Biosensor based on silicon-platinum nanocomposite and its preparation method and application

Abstract

The present invention discloses a biosensor based on a silicon-platinum nanocomposite and its preparation method, and applies it to the detection of Escherichia coli. The one-dimensional silicon-platinum nanocomposite prepared by chemical etching and reduction methods can significantly improve the linear range of biosensor detection. The one-dimensional silicon-platinum nanocomposite is loaded on the working electrode to obtain a carrier, and the biosensor obtained by modifying the carrier with an Escherichia coli aptamer can specifically adsorb Escherichia coli in the bacterial solution, and due to the steric hindrance effect, a current response with a good linear relationship with the logarithm of the bacterial solution concentration is generated. The detection time of the biosensor based on the silicon-platinum nanocomposite disclosed by the present invention is about 30 minutes, the linear range is 1.3×10<supgt;2< / supgt> cfu / mL - 3.6×10<supgt;5< / supgt> cfu / mL, and it also has good selectivity and repeatability.

Description

Technical Field

[0001] The present invention relates to the field of biosensors, and in particular to a biosensor based on silicon-platinum nanocomposite and a preparation method and application thereof. Background Art

[0002] In the field of food safety, the detection method for foodborne pathogens such as Escherichia coli is an important topic. At present, the detection methods for foodborne Escherichia coli are mainly plate culture method, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The traditional methods are cumbersome and time-consuming. Therefore, it is urgent to develop effective on-site rapid detection technology for such bacteria, which is of great significance to human health.

[0003] Nanobiosensor technology is an emerging technology that is obtained by applying nanomaterials to the field of biosensing. The application of special properties such as surface effects and quantum size effects unique to nanomaterials can effectively improve the detection performance of biosensors, especially in terms of equipment miniaturization, analysis speed, and ease of operation. However, when nanobiosensors are used to detect Escherichia coli, the surface of the working electrode is modified with platinum particles, and then the Escherichia coli aptamers are modified on the platinum particles for the detection of Escherichia coli. However, this biosensor is easily interfered by other pathogens during detection, making the detection results unstable, the sensitivity low, and the detection range limited. Therefore, it is necessary to provide a biosensor with good selectivity for Escherichia coli and a large detection range. Summary of the invention

[0004] In order to solve the problem in the prior art that platinum particles are directly modified on the working electrode and then the Escherichia coli aptamers are modified on the platinum particles, which is easily interfered by other pathogenic bacteria during detection, resulting in unstable detection results, low sensitivity and limited detection range, the purpose of the present invention is to provide a biosensor based on silicon-platinum nanocomposite with high sensitivity, fast detection, wide detection range and good selectivity, as well as a preparation method and application thereof.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows.

[0006] A biosensor based on silicon-platinum nanocomposite, wherein the biosensor is obtained by loading a one-dimensional silicon-platinum nanocomposite on a working electrode to obtain a carrier, and modifying an Escherichia coli aptamer on the carrier; wherein the Escherichia coli aptamer is a thiolated Escherichia coli aptamer;

[0007] The one-dimensional silicon-platinum nanocomposite is obtained by modifying platinum ions in a platinum salt precursor solution on silicon nanowires under the action of a reducing agent.

[0008] Furthermore, the specific preparation process of the one-dimensional silicon-platinum nanocomposite is as follows:

[0009] S1, preparing silicon nanowires;

[0010] S2, dispersing silicon nanowires in water to obtain a silicon nanowire dispersion; then mixing the silicon nanowire dispersion with a platinum salt precursor solution, adding a reducing agent, stirring for reaction, and centrifuging after the reaction is completed to obtain a one-dimensional silicon-platinum nanocomposite.

[0011] Furthermore, in S2, the platinum salt precursor solution is a chloroplatinic acid solution with a mass percentage of 1-3%, and the reducing agent is sodium borohydride with a mass percentage of 0.3-0.5%; the volume ratio of the platinum salt precursor solution to the reducing agent is 2:5-7.

[0012] Furthermore, in S1, the specific preparation of silicon nanowires is as follows:

[0013] The pre-treated silicon wafer is immersed in a corrosive solution for corrosion treatment, and silicon nanowires are obtained after cleaning.

[0014] Furthermore, the specific preparation of the pre-treated silicon wafer is as follows:

[0015] The silicon wafer is sequentially immersed in a mixed solution of concentrated sulfuric acid and hydrogen peroxide, a hydrofluoric acid solution, and a mixed solution of hydrofluoric acid and silver nitrate, and then cleaned to obtain a pretreated silicon wafer with silver nanoparticles loaded on the surface, wherein the silver nanoparticles can serve as a catalyst for corrosion reactions.

[0016] Furthermore, the specific operation of the corrosion treatment is as follows:

[0017] The pretreated silicon wafer is immersed in a mixed solution of hydrogen peroxide and hydrofluoric acid for 20-40 minutes, taken out and then immersed in a nitric acid solution for 1-2 hours.

[0018] Furthermore, in the mixed solution of hydrogen peroxide and hydrofluoric acid aqueous solution, the concentration of hydrogen peroxide is 0.27-0.30M, the mass percentage of hydrofluoric acid aqueous solution is 10-15%, and the mass percentage of nitric acid solution is 60-70%.

[0019] The present invention also provides a method for preparing the biosensor according to claim 2, comprising the following steps:

[0020] Step 1: Prepare a one-dimensional silicon-platinum nanocomposite. The specific method is as follows:

[0021] S1, preparing silicon nanowires;

[0022] S2, dispersing silicon nanowires in water to obtain a silicon nanowire dispersion; then mixing the silicon nanowire dispersion with a platinum salt precursor solution, adding a reducing agent, stirring for reaction, and centrifuging after the reaction is completed to obtain a one-dimensional silicon-platinum nanocomposite;

[0023] The silicon nanowire dispersion also includes a Nafion solution, which is used for film formation of a one-dimensional silicon-platinum nanocomposite dispersion.

[0024] Step 2, adding the one-dimensional silicon-platinum nanocomposite into water and dispersing the water to obtain a dispersion of the one-dimensional silicon-platinum nanocomposite;

[0025] Step 3, adding the dispersion of the one-dimensional silicon-platinum nanocomposite and the Escherichia coli aptamer to the pretreated working electrode in sequence, and after drying, obtaining a biosensor based on the silicon-platinum nanocomposite.

[0026] Furthermore, the specific preparation method of the pretreated working electrode is as follows: the glassy carbon electrode is placed on suede with 1μm, 0.3μm, and 0.05μm alumina to polish it until it is mirror smooth, and then ultrasonically cleaned with deionized water, a mixture of ethanol and acetone, and deionized water respectively, and finally blown dry with nitrogen.

[0027] Furthermore, after drying, step 3 further comprises: adding 2-mercaptoethanol to seal free platinum ions.

[0028] The invention also provides an application of a biosensor in quantitative detection of Escherichia coli.

[0029] The biosensor of the present invention can specifically adsorb Escherichia coli on the surface of a working electrode, and the steric hindrance effect of Escherichia coli when adsorbed on the surface of the working electrode can make the glassy carbon electrode modified with a one-dimensional silicon-platinum nanocomposite generate a current response (ΔI), and the current response (ΔI) of a series of standard Escherichia coli bacterial solutions is linearly related to the concentration (C) of the series of standard Escherichia coli bacterial solutions, thereby obtaining a standard working curve; then the current response of the bacterial solution to be tested is detected to obtain the current response of the bacterial solution to be tested, and the current response is substituted into the standard working curve to obtain the concentration value of the bacterial solution to be tested; thereby achieving quantitative detection of Escherichia coli.

[0030] The silicon-platinum nanocomposite in the present invention can enhance the specific adsorption of the detected bacteria and enhance the electrical signal. The biosensor obtained by sequentially modifying the silicon-platinum nanocomposite and the Escherichia coli aptamer on the glassy carbon motor has high sensitivity, short detection time, good repeatability, and good selectivity for Escherichia coli.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The biosensor in the present invention has the effects of fast detection and sensitive response to Escherichia coli. Among them, the one-dimensional silicon-platinum nanocomposite is accumulated on the surface of the glassy carbon electrode to form a three-dimensional micro-nano network structure, in which the platinum nanoparticles as active sites have a multi-layer spatial distribution effect. When the biosensor is incubated in the target bacterial solution, the spatial steric effect of the three-dimensional network structure composed of the one-dimensional silicon-platinum nanocomposite plays a buffering role in the diffusion of the target bacteria, and the multi-layer spatial arrangement of the platinum nanoparticles greatly enhances this spatial steric effect. Under the synergistic effect of the two, the number of target bacteria specifically adsorbed on the platinum nanoparticles and the final electron transfer rate on the surface of the glassy carbon electrode will have a higher correlation with the concentration of the target bacteria in the detection solution. Therefore, the one-dimensional silicon-platinum nanocomposite in the present invention can improve the electrical signal of the biosensor, so that it has a better linear range.

[0033] (2) The present invention adopts a chemical etching method to prepare silicon nanowires, and uses a reduction method to modify platinum nanoparticles to obtain a one-dimensional silicon-platinum nanocomposite material. A pretreated silicon wafer with silver nanoparticles on the surface is obtained by pretreating the silicon wafer, wherein the silver nanoparticles act as a catalyst for the corrosion reaction, thereby accelerating the reaction speed and reaction effect. After chemical etching, a silicon nanowire array distributed perpendicular to the silicon wafer is obtained, and then the silicon wires are peeled off from the surface of the silicon wafer, and platinum nanoparticles are modified thereon by a reduction method, thereby obtaining a one-dimensional silicon-platinum nanocomposite material. The one-dimensional silicon-platinum nanocomposite material can provide a huge surface area for the electrode, thereby increasing the chemical adsorption effect of the platinum nanoparticles as active sites and amplifying the electrical signals generated by electron transfer. Moreover, after the one-dimensional silicon-platinum nanocomposite material in the present invention is deposited on the electrode surface, the active sites composed of the platinum nanoparticles have a multi-layer spatial distribution structure, which further enhances the effects of chemical adsorption and enhancing electrical signals. In addition, in aqueous solution, the silanol groups on the surface of silicon nanowires can be partially ionized to make their surface negatively charged, which can produce electrostatic adsorption on the positively charged amino residues in the outer membrane protein of Escherichia coli due to hydrolysis, which is beneficial to the enrichment of Escherichia coli on the electrode surface, which will further enhance the adsorption and detection effects.

[0034] (3) The biosensor based on silicon-platinum nanocomposite in the present invention has good selectivity for Escherichia coli, and other foodborne pathogens will not cause signal interference, and has high selectivity in the detection application of Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The following are scanning electron microscope images of the silicon nanowire array and the one-dimensional silicon-platinum nanocomposite prepared in Example 1 of the present invention. A is a SEM image of the silicon nanowire array obtained by chemical etching, and B is a SEM image of the one-dimensional silicon-platinum nanocomposite.

[0036] Figure 2 CV diagrams of electrodes in different steps in potassium ferrocyanide solution in an embodiment of the present invention.

[0037] Figure 3 The graphs and working curves of the biosensor in Example 4 of the present invention for the response to different concentrations of E. coli solutions are shown in Figure 1. A is the cyclic voltammetric response of a biosensor based on silicon-platinum nanocomposite to different concentrations of E. coli solutions; B is a graph of the response current at +0.45V of a biosensor based on silicon-platinum nanocomposite versus the logarithm of the E. coli concentration.

[0038] Figure 4 It is a working curve diagram of the response of the platinum-modified glassy carbon electrode with E. coli capture probes immobilized on the surface to E. coli solutions of different concentrations in Comparative Example 1 of the present invention.

[0039] Figure 5 This is a comparison chart of the response signal intensity of the biosensor in Example 4 of the present invention to different types of foodborne pathogenic bacteria solutions.

[0040] Figure 6 The biosensor in Example 4 of the present invention is a concentration pair of 1.6×10 4 Response signal diagram of CFU / ml E. coli solution after 40 consecutive tests. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] The specific purchase conditions of the reagents and materials described in the following examples are as follows:

[0043] The thiolated E. coli aptamer (sequence: 5′HS-(CH2)6-ACC AGT AGA CTT TCA ACT TTA CTGCCA TCG TGT GCC CTAA-3′) was synthesized by Shanghai Sangon Biotechnology Technology Service Co., Ltd., and E. coli ATCC25922, Staphylococcus aureus ATCC 29213, Salmonella ATCC 10420, Vibrio parahaemolyticus ATCC 17802, and Shigella ATCC 12022 were from the American Type Culture Collection.

[0044] % (wt.) in the following examples all represent mass percentage.

[0045] Example 1

[0046] A method for preparing a silicon-platinum nanocomposite comprises the following steps:

[0047] S1, immersing the pretreated silicon wafer into a corrosive solution for corrosion, and obtaining silicon nanowires after immersion;

[0048] S1.1 Pretreatment of silicon wafer: immerse the silicon wafer in a mixture of concentrated sulfuric acid and hydrogen peroxide for 10 minutes, take it out and put it in a 5% (wt.) hydrofluoric acid solution and soak it for 10 minutes, then take it out and put it in 10mL 5% (wt.) hydrofluoric acid and 8.3mM silver nitrate aqueous solution and soak it for 60 seconds to obtain silver nanoparticles on the silicon wafer, and wash it with a large amount of water to obtain a pretreated silicon wafer with silver nanoparticles loaded on the surface;

[0049] The mixed solution of concentrated sulfuric acid and hydrogen peroxide is obtained by mixing 30% (wt.) hydrogen peroxide and 98% (wt.) concentrated sulfuric acid in a volume ratio of 7:3;

[0050] S1.2 The pretreated silicon wafer is placed in a mixed solution of 0.27M hydrogen peroxide and 10% (wt.) hydrofluoric acid aqueous solution for etching for 30 minutes, and then placed in a 60% (wt.) nitric acid solution for 2 hours to obtain silicon nanowires;

[0051] S2. Ultrasonicate the silicon nanowires in S1 for 10 minutes to obtain a silicon nanowire dispersion, mix it with a 1% (wt.) chloroplatinic acid solution, add 0.3% (wt.) sodium borohydride and stir for 30 minutes, the volume ratio of chloroplatinic acid solution to sodium borohydride is 2:5, and centrifuge to obtain a one-dimensional silicon-platinum nanocomposite.

[0052] Example 2

[0053] A method for preparing a silicon-platinum nanocomposite comprises the following steps:

[0054] S1, immersing the pretreated silicon wafer into a corrosive solution for corrosion, and obtaining silicon nanowires after immersion;

[0055] S1.1 Pretreatment of silicon wafer: immerse the silicon wafer in a mixture of concentrated sulfuric acid and hydrogen peroxide for 8 minutes, take it out and put it in a 6% (wt.) hydrofluoric acid solution and soak it for 8 minutes, then take it out and put it in 10mL 6% (wt.) hydrofluoric acid and 8.0mM silver nitrate aqueous solution and soak it for 50 seconds to obtain silver nanoparticles on the silicon wafer, and wash it with a large amount of water to obtain a pretreated silicon wafer with silver nanoparticles loaded on the surface;

[0056] The mixed solution of concentrated sulfuric acid and hydrogen peroxide is obtained by mixing 30% (wt.) hydrogen peroxide and 98% (wt.) concentrated sulfuric acid in a volume ratio of 7:3;

[0057] S1.2 The pretreated silicon wafer is placed in a mixed solution of 0.30M hydrogen peroxide and 15% (wt.) hydrofluoric acid aqueous solution for etching for 20 minutes, and then placed in a 65% (wt.) nitric acid solution for 1 hour to obtain silicon nanowires;

[0058] S2. Ultrasonicate the silicon nanowires in S1 for 10 minutes to obtain a silicon nanowire dispersion, mix it with a 2% (wt.) chloroplatinic acid solution, add 0.4% (wt.) sodium borohydride and stir for 30 minutes, the volume ratio of chloroplatinic acid solution to sodium borohydride is 2:6, and centrifuge to obtain a one-dimensional silicon-platinum nanocomposite.

[0059] Example 3

[0060] A method for preparing a silicon-platinum nanocomposite comprises the following steps:

[0061] S1, immersing the pretreated silicon wafer into a corrosive solution for corrosion, and obtaining silicon nanowires after immersion;

[0062] S1.1 Pretreatment of silicon wafer: immerse the silicon wafer in a mixture of concentrated sulfuric acid and hydrogen peroxide for 12 minutes, take it out and put it in a 7% (wt.) hydrofluoric acid solution and soak it for 12 minutes, then take it out and put it in 10mL 7% (wt.) hydrofluoric acid and 8.5mM silver nitrate aqueous solution and soak it for 50 seconds to obtain silver nanoparticles on the silicon wafer, and wash it with a large amount of water to obtain a pretreated silicon wafer with silver nanoparticles loaded on the surface;

[0063] The mixed solution of concentrated sulfuric acid and hydrogen peroxide is obtained by mixing 30% (wt.) hydrogen peroxide and 98% (wt.) concentrated sulfuric acid in a volume ratio of 7:3;

[0064] S1.2 The pretreated silicon wafer is placed in a mixed solution of 0.30M hydrogen peroxide and 15% (wt.) hydrofluoric acid aqueous solution for corrosion for 40 minutes, and then placed in a 70% (wt.) nitric acid solution for 2 hours to obtain silicon nanowires;

[0065] S2. Ultrasonicate the silicon nanowires in S1 for 10 minutes to obtain a silicon nanowire dispersion, mix it with a 3% (wt.) chloroplatinic acid solution, add 0.5% (wt.) sodium borohydride and stir for 30 minutes, the volume ratio of chloroplatinic acid solution to sodium borohydride is 2:7, and centrifuge to obtain a one-dimensional silicon-platinum nanocomposite.

[0066] The one-dimensional silicon-platinum nanocomposites prepared in the above examples 1-3 all have similar effects. The structure of the one-dimensional silicon-platinum nanocomposites prepared in example 1 is characterized. The results are as follows: Figure 1 As shown, Figure 1(A) in the figure shows the silicon wire obtained by chemical etching, and (B) is a SEM photo of a one-dimensional silicon-platinum nanocomposite. From (A), it can be seen that after chemical etching of the silicon wafer, a silicon nanowire array distributed perpendicular to the silicon wafer is obtained, and the length of the silicon wire is about 8.2 microns. The silicon wire is then peeled off from the surface of the silicon wafer, and platinum nanoparticles are modified on it using a reduction method, thus obtaining a one-dimensional silicon-platinum nanocomposite. Figure 1 (B) The SEM photo shows that platinum nanoparticles with a diameter of about 40 nanometers are evenly dispersed on the surface of the silicon wire with a diameter of about 1 micron, which shows that the platinum nanoparticles are successfully modified on the surface of the silicon wire. This structural characteristic of the one-dimensional silicon-platinum nanocomposite can provide a huge surface area for the electrode, thereby increasing the chemical adsorption effect of the platinum nanoparticles as active sites and amplifying the electrical signals generated by electron transfer. Moreover, after the nanocomposite is accumulated on the electrode surface, the active sites composed of platinum nanoparticles have a multi-layer spatial distribution structure, which has the effect of further enhancing chemical adsorption and enhancing electrical signals.

[0067] The above Examples 1-3 all prepared one-dimensional silicon-platinum nanocomposites with similar effects. The one-dimensional silicon-platinum nanocomposites prepared in Example 1 are used as an example to prepare a biosensor based on silicon-platinum nanocomposites.

[0068] Example 4

[0069] A method for preparing a biosensor based on a silicon-platinum nanocomposite comprises the following steps:

[0070] S1, dispersing the one-dimensional silicon-platinum nanocomposite prepared according to the preparation method of Example 1 in 5 mL of water, adding 0.25% (wt.) Nafion solution to obtain a dispersion of the one-dimensional silicon-platinum nanocomposite;

[0071] S2, sequentially adding a dispersion of a one-dimensional silicon-platinum nanocomposite and a thiolated Escherichia coli aptamer to the pretreated glassy carbon electrode, drying the electrode, blocking free platinum ions with 2-mercaptoethanol, obtaining a biosensor based on a silicon-platinum nanocomposite, and storing the biosensor in a refrigerated PBS buffer solution for later use;

[0072] S2.1. Pretreatment of glassy carbon electrode: The pretreatment of glassy carbon electrode with a diameter of 3 mm is to place 1 μm, 0.3 μm, and 0.05 μm aluminum oxide on suede and polish it to a mirror smooth surface, then ultrasonically clean it with deionized water, ethanol and acetone mixture, and deionized water for 3 minutes each, and finally blow dry with nitrogen to obtain a pretreated glassy carbon electrode;

[0073] S2.2, using a microinjector to draw 5 μL of one-dimensional silicon-platinum nanocomposite and drip it onto the pretreated glassy carbon electrode, and then put it into a desiccator to dry for later use, thereby obtaining a glassy carbon electrode modified with silicon-platinum nanocomposite;

[0074] S2.3. Use a microinjector to draw 5 μL of thiolated Escherichia coli aptamer and drop it on the glassy carbon electrode modified with silicon-platinum nanocomposite. After drying, use 0.1 mol / L 2-mercaptoethanol to block the free platinum ions for 1.5 h. The biosensor based on silicon-platinum nanocomposite is obtained and stored in PBS buffer under refrigerated conditions for use.

[0075] Comparative Example 1

[0076] The same glassy carbon electrode pretreatment method as in Example 4 was used to obtain a pretreated glassy carbon electrode, platinum nanoparticles were modified on the pretreated glassy carbon electrode to obtain a glassy carbon electrode modified with platinum nanoparticles, and 5 μL of thiolated Escherichia coli aptamer was added to the glassy carbon electrode modified with platinum nanoparticles to obtain a glassy carbon electrode modified with platinum nanoparticles having Escherichia coli capture probes fixed on its surface.

[0077] We conducted the following experiments to demonstrate that the biosensor based on silicon-platinum nanocomposite has fast detection, sensitive response, specific adsorption to Escherichia coli, and that one-dimensional silicon-platinum nanocomposite can improve the sensitivity of the sensor.

[0078] 1. Electrochemical characterization

[0079] The glassy carbon electrode modified with the one-dimensional silicon-platinum nanocomposite having the E. coli capture probe fixed on its surface, the glassy carbon electrode modified with the one-dimensional silicon-platinum nanocomposite, and the unmodified bare glassy carbon electrode in Example 4 were placed in potassium ferrocyanide solution for CV scanning. The scanning results are shown in FIG. Figure 2 shown. Figure 2 (a) is an unmodified bare glassy carbon electrode, (b) is a glassy carbon electrode modified with a one-dimensional silicon-platinum nanocomposite, and (c) is a glassy carbon electrode modified with a one-dimensional silicon-platinum nanocomposite with an E. coli capture probe fixed on the surface. When the unmodified bare glassy carbon electrode was subjected to CV scanning in potassium ferrocyanide solution, two reversible redox peaks appeared at +0.25V and +0.19V, while the oxidation peak of the glassy carbon electrode modified with a one-dimensional silicon-platinum nanocomposite moved to +0.29V and the reduction peak moved to +0.15V, and the peak current increased. This is because the platinum nanoparticles in the one-dimensional silicon-platinum nanocomposite have high electrocatalytic activity and dispersion density, so signal amplification can be achieved. It was found that [Fe(CN)6] 3- and [Fe(CN)6] 4- The redox peak current of the Pt nanoparticles decreased because the active sites on the Pt nanoparticles were occupied by the E. coli aptamers. Due to the steric hindrance effect and the E. coli aptamers and [Fe(CN)6] 3- and [Fe(CN)6]4- The electrostatic repulsion between [Fe(CN)6] 3- and [Fe(CN)6] 4- Diffusion to the electrode surface and decreased electron transfer efficiency led to a decrease in peak current, indicating that the E. coli aptamer was successfully modified on the surface of the glassy carbon electrode modified with one-dimensional silicon-platinum nanocomposite.

[0080] 2. Response to E. coli

[0081] Calibration: Take the original strain of Escherichia coli, inoculate it into LB liquid culture medium under sterile conditions, and place it at 37°C for 12 hours for activation and growth; take 25mL of Escherichia coli sample liquid and place it in a sterile conical flask containing 225mL of normal saline, and shake it thoroughly to make the original Escherichia coli liquid; under sterile conditions, take 100μL of the original liquid and slowly inject it into a sterile centrifuge tube containing 900μL of sterile normal saline, shake it to make it fully mixed, and make a 1:10 sample liquid; repeat the previous steps to perform a ten-fold concentration gradient dilution, and ensure strict sterility during the operation; select a dilution factor of 10 -5 -10 -7 Count three plates with different concentrations. Pipette 100μL of E. coli solution and inject it on LB solid plate culture medium to plate. Plate three plates for each concentration in parallel. Culture at 37℃, observe and count, and record the dilution multiple and the corresponding number of colonies. The total number of colonies is expressed in colony-forming units (cfu). When counting, select a plate with a colony count between 30-300cfu and no spreading colony growth. The concentration of the original bacterial solution can be calculated based on the dilution multiple and the number of colonies.

[0082] Before the test, the E. coli solution was diluted with sterile saline and calibrated using the above method; at a constant temperature of 37°C, the glassy carbon electrode modified with the one-dimensional silicon-platinum nanocomposite having the E. coli capture probe fixed on its surface in Example 4 was incubated in the diluted E. coli solution for 30 minutes, and then the electrode was used as the working electrode, and cyclic voltammetry was performed using a three-electrode system (with a platinum wire as the counter electrode and silver / silver chloride as the reference electrode). The electrode surface was rinsed with ultrapure water before and after each measurement. Cyclic voltammetry was used to study the response of the glassy carbon electrode modified with the one-dimensional silicon-platinum nanocomposite having the E. coli capture probe fixed on its surface to different concentrations of E. coli. The results are as follows. Figure 3 shown. Figure 3(A) shows the cyclic voltammetric response of the biosensor of the silicon-platinum nanocomposite to different concentrations of E. coli; (B) shows the response current of the biosensor of the silicon-platinum nanocomposite at +0.45V versus the logarithm of the E. coli concentration. As can be seen from (A), in the range of 0.2-0.6V of the cyclic voltammetric curve, the oxidation current decreases as the concentration of E. coli increases. This is because the surface of the biosensor of the silicon-platinum nanocomposite is modified with an E. coli aptamer. When incubated in an E. coli bacterial solution, the E. coli aptamer will specifically capture the target bacteria on the surface of the biosensor. Due to the steric hindrance effect, the adsorption amount of the bacteria will affect the electron transfer efficiency between the biosensor and the electrolyte, resulting in a decrease in the current value. Therefore, E. coli can be quantitatively detected by the current value. Figure 3 (b) is a graph showing the response current of the biosensor at +0.45 V versus the concentration of E. coli in Example 4. 2 cfu / mL-3.6×10 5 In the range of cfu / mL, the peak current value and the logarithm of the E. coli concentration showed a good linear relationship, and the linear equation was: Y = -1.17X + 12.78 (R 2 =0.9967), and its sensitivity is 1.17μA / lg C E.coli .

[0083] 3. Study on the effect of one-dimensional silicon-platinum nanocomposites

[0084] A comparative experiment was conducted between the platinum-modified glassy carbon electrode with the Escherichia coli capture probe immobilized on the surface in Comparative Example 1 and the one-dimensional silicon-platinum nanocomposite-modified glassy carbon electrode with the Escherichia coli capture probe immobilized on the surface in Example 4.

[0085] After the platinum nanoparticles and the glassy carbon electrode modified with the E. coli capture probe in Comparative Example 1 were incubated in the E. coli sample solution for 30 min, the electrode was studied using cyclic voltammetry. The results are as follows: Figure 4 shown. Figure 4 The graph in the middle is the response current of the platinum-modified glassy carbon electrode with E. coli capture probe fixed on the surface in Comparative Example 1 at +0.45V versus the concentration of E. coli. The upper limit of its linear range is only 1.2×10 3 CFU / mL(R 2 =0.768), the sensitivity is only 0.39μA / lg C E.coli It can be seen that the linear range and sensitivity of Comparative Example 1 are much lower than the linear range of the response current of the glassy carbon electrode at +0.45V to the concentration of E. coli (1.3×10 2 cfu / mL-3.6×10 5cfu / mL) and sensitivity (1.17μA / lg C E.coli ). This shows that the one-dimensional silicon-platinum nanocomposite has a significant improvement effect on the linear range of the glassy carbon electrode. This is because the one-dimensional silicon-platinum nanocomposite accumulates on the surface of the glassy carbon electrode to form a three-dimensional micro-nano network structure, and the platinum nanoparticles as active sites have a multi-layer spatial distribution effect. When the glassy carbon electrode modified with the one-dimensional silicon-platinum nanocomposite is incubated in the target bacterial solution, the steric hindrance effect of the three-dimensional network structure composed of the one-dimensional silicon-platinum nanocomposite plays a buffering role in the diffusion of the target bacteria, and the multi-layer spatial arrangement of the platinum nanoparticles greatly enhances this steric hindrance effect. In addition, in aqueous solution, the silanol on the surface of the silicon nanowire can be partially ionized to make its surface negatively charged, which can produce electrostatic adsorption on the amino residues in the outer membrane protein of Escherichia coli that are positively charged due to hydrolysis, which is conducive to enriching Escherichia coli on the electrode surface, which will further enhance the adsorption and detection effect. In short, under the synergistic effect of multiple parties, the electrical signal of the glassy carbon electrode modified with the one-dimensional silicon-platinum nanocomposite shows a better linear range, greater sensitivity and better correlation, which proves the important role of the one-dimensional silicon-platinum nanocomposite in improving the detection performance.

[0086] 4. Selective Experiment

[0087] Escherichia coli, Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus and Shigella are all common foodborne pathogens. The electrode modified with the one-dimensional silicon-platinum nanocomposite having the Escherichia coli capture probe fixed on the surface in Example 4 was mixed with a concentration of 1.6×10 4 After incubating each bacterial species with CFU / ml for 30 minutes, cyclic voltammetry was used to study the response of the electrode to different bacterial species. The response current obtained at +0.45V for each bacterial species was deducted from the response current of the electrode in a blank solution with a bacterial species concentration of 0 to obtain △I, and then -△I was used as the response signal of the electrode to the bacterial species. The results are shown in the figure. Figure 5 As shown, a is the current response value of the electrode modified with one-dimensional silicon-platinum nanocomplex with E. coli capture probes immobilized on the surface in E. coli ATCC25922, b is the current response value of the electrode modified with one-dimensional silicon-platinum nanocomplex with E. coli capture probes immobilized on the surface in Staphylococcus aureus ATCC 29213, c is the current response value of the electrode modified with one-dimensional silicon-platinum nanocomplex with E. coli capture probes immobilized on the surface in Salmonella ATCC 10420, d is the current response value of the electrode modified with one-dimensional silicon-platinum nanocomplex with E. coli capture probes immobilized on the surface in Vibrio parahaemolyticus ATCC 17802, and e is the current response value of the electrode modified with one-dimensional silicon-platinum nanocomplex with E. coli capture probes immobilized on the surface in Shigella ATCC 12022. Figure 5It can be seen that under the same dilution factor, the response signal -ΔI obtained by the biosensor in Example 4 in the E. coli liquid is 3.18 μA, which is much larger than the response signal (0.25-0.34 μA) for other bacterial species. This indicates that the E. coli aptamer can specifically adsorb E. coli, and other bacterial species will not cause signal interference. Therefore, the biosensor based on silicon-platinum nanocomposite in the present invention has a high selectivity for the detection of E. coli.

[0088] 5. Repeatability Experiment

[0089] The concentration of 1.6×10 4 CFU / ml of E. coli was used to characterize the repeatability of the biosensor in Example 4, and the test was repeated 40 times. Figure 6 The relationship between the response signal and the number of scans is shown. Figure 6 It can be seen that the concentration of the glassy carbon electrode modified with one-dimensional silicon-platinum nanocomposite with E. coli capture probe fixed on the surface is 1.6×10 4 After 40 consecutive repeated detections of E. coli CFU / ml, the response signal only decreased by 4.97%, and the overall relative standard deviation RSD was calculated to be 1.9%. This indicates that the biosensor based on the silicon-platinum nanocomposite in the present invention has good durability, the electron transfer efficiency between the silicon-platinum nanocomposite and the electrolyte, and the glassy carbon electrode modified with the silicon-platinum nanocomposite having the E. coli probe fixed on the surface has good stability in the specific adsorption of E. coli.

[0090] It can be seen from the above results that the silicon-platinum nanocomposite in the present invention can enhance the specific adsorption of the bacterial species to be tested and enhance the electrical signal. The biosensor obtained by sequentially modifying the silicon-platinum nanocomposite and the Escherichia coli aptamer on the glassy carbon motor has high sensitivity, short detection time, good repeatability, and good selectivity for Escherichia coli.

[0091] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A biosensor based on silicon-platinum nanocomposite for quantitative detection of Escherichia coli, characterized in that: The biosensor is obtained by loading a one-dimensional silicon-platinum nanocomposite on a working electrode to obtain a carrier, and modifying a thiolated Escherichia coli aptamer on the carrier; the biosensor performs detection through a steric hindrance effect; The one-dimensional silicon-platinum nanocomposite is obtained by modifying platinum ions in a platinum salt precursor solution on silicon nanowires under the action of a reducing agent; the platinum salt precursor solution is a chloroplatinic acid solution with a mass percentage of 1%, and the reducing agent is a sodium borohydride with a mass percentage of 0.3%; the volume ratio of the platinum salt precursor solution to the reducing agent is 2:5; The sequence of the thiolated E. coli aptamer is 5′HS-(CH2)6-ACCAGTAGACTTTCAACTTTACTGCCATCGTGTGCCCTAA-3′.

2. The biosensor based on silicon-platinum nanocomposite for quantitative detection of Escherichia coli according to claim 1, characterized in that: The specific preparation of the silicon nanowires is as follows: The pre-treated silicon wafer is immersed in an etching solution for etching, and the silicon nanowire is obtained after cleaning; The specific preparation of the pretreated silicon wafer is as follows: the silicon wafer is sequentially immersed in a mixed solution of concentrated sulfuric acid and hydrogen peroxide, a hydrofluoric acid solution, and a mixed solution of hydrofluoric acid and silver nitrate, and then washed to obtain a pretreated silicon wafer with silver nanoparticles loaded on the surface.

3. The biosensor based on silicon-platinum nanocomposite for quantitative detection of Escherichia coli according to claim 2, characterized in that: The specific operation of the corrosion treatment is as follows: The pretreated silicon wafer is placed in a mixed solution of hydrogen peroxide and hydrofluoric acid aqueous solution and soaked for 20-40 minutes, and then taken out and transferred to a nitric acid solution and soaked for 1-2 hours; In the mixed solution of hydrogen peroxide and hydrofluoric acid aqueous solution, the concentration of hydrogen peroxide is 0.27-0.30M, the mass percentage of hydrofluoric acid aqueous solution is 10-15%, and the mass percentage of nitric acid solution is 60-70%.

4. The biosensor based on silicon-platinum nanocomposite for quantitative detection of Escherichia coli according to claim 3, characterized in that: The specific preparation process of the biosensor based on silicon-platinum nanocomposite is as follows: Step 1, dispersing the one-dimensional silicon-platinum nanocomposite in water to obtain a dispersion of the one-dimensional silicon-platinum nanocomposite; Step 2: Add the dispersion of one-dimensional silicon-platinum nanocomposite and Escherichia coli aptamer to the pretreated working electrode in sequence, and after drying, obtain a biosensor based on silicon-platinum nanocomposite for quantitative detection of Escherichia coli.

5. The biosensor based on silicon-platinum nanocomposite for quantitative detection of Escherichia coli according to claim 4, characterized in that: After the drying, the method further comprises: adding 2-mercaptoethanol to seal the free platinum ions.

6. Use of the biosensor based on silicon-platinum nanocomposite for quantitative detection of Escherichia coli as claimed in claim 5 in the quantitative detection of Escherichia coli.

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