Preparation method of electrochemical biosensor based on PLL-coated VPAu modified nuclear track membrane

Abstract

The invention provides a preparation method of an electrochemical biosensor based on a PLL-coated VPAu modified nuclear track membrane, and belongs to the field of electrochemical biosensing analysis. The electrochemical biosensor for detecting troponin is constructed by adsorbing a PLL (at) VPAu composite nanomaterial synthesized by an ultrasonic liquid phase assisted method into a nuclear track membrane pore channel through electrostatic interaction and connecting a specific recognition molecule of a target to the surface of the pore channel modified by the PLL (at) VPAu by utilizing a bonding reaction mechanism. After a target object is captured by the recognition molecules connected to the nuclear track membrane, the effective aperture of the nuclear track membrane nanopores is reduced, and the electrochemical workstation can convert the biological behavior into an electric signal, so that high-sensitivity and rapid detection of the target protein is realized. The method has very important theoretical and practical significance in the field of medical diagnosis, and a new direction is provided for application of the nuclear track membrane in the field of analysis and detection.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical biosensors, and in particular relates to a method for preparing an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane. Background Art

[0002] Acute myocardial infarction is a serious cardiovascular disease that causes high morbidity and mortality worldwide, posing a serious threat to people's lives and health. Troponin (cTnI) is considered to be a reliable and important biomarker for accurate diagnosis and effective monitoring of myocardial infarction due to its sensitivity and specificity. Early detection of cTnI can effectively prevent myocardial infarction, reduce related mortality and prolong the life of patients. Therefore, it is of great significance to establish a reliable, selective and sensitive cTnI detection method.

[0003] Electrochemical biosensors are devices that convert biological signals in a system into monitorable electrical signals. They are easy to use and have rapid analysis. They have become an ideal platform for detecting biomarkers. In addition, they have broad application prospects in food safety, biomedicine, and environmental testing. However, the background signal interference is strong, and it is still challenging to achieve trace detection and improve sensitivity. Nanomaterials and specific recognition elements can usually be modified on biosensors to enable them to have the ability to capture the molecules to be tested with high sensitivity. Nuclear pore membranes are considered to be good substrates for electrochemical biosensors due to their superior mechanical and chemical stability, good structure, and easily customizable surface properties. They have attracted widespread attention in recent years. The pores of porous membranes can simultaneously provide rich and good active sites for the fixation of nanomaterials and specific aptamers, which greatly reduces the response of biosensors to non-targeted substances and improves the detection sensitivity. Summary of the invention

[0004] The present invention aims to provide a method for preparing an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane, and belongs to the field of electrochemical biosensor analysis. The present invention adopts a liquid phase ultrasound-assisted method to synthesize a PLL@VPAu composite nanomaterial, and places it in the pore of the nuclear pore membrane. Since the etched nuclear pore membrane pore contains carboxyl groups, the pore is negatively charged, and the prepared VPAu composite material is positively charged after mixing with polylysine (PLL), and PLL@VPAu is connected to the nuclear pore membrane through electrostatic action. At the same time, the specific recognition element of the target is connected to the pore modified by PLL@VPAu by using a bonding reaction mechanism (Au-S), and the remaining active sites are blocked with BSA to prevent nonspecific adsorption, and an electrochemical biosensor for detecting cTnI is constructed. When the target cTnI is captured by the recognition element connected to the nuclear pore membrane, the effective pore size of the nanopore is reduced, resulting in a smaller carrier density in the pore, and the electrochemical workstation converts the biological behavior into an electrical signal. By recording the change in current before and after the addition of cTnI antigen, highly sensitive and rapid quantitative detection can be achieved.

[0005] The detailed preparation steps of the method are:

[0006] (1) Preparation of PLL@VPAu composite nanomaterials:

[0007] Put the purple phosphorus crystals into a mortar for grinding, then transfer the powder to a stripping solvent, transfer it to an ultrasonic cell crusher and sonicate for 8 to 10 hours for stripping, centrifuge the obtained VP dispersion at 4°C to remove the unstripped solids, and take the supernatant for further centrifugation. Rinse the precipitate with ethanol several times and redissolve it in water to prepare a VP dispersion with a concentration of 0.5 to 2 mg / mL. Mix a chloroauric acid solution with a concentration of 20 to 50 mmol / L with the VP solution and sonicate for 10 to 15 minutes to obtain VPAu, then mix VPAu with a PLL solution, the volume ratio of the two solutions is 1: (0.8 to 1.5), and then obtain a PLL@VPAu composite nanomaterial;

[0008] (2) Construction of biosensor based on PLL@VPAu modified nuclear pore membrane:

[0009] The nuclear pore membrane was placed in the above-mentioned PLL@VPAu solution, and the composite material was adsorbed in the pores of the nuclear pore membrane by electrostatic action. After multiple rinses, it was dried and then incubated in an aptamer solution with a concentration of 5-30 μmol / L. Bovine serum albumin (BSA) was used for site blocking to prevent nonspecific adsorption, and PBS was rinsed three times. After vacuum drying at 45°C, an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane was prepared.

[0010] Preferably, the raw material of the nuclear pore membrane in step 1) is usually polycarbonate (PC), polyethylene terephthalate (PET) or polypropylene (PP). The surface pore size of the nuclear pore membrane is 2.0~5.0μm, and the pore channel length is 10~25μm; the VP size is <2.0μm, and the Au size is <13nm; the stripping solvent is N-methylpyrrolidone (NMP), dimethylformamide (DMF), isopropanol (IPA) or water. The ultrasonic power is 600~1000W, the frequency is 20~25KHz, and the ice bath ultrasound is used; the ultrasound and centrifugation temperatures are both 0~4℃; the PLL molecular weight is 20000~50000, and the concentration is 0.5~2mg / mL.

[0011] Preferably, the aptamer sequence in step 2) is 5'-SH-C6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'.

[0012] An electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane as described above is used to establish an electrochemical analysis method for cTnI detection, comprising the following steps:

[0013] Detection program setting: Place the modified nuclear pore membrane between the clamps of the electrolytic cell, inject a certain concentration of electrolyte solution into the electrolytic cell, insert electrodes at both ends of the electrolytic cell, use an electrochemical workstation to apply a pulse voltage, and record the response current changes before and after the electrochemical biosensor based on the PLL@VPAu modified nuclear pore membrane captures different concentrations of cTnl.

[0014] Drawing of standard curve: The solution to be tested is placed in a functionalized nucleopore membrane for incubation, and then cTnl that is not bound to the specific aptamer is removed to obtain an electrochemical biosensor based on the test object. The current change before and after the addition of cTnI antigen is recorded by an electrochemical workstation to achieve quantitative detection of cTnI, and a standard curve with the horizontal and vertical coordinates of cTnI concentration and the current relative change rate (I0-I) / I0 is drawn, where I0 is the response current before adding the target, and I is the response current after adding the target;

[0015] Preferably, the electrolyte solution in step 1) is a 1×PBS standard solution with a pH of 6 to 8; the electrode material is Ag / AgCl; and the electrochemical test voltage range is -2 to 2V.

[0016] Preferably, in step 2), the incubation time of the antigen and the aptamer is 5-25 min, and the incubation temperature is 25-40°C.

[0017] The technical principle of the present invention is: since the pores of the nuclear pore membrane carry carboxyl groups after etching, its surface is negatively charged, and the positively charged PLL@VPAu nanomaterials can be adsorbed through electrostatic action to prepare an effective electrochemical biosensor substrate, and by introducing specific recognition molecular aptamers, an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane is constructed for efficient and sensitive detection of biomarkers. When the target is present, it is captured by the aptamer connected to the nuclear pore membrane, resulting in pore blockage, and the electron transfer rate in the system drops sharply due to the increase in steric hindrance, thereby achieving ultra-sensitive quantitative detection of target molecules. The present invention mainly uses the electrochemical signal amplification effect of the VPAu material with a two-dimensional layered structure to improve sensitivity, and improves selectivity through aptamer specific capture, thereby achieving ultra-sensitive rapid quantitative detection of cTnI.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects:

[0019] The electrochemical biosensor for cTnI antigen detection provided by the present invention adopts two-dimensional nanomaterials as a signal amplification strategy and uses specific recognition molecules to selectively capture the target to be detected, so that the sensor has high sensitivity and its detection limit can reach 0.1 ng / mL.

[0020] The electrochemical biosensor for cTnI antigen detection provided by the present invention has high specificity and has no response to other non-targeted proteins.

[0021] The electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane provided by the present invention is easy to use and has fast analysis, and the detection can be completed within 15 minutes, which is suitable for the actual needs in the industrialization of electrochemical biosensors.

[0022] The present invention can be extended to the detection of other targets by replacing specific recognition molecules, providing valuable insights for future applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0024] Figure 1 This is a schematic diagram of the detection device of the biosensor for cTnI antigen detection;

[0025] Figure 2 The electrochemical sensing principle diagram of the electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane to detect the target;

[0026] Figure 3Optimization of experimental conditions (optimization of aptamer concentration, antigen-aptamer incubation time, and pH value);

[0027] Figure 4 IV curves of the responses to different concentrations of cTnI;

[0028] Figure 5 Standard curve of the current change rate curve in response to different concentrations of cTnI;

[0029] Figure 6 Repeatability, reproducibility and stability bar graph (current relative rate of change bar graph);

[0030] Figure 7 Specificity bar graph (bar graph of relative current change rate corresponding to different antigens). Specific implementation methods

[0031] The present invention is further described below in conjunction with specific embodiments and drawings, but the present invention is not limited to the following embodiments.

[0032] Example 1

[0033] A method for preparing an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane, wherein the method is as follows: Figure 1 As shown, the following steps are included:

[0034] Preparation of PLL@VPAu composite nanomaterials: 100 mg of purple phosphorus crystals were placed in a mortar for grinding, and then the powder was transferred to 100 mL of NMP solution, and then transferred to an ultrasonic cell crusher for 8 hours for exfoliation. The obtained VP dispersion was centrifuged at 3000 rpm for 20 minutes at a temperature of 4°C, and the supernatant was further centrifuged at 11000 rpm for 15 minutes. The precipitate was rinsed with ethanol several times and then redissolved in water to prepare a VP dispersion with a concentration of 1 mg / mL. 10 μL of 50 mmol / L chloroauric acid solution was mixed with 10 μL of the above VP solution and ultrasonicated for 10 minutes to obtain VPAu, and then VPAu was mixed with PLL solution, and the volume ratio of the two solutions was 1:1 to obtain PLL@VPAu composite nanomaterials.

[0035] Construction of biosensor based on PLL@VPAu modified nuclear pore membrane:

[0036] The nucleopore membrane was placed in the above-mentioned PLL@VPAu solution for 60 min. The composite material was adsorbed in the pores of the nucleopore membrane by electrostatic action. After multiple rinses, it was dried and then incubated in a 15 μmol / L aptamer solution for 60 min. The site was blocked with 1% bovine serum albumin (BSA) to prevent nonspecific adsorption, and PBS was rinsed three times. After vacuum drying at 45°C, an electrochemical biosensor based on PLL@VPAu modified nucleopore membrane was prepared. The prepared nucleopore membrane was immersed in cTnI solutions of different concentrations and incubated for 1 hour. Among them, the optimized parameters ( Figure 3 ) including aptamer concentration, antigen-aptamer incubation time and pH optimization.

[0037] Example 2

[0038] An application of an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane comprises the following steps:

[0039] Detection program setting: Place the modified nucleoporation membrane between the clamps of the electrolytic cell, inject 1×PBS electrolyte solution into the electrolytic cell, insert Ag / AgCl electrodes at both ends of the electrolytic cell, and use an electrochemical workstation to apply a voltage of -2~2V to obtain the current value I0 when there is no target; place solutions containing different concentrations of target in the functionalized nucleoporation membrane for incubation for 15 minutes, then remove cTnl that is not bound to the specific aptamer, and measure to obtain the current value I( Figure 4 ). The response current changes before and after capturing different concentrations of cTnI through the electrochemical biosensor based on PLL@VPAu modified nucleopore membrane were recorded.

[0040] Drawing of standard curve: With cTnI concentration as the horizontal axis and current relative change rate as the vertical axis, draw the standard curve, where the current relative change rate is (I0-I) / I0. As the concentration of cTnI increases in the range of 0.01~100ng / mL, the relative change rate of current also increases significantly ( Figure 5 ), and the two have a good nonlinear relationship.

[0041] Under the same conditions, the current value of the same electrochemical biosensor was measured six times in succession, and its relative standard deviation (RSD) was about 1.30%; the current value of six electrochemical biosensors based on PLL@VPAu modified nuclear pore membrane prepared in the same batch was monitored, and its RSD was 1.66%; after storage at 4°C for 5 weeks, 91.4% of the response current value was retained compared with the first day ( Figure 6 ).

[0042] Example 3

[0043] The specificity of the electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane for cTnI is shown in Figure 7 As shown, other types of proteins were selected at the same concentration, such as cardiac troponin C (cTnC), prostate antigen (PSA), human serum albumin (HSA), glucose oxidase (GO X ) and human mucin 1 (MUC1) were used as interfering substances. The nuclear pore membranes were incubated in different solutions for 15 min, and the relative change rates of the current before and after incubation were compared. X When the target is cTnI and MUC1, the relative change rate of the current of the sensing platform is about 5.93±0.45%. When the target is cTnI, the relative change rate of the current of the entire system is about 62.3±4.53%.

[0044] The above description is only the best embodiment of the present invention and is not intended to limit the spirit and principle of the present invention. Any modifications, equivalent substitutions, improvements, etc. made should be included in the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane, characterized in that: The PLL@VPAu composite material was placed in the pores of the nuclear pore membrane to obtain the PLL@VPAu functionalized nuclear pore membrane. Then, specific recognition molecules were introduced to successfully construct an electrochemical biosensor based on the PLL@VPAu modified nuclear pore membrane.

2. The method for preparing an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane according to claim 1, characterized in that: The specific recognition molecules include antibodies, polypeptides, enzymes, aptamers or other nucleic acid molecules.

3. The method for preparing an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane according to claim 1, characterized in that: Using the specific aptamer as the recognition molecule and cTnI as the analyte, the specific preparation steps are as follows: Step 1) grinding purple phosphorus (VP) crystals, then placing the powder in a stripping solvent for ultrasonic stripping, centrifuging the obtained VP dispersion to remove unstripped solids, and taking the supernatant for further centrifugation; washing the precipitate several times and then redissolving it in water to prepare a VP dispersion; mixing the chloroauric acid solution with the VP solution and ultrasonicating it for a period of time; A VPAu nanocomposite material is obtained, and then VPAu is mixed with a PLL solution to obtain a PLL@VPAu composite nanomaterial; Step 2) Construction of electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane: The nuclear pore membrane is placed in the above-mentioned PLL@VPAu solution, and the composite material is adsorbed in the pores of the nuclear pore membrane by electrostatic action. After multiple rinses, the composite material is dried and then incubated in the aptamer solution. The site is blocked with bovine serum albumin (BSA) to prevent nonspecific adsorption. After multiple rinses with PBS and vacuum drying, an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane is prepared.

4. The method for preparing an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane according to claim 3, characterized in that: Step 1) The raw material of the nuclear pore membrane is usually polycarbonate (PC), polyethylene terephthalate (PET) or polypropylene (PP); the surface pore size of the nuclear pore membrane is larger than the VP size; the stripping solvent is N-methylpyrrolidone (NMP), dimethylformamide (DMF), isopropyl alcohol (IPA) or water.

5. The method for preparing an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane according to claim 3, characterized in that: The aptamer sequence in step 2) is 5'-SH-C6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'.

6. The method for preparing an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane according to claims 1 to 5, characterized in that: The electrochemical analysis method for cTnI detection was established, comprising the following steps: 1) Detection program setting: Place the modified nucleopore membrane between the clamps of the electrolytic cell, inject a certain concentration of electrolyte solution into the electrolytic cell, insert electrodes at both ends of the electrolytic cell, use an electrochemical workstation to apply a pulse voltage, and record the response current values ​​before and after the electrochemical biosensor based on the PLL@VPAu modified nucleopore membrane captures different concentrations of cTnl; 2) Drawing of the standard curve: The test solution is placed in a functionalized nucleopore membrane for incubation, and then cTnl that is not bound to the specific aptamer is removed to obtain an electrochemical biosensor based on the test object; the current change before and after the addition of cTnI antigen is recorded by an electrochemical workstation to achieve quantitative detection of cTnI, and a standard curve is drawn with the horizontal and vertical coordinates being the cTnI concentration and the current relative change rate (I0-I) / I0, respectively, where I0 is the response current before the addition of the target object, and I is the response current after the addition of the target object.

7. The method for preparing an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane according to claim 6, characterized in that: The electrolyte solution is a PBS (phosphate buffer solution) standard solution with a pH of 6-8; the electrode material is Ag / AgCl; the electrochemical test voltage range is -2-2V; the antigen incubation time is 5-25min, and the incubation temperature is 25-40°C.

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