Biosensor based on VS2 nano composite material, preparation method and application

Abstract

The invention relates to the technical field of inspection and detection of food-borne pathogenic bacteria, in particular to a biosensor based on a VS2 nano composite material, a preparation method and application. According to the method, a biosensor is constructed, the biosensor comprises an electrode part and a probe part, the electrode part is GCE / AuNPs / Ab1 / BSA, the probe part is VS2 / AuNPs / PEI-Fc, a to-be-detected solution and the electrode part are mixed and incubated, and GCE / AuNPs / Ab1 / BSA / SH is obtained after washing with a PBS buffer solution; gCE / AuNPs / Ab1 / BSA / SH / Ab2 / PEI-Fc / AuNPs / VS2 is taken as a substrate, then the GCE / AuNPs / Ab1 / BSA / SH / Ab2 / PEI-Fc / AuNPs / VS2 is mixed and incubated with an electrode part and washed with a PBS buffer solution to obtain the biosensor GCE / AuNPs / Ab1 / BSA / SH / Ab2 / PEI-Fc / AuNPs / VS2, the obtained biosensor is placed in the PBS solution, electric signal measurement is conducted through differential pulse voltammetry (DPV), detection of a target object can be achieved, and the signal value of the biosensor and the concentration of the target object are in a positive correlation relation. The biosensor is high in detection sensitivity, the detection limit is as low as 1.1 pg / mL in the range of 0-1 microgram / mL, and the linear correlation coefficient of a prediction model reaches 0.958.

Description

Technical Field

[0001] The present invention relates to the technical field of inspection and detection of foodborne pathogenic bacteria, and in particular to a biosensor based on VS2 nanocomposite materials, a preparation method and applications thereof. Background Art

[0002] Salmonella is a Gram-negative enteric bacillus that is widely found in meat, eggs and raw egg products, 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 in the world; therefore, effective strategies to detect Salmonella are urgently needed.

[0003] Electrochemical biosensors have the advantages of small sample size, high sensitivity, low cost, micro-integration, and suitability for portable devices, which broadens their detection application scenarios beyond the laboratory and becomes an ideal tool with development potential for Salmonella detection. Signal amplification strategy based on nanocomposites is one of the effective means to improve the detection sensitivity of electrochemical biosensors. VS2 is a typical TMD material. Compared with other TMD materials, it has inherent metallic properties and a layered graphite structure. At the same time, in order 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 structure to form a layered structure, which can increase the specific surface area while showing excellent conductive properties.

[0004] Gold nanoparticles (AuNPs) have been used in electrochemical biosensors to further increase the electron transfer rate by loading them onto nanomaterials, and to immobilize biomaterials. Ferrocene (Fc) and its derivatives are often used as electrochemical signal probes for sensors due to their good biocompatibility, high anti-interference ability, and enhanced electron exchange capacity. In order to fix it well to the material, it is usually combined with a polymer. Polyethyleneimine (PEI) is a non-toxic polymer with many hydrophilic amine groups that can be covalently bound to Fc through a Schiff base reaction to form ferrocene-grafted polyethyleneimine (PEI-Fc). PEI-Fc can provide more sites to effectively anchor nanomaterials and biomolecules.

[0005] Therefore, the present invention develops a composite material composed of flower-like vanadium disulfide loaded with gold nanoparticles (AuNPs) and / or polyethyleneimine-ferroxyl (PEI-Fc) for current signal amplification, thereby constructing a new type of high-sensitivity electrochemical sensor for detecting Salmonella, providing a reference method for high-sensitivity detection of Salmonella in samples. Summary of the invention

[0006] The purpose of the present invention is to provide a biosensor based on VS2 nanocomposite materials, a preparation method and an application thereof, so as to achieve the purpose of high-sensitivity detection of a target object.

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

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

[0009] The probe part includes 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 detected respectively.

[0011] Furthermore, the signal amplifier is gold nanoparticles.

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

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

[0014] The preparation of the biosensor includes the preparation of the electrode part and the preparation of the probe part.

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

[0016] S11, immersing the glassy carbon electrode in HAuCl4 solution, depositing gold nanoparticles on the glassy carbon electrode at -0.2V, and washing with deionized water to obtain GCE / AuNPs;

[0017] S12, adding the first antibody to GCE / AuNPs, incubating overnight in a 4°C refrigerator, and washing with PBS buffer to obtain GCE / AuNPs / Ab1;

[0018] S13. Add BSA solution onto 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 comprises the following steps:

[0020] S21, dissolving sodium orthovanadate and thioacetamide in deionized water and stirring vigorously, reacting in an autoclave, cooling, filtering, washing, and freeze-drying to obtain flower-like VS2;

[0021] S22, taking flower-shaped VS2 and dispersing it in ultrapure water by ultrasonic, adding HAuCl4 solution, stirring evenly at room temperature and heating to boiling, adding trisodium citrate dropwise to the boiling solution to continue the reaction, stopping heating after the reaction is completed, cooling, centrifuging, filtering, washing, and freeze-drying to obtain VS2 / AuNPs;

[0022] S23, dissolving linear polyethyleneimine in methanol, adding ferrocene formaldehyde solution dropwise under stirring, adding NaCNBH3 powder after continuous stirring to continue the reaction, and after the reaction is completed, removing methanol, extracting the residue with ether, filtering, and rotary evaporation to obtain PEI-Fc;

[0023] S24, dissolving the PEI-Fc prepared in S23 into PBS buffer, adding the VS2 / AuNPs prepared in S22 after ultrasonic dispersion, stirring for reaction, and obtaining VS2 / AuNPs / PEI-Fc;

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

[0025] The biosensor is used 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 in the range of 0 to 1 μg / mL of Salmonella H antigen, and the linear correlation coefficient of the prediction model reaches 0.958.

[0028] Beneficial effects of the present invention:

[0029] 1. In the VS2 / AuNPs composite material designed and synthesized by the method of the present invention, the flower-shaped VS2 with excellent conductive properties 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, and the synergistic advantage can be used to effectively improve the detection sensitivity of the sensor;

[0030] 2. The biosensor constructed by the present 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1is a schematic diagram of the biosensor for detecting Salmonella H antigen in Example 6 of the present invention;

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

[0033] Figure 3 is a schematic diagram of the biosensor for detecting Salmonella H antigen in Example 10 of the present invention;

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

[0035] Figure 5 It is a test report chart of the manufacturer of Ab1 antibody (first antibody) involved in the embodiment of the present invention;

[0036] Figure 6 It is a test report chart of the manufacturer of Ab2 antibody (second antibody) involved in the embodiment of the present invention. DETAILED DESCRIPTION

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

[0038] The principle of the present invention is:

[0039] like Figure 1 As shown in FIG3 , the present invention constructs an electrochemical biosensor with a “sandwich” structure for detecting Salmonella H antigen.

[0040] First, the electrode surface is coated with AuNPs S The modification was performed to increase its electron transfer rate, enhance conductivity, and increase electrical signals; then, the monoclonal antibody Ab1 was anchored to the electrode surface; subsequently, the nonspecific adsorption sites on the electrode surface were blocked with BSA (bovine serum albumin).

[0041] After modifying the recognition elements Ab1 and BSA, the current decreased. On the one hand, this is because Ab1 (Ab1 is the first antibody) and BSA are non-conductive biological molecules that hinder the electron transfer at the interface. On the other hand, it is due to the steric hindrance effect. The two effects work together to reduce the rate of electron transfer.

[0042] Next, the test liquid is added to the electrode surface and incubated for a period of time to ensure sufficient antigen-antibody binding; the probe part (such as VS2 / AuNPs / PEI-Fc / Ab2 or VS2 / AuNPs / Ab2, where Ab2 is the second antibody) is continued to be added to the electrode surface, and after incubation for a period of time, the electrical signal is tested.

[0043] When the amount of target in the test liquid is greater, the amount of the probe part on the electrode surface is greater, and the electrical signal is stronger, thereby achieving quantitative detection of the target; the signal value of the sensor is positively correlated with the concentration of the target.

[0044] Example 1

[0045] Preparation of flower-shaped VS2:

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

[0047] Example 2

[0048] Preparation of VS2 / AuNPs composites:

[0049] Take 10 mg of VS2 prepared in Example 1 and dissolve it in 10 mL of ultrapure water. Ultrasonic treatment is performed to completely disperse it. 50 mL of 0.3 mM HAuCl4 solution is added thereto. After stirring evenly at room temperature, it is placed in an oil bath and heated until the mixed solution boils. 2 mL of 40 mM trisodium citrate is added dropwise to the boiling solution. After continuing the reaction for 10 minutes, heating is stopped; stirring is continued until it returns to room temperature, centrifuged, filtered, washed three times with ultrapure water, and freeze-dried 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 of about 70,000)] was dissolved in 10 mL of methanol, and 0.187 g (0.87 mmol) of ferrocene carboxaldehyde solution (dissolved in 3 mL of methanol) was added dropwise thereto under continuous stirring to obtain a dark red mixed solution; after stirring for 2 h, NaCNBH3 (0.055 g, 0.87 mmol) powder was added continuously and stirred at room temperature for 24 h to make the color of the solution lighter; after the reaction was completed, methanol was removed under vacuum, the residue was extracted with ether, filtered, and rotary evaporated; methanol was added again, filtered, and rotary evaporated to remove methanol, and the operation was repeated three times to obtain the final product.

[0053] Example 4

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

[0055] 8 mg of PEI-Fc prepared in Example 3 was dissolved in 2 mL of PBS buffer and ultrasonicated for 20 min. 2 mL of VS2 / AuNPS composite material with a concentration of 4 mg / mL prepared in Example 2 was added thereto and stirred 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 it thoroughly, add 1 mL of Ab2 with a concentration of 10 μg / mL, and react it overnight at 4°C for 12 h; continue to add 30 μL of BSA solution (1wt%), shake it for 30 minutes, centrifuge to remove the supernatant, redisperse it in 1 mL PBS (0.1 M, pH 7.4), and store it at 4°C for later use.

[0059] Example 6

[0060] Construction of Salmonella H antigen electrochemical sensor:

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

[0062] Example 7

[0063] Drawing of the working curve of the electrochemical sensor for 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 the electrochemical signal was measured using the sensor prepared in Example 6; the standard curve for detecting Salmonella H antigen by this method was drawn with the concentration of Salmonella H antigen as the abscissa and the current ΔI (I0-I) as the ordinate. Figure 2 ), the regression equation obtained by linear fitting is y=6.394x-6.432(R 2 =0.958), and the detection limit of this method was calculated to be 1.1 pg / mL, where I0 represents the current value when there is no Salmonella H antigen, and I represents the current value when there is Salmonella H antigen.

[0065] Example 8

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

[0067] 10 mL of milk was diluted 10 times with 0.01 M PBS as the test solution, 3 equal volumes of the test solution were taken, and Salmonella H antigen at concentrations of 5 ng / mL, 15 ng / mL, and 45 ng / mL were added thereto. The test was performed according to the steps in Example 6, and the obtained electrical signal value was substituted into the linear regression equation of the standard working curve of Example 7 to calculate the concentration of Salmonella H antigen in the test sample, and the spike recovery rate was calculated (Table 1).

[0068] Table 1 Recovery of Salmonella H antigen in milk samples

[0069]

[0070] As shown in Table 1, the spiked recovery rate of the method for detecting Salmonella H antigen in actual samples is 82.0% to 108.0%, and the relative standard deviation (RSD) is 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 it thoroughly, add 1 mL of Ab2 with a concentration of 10 μg / mL, and react it overnight at 4°C for 12 h; continue to add 30 μL of BSA solution (1wt%), shake it for 30 minutes, centrifuge to remove the supernatant, redisperse it in 1 mL PBS (0.1 M, pH 7.4), and store it at 4°C for later use.

[0074] Example 10

[0075] Construction of Salmonella H antigen electrochemical sensor:

[0076] The glassy carbon electrode (GCE) was immersed in a 10 mM HAuCl4 solution, and AuNPs were deposited on the electrode at -0.2 V. The process lasted for 30 s to obtain GCE / AuNPs, which were washed with deionized water. 10 μL of Ab1 antibody with a concentration of 10 μg / mL was added to the GCE / AuNPs, incubated overnight in a refrigerator at 4 °C, and washed with 0.01 M PBS to obtain GCE / AuNPs / Ab1. 10 μL of Ab1 was added to the electrode surface. BSA solution (1wt%) was incubated at room temperature for 30 min, and then washed with 0.01M PBS to obtain GCE / AuNPs / Ab1 / BSA; 10 μL of the test solution (containing Salmonella H antigen SH) was added dropwise to the electrode surface, incubated at room temperature for 30 min, and then washed with 0.01M 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, and then washed with 0.01M 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 Salmonella H antigen was detected by measuring the electrical signal in a solution (containing 0.1 M KCl) using differential pulse voltammetry (DPV).

[0077] Embodiment 11

[0078] Drawing of the working curve of the electrochemical sensor for 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 the electrochemical signal was measured using the sensor prepared in Example 10; the standard curve for detecting Salmonella H antigen by this method was drawn with the concentration of Salmonella H antigen as the abscissa and the current ΔI (I0-I) as the ordinate. Figure 4 , where A is the test data graph of DPV and B is the standard curve graph), the regression equation obtained by linear fitting is y=19.02x+1.82(R 2 =0.968), and the detection limit of this method was calculated to be 2.5 pg / mL, where I0 represents the current value when there is no Salmonella H antigen, and I represents the current value when there is Salmonella H antigen.

[0080] Example 12

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

[0082] 10 mL of milk was diluted 10 times with 0.01 M PBS as the test solution, 3 equal volumes of the test solution were taken, and Salmonella H antigen at concentrations of 5 ng / mL, 15 ng / mL, and 45 ng / mL were added thereto. The test was performed according to the steps in Example 10, and the obtained electrical signal value was substituted into the linear regression equation of the standard working curve of Example 11 to calculate the concentration of Salmonella H antigen in the test sample, and the spike recovery rate was calculated (Table 2).

[0083] Table 2 Recovery of Salmonella H antigen spiked in milk samples

[0084]

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

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

[0087] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. Biosensor based on VS2 nanocomposite material, characterized by: It includes an electrode part and a probe part; The electrode part includes a glassy carbon electrode, a signal amplifier, a first antibody and a blocking agent; The probe part includes a flower-shaped vanadium disulfide-gold nanoparticle substrate, a second antibody and a blocking agent; The first antibody and the second antibody specifically bind to the antigen to be detected respectively.

2. The biosensor based on VS2 nanocomposite material according to claim 1, characterized in that: The signal amplifier is gold nanoparticles.

3. The biosensor based on VS2 nanocomposite material according to claim 1, characterized in that: The blocking agent is a bovine serum albumin solution.

4. The biosensor based on VS2 nanocomposite material according to claim 1, characterized in that: The probe part also includes a signal probe, which is a ferrocene-polyethyleneimine grafted product.

5. The method for preparing a biosensor according to any one of claims 1 to 4, characterized in that: It includes the preparation of the electrode part and the preparation of the probe part.

6. The method for preparing a biosensor according to claim 5, characterized in that: The preparation of the electrode part comprises the following steps: S11, immersing the glassy carbon electrode in HAuCl4 solution, depositing gold nanoparticles on the glassy carbon electrode at -0.2V, and washing with deionized water to obtain GCE / AuNPs; S12, adding the first antibody to GCE / AuNPs, incubating overnight in a 4°C refrigerator, and washing with PBS buffer to obtain GCE / AuNPs / Ab1; S13. Add BSA solution onto 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.

7. The method for preparing a biosensor according to claim 5, characterized in that: The preparation of the probe part comprises the following steps: S21, dissolving sodium orthovanadate and thioacetamide in deionized water and stirring vigorously, reacting in an autoclave, cooling, filtering, washing, and freeze-drying to obtain flower-like VS2; S22, taking flower-shaped VS2 and dispersing it in ultrapure water by ultrasonic, adding HAuCl4 solution, stirring evenly at room temperature and heating to boiling, adding trisodium citrate dropwise to the boiling solution to continue the reaction, stopping heating after the reaction is completed, cooling, centrifuging, filtering, washing, and freeze-drying to obtain VS2 / AuNPs; S23, dissolving linear polyethyleneimine in methanol, adding ferrocene formaldehyde solution dropwise under stirring, adding NaCNBH3 powder after continuous stirring to continue the reaction, and after the reaction is completed, removing methanol, extracting the residue with ether, filtering, and rotary evaporation to obtain PEI-Fc; S24, dissolving the PEI-Fc prepared in S23 into PBS buffer, adding the VS2 / AuNPs prepared in S22 after ultrasonic dispersion, stirring for reaction, and obtaining VS2 / AuNPs / PEI-Fc; S25. Take VS2 / AuNPs / PEI-Fc, add the second antibody after sufficient shaking, add BSA solution after overnight reaction at 4°C, shake, centrifuge, disperse with PBS buffer, and store at 4°C for later use. This is the probe part of the biosensor.

8. Use of the biosensor according to any one of claims 1 to 7 in detecting Salmonella H antigen.

9. Use of the biosensor according to claim 8 in detection of Salmonella H antigen, characterized in that: The first antibody and the second antibody are antibodies that specifically bind to the Salmonella H antigen respectively.

10. Use of the biosensor according to claim 8 in 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 to 1 μg / mL of Salmonella H antigen, and a linear correlation coefficient of the prediction model reaches 0.958.

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