Preparation method and application of SERS (Surface Enhanced Raman Scattering) biosensor based on nucleic acid aptamer

Abstract

The invention discloses a preparation method and application of an SERS (Surface Enhanced Raman Scattering) biosensor based on a nucleic acid aptamer. Belongs to the technical field of SERS. The method comprises the following steps: synthesizing an Au (at) Ag (at) DTNB nanoparticle solution, and preparing Au (at) Ag (at) DTNB nanoparticles modified by a capture probe; synthesizing a single-layer Au film coated on the silicon wafer, and preparing a rhodamine 6G and complementary probe modified Au film; the SERS biosensor is constructed by combining the capture probe modified Au (at) Ag (at) DTNB nanoparticles with rhodamine 6G and a complementary probe modified Au film. The invention adopts a widely applied portable Raman spectrometer to combine with an aptamer to construct an SERS biosensor, and aims to provide a preparation method of a biosensor for rapidly and conveniently indirectly detecting escherichia coli by weakening a surface enhanced Raman signal.

Description

Technical Field

[0001] The present invention relates to the field of SERS technology, and more particularly to a preparation method and application of a SERS biosensor based on nucleic acid aptamers. Background Art

[0002] Surface-enhanced Raman spectroscopy (SERS) technology refers to the phenomenon that light irradiates the rough surface of a nano-substrate, causing chemical or physical changes, thereby causing a significant enhancement of the Raman signal. It combines Raman technology with nanotechnology to construct SERS metal nano-substrate materials to regulate signal enhancement. This technology has higher sensitivity and spectral resolution, and can achieve 10 14 The single-molecule Raman signal is enhanced by several times, so it has greater application potential in the detection of trace substances.

[0003] SERS-active nanosubstrates can generally be divided into two categories: colloidal substrates and solid surface substrates. Using colloidal substrates is the most direct way to obtain enhanced Raman signals, but it is not easy to control the assembly of metal nanoparticles and evaluate the exact location of target analytes in aqueous solution detection systems. In contrast, solid surface-based substrates can well control the generation of hot spots and obtain the exact location of target analytes, so they show better reproducibility and high sensitivity.

[0004] Escherichia coli is a common bacterium that is widely distributed in nature, especially in the intestines of warm-blooded animals. Although most strains of E. coli are harmless to humans, since the middle of the last century, it has been recognized that some special E. coli can produce enterotoxins that can cause abdominal pain, diarrhea, inflammation, ulcers, and even serious diseases such as hemorrhagic enteritis and hemolysis, especially in infants and young children. Therefore, quantitative detection of E. coli has always been an important topic in the fields of environment, medicine, pharmaceuticals, and food safety.

[0005] At present, the most common detection methods for pathogenic Escherichia coli are bacterial culture, microscopic observation and molecular biology. These methods are relatively simple to operate and low in cost, but the detection cycle is long and requires professional equipment and technicians to operate. As a promising bacterial detection method, biosensors have high selectivity and sensitivity and can achieve rapid and accurate detection of bacteria. SERS biosensors detect targets through the interaction between electromagnetic radiation and plasma matrix, and have the advantages of high enhanced activity and good reproducibility. However, it has been reported that the particles on the aptamer sensors commonly used in SERS have obvious agglomeration and poor distribution uniformity.

[0006] In summary, how to provide a nanocomposite material with good distribution uniformity and stable structure as an aptamer sensor for detecting Escherichia coli is a problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a preparation method of a SERS biosensor based on nucleic acid aptamers and its application. The preparation process of the method of the present invention is simple and low in cost, and the prepared SERS biosensor has the advantages of good distribution uniformity, stable structure, low detection limit, high sensitivity, etc.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for preparing a SERS biosensor based on nucleic acid aptamers comprises the following steps:

[0010] (1) Preparation of gold nanoparticle solution:

[0011] Gold nanoparticle solution was prepared using sodium citrate and chloroauric acid as raw materials;

[0012] (2) Preparation of Au@Ag@DTNB NPs:

[0013] PVP solution, ascorbic acid solution and AgNO3 solution were sequentially added to the gold nanoparticle solution to obtain Au@AgNPs, and then DTNB solution was added to obtain Au@Ag@DTNB NPs;

[0014] (3) Preparation of Au@Ag@DTNB NPs modified capture probes:

[0015] The nucleotide sequence of the capture probe is shown in SEQ ID No. 1;

[0016] 5'-GCA ATG GTA CGG TAC TTC CCC ATG AGT GTT GTG AAA TGT TGG GAC ACTAGG TGG CAT AGA GCC GCA AAA GTG CAC GCT ACT TTG CTA A-SH-3', SEQ ID No. 1;

[0017] (4) Preparation of single-layer gold film:

[0018] The gold nanoparticle solution of step (1) is dispersed in a PVP-ethanol solution and an ethanol solution in turn to obtain an Au-PVP solution; then dichloromethane and ultrapure water are added and rapidly shaken; then n-hexane is added to obtain a single-layer gold film;

[0019] (5) Preparation of single-layer gold film modified with rhodamine 6G and complementary probe:

[0020] The single-layer gold film is immersed in rhodamine 6G and complementary probe successively to obtain a single-layer gold film modified by rhodamine 6G and complementary probe;

[0021] The nucleotide sequence of the complementary probe is shown in SEQ ID No. 2;

[0022] 5'-SH-TTA GCA AAG TAG CGT GCA CTT TTG-3', SEQ ID No. 2;

[0023] (6) Preparation of nucleotide aptamer biosensor:

[0024] The product of step (5) is immersed in the product of step (3) to obtain a nucleotide aptamer biosensor.

[0025] Furthermore, the specific operation of step (1) is: heating a 0.01% chloroauric acid solution to boiling, then adding a 1% sodium citrate solution, and continuing to heat for 15 minutes after the solution gradually turns wine red, and then cooling;

[0026] The volume ratio of the chloroauric acid solution to the sodium citrate solution is 100:1.4.

[0027] Furthermore, the specific operation of step (2) is as follows:

[0028] (21) centrifuging the gold nanoparticle solution obtained in step (1) and dispersing it in deionized water to obtain solution A, wherein the volume ratio of the gold nanoparticle solution to deionized water is 5:1;

[0029] (22) Solution A was mixed with a 1% PVP solution and stirred, then a 10 mM ascorbic acid solution was added and stirred, and finally a 10 mM AgNO3 solution was added dropwise and stirred for 30–40 min to obtain Au@Ag NPs.

[0030] Among them, the volume ratio of solution A, PVP solution, ascorbic acid solution, and AgNO3 solution is 2:1:2:0.5;

[0031] (23) 1 mM DTNB solution was added to Au@Ag NPs and stirred. After centrifugation, the solution was dispersed in deionized water to obtain Au@Ag@DTNB NPs.

[0032] Furthermore, the specific operation of step (3) is as follows:

[0033] 10 uM capture probe was added to Au@Ag@DTNB NPs, and mixed and cultured at room temperature for 2 hours, and then incubated in sodium citrate solution, washed with HEPES buffer, and dispersed in phosphate buffer to obtain Au@Ag@DTNB NPs solution modified with capture probe;

[0034] Among them, the volume ratio of Au@Ag@DTNB NPs to capture probes was 20:1.

[0035] Furthermore, the specific operation of step (4) is as follows:

[0036] (41) The gold nanoparticle solution of step (1) is centrifuged and dispersed in a PVP-ethanol solution and allowed to stand overnight, and then centrifuged again and dispersed in an ethanol solution to obtain an Au-PVP solution;

[0037] The volume ratio of the gold nanoparticle solution, the PVP-ethanol solution, and the ethanol solution is 10:1:1;

[0038] (42) The Au-PVP solution, dichloromethane, and ultrapure water were mixed and rapidly shaken, and then n-hexane was added to obtain a single-layer gold film.

[0039] Furthermore, in step (5), the concentration of rhodamine 6G is 10 -4 M, the concentration of complementary probe was 1 uM.

[0040] Furthermore, in the step (6), the sample is taken out after immersion for 1 hour and baked at 60° C. for 10 minutes.

[0041] Application of the nucleotide aptamer biosensor prepared by the above preparation method in detecting Escherichia coli.

[0042] In this scheme, the SERS probe is first bound to the Au NS silicon wafer and then immersed in the E. coli solution. Since the capture probe has a stronger binding ability with E. coli, the SERS probe is separated from the Au NS, thus forming a competitive assay. Therefore, through analytical determination, the SERS signal intensity gradually decreases because the number of SERS probes adsorbed by the SERS active substrate decreases with the increase of E. coli concentration.

[0043] A method for detecting Escherichia coli, the specific operating steps are as follows:

[0044] The aptamer SERS sensor was immersed in it for 2 hours, taken out and rinsed with deionized water, dried at 60℃ for 10 minutes, and then the SERS signal of the aptamer SERS sensor was detected. Test conditions: portable Raman spectrometer, laser wavelength of 785 nm, power of 300 mW, integration time of 5 s, scanning range of 550~1800 cm-1 The classic Raman peak 1329 cm of the Raman label DTNB -1 The detection result is determined by the Raman spectrum obtained by comparing the average SERS intensity at the location with the logarithm of the Escherichia coli concentration and the corresponding linear correlation.

[0045] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0046] (1) The silver-coated gold core-shell nanoparticles prepared by the present invention have the characteristics of stable structure, good reproducibility and enhanced Raman signal.

[0047] (2) The Au film prepared in the present invention can overcome the electrostatic repulsion between Au NSs and form a highly dense monolayer film with uniform surface distribution and uniform particle size, which can enhance the Raman signal as a SERS substrate.

[0048] (3) The SERS biosensor synthesized by the present invention has a stable structure, good accuracy, and high sensitivity for detecting Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0050] Figure 1 Schematic diagram of the SERS biosensor based on nucleic acid aptamers for detecting Escherichia coli in the present invention;

[0051] Figure 2 The SEM images of Au@Ag NPs prepared in each embodiment of Experiment 1 of the present invention (scale 100 nm), wherein a is the result of the sample of Example 1, b is the result of the sample of Example 2, c is the result of the sample of Example 3, and d is the result of the sample of Example 4;

[0052] Figure 3 The ultraviolet absorption spectra of Au@Ag NPs prepared in each embodiment of Experiment 2 of the present invention;

[0053] Figure 4 Surface enhanced Raman spectra of Au@Ag NPs prepared in each embodiment of Experiment 3 of the present invention;

[0054] Figure 5 The surface enhanced Raman spectra of Au@Ag@DTNB NPs prepared in each embodiment of Experiment 3 of the present invention;

[0055] Figure 6 This is the surface enhanced Raman spectrum of Au NS modified by rhodamine 6G in Experiment 3 of the present invention;

[0056] Figure 7 The results of Experiment 4 of the present invention are shown in the left figure. The left figure is the surface enhanced Raman spectrum of the nucleic acid aptamer sensor used to detect Escherichia coli; the right figure is the Raman characteristic peak at 1329 cm -1 Quantitative analysis fitting diagram of SERS signal intensity-concentration at ;

[0057] Figure 8 The results of Experiment 5 of the present invention are shown in the left figure. The left figure shows the surface enhanced Raman spectra of the aptamer sensor used to detect three interfering bacteria; the right figure shows the Raman characteristic peak at 1329 cm -1 SERS intensity bar comparison chart at . DETAILED DESCRIPTION

[0058] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0060] Example 1

[0061] Preparation of AgNO3 solution with a volume of 0.1 ml

[0062] Step 1: Preparation of gold nanoparticle solution (AuNPs):

[0063] First, weigh 0.1 g of sodium citrate and completely dissolve it in 9.9 ml of deionized water to obtain a sodium citrate aqueous solution with a mass concentration of 1%; secondly, weigh 0.1 g of chloroauric acid and completely dissolve it in 9.9 ml of deionized water to obtain a chloroauric acid solution with a mass concentration of 1%; add 99 ml of deionized water and 1 ml of 1% chloroauric acid solution into a three-necked flask, heat and stir in an oil bath until the solution boils, then maintain the boiling temperature, quickly add 1.4 ml of 1% sodium citrate aqueous solution, and continue heating for 15 minutes after the color of the solution gradually turns wine red, turn off the oil bath, and obtain the AuNPs solution after naturally cooling to room temperature.

[0064] Step 2: Preparation of silver-coated gold nanoparticle solution (Au@Ag NPs) and silver-coated gold nanoparticles modified with 5,5'-dithiobis(2-nitrobenzoic acid) (Au@Ag@DTNB NPs):

[0065] Take 10 ml of AuNPs solution in step 1, centrifuge at 8000 rpm for 15 minutes and disperse in 2 ml of deionized water; take a 25 ml beaker and place it on a magnetic stirrer, add 2 ml of AuNPs prepared above and 1 ml of 1% PVP solution, stir at 600 rpm for 8-10 minutes; then add 2 ml of 10 mM ascorbic acid solution and stir for 20-25 minutes; finally, add 0.1 ml of 10 mM AgNO3 solution dropwise and stir for 30-40 minutes to obtain Au@Ag NPs; add 10 ul of 1 mM DTNB to Au@Ag NPs and stir for 2 hours, centrifuge at 7500 rpm for 15 minutes, disperse in the same volume of deionized water to obtain Au@Ag@DTNB NPs solution.

[0066] Step 3: Preparation of Au@Ag@DTNB NPs modified capture probes:

[0067] Take 400 ul Au@Ag@DTNB NPs, add 20 ul 10 uM capture probe, and mix and incubate at room temperature for 2 hours; then add 8 ul 10 mM sodium citrate solution (PH=3), vortex briefly, and incubate at room temperature for 8 minutes; then add 12 ul 1M HEPES buffer (PH=7.6), centrifuge at 9000 rpm for 15 minutes, remove the supernatant, and then redisperse in 400 ul 10 mM phosphate buffer to obtain Au@Ag@DTNB NPs solution modified with capture probe (SERS probe);

[0068] The capture probe is a thiol-modified nucleic acid aptamer: 5'-GCA ATG GTA CGG TAC TTC CCC ATGAGT GTT GTG AAA TGT TGG GAC ACT AGG TGG CAT AGA GCC GCA AAA GTG CAC GCT ACTTTG CTA A-SH-3', SEQ ID No.1.

[0069] Step 4: Preparation of single-layer gold film (Au NS):

[0070] Take 10 ml of AuNPs solution in step 1, centrifuge at 8000 rpm for 15 minutes, disperse in 1 ml of PVP-ethanol solution and let stand overnight, then centrifuge at 9000 rpm for 15 minutes, and disperse in 1 ml of ethanol solution to obtain Au-PVP solution; take a 5 ml centrifuge tube, add 200 ul of Au-PVP solution, 800 ul of dichloromethane and 2 ml of ultrapure water, and shake rapidly for 30 seconds; then add 400 ul of n-hexane to obtain a single-layer gold film, and attach the obtained single-layer gold film to a silicon wafer.

[0071] Step 5: Preparation of Au NS modified rhodamine 6G and complementary probes:

[0072] The Au NS was immersed in 200 ul 10 -4 M rhodamine 6G and 200 ul 1 uM complementary probe to obtain Au NS modified with rhodamine 6G and complementary probe;

[0073] The complementary probe is a thiol-modified nucleic acid aptamer: 5'-SH-TTA GCA AAG TAG CGT GCA CTTTTG-3', SEQ ID No.2.

[0074] Step 6: Preparation of nucleic acid aptamer sensor:

[0075] The Au NS modified in step five was immersed in the Au@Ag@DTNB NPs solution modified with the capture probe described in step three, and after standing for 1 hour, it was taken out and dried at 60° C. for 10 minutes to obtain a nucleic acid aptamer sensor.

[0076] Wherein, the nucleic acid aptamer sensor comprises:

[0077] The capture probe is connected to the surface of silver-coated gold nanoparticles through SH-Au bonds, and the complementary probe hybridizes with the capture probe to form a double chain.

[0078] Example 2

[0079] Preparation of AgNO3 solution with a volume of 0.3 ml

[0080] In step 2, the amount of AgNO3 solution added is 0.3 ml, and the remaining operations are the same as in Example 1.

[0081] Example 3

[0082] Preparation of AgNO3 solution with a volume of 0.5 ml

[0083] In step 2, the amount of AgNO3 solution added is 0.5 ml, and the remaining operations are the same as in Example 1.

[0084] Example 4

[0085] Preparation of AgNO3 solution with a volume of 0.7 ml

[0086] In step 2, the amount of AgNO3 solution added is 0.7 ml, and the remaining operations are the same as in Example 1.

[0087] Example 5

[0088] Preparation of 0.01 mM DTNB solution

[0089] In step 2, the amount of AgNO3 solution added is 0.5 ml, the concentration of DTNB solution is 0.01 mM, and the remaining operations are the same as in Example 1.

[0090] Example 6

[0091] Preparation of 0.1 mM DTNB solution

[0092] In step 2, the amount of AgNO3 solution added is 0.5 ml, the concentration of DTNB solution is 0.1 mM, and the remaining operations are the same as in Example 1.

[0093] Example 7

[0094] Preparation of 1 mM DTNB solution

[0095] Same as Example 3.

[0096] Example 8

[0097] Preparation of 10 mM DTNB solution

[0098] In step 2, the amount of AgNO3 solution added is 0.5 ml, the concentration of DTNB solution is 10 mM, and the remaining operations are the same as in Example 1.

[0099] Experiment 1

[0100] High magnification scanning electron micrographs were taken using a scanning electron microscope (HITACHI, SU 8010 FE-SEM) at an accelerating voltage of 10.0 kV to analyze the size and morphology of the product samples.

[0101] The results are as follows Figure 2 As shown in the figure, it can be seen that with the increase of the volume of AgNO3 solution, the thickness of the silver shell gradually increases, among which the Au@Ag NPs prepared with a volume of 0.5 ml AgNO3 solution are evenly distributed and have uniform particle size.

[0102] Experiment 2

[0103] The UV / visible absorption spectra were obtained using a spectrophotometer (UVmini-1280) with a scanning range of 300–900 nm and a step size of 0.5 nm.

[0104] The results are as follows Figure 3 As shown in the figure, the absorption peak of AuNPs is at 530 nm. As the amount of silver nitrate added increases, the ultraviolet absorption peak blue-shifts, and as the thickness of the silver shell increases, the absorption peak gradually red-shifts.

[0105] Experiment 3

[0106] Raman spectroscopy was performed using a portable Raman spectrometer, with a laser operating at a wavelength of λ=785 nm as the excitation source and a laser power of 300 mW. All spectra were referenced to the Raman characteristic peak of the silicon wafer at 520 cm -1 Instrument calibration was performed and all Raman spectra were in the range of 550-1800 cm -1 The integration time within the range was 5 seconds and the acquisition was performed by scanning 3 times.

[0107] The surface enhanced Raman spectra of Au@Ag NPs prepared in each example are shown in FIG. Figure 4 As shown in the figure, for the construction of Au@Ag NPs core-shell structure, the thickness of the silver shell was optimized by adjusting the amount of silver nitrate added, thereby obtaining the best SERS signal enhancement effect.

[0108] The surface enhanced Raman spectra of Au@Ag@DTNB NPs prepared in each example are shown in Figure 2. Figure 5 As shown in Figure 2, by adding different concentrations of DTNB to modify Au@Ag NPs, the SERS signal can be enhanced. However, too high a concentration will cause Au@Ag NPs to aggregate and reduce their stability. Figure 5 It can be seen that the SERS signal is better when the DTNB concentration is 1 mM.

[0109] The surface enhanced Raman spectrum of Au NS modified with rhodamine 6G prepared in step 5 is shown in Figure 6 As shown, from Figure 6 It can be seen that the gold film has the effect of enhancing the SERS signal.

[0110] Experiment 4

[0111] Detection scheme of Escherichia coli at different concentration gradients based on SERS biosensor with optimal condition aptamer

[0112] The material used in this experiment is the nucleic acid aptamer sensor prepared by the preparation method of Example 3.

[0113] The aptamer sensor was immersed in water with a concentration of 0-10 8CFU / ml Escherichia coli ATCC 25922 solution, stand at 37°C for 2 hours, take out, dry at 60°C for 10 minutes and then take out for Raman testing. Test conditions: portable Raman spectrometer, laser wavelength of 785 nm, power of 300 mW, integration time of 5 s, scanning range of 550~1800 cm -1 .

[0114] The results are as follows Figure 7 As shown, from Figure 7 It can be seen from the figure that after the addition of E. coli, the capture probe Aptamer is separated from the complementary probe cDNA due to the stronger hybridization ability between the capture probe and E. coli, so that the SERS probe is detached from the Au NS and the SERS signal of the Raman tag DTNB is weakened. In addition, the Raman characteristic peak at 1329 cm -1 Quantitative analysis model of SERS signal intensity-concentration at: Y=9.24.64*Log(C)+9376.97, C is the concentration of Escherichia coli.

[0115] Experiment 5

[0116] The detection concentration of SERS biosensor based on the optimal condition aptamer is 10 8 Three interfering bacteria detection schemes for CFU / ml

[0117] The material used in this experiment is the nucleic acid aptamer sensor prepared by the preparation method of Example 3.

[0118] The nucleic acid aptamer sensors were immersed in 10 8 CFU / ml Salmonella SC-H1910-0063, Vibrio parahaemolyticus ATCC17802, Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 solutions and the mixed solutions of the above four pathogens were placed at 37°C for 2 hours and then taken out and dried at 60°C for 10 minutes before taking out for Raman testing. The test conditions were as follows: portable Raman spectrometer, laser wavelength of 785 nm, power of 300 mW, integration time of 5 s, scanning range of 550~1800 cm -1 .

[0119] The results are as follows Figure 8 As shown, from Figure 8 It can be seen that even in the presence of high concentrations of the three interfering strains, the assembled aptamer sensor has good selectivity for Escherichia coli, indicating that the assembled aptamer sensor has superior specific recognition ability and excellent anti-interference ability.

[0120] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0121] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a SERS biosensor based on nucleic acid aptamers, characterized in that: The steps include: (1) Preparation of gold nanoparticle solution: Gold nanoparticle solution was prepared using sodium citrate and chloroauric acid as raw materials; (2) Preparation of Au@Ag@DTNB NPs: PVP solution, ascorbic acid solution and AgNO3 solution were sequentially added to the gold nanoparticle solution to obtain Au@Ag NPs, and then DTNB solution was added to obtain Au@Ag@DTNB NPs; (3) Preparation of Au@Ag@DTNB NPs modified capture probes: The nucleotide sequence of the capture probe is shown in SEQ ID No. 1; 5'-GCA ATG GTA CGG TAC TTC CCC ATG AGT GTT GTG AAA TGT TGG GAC ACT AGGTGG CAT AGA GCC GCA AAA GTG CAC GCT ACT TTG CTA A-SH-3', SEQ ID No. 1; (4) Preparation of single-layer gold film: The gold nanoparticle solution of step (1) is dispersed in a PVP-ethanol solution and an ethanol solution in turn to obtain an Au-PVP solution; then dichloromethane and ultrapure water are added and rapidly shaken; then n-hexane is added to obtain a single-layer gold film; (5) Preparation of single-layer gold film modified with rhodamine 6G and complementary probe: The single-layer gold film is immersed in rhodamine 6G and complementary probe successively to obtain a single-layer gold film modified by rhodamine 6G and complementary probe; The nucleotide sequence of the complementary probe is shown in SEQ ID No. 2; 5'-SH-TTA GCA AAG TAG CGT GCA CTT TTG-3', SEQ ID No. 2; (6) Preparation of nucleotide aptamer biosensor: The product of step (5) is immersed in the product of step (3) to obtain a nucleotide aptamer biosensor.

2. The preparation method according to claim 1, characterized in that The specific operation of step (1) is: heating a 0.01% chloroauric acid solution to boiling, then adding a 1% sodium citrate solution, and continuing to heat for 15 minutes after the solution gradually turns wine red, and then cooling; The volume ratio of the chloroauric acid solution to the sodium citrate solution is 100:1.

4.

3. The preparation method according to claim 1, characterized in that: The specific operations of step (2) are: (21) centrifuging the gold nanoparticle solution obtained in step (1) and dispersing it in deionized water to obtain solution A, wherein the volume ratio of the gold nanoparticle solution to deionized water is 5:1; (22) Solution A was mixed with a 1% PVP solution and stirred, then a 10 mM ascorbic acid solution was added and stirred, and finally a 10 mM AgNO3 solution was added dropwise and stirred for 30–40 min to obtain Au@Ag NPs. Among them, the volume ratio of solution A, PVP solution, ascorbic acid solution, and AgNO3 solution is 2:1:2:0.5; (23) 1 mM DTNB solution was added to Au@Ag NPs and stirred. After centrifugation, the solution was dispersed in deionized water to obtain Au@Ag@DTNB NPs.

4. The preparation method according to claim 1, characterized in that: The specific operations of step (3) are: 10 uM capture probe was added to Au@Ag@DTNB NPs, and mixed and cultured at room temperature for 2 hours, and then incubated in sodium citrate solution, washed with HEPES buffer, and dispersed in phosphate buffer to obtain Au@Ag@DTNB NPs solution modified with capture probe; Among them, the volume ratio of Au@Ag@DTNB NPs to capture probes was 20:

1.

5. The preparation method according to claim 1, characterized in that: The specific operations of step (4) are: (41) The gold nanoparticle solution of step (1) is centrifuged and dispersed in a PVP-ethanol solution and allowed to stand overnight, and then centrifuged again and dispersed in an ethanol solution to obtain an Au-PVP solution; The volume ratio of the gold nanoparticle solution, the PVP-ethanol solution, and the ethanol solution is 10:1:1; (42) The Au-PVP solution, dichloromethane, and ultrapure water were mixed and rapidly shaken, and then hexane was added to obtain a single-layer gold film.

6. The preparation method according to claim 1, characterized in that: In step (5), the concentration of rhodamine 6G is 10 -4 M, the concentration of complementary probe was 1 uM.

7. The preparation method according to claim 1, characterized in that: In the step (6), the sample is taken out after immersion for 1 hour and baked at 60° C. for 10 minutes.

8. Use of the nucleotide aptamer biosensor prepared by the preparation method according to any one of claims 1 to 7 in detecting Escherichia coli.

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