CN113899731B - One-step detection method for vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters - Google Patents
One-step detection method for vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters Download PDFInfo
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Abstract
The invention discloses a one-step detection method of vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters, which comprises the following steps: (1) synthesizing gold nanoclusters; (2) combining the aptamer with the gold nanocluster through hydrogen bond and electrostatic interaction to obtain a compound of the aptamer and the gold nanocluster; (3) placing the complex of the aptamer and the gold nanocluster in H2O2In the TMB color development system; (4) mixing and incubating a sample to be detected, the aptamer and the gold nanocluster in sequence and adding H2O2In the TMB color development system, H added with the sample solution to be detected and H not added with the sample solution to be detected are measured2O2Establishing a standard curve by the absorbance difference between TMB color development systems, and quantitatively determining the content of the vibrio parahaemolyticus in the sample solution. The invention utilizes H based on the difference of affinity of aptamer to target bacteria and gold nanoclusters2O2The TMB color development system can detect the vibrio parahemolyticus in one step and meet the requirement of quickly and efficiently detecting the food-borne pathogenic bacteria.
Description
Technical Field
The invention relates to the technical field of pathogenic bacteria detection, in particular to a one-step detection method of vibrio parahaemolyticus based on affinity difference of a nucleic acid aptamer to a target bacterium and a gold nanocluster.
Background
Vibrio parahaemolyticus (vibrio parahaemolyticus, v. parahaemolyticus) is a marine bacterium, mainly derived from marine products such as fish, shrimp, crab, shellfish and seaweed, and is one of the common food-borne pathogenic bacteria. In recent years, the number of food safety events caused by vibrio parahaemolyticus contamination is increasing, and therefore, the monitoring and control work for vibrio parahaemolyticus needs to be strengthened.
The traditional plate colony counting method and the physiological and biochemical identification method are accurate and universal technologies for detecting pathogenic bacteria at present, but have the biggest weaknesses of time and labor consumption and obvious hysteresis, and cannot meet the requirement of quickly monitoring and early warning food-borne pathogenic bacteria pollution. With the continuous progress of nanotechnology, immunology technology and molecular biology technology, researchers at home and abroad develop novel detection technologies such as electrochemical luminescence immunosensor (ECL), surface enhanced Raman scattering immunosensor (SERS), multiplex real-time PCR (polymerase chain reaction) and loop-mediated isothermal amplification (LAMP), and the like, so that the rapid detection of pathogenic microorganisms is realized. The new detection technologies have the advantages of high sensitivity, strong specificity, good reliability and the like, but the detection process needs to execute complex operation and the adopted equipment is expensive, so that the requirements of daily field detection are difficult to meet, and the practical application feasibility is low. The colorimetric biosensing technique determines the content of a component to be measured by comparing or measuring the color depth of a colored substance based on a color reaction that generates a colored compound visible to the naked eye. Compared with the above technology, the colorimetric biosensing technology has the advantages of simple and convenient operation, low cost, short reaction time and visual result, can meet the requirement of on-site rapid detection, and has better development prospect in practical application. The current colorimetric sensing technology is typically focused on two aspects: on one hand, the target substance recognition is responded based on controlling the dispersity and aggregability of the gold nanoparticles, for example, the current document discloses that the visible detection of salmonella and escherichia coli is realized by utilizing the color change of a solution caused by the agglomeration of the gold nanoparticles in a high-salt environment; on the other hand, the detection method is based on the enzymatic color reaction of peroxidase to respond to the recognition of target substances, for example, the existing documents disclose that the high-sensitivity detection of salmonella typhimurium and tetracycline antibiotics is successfully realized by using the color development of horseradish peroxidase. However, the diversity of food materials determines that the food contains various interference components, which easily affect the dispersion and aggregation state of the gold nanoparticles or induce the denaturation and inactivation of the biological enzyme, thereby affecting the accuracy and sensitivity of the detection result. Gold nanoclusters are molecular-like cluster structures consisting of several to tens of gold atoms. Due to the quantum size effect, the gold nanoclusters lose the local surface plasmon resonance characteristic of the traditional gold nanoparticles and have oxidase and peroxidase catalytic activities. Compared with biological enzymes, the artificial nano enzyme has more stable properties and can be used as a substitute of the biological enzymes. Meanwhile, the catalytic activity of the gold nanoclusters is easy to adjust so as to meet the color development requirement of actual detection. Because the nanoenzyme reaction occurs on the surface of the nanomaterial, the surface modification is an effective method for regulating the enzyme activity, wherein the catalytic capability of the nanoenzyme is changed by the thickness of the surface coating, the type of the coating functional group, the surface charge and the like.
The pathogenic bacteria are recognized based on the immune reaction between antigen and antibody, and although the sensitivity meets the requirement of practical application, the pathogenic bacteria have the problem of poor selectivity, and most of the antibodies need specific conditions for storage and treatment to prevent the antibodies from being denatured. The aptamer (aptamer) is a single-stranded DNA or RNA with the length of about 25-90bp, can be combined with a target object with high affinity and high selectivity, and can be used as a suitable substitute of an antibody. Compared with an antibody, the aptamer has the advantages of simple synthesis, low price, high stability, low denaturation sensitivity, easy modification of various functional groups and the like, so the problem of specific recognition of the biological recognition element on the food-borne pathogenic bacteria in practical research and application can be effectively solved by using the aptamer.
At present, the colorimetric sensing technology based on aptamer or antibody is usually a two-step method, and two target bacteria recognition elements need to be designed, wherein one aptamer or antibody recognizes and enriches the target bacteria, and the other aptamer or antibody connected with the colorimetric element outputs a visible signal, for example, Chinese patent publication No. CN110470840A discloses a method for detecting Vibrio parahaemolyticus by using antibody fixed on nitrocellulose membrane and Fe3O4@ Au @ Pt-antibody-target bacterium complex binding formationSandwich structure, showing Fe3O4Color of @ Au @ Pt nanoparticles. Chinese patent publication No. CN108169483A discloses a method for detecting Vibrio parahaemolyticus based on aptamer-molecular motor biosensing, and establishes a system of F0F1-ATPase molecular motor-epsilon subunit antibody-biotin-avidin-5' -end biotinylation of Vibrio parahaemolyticus aptamer. At present, target bacteria can be identified and colorimetric elements can be activated at the same time through only one aptamer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a vibrio parahaemolyticus one-step detection method based on the affinity difference of a nucleic acid aptamer to a target bacterium and a gold nanocluster, and the invention utilizes H to regulate the activity of gold nanocluster peroxidase based on the affinity difference of the nucleic acid aptamer to the target bacterium and the gold nanocluster and the advantage of the nucleic acid aptamer in regulating and controlling the activity of the gold nanocluster peroxidase2O2The TMB color development system develops a one-step colorimetric detection method for vibrio parahaemolyticus, simplifies the detection steps and meets the requirement of rapid and efficient detection of food-borne pathogenic bacteria.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a one-step detection method of vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters comprises the following steps:
(1) synthesizing gold nanoclusters;
(2) combining the aptamer with the gold nanocluster through hydrogen bonds and electrostatic interaction to obtain a compound of the aptamer and the gold nanocluster;
(3) placing the complex of the aptamer and the gold nanocluster in H2O2In the TMB color development system;
(4) mixing and incubating a sample to be detected, the aptamer and the gold nanocluster in sequence and adding H2O2In the TMB color development system, H added into a sample solution to be detected is measured2O2TMB color development system and H without adding to-be-detected sample solution2O2The absorbance difference between the/TMB color development systems, establishing a standard curve and determiningThe content of Vibrio parahaemolyticus in the sample solution was quantitatively determined.
As a further improvement of the above aspect, the step (1) includes: placing a 20mL sample bottle on a magnetic stirrer, wherein the rotating speed of the magnetic stirrer is set to be 300-400 rpm/min; HAuC l freshly prepared from 0.5mL of ultrapure water4Adding the solution and 4.7mL of 60% ethanol into a sample bottle, fully and uniformly mixing, adding 4mL of glutathione solution and 0.6mL of NaOH solution, uniformly mixing again, and finally adding 0.2mL of NaBH4The solution is used as a reducing agent and is stirred for 6-7 hours; and after stirring, injecting all the solution in the sample bottle into a dialysis bag, placing the dialysis bag in distilled water for dialysis for 24-28 days, pouring out the solution to obtain the gold nanocluster taking the glutathione as the ligand, and finally storing the gold nanocluster at 1-4 ℃ for later use.
As a further improvement of the above, the HAuCl4The concentration of the solution is 20 mM; the concentration of the glutathione solution is 5 mM; the concentration of the NaOH solution is 0.2M; the cut-off molecular weight of the dialysis bag is 3000 Da.
As a further improvement of the above scheme, the step (2) comprises: and (2) fully and uniformly mixing the newly prepared aptamer solution and the gold nanoclusters synthesized in the step (1), then incubating for 10-60 min at the temperature of 25-45 ℃ to form a compound of the aptamer and the gold nanoclusters, and finally refrigerating at the temperature of 1-4 ℃ for later use.
As a further improvement of the scheme, in the step (2), the concentration of the aptamer is 0.5-15 μ M, and the aptamer solution and the gold nanoclusters are fully and uniformly mixed and then incubated at 35 ℃ for 30 min; in the step (3), H2O2In the color system of/TMB, H2O2The concentration of the (D) is 50-300 mM, the concentration of the TMB is 5-30 mM, the pH value of the system is 3.8-5.0, the reaction temperature is 25-45 ℃, and the reaction time is 10-60 min.
As a further improvement of the above scheme, in the step (4), the concentration of the aptamer is 5 μ M, the concentration of the gold nanocluster is 4.8 μ M, and the sample to be tested, the aptamer and the gold nanocluster are mixed in sequence and then incubated at 35 ℃ for 30 min; h2O2In the color system of/TMB, H2O2Was 100mM, TMB was 20mM, the pH of the system was 4.2, the reaction temperature was 35 ℃ and the reaction time was 2 min.
As a further improvement of the scheme, in the step (4), the absorbance of the solution of the sample to be detected at 652nm is recorded by a microplate reader, and the H not added with the solution of the sample to be detected is compared2O2Calculating the absorbance value of the TMB color development system at 652nm to obtain the absorbance value652nmDrawing a standard curve, and quantitatively determining the content of the vibrio parahaemolyticus in the sample solution.
The inventor finds that the single-stranded DNA also has high affinity with the gold nanoclusters, and meanwhile, the surface property of the gold nanoclusters can be changed by the assembly of the DNA on the gold nanoclusters, so that the catalytic capability of the gold nanoclusters is changed. Therefore, the invention develops the one-pot method for colorimetric detection of vibrio parahaemolyticus based on the difference of the affinity of the aptamer to the target bacteria and the gold nanocluster artificial enzyme and the activity regulation and control of the aptamer to the gold nanocluster artificial enzyme, thereby simplifying the detection steps and meeting the requirement of rapid on-site detection of food-borne pathogenic bacteria. Meanwhile, adverse effects caused by food material effects are reduced under the condition that specific recognition target objects are not influenced, the on-site detection requirements of the vibrio parahaemolyticus are better met, and the vibrio parahaemolyticus has a better application prospect.
The invention synthesizes gold nanoclusters (GSH @ AuNCs) with the particle size of about 2nm by taking Glutathione (GSH) as a ligand through a reduction method. The aptamer (Apt) is combined with the GSH @ AuNCs through hydrogen bonds and electrostatic interaction to obtain an Apt @ GSH @ AuNCs compound. Compared with gold nanoclusters (GSH @ AuNCs), the peroxidase activity of the Apt @ GSH @ AuNCs compound is greatly improved, and H can be catalyzed more quickly2O2The reaction of TMB color development. Due to the difference of the affinity of the aptamer to the target bacteria and the affinity of the gold nanoclusters, when the target bacteria vibrio parahaemolyticus (V.parahaemolyticus) exists, the stronger interaction of the aptamer and the target bacteria leads to the tendency of forming Apt @ V.parahaemolyticus complexes, the conversion towards the Apt @ GSH @ AuNCs complexes is reduced, the influence on the GSH @ AuNCs is reduced, and the H is weakened2O2Degree of TMB color reaction. Therefore, for Apt and GSH @ AuNCs with fixed concentrations, different concentrations of side solutions are introducedVibrio haemolyticus (V.parahaemolyticus) reduces the amount of Apt bound to GSH @ AuNCs accordingly, and H is catalyzed by measuring the introduced target bacteria system (Apt @ V.parahaemolyticus-GSH @ AuNCs) and the non-introduced target bacteria system (Apt @ GSH @ AuNCs)2O2The absorbance difference of TMB color development can establish a standard curve to quantitatively determine the content of V. For non-target bacteria except V.parahaemolyticus, because the non-target bacteria are not combined with Apt, Apt tends to be completely combined with GSH @ AuNCs to obtain high catalytic activity, so the method can realize specific detection.
The second aspect of the invention provides application of the compound of the aptamer and the gold nanocluster in preparation of a vibrio parahaemolyticus detection kit.
As a further improvement of the scheme, the color development system of the vibrio parahaemolyticus detection kit is H2O2a/TMB color system.
The third aspect of the invention provides a vibrio parahaemolyticus detection kit, which comprises a compound of a nucleic acid aptamer and a gold nanocluster, and a color development system of the detection kit is H2O2a/TMB color system.
Compared with the prior art, the invention has the beneficial effects that:
1. based on the difference of the affinity of the aptamer to the target bacteria and the gold nanoclusters and the advantage of the aptamer to the activity regulation of the gold nanocluster peroxidase, H is utilized2O2Compared with the two-step detection method of the common double-target recognition element, the detection method is simpler, has shorter detection time, can ensure high detection sensitivity, reduces adverse effects caused by food matrix effect, pays attention to the popularity and applicability of the new method, and combines and applies advanced analysis technology to better meet the field detection requirement of the vibrio parahemolyticus;
2. the aptamer is modified on the surface of the gold nanocluster (GSH @ AuNCs) through non-covalent bond forces such as hydrogen bonds and electrostatic interaction, and the like to form the aptamer and the gold nanoclusterCompared with gold nanoclusters (GSH @ AuNCs), the peroxidase activity of the Apt @ GSH @ AuNCs complex is obviously enhanced, and H can be catalyzed more rapidly2O2the/TMB system develops color.
Drawings
FIG. 1A is a graph showing statistics of zeta potential values of Apt, GSH @ AuNCs and Apt @ GSH @ AuNCs in example 2 of the present invention;
FIG. 1B is a TEM image of Apt @ GSH @ AuNCs in example 2 of the present invention;
FIG. 2A, D shows TMB and H in example 3 of the present invention, respectively2O2The Michaelis-Menten equation of (A) corresponds to a result graph;
FIG. 2B, C, E, F is a diagram of the reciprocal double Lineweaver-Burk of the Michaelis-Menten equation in example 3 of the present invention;
FIGS. 3A to F are graphs showing aptamer concentration, incubation time, reaction temperature, and H, respectively, in example 4 of the present invention2O2Concentration, TMB concentration and reaction System pH vs Apt @ GSH @ AuNCs catalytic H2O2Experimental result statistical chart of TMB color development influence;
FIG. 4A is a graph showing the Δ A measured at different concentrations of V.parahaemolyticus in example 5 of the present invention652nmA statistical chart;
FIG. 4B shows Δ A in example 5 of the present invention652nmA standard curve plotted against v.parahaemolyticus concentration;
FIG. 5A is a graph showing the results of detection of different target bacteria in example 5 of the present invention;
FIG. 5B is the GSH @ AuNCs catalytic H of different strains in the absence of Apt recognition target bacteria in example 5 of the present invention2O2Statistical chart of the effect of color reaction of/TMB system.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
In the following, unless otherwise specified, the unit M represents mol/L, mM represents mmol/L, nM represents nmol/L, and. mu.M represents. mu. mol/L.
Example 1:
synthesis of gold nanoclusters (GSH @ AuNCs)
Placing a 20mL sample bottle on a magnetic stirrer, and setting the rotating speed to be 300 rpm/min; then 0.5mL of HAuCl freshly prepared with ultrapure water was taken4Adding the solution (with concentration of 20mM) and 4.7mL of 60% ethanol into a sample bottle, mixing, adding 4mL of Glutathione (GSH) solution (with concentration of 5mM) and 0.6mL of NaOH solution (with concentration of 0.2M), mixing, and adding 0.2mL of NaBH4The solution was stirred for 6h as a reducing agent. After stirring, injecting all the solution in the sample bottle into a dialysis bag with the molecular weight cutoff of 3000Da, placing the dialysis bag in distilled water for dialysis for 24d, pouring out the solution to obtain gold nanoclusters (GSH @ AuNCs) with GSH as a ligand, and finally storing the gold nanoclusters at 4 ℃ for later use.
Example 2:
aptamer (Apt) modified gold nanocluster forming complex (Apt @ GSH @ AuNCs)
Fully and uniformly mixing a freshly prepared aptamer (Apt) solution with the gold nanocluster (GSH @ AuNCs) solution prepared in the example 1, incubating at 35 ℃ for 30min to form a compound (Apt @ GSH @ AuNCs compound) of the aptamer and the gold nanocluster, and storing the compound in a refrigerator (the ambient temperature is 4 ℃) for later use. FIG. 1A shows zeta potentials of Apt, GSH @ AuNCs and Apt @ GSH @ AuNCs, and as can be seen from FIG. 1A, the potential values of Apt @ GSH @ AuNCs are lower than those of Apt and GSH @ AuNCs, indicating that Apt forms a complex with GSH @ AuNCs. The morphology and dimensions of the Apt @ GSH @ AuNCs complex were observed by TEM and the size of the Apt @ GSH @ AuNCs complex was about 2nm as shown in FIG. 1B.
Example 3:
GSH @ AuNCs and Apt @ GSH @ AuNCs catalytic H2O2Steady-state kinetic study of/TMB color development System
Recording H in scanning dynamics mode using UV spectrophotometer2O2The change of the absorbance value at 652nm of the TMB system researches the steady-state dynamics of the GSH @ AuNCs and Apt @ GSH @ AuNCs peroxidase. Test different concentrations of H were tested by fixing the TMB concentration2O2Reaction rate of (2), immobilization of H2O2Concentrations to test the reaction rates of various concentrations of TMB,on the basis, the michaelis constant K of the substrate is obtained by calculating by using a kinetic calculation formula (formula 1)mAnd a maximum reaction rate Vmax. Wherein formula 1: v is the initial velocity, VmaxFor maximum reaction rate, [ S ]]As substrate concentration, KmThe value is the Michaelis constant.
FIG. 2 shows the steady state kinetic results for GSH @ AuNCs and Apt @ GSH @ AuNCs. Wherein A, D represents TMB and H, respectively2O2The Michaelis-Menten equation of (a); B. c, E, F is a double reciprocal Lineweaver-Burk plot of the Michaelis-Menten equation. From the results of FIG. 2, Apt @ GSH @ AuNCs was found to be at H2O2The reaction rate and the reaction degree in the/TMB system are obviously higher than GSH @ AuNCs.
Example 4:
apt @ GSH @ AuNCs catalytic H2O2Optimization of conditions of TMB color development system
In order to obtain high sensitivity in the biosensing process without influencing the specificity of the reaction, Apt @ GSH @ AuNC is catalyzed by2O2Optimization of relevant parameters of the TMB color development system, wherein the parameters comprise aptamer concentration, reaction temperature, incubation time, H2O2Concentration, TMB concentration and pH of the reaction system. The aptamer concentrations (500nM, 800nM, 1. mu.M, 5. mu.M, 10. mu.M, 15. mu.M), the temperature (25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃), the incubation times (10min, 20min, 30min, 40min, 50min, 60min), H, were examined separately2O2Effect of concentration (50mM, 100mM, 150mM, 200mM, 250mM, 300mM), TMB concentration (5mM, 10mM, 15mM, 20mM, 25mM, 30mM) and pH (3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0) on the reaction. The change in absorbance at 652nm was recorded in the scanning kinetics mode of the UV spectrophotometer and the effect on Apt @ GSH @ AuNCs peroxidase activity was compared to determine the optimal reaction parameter concentration.
FIGS. 3A-F show aptamer concentration, incubation time, reaction temperature, H, respectively2O2Concentration, TMB concentration and reaction System pH vs Apt @ GSH @ AuNCs catalytic H2O2Effect of TMB color development. As can be seen from FIGS. 3A to F, in the case where the incubation time was 30min and the reaction temperature was 35 ℃, 5. mu.MApt, 20mM TMB and 100mMH were selected2O2And the optimal detection condition parameter of the novel colorimetric biosensor is obtained by a 0.2MNaAc buffer system and the pH value is 4.2.
Example 5:
novel colorimetric biosensor for evaluating detection performance and verifying specificity of vibrio parahaemolyticus
Resuscitating original standard strain of Vibrio parahaemolyticus (ATCC17802), centrifuging 1mL of bacterial suspension when bacteria grow to logarithmic phase, discarding supernatant, collecting thallus, washing with sterile water for 2 times, and resuspending in 1mL of 1 Xbinding buffer solution (including 50mM Tris-HCl, 5mM KCl, 100mM NaCl, 1mM MgCl)2) And adjusting the concentration range of the bacterial suspension to be 102-108CFU/mL, for subsequent experiments. 50 μ L of parahemolytic vibrio suspension (concentration 10)2-108CFU/mL) was mixed with 50. mu.L of freshly prepared Apt solution (concentration 5. mu.M) and 25. mu.L of LGSH @ AuNCs solution (concentration 4.8. mu.M) in this order, incubated at 35 ℃ for 30min, and then 100. mu.L of NaAC buffer (concentration 0.2M) and 20. mu.L of LH were added2O2Mixing the solution (concentration 100mM) and 25 μ LTMB solution (concentration 10mM), developing at 35 deg.C for 5min, recording solution absorbance at 652nm with microplate reader, comparing absorbance of reaction system without adding bacterial suspension, and calculating Δ A652nmAnd drawing a standard curve. Based on the same concentration (10) under the same experimental conditions8CFU/mL) for several other pathogens including E.coli O157H7, staphylococcus aureus, salmonella typhimurium, and listeria monocytogenes) to evaluate the selectivity specificity of the novel colorimetric biosensor for the detection of vibrio parahaemolyticus v.
As shown in FIG. 4A, as the concentration of V.parahaemolyticus increases, the absorbance at 652nm gradually decreases, and the color of the solution gradually transitions from deep blue to light blue, thus Δ A652nmAnd gradually increases. Such asFIG. 4B shows that FIG. 4B is Δ A652nmThe detection limit of the novel colorimetric biosensor on V.parahaemolyticus is 100CFU/mL, as can be seen from a standard curve drawn relative to the concentration of V.parahaemolyticus.
As shown in FIG. 5A, the present invention was used to treat several other common pathogenic bacteria (Escherichia coli O)157H7, staphylococcus aureus, salmonella typhimurium and listeria monocytogenes) were not reactive, and the absorbance was similar to that of the control group without the addition of the bacterial suspension, indicating good specificity and selectivity for the detection of v. As shown in FIG. 5B, five strains catalyzed H by GSH @ AuNCs without introduction of Apt-recognizing target bacteria2O2The influence of the color reaction of the/TMB system is similar, which shows that the specific detection of V.parahaemolyticus can be realized only by introducing Apt.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and substitutions can be made without departing from the technical principle of the present invention, and these modifications and substitutions should also be regarded as the protection scope of the present invention.
Claims (7)
1. A one-step detection method of vibrio parahaemolyticus based on affinity difference of aptamer to target bacteria and gold nanoclusters is characterized by comprising the following steps:
(1) synthesizing gold nanoclusters;
(2) combining the aptamer with the gold nanocluster through hydrogen bond and electrostatic interaction to obtain a compound of the aptamer and the gold nanocluster;
(3) placing the complex of the aptamer and the gold nanocluster in H2O2In the TMB color development system;
(4) mixing and incubating a sample to be detected, the aptamer and the gold nanocluster in sequence and adding H2O2TMB color system by measuring H added to sample solution to be measured2O2TMB color development system and H without adding sample solution to be detected2O2The absorbance difference between TMB color development systems, a standard curve is established,quantitatively determining the content of the vibrio parahaemolyticus in the sample solution.
2. The method for detecting vibrio parahaemolyticus according to claim 1, wherein the step (1) comprises: placing a 20mL sample bottle on a magnetic stirrer, wherein the rotating speed of the magnetic stirrer is set to be 300-400 rpm/min; HAuCl freshly prepared from 0.5mL of ultrapure water4Adding the solution and 4.7mL of 60% ethanol into a sample bottle, fully and uniformly mixing, adding 4mL of glutathione solution and 0.6mL of NaOH solution, uniformly mixing again, and finally adding 0.2mL of NaBH4The solution is used as a reducing agent and is stirred for 6-7 hours; and after stirring, injecting all the solution in the sample bottle into a dialysis bag, placing the dialysis bag in distilled water for dialysis for 24-28 days, pouring out the solution to obtain the gold nanocluster taking the glutathione as the ligand, and finally storing the gold nanocluster at 1-4 ℃ for later use.
3. The method for detecting Vibrio parahaemolyticus according to claim 2, wherein the HAuCl is a single step detection method based on the difference in affinity of aptamers for target bacteria and gold nanoclusters4The concentration of the solution is 20 mM; the concentration of the glutathione solution is 5 mM; the concentration of the NaOH solution is 0.2M; the cut-off molecular weight of the dialysis bag is 3000 Da.
4. The method for detecting vibrio parahaemolyticus according to claim 1, wherein the step (2) comprises: and (2) fully and uniformly mixing the newly prepared aptamer solution and the gold nanoclusters synthesized in the step (1), then incubating for 10-60 min at the temperature of 25-45 ℃ to form a compound of the aptamer and the gold nanoclusters, and finally refrigerating at the temperature of 1-4 ℃ for later use.
5. The method for detecting Vibrio parahaemolyticus according to claim 1, wherein in step (2), the aptamer is used for detecting the affinity difference between the target bacteria and the gold nanoclustersThe concentration of the aptamer is 0.5-15 mu M, and the aptamer solution and the gold nanocluster are fully and uniformly mixed and then incubated for 30min at the temperature of 35 ℃; in the step (3), H2O2In the color system of/TMB, H2O2The concentration of the (D) is 50-300 mM, the concentration of the TMB is 5-30 mM, the pH value of the system is 3.8-5.0, the reaction temperature is 25-45 ℃, and the reaction time is 10-60 min.
6. The method for detecting vibrio parahaemolyticus according to claim 1, wherein in step (4), the concentration of the aptamer is 5 μ M, the concentration of the gold nanocluster is 4.8 μ M, and the sample to be detected, the aptamer and the gold nanocluster are mixed in sequence and then incubated at 35 ℃ for 30 min; h2O2In the color system of/TMB, H2O2Was 100mM, TMB was 20mM, the pH of the system was 4.2, the reaction temperature was 35 ℃ and the reaction time was 2 min.
7. The method for detecting vibrio parahaemolyticus based on affinity difference between aptamer and target bacteria and gold nanocluster in claim 1, wherein in step (4), the absorbance of the sample solution to be detected at 652nm is recorded by an enzyme-linked immunosorbent assay, and the absorbance is compared with the absorbance of H which is not added with the sample solution to be detected2O2Calculating the absorbance value of the TMB color development system at 652nm to obtain the absorbance value652nmDrawing a standard curve, and quantitatively determining the content of the vibrio parahaemolyticus in the sample solution.
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