In-situ preparation method of silicon-gold-aptamer nanowire array for escherichia coli detection

Abstract

The invention belongs to the field of nano biosensors, and discloses an in-situ preparation method of a silicon-gold-aptamer nanowire array for escherichia coli detection, which comprises the following steps: preparing a nanowire array material on a silicon wafer by using an in-situ synthesis method, loading gold nanoparticles and escherichia coli aptamers on the nanowire array in situ by using a chemical reduction method, and preparing the silicon-gold-aptamer nanowire array. A metal film is plated on the back surface of the silicon wafer, and the surface is connected with a circuit when the silicon wafer is used, so that an electric signal generated by the silicon-gold-aptamer nanowire array in a test is input into the circuit through conduction of the silicon wafer. The escherichia coli aptamer can specifically adsorb escherichia coli in a test solution to the surface of a material and generate current response linearly related to the concentration of a bacterial solution, and the sensor has good sensitivity due to the huge surface area of the in-situ synthesized electrode with the nano array structure. The high-performance nano biosensor provides a novel practical method for on-site rapid detection of food-borne pathogenic bacteria.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical biosensors, and in particular relates to an in-situ preparation method of a silicon-gold-aptamer nanowire array for detecting Escherichia coli. Background Art

[0002] Microbial testing in food is crucial for food safety. The commonly used methods for detecting foodborne Escherichia coli, such as plate culture, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) and other traditional methods, are cumbersome and time-consuming. Electrochemical detection methods have emerged. Electrochemical detection is widely used in the field of analytical detection due to its high sensitivity and simple operation. For example, Chinese patent CN109060917A constructs a nucleic acid aptamer electrochemical sensor by fixing the capture probe on the surface of the gold electrode with S-Au bonds and hybridizing with the capture probe to form a double-stranded signal probe for the detection of enteropathogenic Escherichia coli, but the preparation method of the sensor is complicated and the detection sensitivity needs to be improved.

[0003] Silicon nanowire arrays (SiNWAs) are tiny nanoarrays formed by neatly arranging on the surface of materials in a certain orientation, and have broad applications in energy, catalysis, environment and biology. For example, in the field of biological detection, silicon nanowire field effect transistor biosensors are usually prepared, and the change of the conductivity of the conductive channel of the field effect transistor by biological molecules is used to change the source and drain current of the field effect transistor, thereby realizing the detection of biological molecules. For example, Chinese patent CN114384141A cross-links modified polyethylene glycol with different molecular weights and silane groups and amino groups at both ends with hydroxylated silicon nanowire field effect transistors to obtain modified polyethylene glycol-modified silicon nanowire field effect transistors; a single-stranded DNA probe with a carboxyl group at the end is coupled to the modified polyethylene glycol-modified silicon nanowire field effect transistor by dehydration condensation reaction to obtain the silicon nanowire biosensor. Chinese patent CN113960128A adopts COMS-compatible silicon nanowire field effect tubes, modifies the surface of the silicon nanowires with aptamers connected to gold nanoparticles at the 3' end, and obtains a silicon nanowire field effect tube biosensor modified with potassium ion aptamers.

[0004] The electrochemical test method of the classic three-electrode system has the characteristics of fast detection, low cost and high sensitivity. At present, there are few research reports on the three-electrode system constructed by silicon nanowire array electrodes. The present invention is committed to establishing a new technology based on the electrode method suitable for on-site detection of Escherichia coli. Summary of the invention

[0005] In order to solve the above technical problems, the object of the present invention is to provide an in-situ preparation method of silicon-gold-aptamer nanowire array for Escherichia coli detection.

[0006] The present invention first provides an in-situ preparation method of a silicon-gold-aptamer nanowire array, comprising the following steps: (1) Preparation of silicon nanowire arrays on the polished surface of a single-sided polished silicon wafer by metal-assisted chemical etching; (2) immersing the silicon wafer obtained in step (1) in a chloroauric acid solution, adding sodium borohydride and mixing thoroughly to react, thereby obtaining a silicon-gold nanowire array on the polished surface of the silicon wafer; (3) modifying the surface of the silicon-gold nanowire array with a thiolated aptamer and blocking it with 2-mercaptoethanol to obtain a silicon-gold-aptamer nanowire array; (4) A metal film is coated on the back of the silicon-gold-aptamer nanowire array to obtain a silicon-gold-aptamer nanowire array modified electrode.

[0007] The specific method for preparing the silicon nanowire array on the polished surface of a single-sided polished silicon wafer in step (1) is as follows: the cleaned silicon wafer is moved into a 3-8% hydrofluoric acid, 5-10 mM silver nitrate aqueous solution, immersed for 45-75 seconds to obtain metal nanoparticles on the silicon wafer, and then washed with a large amount of water; the sample is placed in a 0.15-0.4M hydrogen peroxide, 8-15% hydrofluoric acid aqueous solution for chemical etching for 15-45 minutes, and then immersed in a nitric acid solution to obtain silicon nanowires on the polished surface of the silicon wafer.

[0008] Preferably, in the process of preparing the metal nanoparticles, the immersion time is 75 seconds; and the chemical etching time is 45 minutes.

[0009] The cleaned silicon wafer is obtained by the following method: immersing a single-side polished silicon wafer in a mixture of concentrated sulfuric acid and hydrogen peroxide for 10 minutes, then taking it out and immersing it in a 1-10% hydrofluoric acid solution for 10 minutes to clean the silicon wafer and obtain a cleaned silicon wafer.

[0010] The specific method of step (2) is as follows: immersing the sample in a 0.5-1.5% by mass chloroauric acid solution, adding 0.25-0.75% by mass sodium borohydride and shaking to fully mix the solution, and continuing the reaction for 20-45 minutes to obtain a silicon-gold nanowire array on the polished surface of the silicon wafer.

[0011] Preferably, the sample is immersed in a chloroauric acid solution with a mass fraction of 1-1.5%, and then 0.5-0.75% sodium borohydride is added and shaken to mix thoroughly. After the reaction is continued for 30-45 minutes, a silicon-gold nanowire array can be obtained on the polished surface of the silicon wafer.

[0012] Wherein, the aptamer described in step (3) is an Escherichia coli aptamer, and the nucleotide sequence of the thiolated Escherichia coli aptamer is: 5'-SH-(CH2)6-GCAAT GGTAC GGTAC TTCCC CATGA GTGTT GTGAA ATGTT GGGACACTAG GTGGC ATAGA GCCGC AAAAG TGCAC GCTAC TTTGC TAA-3'.

[0013] The thiolated E. coli aptamer solution was prepared with TE buffer (10 mmol / L Tris-HCl, 1 mmol / LEDTA, pH=7.5) at a concentration of 2.5 μmol / L and a modification amount of 0.1-1.2 mL / cm 2 .

[0014] The concentration of the 2-mercaptoethanol is 0.05-0.5 mol / L, and the blocking time is 0.5-2h.

[0015] Wherein, the metal film coated in step (4) has a thickness of 10-500 nanometers; the metal material is aluminum, copper, platinum, nickel, titanium, cobalt, palladium, tungsten, etc.; the method of coating the metal film includes but is not limited to vacuum evaporation, sputtering, chemical vapor deposition, etc.

[0016] The second aspect of the present invention provides the use of the silicon-gold-aptamer nanowire array modified electrode obtained by the above preparation method in the electrochemical detection of Escherichia coli.

[0017] Specifically, a silicon-gold-aptamer nanowire array modified electrode was used as the working electrode to form a three-electrode system with a counter electrode and a reference electrode. The current response of different concentrations of Escherichia coli was determined by cyclic voltammetry, and the concentration of Escherichia coli in the sample to be tested was calculated using the standard curve method.

[0018] The counter electrode is a platinum wire electrode, and the reference electrode is a silver / silver chloride electrode.

[0019] The parameters of the cyclic voltammetry are: potential window -0.1~+0.7mV, scanning speed 50mV / s.

[0020] When in use, the metal-plated film layer of the modified electrode is connected to the circuit (such as welding), so that the electrical signal generated by the silicon-gold-aptamer nanowire array in the test is input into the circuit through the conduction of the silicon chip.

[0021] Beneficial effects of the present invention: The present invention proposes an in-situ preparation method of a silicon-gold-aptamer nanowire array for detecting Escherichia coli, which can be used as an electrode-type biosensor material to detect Escherichia coli. The huge surface area of ​​the in-situ synthesized nanoarray structure electrode enables the sensor to have good sensitivity. These provide a new practical method for on-site rapid testing of foodborne pathogens for food inspection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning electron microscope photograph of the silicon-gold-aptamer nanowire array in Example 1.

[0023] Figure 2 This is the result of detecting Escherichia coli using the silicon-gold-aptamer nanowire array in Example 1.

[0024] Figure 3 This is the result of detecting Escherichia coli using the silicon-gold-aptamer nanowire array in Example 2.

[0025] Figure 4 This is the result of detecting Escherichia coli using the silicon-gold-aptamer nanowire array in Example 3.

[0026] Figure 5 This is the result of detecting Escherichia coli using the silicon-gold-aptamer nanowire array in Example 4.

[0027] Figure 6 This is the result of detecting Escherichia coli using the silicon-gold-aptamer nanowire array in Example 5.

[0028] Figure 7 This is the CV test result of the silicon-gold-aptamer nanowire array on Escherichia coli in Example 5. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] The E. coli concentration calibration method used in the following embodiments of the present invention is as follows: take the original strain of E. coli (strain source: E. coli ATCC 25922, from the American Type Culture Collection (ATCC)), inoculate it into LB liquid culture medium under sterile conditions, and place it at 37°C for 12 hours for activation and bacterial growth; take 25mL of E. coli sample liquid and place it in a sterile conical flask containing 225mL of normal saline, and shake it thoroughly to prepare the original E. coli bacterial liquid; under sterile conditions, take 100μL of the original bacterial liquid and slowly inject it into a sterile centrifuge tube containing 900μL of sterile normal saline, shake it to make it fully mixed, and prepare a 1:10 sample bacterial liquid; repeat the previous operation steps to perform a ten-fold concentration gradient dilution, and ensure strict sterility during the operation; select the dilution multiple 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 spread on the plate. Spread 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 plates with colony counts between 30-300 cfu 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. Dilute the E. coli solution with sterile saline before testing and calibrate using the above method.

[0031] The electrochemical test method used in the above specific embodiments of the present invention is as follows: at a constant temperature of 37°C, the silicon-gold-aptamer nanowire array modified electrode is incubated in a diluted Escherichia coli solution for 30 minutes, then the material is taken out and rinsed with ultrapure water, and then used as a working electrode, using a three-electrode system (with a platinum wire as a counter electrode and silver / silver chloride as a reference electrode) in a 0.2 M phosphate buffer solution (pH 6.8, containing 5 mM MgCl2) for cyclic voltammetry measurement. The electrode material made of the silicon-gold-aptamer nanowire array with Escherichia coli capture probes fixed on the surface in this method is disposable. For each measurement, an unused new electrode needs to be selected and incubated in the test solution, and then cleaned after incubation, and then the electrode response to Escherichia coli in the test solution is studied by cyclic voltammetry in the above phosphate buffer solution.

[0032] The nucleotide sequence of the thiolated Escherichia coli aptamer used in the following specific embodiments of the present invention is: 5'-SH-(CH2)6-GCAAT GGTAC GGTAC TTCCC CATGA GTGTT GTGAA ATGTT GGGAC ACTAG GTGGCATAGA GCCGC AAAAG TGCAC GCTAC TTTGC TAA-3', which was synthesized by Shanghai Sangon Biotechnology Technology Service Co., Ltd. The thiolated Escherichia coli aptamer test solution was prepared with TE buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH=7.5) and its concentration was 2.5 μM.

[0033] It should be noted that the raw materials or reagents not specifically described in the following examples of the present invention are all common commercially available products. The preparation methods or electrochemical detection methods not specifically described are conventional methods in the art. Example 1

[0034] An in-situ preparation method of a silicon-gold-aptamer nanowire array for Escherichia coli detection comprises the following steps: (1) Immerse a single-side polished silicon wafer (size: 1.2x1.2cm) in a mixture of concentrated sulfuric acid and hydrogen peroxide for 10 minutes, then take it out and soak it in a 3% hydrofluoric acid solution for 10 minutes, then transfer 10mL of 3% hydrofluoric acid and 5 mM silver nitrate aqueous solution and soak it for 45 seconds to obtain metal nanoparticles on the silicon wafer, and then wash it with a large amount of water; chemically etch the sample in a 0.15M hydrogen peroxide and 8% hydrofluoric acid aqueous solution for 15 minutes, and then soak it in a nitric acid solution for 5 minutes to obtain silicon nanowires on the polished surface of the silicon wafer.

[0035] (2) The sample is then immersed in 0.2 mL of 1.0% chloroauric acid solution, and then 0.5 mL of 0.5% sodium borohydride is added and shaken to mix thoroughly. After the reaction is continued for 30 minutes, a silicon-gold nanowire array can be obtained on the polished surface of the silicon wafer.

[0036] (3) Then, 0.3 mL of the thiolated Escherichia coli aptamer test solution was dropped onto the surface of the silicon-gold nanowire array electrode. After drying at room temperature, the electrode was blocked with 0.1 mol / L 2-mercaptoethanol for 0.5 h to obtain the silicon-gold aptamer nanowire array.

[0037] (4) The back side is modified with 300 nm thick aluminum by sputtering coating.

[0038] Figure 2The response current of the biosensor in this embodiment at +0.42V is plotted against the concentration of E. coli. The figure shows that within the test range, the peak current value and the logarithm of the concentration of E. coli have a good linear relationship, and its sensitivity is -2.19μA / lg C E. coli . Example 2

[0039] An in-situ preparation method of a silicon-gold-aptamer nanowire array for Escherichia coli detection comprises the following steps: (1) Immerse a single-side polished silicon wafer in a mixture of concentrated sulfuric acid and hydrogen peroxide for 10 minutes, then take it out and soak it in a 5% hydrofluoric acid solution for 10 minutes, then transfer 10 mL of 5% hydrofluoric acid and 8.3 mM silver nitrate aqueous solution and soak it for 60 seconds to obtain metal nanoparticles on the silicon wafer, and then wash it with a large amount of water; chemically etch the sample in a 0.27 M hydrogen peroxide and 10% hydrofluoric acid aqueous solution for 30 minutes, and then soak it in a nitric acid solution for 5 minutes to obtain silicon nanowires on the polished surface of the silicon wafer.

[0040] (2) The sample is then immersed in 0.2 mL of 1.0% chloroauric acid solution, and then 0.5 mL of 0.5% sodium borohydride is added and shaken to mix thoroughly. After the reaction is continued for 30 minutes, a silicon-gold nanowire array can be obtained on the polished surface of the silicon wafer.

[0041] (3) Then, 0.5 mL of the thiolated Escherichia coli aptamer test solution was dropped onto the surface of the silicon-gold nanowire array electrode. After drying at room temperature, the electrode was blocked with 0.1 mol / L 2-mercaptoethanol for 1.5 h to obtain the silicon-gold aptamer nanowire array.

[0042] (4) Same as Example 1.

[0043] Figure 3 The response current of the biosensor at +0.42V in this example is plotted against the concentration of E. coli. The figure shows that within the test range, the peak current value has a good linear relationship with the logarithm of the concentration of E. coli, and its sensitivity is -5.83μA / lg C. E. coli . Example 3

[0044] An in-situ preparation method of a silicon-gold-aptamer nanowire array for Escherichia coli detection comprises the following steps: (1) Immerse a single-side polished silicon wafer in a mixture of concentrated sulfuric acid and hydrogen peroxide for 10 minutes, then take it out and soak it in a 10% hydrofluoric acid solution for 10 minutes, then transfer 10mL of 8% hydrofluoric acid and 10 mM silver nitrate aqueous solution and soak it for 75 seconds to obtain metal nanoparticles on the silicon wafer, and then wash it with a large amount of water; place the sample in a 0.4 M hydrogen peroxide and 15% hydrofluoric acid aqueous solution for chemical etching for 45 minutes, and then soak it in a nitric acid solution for 5 minutes to obtain silicon nanowires on the polished surface of the silicon wafer.

[0045] (2) The sample is then immersed in 0.2 mL of 1.0% chloroauric acid solution, and then 0.5 mL of 0.5% sodium borohydride is added and shaken to mix thoroughly. After the reaction is continued for 45 minutes, a silicon-gold nanowire array can be obtained on the polished surface of the silicon wafer.

[0046] (3) Then, 0.5 mL of the thiolated Escherichia coli aptamer test solution was dropped onto the surface of the silicon-gold nanowire array electrode. After drying at room temperature, the electrode was blocked with 0.4 mol / L 2-mercaptoethanol for 1.5 h to obtain the silicon-gold aptamer nanowire array.

[0047] (4) The back side is modified with 400 nm thick aluminum by sputtering coating.

[0048] Figure 4 The response current of the biosensor in this embodiment at +0.42V is plotted against the concentration of E. coli. The figure shows that within the test range, the peak current value and the logarithm of the concentration of E. coli have a good linear relationship, and its sensitivity is -7.63μA / lg C E. coli . Example 4

[0049] An in-situ preparation method of a silicon-gold-aptamer nanowire array for Escherichia coli detection comprises the following steps: (1) Silicon nanowires were obtained on the polished surface of a silicon wafer according to the method of Example 3.

[0050] (2) The sample is then immersed in 0.2 mL of 0.5% chloroauric acid solution, and then 0.5 mL of 0.25% sodium borohydride is added and shaken to mix thoroughly. After the reaction is continued for 20 minutes, a silicon-gold nanowire array can be obtained on the polished surface of the silicon wafer.

[0051] (3) and (4) are the same as in Example 3.

[0052] Figure 5The response current of the biosensor in this embodiment at +0.42V is plotted against the concentration of E. coli. The figure shows that within the test range, the peak current value and the logarithm of the concentration of E. coli have a good linear relationship, and its sensitivity is -6.89μA / lg C E. coli . Example 5

[0053] An in-situ preparation method of a silicon-gold-aptamer nanowire array for Escherichia coli detection comprises the following steps: (1) Silicon nanowires were obtained on the polished surface of a silicon wafer according to the method of Example 3.

[0054] (2) The sample is then immersed in 0.2 mL of 1.5% chloroauric acid solution, and then 0.5 mL of 0.75% sodium borohydride is added and shaken to mix thoroughly. After the reaction is continued for 45 minutes, a silicon-gold nanowire array can be obtained on the polished surface of the silicon wafer.

[0055] (3) and (4) are the same as in Example 3.

[0056] Figure 6 The response current of the biosensor in this embodiment at +0.42V is plotted against the concentration of E. coli. The figure shows that within the test range, the peak current value and the logarithm of the concentration of E. coli have a good linear relationship, and its sensitivity is -10.71μA / lg C E. coli .

[0057] From the above examples, it can be seen that the silicon-gold-aptamer nanowire array obtained by this method has high sensitivity in detecting Escherichia coli. In addition, during the preparation process, the sensitivity of detecting Escherichia coli can be controlled by changing parameters such as the chemical etching time of the silicon wire and the gold loading amount. For example, in Examples 1-3, by changing the chemical etching conditions of the silicon wire, the sensitivity of the silicon-gold-aptamer nanowire array electrode in detecting Escherichia coli was increased from -2.19μA / lg C to 1.5 μA / lg C. E. coli To -7.63μA / lg C E. coli In Example 3-5, by changing the gold loading conditions, the sensitivity of the silicon-gold-aptamer nanowire array electrode to detect Escherichia coli was increased from -6.89μA / lg C E. coli To -10.71μA / lg C E. coli control.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An in-situ preparation method of a silicon-gold-aptamer nanowire array, characterized in that: The steps include: (1) Preparation of silicon nanowire arrays on the polished surface of a single-sided polished silicon wafer by metal-assisted chemical etching; (2) immersing the silicon wafer obtained in step (1) in a chloroauric acid solution, adding sodium borohydride and mixing thoroughly to react, thereby obtaining a silicon-gold nanowire array on the polished surface of the silicon wafer; (3) modifying the surface of the silicon-gold nanowire array with a thiolated aptamer and blocking it with 2-mercaptoethanol to obtain a silicon-gold-aptamer nanowire array; (4) A metal film is coated on the back of the silicon-gold-aptamer nanowire array to obtain a silicon-gold-aptamer nanowire array modified electrode.

2. The in-situ preparation method of silicon-gold-aptamer nanowire array according to claim 1, characterized in that: The specific method of step (1) is as follows: the cleaned silicon wafer is moved into a 3-8% hydrofluoric acid, 5-10 mmol / L silver nitrate aqueous solution, immersed for 45-75 seconds to obtain metal nanoparticles on the silicon wafer, and then washed with a large amount of water; the sample is placed in a 0.15-0.4 mol / L hydrogen peroxide, 8-15% hydrofluoric acid aqueous solution for chemical etching for 15-45 minutes, and then immersed in a nitric acid solution to obtain silicon nanowires on the polished surface of the silicon wafer.

3. The in-situ preparation method of silicon-gold-aptamer nanowire array according to claim 1, characterized in that: The specific method of step (2) is: immersing the silicon wafer obtained in step (1) in a 0.5-1.5% by mass chloroauric acid solution, then adding 0.25-0.75% by mass sodium borohydride and shaking to fully mix, and continuing the reaction for 20-45 minutes to obtain a silicon-gold nanowire array on the polished surface of the silicon wafer.

4. The in-situ preparation method of silicon-gold-aptamer nanowire array according to claim 1, characterized in that: The aptamer described in step (3) is an Escherichia coli aptamer, and the nucleotide sequence of the thiolated Escherichia coli aptamer is: 5'-SH-(CH2)6-GCAAT GGTAC GGTAC TTCCC CATGA GTGTT GTGAA ATGTT GGGAC ACTAG GTGGC ATAGAGCCGC AAAAG TGCAC GCTAC TTTGC TAA-3'.

5. The in-situ preparation method of silicon-gold-aptamer nanowire array according to claim 1, characterized in that: The concentration of the thiolated E. coli aptamer solution was 2.5 μmol / L, and the modification amount was 0.1-1.2 mL / cm 2 .

6. The in-situ preparation method of silicon-gold-aptamer nanowire array according to claim 1, characterized in that: The concentration of the 2-mercaptoethanol is 0.05-0.5 mol / L, and the blocking time is 0.5-2h.

7. The in-situ preparation method of silicon-gold-aptamer nanowire array according to claim 1, characterized in that: The thickness of the metal film in step (4) is 10-500 nanometers; the metal material is any one or more of aluminum, copper, platinum, nickel, titanium, cobalt, palladium, and tungsten.

8. Use of the silicon-gold-aptamer nanowire array modified electrode prepared according to the method according to any one of claims 1 to 7 in electrochemical detection of Escherichia coli.

9. The use according to claim 8, characterized in that: The silicon-gold-aptamer nanowire array modified electrode was used as the working electrode to form a three-electrode system with the counter electrode and the reference electrode. The current response of Escherichia coli at different concentrations was determined by cyclic voltammetry, and the concentration of Escherichia coli in the test sample was calculated using the standard curve method.

10. The use according to claim 9, characterized in that: The parameters of the cyclic voltammetry are: potential window -0.1~+0.7mV, scanning speed 50mV / s.

Images