it at 4°C for 2 h, and after the reaction, wash it with PBS and ultrapure water.

[0056] Immerse the activated nuclear pore membrane in a 5 mg / ml PEI solution, with the solvent being 1x PBS solution, react at 37°C for 4 h, and after the reaction, wash it with PBS solution and ultrapure water to obtain an adsorption membrane.

[0057] Immerse the adsorption membrane in a 100 μg / ml anti-p-tau-181 antibody solution, react on a shaker at 37°C for 1 h, and after the reaction, wash it with PBS and ultrapure water to obtain an antibody adsorption membrane.

[0058] Immerse the washed antibody adsorption membrane in a 1% BSA solution, react on a shaker at 37°C for 1 h, and after the reaction, wash it with PBS and ultrapure water to obtain a coated membrane. Seal and store the coated membrane in a 4°C refrigerator for later use.

[0059] The inner diameter of the insulating gasket is 10 mm, the outer diameter is 15 mm, and the thickness is 2.5 mm; the length of the upper and lower electrode grooves is 36 mm, the width is 15 mm, and the height is 1 mm.

[0060] Connect the upper electrode groove, insulating gasket, coated membrane, insulating gasket, and lower electrode groove in sequence. The two sides of the insulating gasket are coated with hydrophobic insulating glue, and it is pressed between the upper and lower electrode grooves and the coated membrane to form a closed detection area.

[0061] Example 2 Sensitivity test of the electrochemical sensor

[0062] Step 1, slowly inject the test solution into the sensor through the lower leak-proof plug, so that the air in the sensor is completely discharged through the exhaust hole above the sensor by the test solution;

[0063] Step 2, connect the sensor to an electrochemical workstation, where the working electrode is connected to the upper electrode groove of the sensor, and the counter electrode and reference electrode are connected to the lower electrode groove of the sensor. Use chronopotentiometry, set the test voltage to 1 V, and the time to 200 s;

[0064] Step 3: Using chronopotentiometry, set the test voltage to -1V and the time to 100s;

[0065] Step 4: Using cyclic voltammetry, the initial potential is 0V, the end potential is 0V, the upper limit voltage is 250mV, the lower limit voltage is -250mV, the scan rate is 10mV / s, and the number of cycles is 6;

[0066] Step 5: Using chronopotentiometry, set the test voltage to 2V and the time to 200s.

[0067] Take the current value after stabilization in Step 2 as the baseline value and plot the concentration-baseline current curve. Considering the complex protein components in plasma during actual detection, to correct for endogenous interference, take the current value after stabilization in Step 5 as the response value, calculate the current change rate, and substitute it into the quantification curve to obtain the concentration of the analyte in the test solution. If the results obtained from the two curves are inconsistent, it indicates that there is significant endogenous interference in the sample and further testing is required.

[0068] Using the sensor prepared in Example 1, according to the above detection method, test analytes at different concentrations to plot the quantification curve. The experimental results are shown in the following table:

[0069] Where the concentration is the concentration of the analyte, the group is the control group at the same concentration, the baseline value is the current value obtained in Detection Step 2, the response value is the current value obtained in Detection Step 5, SD is the standard deviation, and CV is the coefficient of variation.

[0070] The results are as Figure 3 The concentration-baseline current curve of the electrochemical sensor for detecting p-tau-181 and Figure 4 The quantification curve of the electrochemical sensor for detecting p-tau-181 show that the detection results of the sensor for p-tau-181 are significantly linearly related in the range of 0 - 15 pg / ml, indicating that the sensor has excellent detection performance for the content range of p-tau-181 in human plasma.

[0071] Specificity experiment of the electrochemical sensor in Example 3.

[0072] Using the sensor prepared in Example 1, according to the detection method in Example 2, take unphosphorylated tau-181 and p-tau-217 as the control groups to test the specificity of the electrochemical sensor. The concentration of the test solution is 10 pg / ml. The test results are shown in the following table:

[0073] Where the group is the control group under the same analyte, SD is the standard deviation, and CV is the coefficient of variation.

[0074] The test results are as follows Figure 5 shown. Compared with the test results of p-tau-181 at 10 pg / ml in Example 2, it shows that the sensor has good specificity.

[0075] Stability experiment of the electrochemical sensor in Example 4.

[0076] The prepared electrochemical sensor in Example 1 was stored at -20 °C. Using the analyte with a concentration of 10 pg / ml, it was tested according to the test method in Example 2 on the 1st, 2nd, 3rd, 5th, 7th, 10th, 14th, 21st, 28th, 42nd, and 56th days respectively, and then repeated every four weeks until either the CV value of the baseline value or the baseline / response value was greater than 5%. The test results are shown in the following table:

[0077] Among them, the number of days is the storage time of the sensors prepared in the same batch, and CV is the coefficient of variation.

[0078] The test results show that when the standard is CV < 5%, the sensor has a shelf life of at least 112 days and has good stability.

Claims

1. An electrochemical sensor for detecting p-tau-181 protein based on electrophoretic release, characterized in that: It includes an upper electrode groove, an insulating sealing pad, a coating film, and a lower electrode groove which are overlapped in sequence; The upper groove of the electrode is divided into a conductive area and a detection area, and an exhaust hole is arranged at the edge of the detection area, and a conductive slurry is coated to form an electrode that penetrates the conductive area and the detection area; The insulating sealing pad is made of a circular insulating sheet of plastic or rubber material, and the upper and lower parts of the ring are coated with insulating hydrophobic glue, which is used to connect the coating membrane and the electrode groove; The coating membrane is made of an insulating membrane with a uniform pore structure, and the interior of the pores is modified with biological probes that specifically capture the object to be detected; The lower groove of the electrode is divided into a conductive area and a detection area. An injection hole is arranged at the edge of the detection area. The injection hole is provided with a rubber anti-leakage plug. Conductive slurry is coated to form an electrode that penetrates the conductive area and the detection area.

2. The detection zone according to claim 1, characterized in that The detection area is a circular area with a diameter of 3-20 mm. The top of the upper groove and the bottom of the lower groove in the detection area are coated with electrode materials to form detection electrodes.

3. The insulating sealing pad according to claim 1, characterized in that: The insulating sealing gasket is in the shape of a ring with an inner diameter of 3-20mm and an outer diameter of 5-30mm, and a thickness of 0.1-3mm.

4. The upper electrode tank and the lower electrode tank according to claim 1, characterized in that: The length of the upper electrode groove and the lower electrode groove is 30-60mm, the width is 5-20mm, the height is 0.1-3mm, and the material is insulating plastic.

5. The coating film according to claim 1, characterized in that The substance that specifically captures the analyte is modified on the inner wall of the pore of the coating membrane to achieve specific capture of the analyte.

6. The electrochemical sensor according to claim 1, characterized in that The preparation method of the electrochemical sensor is to bond and fix the insulating sealing pad to the detection areas of the upper electrode groove and the lower electrode groove respectively, and bond and fix the coating film to the center of the insulating sealing pad of the upper electrode groove and the lower electrode groove to obtain the electrochemical sensor.

7. A detection method of an electrochemical sensor based on electrophoretic release, characterized in that: The test steps are: Step 1, slowly inject the test liquid into the sensor through the anti-leakage plug at the bottom, so that the test liquid completely discharges the air in the sensor through the exhaust hole at the top of the sensor; Step 2: Connect the sensor to the electrochemical workstation, with the working electrode connected to the upper tank electrode of the sensor, and the counter electrode and reference electrode connected to the lower tank of the sensor. Use the chronopotentiometry method, set the test voltage to 0.5-3V, and the time to 30-600s. Step 3: Use the chronopotentiometry method, set the test voltage to -0.5 – -1V, and the time to 10 – 300s. Step 4: Cyclic voltammetry was used with an initial potential of 0 V, an end potential of 0 V, an upper voltage of 50 – 500 mV, a lower voltage of -50 – -500 mV, a scan rate of 1 – 100 mV / s, and a cycle number of 3 – 20 times. Step 5, using the chronopotentiometry, set the test voltage to 1-3V, the time to 15-600s, and after the current stabilizes, record the current value as the response value; The current value stabilized in step 2 is used as the baseline value, and a concentration-baseline current quantification curve is drawn; the current value stabilized in step 5 is used as the response value, the current change rate is calculated, and a concentration-current change rate quantification curve is drawn to obtain the concentration of the analyte in the test solution; It provides a means of identifying false positives. If the results of the two quantitative curves are inconsistent, it indicates that the sample has a large endogenous interference. It is necessary to exclude the interfering factors, such as the increase of rheumatoid factor, before further testing. The purpose of steps 2-4 is to use electrophoresis technology to regulate the enrichment site of the analyte in the sensor and improve the incubation efficiency of the analyte; The purpose of step 5 is to utilize a strong electric field force to desorb the biological probe and the substance to be detected from the electrostatic interaction in the pores of the nuclear pore membrane, and then enrich them on the electrode surface.

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