Biosensors based on VS2 nanocomposite materials, preparation methods and applications

Abstract

The present application relates to the technical field of detection of foodborne pathogenic bacteria, and particularly relates to a biosensor based on VS2 nanocomposite material, a preparation method and application. The biosensor is constructed, wherein the biosensor comprises an electrode part and a probe part, the electrode part is GCE / AuNPs / Ab1 / BSA, and the probe part is VS2 / AuNPs / PEI-Fc. The electrode part is mixed with a to-be-tested liquid for incubation, and after washing with PBS buffer, GCE / AuNPs / Ab1 / BSA / SH is obtained. Then, the electrode part is mixed with the to-be-tested liquid for incubation, and after washing with PBS buffer, the biosensor GCE / AuNPs / Ab1 / BSA / SH / Ab2 / PEI-Fc / AuNPs / VS2 is obtained. The obtained biosensor is placed in a PBS solution, and an electric signal is measured by using a differential pulse voltammetry (DPV) method, so that the target object can be detected. The signal value of the biosensor is positively correlated with the concentration of the target object. The biosensor has high detection sensitivity, and in the range of 0-1 μg / mL, the detection limit is as low as 1.1 pg / mL, and the linear correlation coefficient of the prediction model reaches 0.958.

Description

Technical Field

[0001] This invention relates to the field of detection and testing technology for foodborne pathogens, specifically to a biosensor based on VS2 nanocomposite materials, its preparation method, and its application. Background Technology

[0002] Salmonella is a Gram-negative enteric bacillus widely found in meat, eggs and raw egg products, as well as unpasteurized milk and beverages. According to the European Centre for Disease Prevention and Control (ECDC), Salmonella is the second most common cause of food poisoning worldwide; therefore, effective strategies for detecting Salmonella are urgently needed.

[0003] Electrochemical biosensors offer advantages such as small sample size, high sensitivity, low cost, miniaturization and integrability, and suitability for portable devices, expanding their application scenarios beyond the laboratory and making them a promising tool for Salmonella detection. Signal amplification strategies based on nanocomposite materials are one effective means to improve the detection sensitivity of electrochemical biosensors. VS2 is a typical TMD material, possessing inherent metallic properties and a layered graphite-like structure compared to other TMD materials. Furthermore, to avoid the problems of small specific surface area and poor conductivity caused by the stacking of layered materials, the material is usually prepared into a nanoflower-like layered structure, increasing the specific surface area while exhibiting excellent conductivity.

[0004] Gold nanoparticles (AuNPs) have been used in electrochemical biosensors, further enhancing electron transfer rates by loading them onto nanomaterials and for immobilizing biomaterials. Ferrocene (Fc) and its derivatives are commonly used as electrochemical signal probes for sensors due to their good biocompatibility, high anti-interference ability, and enhanced electron exchange capacity. To effectively immobilize them onto materials, they are often combined with polymers. Polyethyleneimine (PEI), a non-toxic polymer with many hydrophilic amine groups, can be covalently bound to Fc via a Schiff base reaction to form ferrocene-grafted polyethyleneimine (PEI-Fc). PEI-Fc provides more sites for effectively anchoring nanomaterials and biomolecules.

[0005] Therefore, this invention develops a composite material of flower-shaped vanadium disulfide-loaded gold nanoparticles (AuNPs) and / or polyethyleneimine-ferryl (PEI-Fc) for current signal amplification, thereby constructing a novel high-sensitivity electrochemical sensor for detecting Salmonella, providing a reference method for highly sensitive detection of Salmonella in samples. Summary of the Invention

[0006] The purpose of this invention is to provide a biosensor based on VS2 nanocomposite materials, its preparation method, and its application, so as to achieve high-sensitivity detection of target analytes.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a biosensor based on VS2 nanocomposite material, comprising an electrode part and a probe part;

[0008] The electrode portion includes a glassy carbon electrode, a signal amplifying agent, a first antibody, and a blocking agent;

[0009] The probe portion comprises a flower-shaped vanadium disulfide-gold nanoparticle substrate, a second antibody, and a blocking agent;

[0010] The first antibody and the second antibody specifically bind to the antigen to be tested.

[0011] Furthermore, the signal amplifying agent is gold nanoparticles.

[0012] Furthermore, the sealing agent is bovine serum albumin solution.

[0013] Furthermore, the probe portion also includes a signal probe, which is a ferrocene-polyethyleneimine graft.

[0014] The fabrication of the biosensor includes the fabrication of the electrode portion and the fabrication of the probe portion.

[0015] Furthermore, the preparation of the electrode portion includes the following steps:

[0016] S11. Immerse the glassy carbon electrode in HAuCl4 solution, deposit gold nanoparticles on the glassy carbon electrode at -0.2V, wash with deionized water to obtain GCE / AuNPs;

[0017] S12. Add the first antibody to GCE / AuNPs, incubate overnight at 4°C, wash with PBS buffer to obtain GCE / AuNPs / Ab1;

[0018] S13. Add BSA solution to GCE / AuNPs / Ab1, incubate at room temperature, and wash with PBS buffer to obtain GCE / AuNPs / Ab1 / BSA, which is the electrode part of the biosensor.

[0019] Furthermore, the preparation of the probe portion includes the following steps:

[0020] S21. Dissolve sodium vanadate and thioacetamide in deionized water and stir vigorously. After reaction in a high-pressure reactor, cool, filter, wash, and freeze dry to obtain flower-shaped VS2.

[0021] S22. Take flower-shaped VS2 in ultrapure water and disperse it by ultrasonication. Add HAuCl4 solution, stir evenly at room temperature, and heat to boiling. Add trisodium citrate dropwise to the boiling solution to continue the reaction. After the reaction is completed, stop heating, cool, centrifuge, filter, wash, and freeze dry to obtain VS2 / AuNPs.

[0022] S23. Linear polyethyleneimine was dissolved in methanol, and ferrocene formaldehyde solution was added dropwise under stirring. After continuous stirring, NaCNBH3 powder was added to continue the reaction. After the reaction was completed, methanol was removed, and the residue was extracted with diethyl ether, filtered, and rotary evaporated to obtain PEI-Fc.

[0023] S24. Dissolve the PEI-Fc prepared in S23 into PBS buffer, disperse by sonication, add the VS2 / AuNPs prepared in S22, stir to react, and obtain VS2 / AuNPs / PEI-Fc.

[0024] S25. Take VS2 / AuNPs / PEI-Fc, shake thoroughly, add the second antibody, react overnight at 4°C, add BSA solution, shake, centrifuge, disperse with PBS buffer, and store at 4°C for later use. This is the probe part of the biosensor.

[0025] The application of the described biosensor in the detection of Salmonella H antigen.

[0026] Furthermore, the first antibody and the second antibody are antibodies that specifically bind to Salmonella H antigen, respectively.

[0027] Furthermore, in the detection of Salmonella H antigen, the biosensor has a detection limit as low as 1.1 pg / mL within the range of 0-1 μg / mL for Salmonella H antigen, and the linear correlation coefficient of the prediction model reaches 0.958.

[0028] The beneficial effects of this invention are:

[0029] 1. In the VS2 / AuNPs composite material designed and synthesized by the method of the present invention, flower-shaped VS2 with excellent conductivity and high specific surface area is used as the substrate, which improves the sensitivity of the sensor. In the VS2 / AuNPs / PEI-Fc composite material, Fc with high anti-interference ability and enhanced electron exchange ability is selected as the signal probe. The synergistic advantage can effectively improve the detection sensitivity of the sensor.

[0030] 2. The biosensor constructed in this invention has a detection limit as low as 1.1 pg / mL in the range of 0 to 1 μg / mL, and the linear correlation coefficient of the prediction model reaches 0.958. Attached Figure Description

[0031] Figure 1This is a schematic diagram illustrating the principle of the biosensor for detecting Salmonella H antigen in Embodiment 6 of the present invention.

[0032] Figure 2 This is the standard working curve diagram for detecting Salmonella H antigen in Example 7 of the present invention;

[0033] Figure 3 This is a schematic diagram illustrating the principle of the biosensor for detecting Salmonella H antigen in Embodiment 10 of the present invention.

[0034] Figure 4 This is the standard working curve diagram for detecting Salmonella H antigen in Example 11 of the present invention;

[0035] Figure 5 This is a diagram of the manufacturer's test report for the Ab1 antibody (first antibody) involved in an embodiment of the present invention;

[0036] Figure 6 This is a diagram of the manufacturer's test report for the Ab2 antibody (second antibody) involved in an embodiment of the present invention. Detailed Implementation

[0037] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention.

[0038] The principle of this invention is as follows:

[0039] like Figure 1 As shown in Figure 3, the present invention constructs an electrochemical biosensor with a "sandwich" structure to detect Salmonella H antigen.

[0040] First, AuNP is used on the electrode surface. S The electrode was modified to improve its electron transfer rate, enhance conductivity, and increase electrical signal; then, the monoclonal antibody Ab1 was anchored to the electrode surface; subsequently, the non-specific adsorption sites on the electrode surface were blocked with BSA (bovine serum albumin).

[0041] The current decreased after modifying the recognition elements Ab1 and BSA. This is because Ab1 (Ab1 is the primary antibody) and BSA are non-conductive biomolecules that hinder electron transfer at the interface. The steric hindrance effect also contributed to the decrease in the electron transfer rate.

[0042] Next, the test solution is added to the electrode surface and incubated for a period of time to ensure sufficient binding of antigen and antibody; the probe portion (such as VS2 / AuNPs / PEI-Fc / Ab2 or VS2 / AuNPs / Ab2, where Ab2 is the secondary antibody) is then added to the electrode surface and incubated for a period of time before the electrical signal is tested.

[0043] The more target substance in the test solution, the more probe part is on the electrode surface, and the stronger the electrical signal, thus realizing the quantitative detection of the target substance; the signal value of the sensor is positively correlated with the concentration of the target substance.

[0044] Example 1

[0045] Preparation of flower-like VS2:

[0046] Dissolve 6 mmol of sodium orthovanadate (Na3VO4·12H2O) and 32 mmol of thioacetamide (TAA) in 50 mL of deionized water and stir the solution vigorously for 1 h. Transfer the prepared solution to a 100 mL autoclave lined with polytetrafluoroethylene and react at 60 °C for 24 h. Slowly cool the system to room temperature, wash the black precipitate three times with deionized water and anhydrous ethanol, collect it by vacuum filtration, and freeze-dry for 12 h to obtain a black powder solid.

[0047] Example 2

[0048] Preparation of VS2 / AuNPs composite materials:

[0049] Take 10 mg of VS2 prepared in Example 1 and add it to 10 mL of ultrapure water. Sonicate the mixture to disperse it completely. Add 50 mL of 0.3 mM HAuCl4 solution and stir evenly at room temperature. Heat the mixture in an oil bath until it boils. Add 2 mL of 40 mM trisodium citrate dropwise to the boiling solution and continue the reaction for 10 min. Stop heating and continue stirring until the temperature returns to room temperature. Centrifuge, filter, wash three times with ultrapure water, and freeze dry to obtain VS2 / AuNPs.

[0050] Example 3

[0051] Preparation of PEI-Fc:

[0052] 0.252 g of LPEI [i.e., linear polyethyleneimine (LPEI) (average molecular weight approximately 70,000)] was dissolved in 10 mL of methanol, and 0.187 g (0.87 mmol) of ferrocene formaldehyde solution (dissolved in 3 mL of methanol) was added dropwise while stirring continuously to obtain a dark red mixture. After stirring for 2 h, NaCNBH3 powder (0.055 g, 0.87 mmol) was added, and the mixture was stirred at room temperature for 24 h to lighten the color of the solution. After the reaction was completed, methanol was removed under vacuum, and the residue was extracted with diethyl ether, filtered, and rotary evaporated. Methanol was then added again, filtered, and rotary evaporated to remove methanol. This operation was repeated three times to obtain the final product.

[0053] Example 4

[0054] Preparation of VS2 / AuNPs / PEI-Fc composite material:

[0055] Dissolve 8 mg of PEI-Fc prepared in Example 3 in 2 mL of PBS buffer and sonicate for 20 min; add 2 mL of VS2 / AuNPS composite material prepared in Example 2 with a concentration of 4 mg / mL and stir for 30 min to obtain the composite material.

[0056] Example 5

[0057] Preparation of VS2 / AuNPs / PEI-Fc / Ab2:

[0058] Take 2 mL of VS2 / AuNPs / PEI-Fc prepared in Example 4, shake thoroughly, add 1 mL of Ab2 with a concentration of 10 μg / mL, and react overnight at 4°C for 12 h; then add 30 μL of BSA solution (1 wt%), shake for 30 min, centrifuge to remove the supernatant, redisperse in 1 mL of PBS (0.1 M, pH 7.4), and store at 4°C for later use.

[0059] Example 6

[0060] Construction of an electrochemical sensor for Salmonella H antigen:

[0061] A glassy carbon electrode (GCE) was immersed in 10 mM HAuCl4 solution, and AuNPs were deposited on the electrode at -0.2 V for 30 s to obtain GCE / AuNPs, which were then washed with deionized water. 10 μL of 10 μg / mL Ab1 antibody was added to the GCE / AuNPs, and the mixture was incubated overnight at 4 °C. The mixture was then washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1. 10 μL of [unspecified ingredient] was then added to the electrode surface. BSA solution (1 wt%) was incubated at room temperature for 30 min, then washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1 / BSA; 10 μL of the test solution (containing Salmonella H antigen SH) was added to the electrode surface, incubated at room temperature for 30 min, then washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1 / BSA / SH; 10 μL of VS2 / AuNPs / PEI-Fc / Ab2 prepared in Example 5 was incubated at room temperature for 30 min, then washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1 / BSA / SH / Ab2 / PEI-Fc / AuNPs / VS2; the electrode was placed in 0.1 M PBS solution, and the electrical signal was measured using differential pulse voltammetry (DPV) to detect Salmonella H antigen.

[0062] Example 7

[0063] Plotting the operating curve for the electrochemical sensor detecting Salmonella H antigen:

[0064] Different concentrations of Salmonella H antigen (0, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL) were prepared, and their electrochemical signals were measured using the sensor prepared in Example 6. A standard curve for detecting Salmonella H antigen by this method was plotted with Salmonella H antigen concentration on the x-axis and current ΔI(I0-I) on the y-axis. Figure 2 The regression equation obtained through linear fitting is y = 6.394x - 6.432 (R²). 2 =0.958), and the detection limit of this method was calculated to be 1.1 pg / mL, where I0 represents the current value without Salmonella H antigen and I represents the current value with Salmonella H antigen.

[0065] Example 8

[0066] Detection of Salmonella H antigen in milk samples:

[0067] Dilute 10 mL of milk 10 times with 0.01 M PBS to prepare the test solution. Take three equal volumes of the test solution and add Salmonella H antigen at concentrations of 5 ng / mL, 15 ng / mL, and 45 ng / mL to them. Perform the detection according to the steps in Example 6. Substitute the obtained electrical signal values ​​into the linear regression equation of the standard working curve in Example 7 to calculate the concentration of Salmonella H antigen in the test sample and calculate the spiked recovery rate (Table 1).

[0068] Table 1. Spiked recovery of Salmonella H antigen in milk samples

[0069]

[0070] As shown in Table 1, the spiked recoveries of Salmonella H antigen in actual samples using this method ranged from 82.0% to 108.0%, with relative standard deviations (RSD) of 3.83% to 4.09%, indicating that the present invention has good accuracy in detecting Salmonella H antigen in milk samples.

[0071] Example 9

[0072] Preparation of VS2 / AuNPs / Ab2:

[0073] Take 2 mL of VS2 / AuNPs prepared in Example 2, shake thoroughly, add 1 mL of Ab2 with a concentration of 10 μg / mL, and react overnight at 4°C for 12 h; then add 30 μL of BSA solution (1 wt%), shake for 30 min, centrifuge to remove the supernatant, redisperse in 1 mL of PBS (0.1 M, pH 7.4), and store at 4°C for later use.

[0074] Example 10

[0075] Construction of an electrochemical sensor for Salmonella H antigen:

[0076] A glassy carbon electrode (GCE) was immersed in 10 mM HAuCl4 solution, and AuNPs were deposited on the electrode at -0.2 V for 30 s to obtain GCE / AuNPs, which were then washed with deionized water. 10 μL of 10 μg / mL Ab1 antibody was added to the GCE / AuNPs, and the mixture was incubated overnight at 4 °C. The mixture was then washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1. 10 μL of [unspecified ingredient] was then added to the electrode surface. BSA solution (1 wt%) was incubated at room temperature for 30 min, then washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1 / BSA; 10 μL of the test solution (containing Salmonella H antigen SH) was added to the electrode surface, incubated at room temperature for 30 min, then washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1 / BSA / SH; 10 μL of VS2 / AuNPs / Ab2 prepared in Example 9 was incubated at room temperature for 30 min, then washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1 / BSA / SH / Ab2 / AuNPs / VS2; the electrode was placed in 2.5 mM [Fe(CN)6] 4- / 3- The electrical signal was measured using differential pulse voltammetry (DPV) in a solution (containing 0.1 M KCl) to detect Salmonella H antigen.

[0077] Example 11

[0078] Plotting the operating curve for the electrochemical sensor detecting Salmonella H antigen:

[0079] Different concentrations of Salmonella H antigen (0, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL) were prepared, and their electrochemical signals were measured using the sensor prepared in Example 10. A standard curve for detecting Salmonella H antigen by this method was plotted with Salmonella H antigen concentration on the x-axis and current ΔI(I0-I) on the y-axis. Figure 4 (where A is the test data graph of DPV and B is the standard curve graph). The regression equation obtained through linear fitting is y = 19.02x + 1.82 (R²). 2 =0.968), and the detection limit of this method was calculated to be 2.5 pg / mL, where I0 represents the current value without Salmonella H antigen and I represents the current value with Salmonella H antigen.

[0080] Example 12

[0081] Detection of Salmonella H antigen in milk samples:

[0082] Dilute 10 mL of milk 10 times with 0.01 M PBS to prepare the test solution. Take three equal volumes of the test solution and add Salmonella H antigen at concentrations of 5 ng / mL, 15 ng / mL, and 45 ng / mL to them. Perform the detection according to the steps in Example 10. Substitute the obtained electrical signal values ​​into the linear regression equation of the standard working curve in Example 11 to calculate the concentration of Salmonella H antigen in the test sample and calculate the spiked recovery rate (Table 2).

[0083] Table 2. Spiked recovery of Salmonella H antigen in milk samples

[0084]

[0085] As shown in Table 2, the spiked recovery rate of this method for detecting Salmonella H antigen in actual samples was 85.3%–106.1%, and the relative standard deviation (RSD) was 3.12–4.64%, indicating that this embodiment has good accuracy in detecting Salmonella H antigen in milk samples.

[0086] The Ab1 (first antibody) and Ab2 (second antibody) involved in the above embodiments of the present invention are monoclonal antibodies against Salmonella typhi, which were commercially available and purchased from Zhengzhou Saitukang Biotechnology Co., Ltd. The reagents and the test reports issued by the company are as follows: Figure 5-6 As shown.

[0087] This invention is not limited to the preferred embodiments described above. Anyone can derive other forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. A biosensor based on VS2 nanocomposite material, characterized in that: Includes the electrode section and the probe section; The electrode portion includes a glassy carbon electrode, a signal amplifying agent, a first antibody, and a blocking agent; The signal amplifying agent is gold nanoparticles; the blocking agent is bovine serum albumin solution. The probe portion comprises a flower-shaped vanadium disulfide-gold nanoparticle substrate, a second antibody, and a blocking agent; The probe portion further includes a signal probe, which is a ferrocene-polyethyleneimine graft. The first antibody and the second antibody specifically bind to the antigen to be tested.

2. The method for preparing a biosensor according to claim 1, characterized in that: This includes the fabrication of the electrode portion and the fabrication of the probe portion.

3. The method for preparing a biosensor according to claim 2, characterized in that, The preparation of the electrode portion includes the following steps: S11. Immerse the glassy carbon electrode in HAuCl4 solution, deposit gold nanoparticles on the glassy carbon electrode at -0.2V, wash with deionized water to obtain GCE / AuNPs; S12. Add the first antibody to GCE / AuNPs, incubate overnight at 4°C, wash with PBS buffer to obtain GCE / AuNPs / Ab1; S13. Add BSA solution to GCE / AuNPs / Ab1, incubate at room temperature, and wash with PBS buffer to obtain GCE / AuNPs / Ab1 / BSA, which is the electrode part of the biosensor.

4. The method for preparing a biosensor according to claim 2, characterized in that, The preparation of the probe portion includes the following steps: S21. Dissolve sodium vanadate and thioacetamide in deionized water and stir vigorously. After reaction in a high-pressure reactor, cool, filter, wash, and freeze dry to obtain flower-shaped VS2. S22. Take flower-shaped VS2 in ultrapure water and disperse it by ultrasonication. Add HAuCl4 solution, stir evenly at room temperature, and heat to boiling. Add trisodium citrate dropwise to the boiling solution to continue the reaction. After the reaction is completed, stop heating, cool, centrifuge, filter, wash, and freeze dry to obtain VS2 / AuNPs. S23. Linear polyethyleneimine was dissolved in methanol, and ferrocene formaldehyde solution was added dropwise under stirring. After continuous stirring, NaCNBH3 powder was added to continue the reaction. After the reaction was completed, methanol was removed, and the residue was extracted with diethyl ether, filtered, and rotary evaporated to obtain PEI-Fc. S24. Dissolve the PEI-Fc prepared in S23 into PBS buffer, disperse by sonication, add the VS2 / AuNPs prepared in S22, stir to react, and obtain VS2 / AuNPs / PEI-Fc. S25. Take VS2 / AuNPs / PEI-Fc, shake thoroughly, add the second antibody, react overnight at 4°C, add BSA solution, shake, centrifuge, disperse with PBS buffer, and store at 4°C for later use. This is the probe part of the biosensor.

5. The application of the biosensor according to claim 1 in the detection of Salmonella H antigen.

6. The application of the biosensor according to claim 5 in the detection of Salmonella H antigen, characterized in that: The first antibody and the second antibody are antibodies that specifically bind to Salmonella H antigen, respectively.

7. The application of the biosensor according to claim 5 in the detection of Salmonella H antigen, characterized in that: In the detection of Salmonella H antigen, the biosensor has a detection limit as low as 1.1 pg / mL within the range of 0-1 μg / mL, and the linear correlation coefficient of the prediction model reaches 0.958.

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