Preparation method of an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane

Abstract

The application provides a preparation method of an electrochemical biosensor based on a nuclear pore membrane modified by PLL@VPAu, and belongs to the field of electrochemical biosensing analysis. In the application, PLL@VPAu composite nanomaterials synthesized by ultrasonic liquid-phase assisted method are adsorbed in the pore channel of the nuclear pore membrane through electrostatic action, and the specific recognition molecules of the target are connected to the pore channel surface of the PLL@VPAu modification through a bonding reaction mechanism, so as to construct an electrochemical biosensor for detecting troponin. When the target is captured by the recognition molecules connected on the nuclear pore membrane, the effective pore diameter of the nanopore of the nuclear pore membrane is reduced, and the electrochemical workstation can convert the biological behavior into an electrical signal, so that high-sensitivity and rapid detection of the target protein is realized. The application has very important theoretical and practical significance in the field of medical diagnosis, and provides a new direction for the application of the nuclear pore membrane in the field of analysis and detection.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical biosensor technology, specifically relating to a method for preparing an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane. Background Technology

[0002] Acute myocardial infarction is a serious cardiovascular disease, causing high morbidity and mortality worldwide and posing a serious threat to human health. Troponin I (cTnI) is considered 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 patient life. Therefore, establishing a reliable, selective, and sensitive cTnI detection method is of great significance.

[0003] Electrochemical biosensors convert biological signals in a system into measurable electrical signals. They are distinguished by their ease of use and rapid analysis, and have become an ideal platform for detecting biomarkers. Furthermore, they have broad application prospects in food safety, biomedicine, and environmental monitoring. However, strong background interference makes achieving trace detection and improving sensitivity challenging. Typically, nanomaterials and specific recognition elements can be modified onto biosensors to enable them to capture analytes with high sensitivity. Nuclear pore membranes are considered excellent substrates for electrochemical biosensors due to their superior mechanical and chemical stability, favorable structure, and easily customizable surface properties, and have attracted widespread attention in recent years. The porous membrane channels can simultaneously provide abundant and favorable active sites for the immobilization of nanomaterials and specific aptamers, which significantly reduces the biosensor's response to non-target substances and improves detection sensitivity. Summary of the Invention

[0004] This invention aims to provide a method for preparing an electrochemical biosensor based on a PLL@VPAu-modified nuclear pore membrane, belonging to the field of electrochemical biosensing and analysis. This invention employs a liquid-phase ultrasonic-assisted method to synthesize PLL@VPAu composite nanomaterials, which are then placed within the pores of a nuclear pore membrane. Because the etched nuclear pore membrane pores contain carboxyl groups, they are negatively charged. The prepared VPAu composite material, when mixed with poly-L-lysine (PLL), becomes positively charged. PLL@VPAu and the nuclear pore membrane are connected via electrostatic interactions. Simultaneously, a specific recognition element for the target is attached to the PLL@VPAu-modified pores using a bonding reaction mechanism (Au-S), and the remaining active sites are sealed with BSA to prevent non-specific adsorption, thus constructing an electrochemical biosensor for detecting cTnI. When the target analyte cTnI is captured by the recognition element attached to the nuclear pore membrane, the effective pore size of the nanopore decreases, leading to a lower carrier density within the pore. The electrochemical workstation then converts this biological behavior into an electrical signal. By recording the changes 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 as follows:

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

[0007] Purple phosphorus crystals were ground in a mortar, and the powder was then transferred to an exfoliating solvent and sonicated in an ultrasonic cell disruptor for 8-10 hours for exfoliation. The resulting VP dispersion was centrifuged at 4°C to remove unexfoliated solids, and the supernatant was centrifuged again. The precipitate was washed repeatedly with ethanol and then redissolved in water to prepare a VP dispersion with a concentration of 0.5-2 mg / mL. A chloroauric acid solution with a concentration of 20-50 mmol / L was mixed with the VP solution and sonicated for 10-15 min to obtain VPAu. VPAu was then mixed with a PLL solution at a volume ratio of 1:(0.8-1.5) to obtain PLL@VPAu composite nanomaterials.

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

[0009] The nuclear pore membrane was placed in the above-mentioned PLL@VPAu solution. The composite material was adsorbed into the pores of the nuclear pore membrane through electrostatic interaction. After multiple rinsings and air drying, it was incubated in an aptamer solution with a concentration of 5-30 μmol / L. Site blocking was performed with bovine serum albumin (BSA) to prevent non-specific adsorption. After washing three times with PBS and vacuum drying at 45°C, the electrochemical biosensor based on the PLL@VPAu modified nuclear pore membrane was prepared.

[0010] Preferably, the raw material for the nuclear pore membrane in step 1) is typically polycarbonate (PC), polyethylene terephthalate (PET), or polypropylene (PP). The surface pore size of the nuclear pore membrane is 2.0–5.0 μm, and the pore channel length is 10–25 μm; the VP size is <2.0 μm, and the Au size is <13 nm; the stripping solvent is N-methylpyrrolidone (NMP), dimethylformamide (DMF), isopropanol (IPA), or water. The ultrasonic power is 600–1000 W, the frequency is 20–25 kHz, and the ultrasonication is performed in an ice bath; the ultrasonic and centrifugation temperatures are both 0–4 °C; the PLL molecular weight is 20,000–50,000, and the concentration is 0.5–2 mg / mL.

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

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

[0013] Detection procedure setup: The modified nuclear pore membrane was placed between the electrolytic cell clamps, and an electrolyte solution of a certain concentration was injected into the electrolytic cell. After the electrodes were inserted at both ends of the electrolytic cell, a pulse voltage was applied using an electrochemical workstation, and the response current changes of the electrochemical biosensor based on the PLL@VPAu modified nuclear pore membrane before and after capturing different concentrations of cTnl were recorded.

[0014] Plotting the standard curve: The test solution was incubated in a functionalized nuclear pore membrane, and then cTnI that had not bound to the specific aptamer was removed, resulting in an electrochemical biosensor based on the analyte. The change in current before and after the addition of cTnI antigen was recorded using an electrochemical workstation to achieve quantitative detection of cTnI. A standard curve was plotted, with the horizontal and vertical axes representing cTnI concentration and the relative change in current (I0-I) / I0, respectively, where I0 is the response current before the addition of the target analyte, and I is the response current after the addition of the target analyte.

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

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

[0017] The technical principle of this invention is as follows: Since the pores of the nuclear pore membrane have carboxyl groups after etching, their surface is negatively charged. Positively charged PLL@VPAu nanomaterials can be adsorbed via electrostatic interactions to prepare an effective electrochemical biosensor substrate. By introducing aptamers that specifically recognize molecules, an electrochemical biosensor based on a PLL@VPAu-modified nuclear pore membrane is constructed for the efficient and sensitive detection of biomarkers. When the target analyte is present, it is captured by the aptamer attached to the nuclear pore membrane, causing pore blockage. The electron transfer rate within the system decreases sharply due to the increased steric hindrance, thus achieving ultrasensitive quantitative detection of the target molecule. This invention mainly utilizes the electrochemical signal amplification effect of the two-dimensional layered VPAu material to enhance sensitivity and improves selectivity through aptamer-specific capture, thereby achieving ultrasensitive and rapid quantitative detection of cTnI.

[0018] Compared with the prior art, the beneficial effects of the present invention are at least as follows:

[0019] The electrochemical biosensor for cTnI antigen detection provided by this invention uses two-dimensional nanomaterials as a signal amplification strategy and employs specific recognition molecules to selectively capture the target analyte, giving the sensor high sensitivity and a detection limit of up to 0.1 ng / mL.

[0020] The electrochemical biosensor for cTnI antigen detection provided by this invention has high specificity and does not respond to other non-target proteins.

[0021] The electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane provided by this invention is easy to use and provides rapid analysis, with detection completed within 15 minutes, making it suitable for the practical needs of the industrialization of electrochemical biosensors.

[0022] This invention can be extended to the detection of other targets by changing the specific recognition molecule, providing valuable insights for future applications. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

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

[0025] Figure 2 This is a schematic diagram of the electrochemical sensing principle of an electrochemical biosensor based on PLL@VPAu modified nuclear pore membrane for detecting target analytes.

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

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

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

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

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

[0031] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0032] Example 1

[0033] A method for fabricating an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane, the implementation method of which is as follows: Figure 1 As shown, it includes the following steps:

[0034] Preparation of PLL@VPAu composite nanomaterials: 100 mg of purple phosphorus crystals were ground in a mortar, and the powder was then transferred to 100 mL of NMP solution. The powder was then sonicated for 8 h in an ultrasonic cell disruptor for exfoliation. The resulting VP dispersion was centrifuged at 3000 rpm for 20 min at 4 °C, and the supernatant was further centrifuged at 11000 rpm for 15 min. The precipitate was washed repeatedly with ethanol and then redissolved in water to prepare a VP dispersion with a concentration of 1 mg / mL. 10 μL of a 50 mmol / L chloroauric acid solution was mixed with 10 μL of the above VP solution and sonicated for 10 min to obtain VPAu. VPAu was then mixed with a PLL solution at a volume ratio of 1:1 to obtain the PLL@VPAu composite nanomaterials.

[0035] Construction of biosensors based on PLL@VPAu modified nuclear pore membranes:

[0036] The nuclear pore membrane was placed in the above PLL@VPAu solution for 60 min. The composite material was adsorbed into the pores of the nuclear pore membrane by electrostatic interaction. After multiple rinsings and air drying, it was incubated in an aptamer solution with a concentration of 15 μmol / L for 60 min. Site blocking was performed with 1% bovine serum albumin (BSA) to prevent non-specific adsorption. After rinsing three times with PBS and vacuum drying at 45 °C, an electrochemical biosensor based on the PLL@VPAu modified nuclear pore membrane was prepared. The prepared nuclear pore membrane was immersed in cTnI solutions of different concentrations for 1 h. Among them, the optimized parameters ( Figure 3 This includes optimization of aptamer concentration, antigen-aptamer incubation time, and pH.

[0037] Example 2

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

[0039] Detection procedure setup: The modified nuclear pore membrane was placed between the electrolytic cell clamps. A 1×PBS electrolyte solution was injected into the electrolytic cell. Ag / AgCl electrodes were inserted at both ends of the electrolytic cell. A voltage of -2 to 2V was applied using an electrochemical workstation to obtain the current value I0 when the target substance was absent. Solutions containing different concentrations of the target substance were placed in the functionalized nuclear pore membrane and incubated for 15 minutes. Then, cTnl that had not bound to the specific aptamer was removed, and the current value I0 was measured. Figure 4 The changes in response current before and after capturing different concentrations of cTnI were recorded using an electrochemical biosensor based on a PLL@VPAu-modified nuclear pore membrane.

[0040] Plotting the standard curve: A standard curve was plotted with cTnI concentration on the x-axis and the relative change rate of current on the y-axis, where the relative change rate of current is (I0-I) / I0. As the concentration of cTnI increases within the range of 0.01~100 ng / mL, the relative change rate of current also increases significantly. Figure 5 Furthermore, the two exhibit a good nonlinear relationship.

[0041] Under the same conditions, the relative standard deviation (RSD) of six consecutive measurements of the current value of the same electrochemical biosensor was approximately 1.30%; the RSD of monitoring the current values ​​of six electrochemical biosensors based on PLL@VPAu modified nuclear pore membranes prepared in the same batch was 1.66%; after storage at 4°C for 5 weeks, 91.4% of the response current value was retained compared to 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 as follows: Figure 7 As shown, other types of proteins at the same concentration were selected, such as cardiac troponin C (cTnC), prostate antigen (PSA), human serum albumin (HSA), and glucose oxidase (GO). X Human mucin 1 (MUC1) and other interfering substances were used. Nuclear pore membranes were incubated in different solutions for 15 min, and the relative changes in current before and after incubation were compared. The interfering substances cTnC, PSA, HSA, and GO were also analyzed. X For MUC1, the relative rate of change of current in the sensing platform is approximately 5.93 ± 0.45%. However, when the target material is cTnI, the relative rate of change of current in the entire system is approximately 62.3 ± 4.53%.

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

Claims

1. A method for fabricating an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane, characterized in that: PLL@VPAu composite material was placed inside the pores of a nuclear pore membrane to obtain a PLL@VPAu functionalized nuclear pore membrane. Then, a specific recognition molecule was introduced, using a specific aptamer as the recognition molecule and cTnI as the analyte. The specific fabrication steps of the electrochemical biosensor are as follows: Step 1) Grind the purple phosphorus VP crystals, then put the powder into the stripping solvent for ultrasonic stripping, centrifuge the obtained VP dispersion to remove the unstripped solids, and take the supernatant for further centrifugation; wash the precipitate several times and redissolve it in water to prepare the VP dispersion; mix the chloroauric acid solution with the VP solution and sonicate for a period of time. VPAu nanocomposite material was obtained, and then VPAu was mixed with polylysine PLL solution to obtain PLL@VPAu composite nanomaterial. Step 2) The nuclear pore membrane was placed in the above PLL@VPAu composite nanomaterial solution. The PLL@VPAu composite nanomaterial was adsorbed into the pores of the nuclear pore membrane by electrostatic interaction. After rinsing several times and drying, it was incubated in aptamer solution. Bovine serum albumin was used for site blocking to prevent non-specific adsorption. After rinsing several times with PBS and vacuum drying, the electrochemical biosensor based on the PLL@VPAu modified nuclear pore membrane was obtained.

2. The method for preparing an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane according to claim 1, characterized in that: The stripping solvent in step 1) is N-methylpyrrolidone, dimethylformamide, isopropanol, or water.

3. The method for preparing an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane according to claim 1, characterized in that: The raw material for the nuclear pore membrane in step 2) is polycarbonate, polyethylene terephthalate or polypropylene; the surface pore size of the nuclear pore membrane is larger than the VP size; the aptamer sequence is 5'-SH-C6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3'.

4. The method for preparing an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane according to claim 1, characterized in that: The electrochemical analytical method for cTnI detection includes the following steps: 1) Detection program settings: Place the PLL@VPAu modified nuclear pore membrane between the electrolytic cell clamps, inject an electrolyte solution of a certain concentration into the electrolytic cell, insert electrodes at both ends of the electrolytic cell, apply pulse voltage using an electrochemical workstation, and record the response current values ​​of the electrochemical biosensor based on the PLL@VPAu modified nuclear pore membrane before and after capturing different concentrations of cTnl. 2) Construction of standard curve: The test solution was incubated in a PLL@VPAu modified nuclear pore membrane, and then cTnI that did not bind to the specific aptamer was removed; the change in current before and after the addition of cTnI antigen was recorded by an electrochemical workstation, and a standard curve was plotted with cTnI concentration and relative change rate of current (I0-I) / I0 as the x and y axes, respectively, where I0 is the response current before the addition of the target substance and I is the response current after the addition of the target substance.

5. The method for preparing an electrochemical biosensor based on a PLL@VPAu modified nuclear pore membrane according to claim 4, characterized in that: The electrolyte solution is a PBS 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 5-25 min, and the incubation temperature is 25-40℃.

Images