Technology for rapidly determining kanamycin based on three-dimensional DNA walker

Abstract

The invention provides construction of a rapid detection technology based on three-dimensional DNA walker operation and polymerization cutting reaction and application of the rapid detection technology in detection of kanamycin content in animal-derived food, and belongs to the technical field of sensing detection. The method comprises the following steps: preparing gold nanoparticles (AuNPs) by adopting a standard sodium citrate reduction method, then modifying thiolated orbital hairpin DNA and closed walking DNA to the surface of the AuNPs by utilizing the strong Au-S chemical bond action of sulfydryl and the gold surface so as to prepare a three-dimensional DNA walker, and further applying the rapid detection technology to the detection of the kanamycin content in animal-derived food. On one hand, the steric effect of a three-dimensional space structure of AuNPs is utilized, the anti-interference capability of the nucleic acid probe is enhanced, the probability that a nucleic acid chain on the surface is degraded by nuclease is effectively reduced, and the accuracy and credibility of the probe in practical application are improved; and on the other hand, the inherent signal amplification capability of the three-dimensional DNA walker is utilized, and the double amplification of the polymerization cutting reaction is combined, so that the detection speed and sensitivity are improved, and the rapid and sensitive detection of kanamycin in animal-derived food is realized.

Description

Technical Field

[0001] The invention belongs to the field of sensor detection technology, and relates to the development of a visualization rapid detection technology, and specifically to the development of a technology for rapidly determining kanamycin based on a three-dimensional DNA walker.

[0002] Kanamycin is an aminoglycoside antibiotic extracted from Streptomyces kanamycinii. This antibiotic has a broad spectrum of antibacterial properties and can be used to treat a series of infections caused by Gram-negative and Gram-positive bacteria. It is often used in animal husbandry as a veterinary drug and animal growth promoter. However, the irrational and inappropriate use of kanamycin leads to intestinal flora imbalance, metabolic disorders, and reduced immunity in livestock and poultry, which in turn leads to excessive residues in animal-derived foods (eggs, dairy products, and meat). These residual kanamycin enters the human circulatory system through the food chain and accumulates, causing ototoxicity, nephrotoxicity, allergic reactions, drug resistance, and even cancer, which seriously affects human health, and the public health and ecological environment problems caused by this are becoming increasingly serious. At present, the EU and my country's national food safety standards (GB 31650-2019) have imposed restrictions on the maximum residue of kanamycin in milk, for example, 150 μg / kg in the EU and 200 μg / kg in China. Therefore, it is imperative to develop effective regulatory technologies for kanamycin in animal-derived foods and promote the healthy development of this type of food industry.

[0003] At present, a variety of methods including capillary electrophoresis, high performance liquid chromatography, gas chromatography-mass spectrometry, microbial inhibition, immunoassay and molecular imprinting have been developed for the detection of kanamycin. Although these methods have high detection sensitivity and accuracy and can be used to analyze trace components in complex samples, they are difficult to detect on site due to the need to rely on large instruments, professionals, complex operations, time-consuming and labor-intensive, strict sample requirements, long single sample analysis time, and inconvenient to carry. Fluorescence methods are used for the detection of various antibiotics due to their advantages such as rapidity, simplicity, easy automation, high sensitivity and fast analysis speed. For example, researchers used nickase and magnetic nanoparticles to assist DNA recovery to detect kanamycin, or used exonuclease I to assist signal amplification for fluorescence analysis of kanamycin. However, this type of method disperses the DNA probe in the test solution, and the probe is easily degraded by nucleases and becomes ineffective. In addition, self-leakage or non-specific hybridization reactions between multiple probes are prone to produce false positive signals, resulting in poor stability and reliability of the test results. Therefore, it is urgent to develop a method for detecting kanamycin that is rapid, accurate, sensitive, practical, easy to operate, and highly stable.

[0004] Inspired by natural nanomachines, researchers have designed DNA nanomachines that can simulate machine functions based on the precise base complementary pairing principle, sequence programmability and structural controllability of DNA. They are activated by interactions with specific molecules or environmental changes, thereby achieving controllable transformations between different structures and conformations of DNA and performing machine-like movements. At present, researchers have designed and prepared various types of DNA nanomachines, including DNA motors, DNA tweezers, DNA gears, DNA walkers, etc. Among them, DNA walkers are composed of walking components, tracks and fuel molecules or other forms of energy supply components, and can drive specific oligonucleotide walking components to perform step displacement along a pre-set track. If a signal molecule is generated in each step, the DNA walker can accumulate a large number of signal copies during repeated steps. This feature gives it inherent signal amplification capabilities and potential for application in the field of biomolecule detection. Among them, three-dimensional DNA walkers have better amplification performance due to their spacious walking space, good DNA enrichment ability and resistance to nonspecific interactions caused by the use of nanoparticles with large specific surface areas as substrates.

[0005] In view of the shortcomings of the prior art, the present invention constructs a three-dimensional DNA walker using AuNP as a carrier and DNA as a core material, and develops a simple, sensitive, rapid and highly stable antibiotic rapid detection device in combination with the polymerization nicking reaction, thereby realizing the rapid detection of kanamycin in milk. Summary of the invention

[0006] The present invention has developed a technology for rapid determination of kanamycin based on a three-dimensional DNA walker. On the one hand, a three-dimensional DNA walker sensing rapid detection platform is constructed with gold nanoparticles (AuNP) as a carrier and DNA as a core material, and the steric hindrance effect of AuNP is used to reduce the probability of nucleic acid chains being enzymatically hydrolyzed, thereby improving the accuracy and credibility of the probe in practical applications; on the other hand, the DNA walker and the polymerization nicking reaction are double-amplified to improve the speed and sensitivity of detection, thereby realizing rapid and sensitive detection of kanamycin in milk.

[0007] The technical solution of the present invention is a technology for rapid determination of kanamycin based on a three-dimensional DNA walker, characterized in that: first, AuNP is synthesized by sodium citrate reduction method; then, the Au-S bond formed by covalent coupling of the gold atoms on its surface with the thiol groups is used to connect the thiol-functionalized nucleic acid chain to the particle surface to construct a three-dimensional DNA walker; finally, with the assistance of polymerase and nicking enzyme, the operation of the DNA walker and the detection of kanamycin in milk are realized. The specific scheme is as follows: 1) AuNPs were prepared by the standard sodium citrate reduction method. First, HAuCl4•3H2O solution (1.25 mL, 10 mM) was quickly injected into 45 mL of boiling ultrapure water under vigorous stirring. After 2 min, the pre-prepared 1% sodium citrate solution was quickly added. After continuing to stir vigorously for 10 min under boiling conditions, the heating device was removed, and then the mixture was naturally cooled to room temperature under gentle stirring. Finally, the prepared AuNPs colloid was filtered using a 0.22 μm microporous membrane filter and stored in a 4 °C refrigerator for later use; 2) Utilizing the strong Au-S chemical bond between thiol groups and the gold surface, thiolated track hairpin DNA and blocked walking DNA were modified onto the surface of AuNPs to prepare a three-dimensional DNA walker. Briefly, the thiol-modified Walking strand and Blocking strand were first mixed in a 1× Tris-HCl buffer (140 mM NaCl, 20 mMTris, 5.0 mM KCl, pH 7.4) at a certain molar ratio. An excess of Blocking strand was used to ensure that the Walking strand was completely locked. The above mixed solution was heated at 95 °C for 10 min and then slowly cooled to 20 °C. Then, TCEP•HCl solution was added to the Walking strand and the hairpin Track strand, respectively, and the thiol groups were activated at room temperature for 2 h. Subsequently, AuNPs, blocked Walking strand, and hairpin Track strand were mixed at a certain molar ratio, and the mixture was slowly stirred at room temperature for 16 h. Next, 0.2 M PBS buffer (pH 7.4) was injected into the mixture to a final concentration of 0.01 M. Then, 2.0 M NaCl solution was added dropwise under slow stirring to reach a final concentration of 0.2 M within 8 h. After continued slow stirring at room temperature for 24 h, the obtained three-dimensional DNA walker (i.e., AuNP-DNAs) was collected and centrifuged in a centrifuge tube (12500 rpm, 30 min). The unbound DNA was washed three times with 1× Tris-HCl buffer, and finally the obtained AuNP-DNAs were resuspended in the buffer.

[0008] The volume of the sodium citrate solution in step 1) is 0.3 to 2 mL, preferably 1.6 mL; In step 2), the molar ratio of the Walking strand to the Blocking strand is 1:1 to 1:7, preferably 1:5; In step 2), the molar ratio of AuNPs to the closed Walking strand and the hairpin Track strand is 1:30:150 to 1:10:150, preferably 1:15:150.

[0009] In the preparation method, the reagents are all analytically pure; the solutions are all prepared in water unless otherwise specified.

[0010] The application of the technology for rapid determination of kanamycin based on the three-dimensional DNA walker prepared by the above method of the present invention is: for rapid determination of kanamycin content in animal-derived food, and the test method is fluorescence analysis. The details are as follows: 1) Establishment of standard curve: At room temperature, the AuNP-DNAs solution prepared above was added to 50 μL of Tris-HCl buffer containing 3 U Nt.BbvCI endonuclease and 1.5 U KF polymerase, and then kanamycin with a concentration gradient ranging from 5 pM to 500 nM was added, and incubated at 37 ºC for 3 h. The absorbance values ​​were measured at a wavelength of 520 nm to establish a concentration-fluorescence intensity standard curve and obtain the standard curve equation; 2) Detection of test samples: Pre-treat the test samples to obtain dilutions, add AuNP-DNAs, 3 U Nt.BbvCI endonuclease and 1.5 U KF polymerase to the test dilutions, incubate at 37 ºC for 3 h, measure the absorbance at a wavelength of 520 nm, and then calculate the concentration of kanamycin contained in the sample based on the concentration-fluorescence intensity standard curve and the standard curve equation.

[0011] The Tris-HCl buffer solution in step 1) has a pH of 7.4 and a concentration of 140 mM NaCl, 20 mM Tris, and 5.0 mM KCl; Step 2) Test sample: Test the test sample: if it is a solid sample, the cells need to be ultrasonically crushed before soaking in clean water, soak in 3 times the volume of clean water for 24-36 hours, take the soaking supernatant and dilute it 2-6 times with clean water to obtain a dilution; if it is a liquid sample, it is directly diluted 5-12 times with clean water to obtain a dilution.

[0012] The technology of rapid determination of kanamycin based on three-dimensional DNA walker of the present invention adopts fluorescence analysis method to rapidly determine the content of kanamycin in animal-derived food. The measurable kanamycin concentration range is: 5 pM ~ 200 nM, and the detection limit is 1.5 pM.

[0013] The principle of the technical solution of the present invention is explained as follows: The principle of this technique for rapid determination of kanamycin based on a three-dimensional DNA walker is as follows Figure 1As shown. First, the Walking strand and Track strand are modified on the surface of AuNPs through stable Au-S bonds to construct a DNAwalker. Among them, the active center of the Walking strand is pre-locked by the Blocking strand containing the kanamycin aptamer sequence. The Track strand is a specially designed conformational hairpin DNA, in which the complementary sequence of the endonuclease recognition sequence is embedded in its loop. When kanamycin is present, it can specifically bind to its aptamer sequence to unlock the Walking strand, exposing the active center region of the Walking strand. Subsequently, the Walking strand walks along the three-dimensional track, and with the assistance of Nt.BbvCI endonuclease and KF polymerase, the three-dimensional DNA walker continues to run and triggers subsequent cascade polymerization and nicking reactions, releasing a large number of G quadruplex sequences. Subsequently, K + After the addition of kanamycin and N-methylporphyrin dipropionate IX, a large amount of fluorescence signal was generated. However, in the absence of kanamycin, the Walking strand was blocked by the Blocking strand, and the catalytic activity of the Walking strand was inhibited. The operation of the DNA walker and the subsequent polymerization nicking reaction could not be triggered, so a weak background fluorescence signal was obtained.

[0014] from Figure 2 It can be seen that when the technology of rapid determination of kanamycin based on three-dimensional DNA walker detects kanamycin, it shows a good linear relationship in the concentration range of 5.0 pM-200 nM, with a correlation coefficient of 0.9927 and a minimum detection limit of 1.5 pM.

[0015] The main advantages of the technical solution of the present invention are: 1) Utilize the steric effect of the three-dimensional structure of AuNPs to enhance the anti-interference ability of nucleic acid probes and effectively reduce the probability of surface nucleic acid chains being degraded by nucleases, thereby improving the accuracy and credibility of probes in practical applications; 2) Utilize the inherent signal amplification capability of the three-dimensional DNA walker and combine it with the double amplification of the polymerization nicking reaction to improve the speed and sensitivity of detection, thereby achieving rapid and sensitive detection of kanamycin in animal-derived foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the principle of the technology for rapid determination of kanamycin based on three-dimensional DNA walker.

[0017] Figure 2 Standard curve for the rapid determination of kanamycin based on a three-dimensional DNA walker. DETAILED DESCRIPTION

[0018] The present invention is described in conjunction with specific embodiments. Various substitutions or changes made according to common technical knowledge and customary means in the art without departing from the above technical concept of the present invention are all included in the scope of the present invention.

[0019] Example 1 A method for preparing a technology for rapid determination of kanamycin based on a three-dimensional DNA walker, comprising the following steps: 1) AuNPs were prepared by the standard sodium citrate reduction method. First, HAuCl4•3H2O solution (1.25 mL, 10 mM) was quickly injected into 45 mL of boiling ultrapure water under vigorous stirring. After 2 min, 0.3 mL of the pre-prepared 1% sodium citrate solution was quickly added. After continuing to stir vigorously for 10 min under boiling conditions, the heating device was removed, and then the mixture was naturally cooled to room temperature under gentle stirring. Finally, the prepared AuNPs colloid was filtered using a 0.22 µm microporous membrane filter and stored in a 4 ℃ refrigerator for later use; 2) Taking advantage of the strong Au-S chemical bond between thiol groups and the gold surface, thiolated track hairpin DNA and blocked walking DNA were modified onto the surface of AuNPs to prepare a three-dimensional DNA walker. Briefly, the thiol-modified Walking strand and Blocking strand were first mixed in a 1× Tris-HCl buffer (140 mM NaCl, 20 mMTris, 5.0 mM KCl, pH 7.4) at a molar ratio of 1:1. Excess Blocking strand was used to ensure that the Walking strand was completely locked. The mixed solution was heated at 95 °C for 10 min and then slowly cooled to 20 °C. Then TCEP•HCl solution was added to the Walking strand and hairpin Track strand respectively, and the thiol groups were activated at room temperature for 2 h. Subsequently, AuNPs, blocked Walking strand, and hairpin Track strand were mixed at a molar ratio of 1:30:150, and the mixture was slowly stirred at room temperature for 16 h. Next, 0.2 M PBS buffer (pH 7.4) was injected into the mixture to a final concentration of 0.01 M. Then, 2.0 M NaCl solution was added dropwise under slow stirring to reach a final concentration of 0.2 M within 8 h. After continuing to stir slowly at room temperature for 24 h, the obtained three-dimensional DNA walker (i.e., AuNP-DNAs) was collected and centrifuged in a centrifuge tube (12500 rpm, 30 min). The unbound DNA was washed three times with 1× Tris-HCl buffer, and finally the obtained AuNP-DNAs were resuspended in the buffer.

[0020] Example 2 A method for preparing a technology for rapid determination of kanamycin based on a three-dimensional DNA walker, comprising the following steps: 1) AuNPs were prepared by the standard sodium citrate reduction method. First, HAuCl4•3H2O solution (1.25 mL, 10 mM) was quickly injected into 45 mL of boiling ultrapure water under vigorous stirring. After 2 min, 0.3 mL of the pre-prepared 1% sodium citrate solution was quickly added. After continuing to stir vigorously for 10 min under boiling conditions, the heating device was removed, and then the mixture was naturally cooled to room temperature under gentle stirring. Finally, the prepared AuNPs colloid was filtered using a 0.22 μm microporous membrane filter and stored in a 4 °C refrigerator for later use; 2) Taking advantage of the strong Au-S chemical bond between thiol groups and the gold surface, thiolated track hairpin DNA and blocked walking DNA were modified onto the surface of AuNPs to prepare a three-dimensional DNA walker. Briefly, the thiol-modified Walking strand and Blocking strand were first mixed in a 1× Tris-HCl buffer (140 mM NaCl, 20 mMTris, 5.0 mM KCl, pH 7.4) at a molar ratio of 1:3. Excess Blocking strand was used to ensure that the Walking strand was completely locked. The mixed solution was heated at 95 °C for 10 min and then slowly cooled to 20 °C. Then TCEP•HCl solution was added to the Walking strand and hairpin Track strand respectively, and the thiol groups were activated at room temperature for 2 h. Subsequently, AuNPs, blocked Walking strand, and hairpin Track strand were mixed at a molar ratio of 1:20:150, and the mixture was slowly stirred at room temperature for 16 h. Next, 0.2 M PBS buffer (pH 7.4) was injected into the mixture to a final concentration of 0.01 M. Then, 2.0 M NaCl solution was added dropwise under slow stirring to reach a final concentration of 0.2 M within 8 h. After continuing to stir slowly at room temperature for 24 h, the obtained three-dimensional DNA walker (i.e., AuNP-DNAs) was collected and centrifuged in a centrifuge tube (12500 rpm, 30 min). The unbound DNA was washed three times with 1× Tris-HCl buffer, and finally the obtained AuNP-DNAs were resuspended in the buffer.

[0021] Example 3 A method for preparing a technology for rapid determination of kanamycin based on a three-dimensional DNA walker, comprising the following steps: 1) AuNPs were prepared by the standard sodium citrate reduction method. First, HAuCl4•3H2O solution (1.25 mL, 10 mM) was quickly injected into 45 mL of boiling ultrapure water under vigorous stirring. After 2 min, 0.3 mL of the pre-prepared 1% sodium citrate solution was quickly added. After continuing to stir vigorously for 10 min under boiling conditions, the heating device was removed, and then the mixture was naturally cooled to room temperature under gentle stirring. Finally, the prepared AuNPs colloid was filtered using a 0.22 μm microporous membrane filter and stored in a 4 °C refrigerator for later use; 2) Taking advantage of the strong Au-S chemical bond between thiol groups and the gold surface, thiolated track hairpin DNA and blocked walking DNA were modified onto the surface of AuNPs to prepare a three-dimensional DNA walker. Briefly, the thiol-modified Walking strand and Blocking strand were first mixed in a 1× Tris-HCl buffer (140 mM NaCl, 20 mMTris, 5.0 mM KCl, pH 7.4) at a molar ratio of 1:5. Excess Blocking strand was used to ensure that the Walking strand was completely locked. The mixed solution was heated at 95 °C for 10 min and then slowly cooled to 20 °C. Then TCEP•HCl solution was added to the Walking strand and hairpin Track strand respectively, and the thiol groups were activated at room temperature for 2 h. Subsequently, AuNPs, blocked Walking strand, and hairpin Track strand were mixed at a molar ratio of 1:15:150, and the mixture was slowly stirred at room temperature for 16 h. Next, 0.2 M PBS buffer (pH 7.4) was injected into the mixture to a final concentration of 0.01 M. Then, 2.0 M NaCl solution was added dropwise under slow stirring to reach a final concentration of 0.2 M within 8 h. After continuing to stir slowly at room temperature for 24 h, the obtained three-dimensional DNA walker (i.e., AuNP-DNAs) was collected and centrifuged in a centrifuge tube (12500 rpm, 30 min). The unbound DNA was washed three times with 1× Tris-HCl buffer, and finally the obtained AuNP-DNAs were resuspended in the buffer.

[0022] Example 4 A method for preparing a technology for rapid determination of kanamycin based on a three-dimensional DNA walker, comprising the following steps: 1) AuNPs were prepared by the standard sodium citrate reduction method. First, HAuCl4•3H2O solution (1.25 mL, 10 mM) was quickly injected into 45 mL of boiling ultrapure water under vigorous stirring. After 2 min, 0.3 mL of the pre-prepared 1% sodium citrate solution was quickly added. After continuing to stir vigorously for 10 min under boiling conditions, the heating device was removed, and then the mixture was naturally cooled to room temperature under gentle stirring. Finally, the prepared AuNPs colloid was filtered using a 0.22 μm microporous membrane filter and stored in a 4 °C refrigerator for later use; 2) Taking advantage of the strong Au-S chemical bond between thiol groups and the gold surface, thiolated track hairpin DNA and blocked walking DNA were modified onto the surface of AuNPs to prepare a three-dimensional DNA walker. Briefly, the thiol-modified Walking strand and Blocking strand were first mixed in a 1× Tris-HCl buffer (140 mM NaCl, 20 mMTris, 5.0 mM KCl, pH 7.4) at a molar ratio of 1:6. Excess Blocking strand was used to ensure that the Walking strand was completely locked. The mixed solution was heated at 95 °C for 10 min and then slowly cooled to 20 °C. Then TCEP•HCl solution was added to the Walking strand and hairpin Track strand respectively, and the thiol groups were activated at room temperature for 2 h. Subsequently, AuNPs, blocked Walking strand, and hairpin Track strand were mixed at a molar ratio of 1:10:150, and the mixture was slowly stirred at room temperature for 16 h. Next, 0.2 M PBS buffer (pH 7.4) was injected into the mixture to a final concentration of 0.01 M. Then, 2.0 M NaCl solution was added dropwise under slow stirring to reach a final concentration of 0.2 M within 8 h. After continuing to stir slowly at room temperature for 24 h, the obtained three-dimensional DNA walker (i.e., AuNP-DNAs) was collected and centrifuged in a centrifuge tube (12500 rpm, 30 min). The unbound DNA was washed three times with 1× Tris-HCl buffer, and finally the obtained AuNP-DNAs were resuspended in the buffer.

[0023] Example 5 A method for preparing a technology for rapid determination of kanamycin based on a three-dimensional DNA walker, comprising the following steps: 1) AuNPs were prepared by the standard sodium citrate reduction method. First, HAuCl4•3H2O solution (1.25 mL, 10 mM) was quickly injected into 45 mL of boiling ultrapure water under vigorous stirring. After 2 min, 0.3 mL of the pre-prepared 1% sodium citrate solution was quickly added. After continuing to stir vigorously for 10 min under boiling conditions, the heating device was removed, and then the mixture was naturally cooled to room temperature under gentle stirring. Finally, the prepared AuNPs colloid was filtered using a 0.22 μm microporous membrane filter and stored in a 4 °C refrigerator for later use; 2) Taking advantage of the strong Au-S chemical bond between thiol groups and the gold surface, thiolated track hairpin DNA and blocked walking DNA were modified onto the surface of AuNPs to prepare a three-dimensional DNA walker. Briefly, the thiol-modified Walking strand and Blocking strand were first mixed in a 1× Tris-HCl buffer (140 mM NaCl, 20 mMTris, 5.0 mM KCl, pH 7.4) at a molar ratio of 1:7. Excessive Blocking strand was used to ensure that the Walking strand was completely locked. The mixed solution was heated at 95 °C for 10 min and then slowly cooled to 20 °C. Then TCEP•HCl solution was added to the Walking strand and hairpin Track strand respectively, and the thiol groups were activated at room temperature for 2 h. Subsequently, AuNPs, blocked Walking strand, and hairpin Track strand were mixed at a molar ratio of 1:5:150, and the mixture was slowly stirred at room temperature for 16 h. Next, 0.2 M PBS buffer (pH 7.4) was injected into the mixture to a final concentration of 0.01 M. Then, 2.0 M NaCl solution was added dropwise under slow stirring to reach a final concentration of 0.2 M within 8 h. After continuing to stir slowly at room temperature for 24 h, the obtained three-dimensional DNA walker (i.e., AuNP-DNAs) was collected and centrifuged in a centrifuge tube (12500 rpm, 30 min). The unbound DNA was washed three times with 1× Tris-HCl buffer, and finally the obtained AuNP-DNAs were resuspended in the buffer.

[0024] Example 6 A technology based on three-dimensional DNA walker for rapid determination of kanamycin. Rapid detection of kanamycin content in common animal-derived foods and rapid detection of kanamycin content in fresh milk: Randomly take fresh milk samples from the market, pre-treat the samples to be tested to obtain dilutions, add AuNP-DNAs, 3 UNt.BbvCI endonuclease and 1.5 U KF polymerase to the dilutions to be tested, incubate at 37 ºC for 3 h, measure the absorbance at a wavelength of 520nm, and then calculate the concentration of kanamycin contained in the standard curve of concentration-fluorescence intensity and the standard curve equation. Kanamycin in other milk products was measured in this way, and the results are as follows: variety quantity Kanamycin content nM Average value nM Fresh milk 10 2.57—24.22 8.58 Fresh goat milk 8 3.24—4.4 3.83 Fresh horse milk 6 0.32—0.71 0.45 Fresh yogurt 10 2.56—10.96 5.18

[0025] Example 7 A technology based on three-dimensional DNA walker for rapid determination of kanamycin. Rapid detection of kanamycin content in common animal-derived foods. Rapid detection of kanamycin content in fresh eggs: Randomly take egg samples from the market, select a certain part of the egg to prepare the sample with an ultrasonic cell crusher, soak the obtained sample in clean water for a period of time, take the soaking supernatant to dilute a series of concentrations, add AuNP-DNAs, 3 U Nt.BbvCI endonuclease and 1.5 U KF polymerase to the dilution to be tested, incubate at 37 ºC for 3 hours, measure the absorbance value at a wavelength of 520 nm, and then calculate the concentration of kanamycin contained in the standard curve of concentration-fluorescence intensity and the standard curve equation. Kanamycin in other eggs was measured in this way, and the results are as follows: variety quantity Kanamycin content nM Average value nM egg 10 0.28—3.92 1.47 Duck Eggs 8 0.35-4.28 1.59 Goose Egg 6 0.25—3.96 1.62 Quail eggs 12 0.37—3.27 1.78

[0026] Example 8 A technology based on three-dimensional DNA walker for rapid determination of kanamycin. Rapid detection of kanamycin content in common animal-derived foods. Rapid detection of kanamycin content in fresh meat: Fresh pork samples were randomly taken from the market, and a certain part of the fresh pork was selected for pulverization with an ultrasonic cell pulverizer and then soaked. The soaking supernatant was diluted to a series of concentrations, and AuNP-DNAs, 3 U Nt.BbvCI endonuclease and 1.5 U KF polymerase were added to the dilution to be tested, incubated at 37 ºC for 3 h, and the absorbance value was measured at a wavelength of 520 nm. Then, the concentration of kanamycin contained was calculated by combining the concentration-fluorescence intensity standard curve and the standard curve equation. Kanamycin in other meats was measured in this way, and the results are as follows: variety quantity Kanamycin content nM Average value nM pork 6 4.62—26.70 12.4 beef 4 March 13 to 24.28 9.36 mutton 5 2.25—13.96 5.64 muscle 8 5.67—28.78 13.78

Claims

1. A technique for rapid determination of kanamycin based on a three-dimensional DNA walker, characterized by comprising the following steps: Gold nanoparticles (AuNPs) were prepared by the standard sodium citrate reduction method. Then, the strong Au-S chemical bond between the thiol group and the gold surface was used to modify the thiolated track hairpin DNA and the closed walking DNA onto the surface of AuNPs, thereby preparing a three-dimensional DNA walker. Furthermore, this rapid testing technology was applied to the detection of kanamycin content in animal-derived foods.

2. The construction method according to claim 1, characterized in that: The steps are: 1) AuNPs were prepared by the standard sodium citrate reduction method. First, HAuCl4•3H2O solution (1.25 mL, 10 mM) was quickly injected into 45 mL of boiling ultrapure water under vigorous stirring. After 2 min, the pre-prepared 1% sodium citrate solution was quickly added. After continuing to stir vigorously for 10 min under boiling conditions, the heating device was removed, and then the mixture was naturally cooled to room temperature under gentle stirring. Finally, the prepared AuNPs colloid was filtered using a 0.22 μm microporous membrane filter and stored in a 4°C refrigerator for later use; 2) Utilizing the strong Au-S chemical bond between thiol groups and the gold surface, thiolated track hairpin DNA and blocked walking DNA were modified onto the surface of AuNPs to prepare a three-dimensional DNA walker. Briefly, the thiol-modified Walking strand and Blocking strand were first mixed in a 1× Tris-HCl buffer (140 mM NaCl, 20 mMTris, 5.0 mM KCl, pH 7.4) at a certain molar ratio. An excess of Blocking strand was used to ensure that the Walking strand was completely locked. The above mixed solution was heated at 95 °C for 10 min and then slowly cooled to 20 °C. Then, TCEP•HCl solution was added to the Walking strand and the hairpin Track strand, respectively, and the thiol groups were activated at room temperature for 2 h. Subsequently, AuNPs, blocked Walking strand, and hairpin Track strand were mixed at a certain molar ratio, and the mixture was slowly stirred at room temperature for 16 h. Next, 0.2 M PBS buffer (pH 7.4) was injected into the mixture to a final concentration of 0.01 M. Then, 2.0 M NaCl solution was added dropwise under slow stirring to reach a final concentration of 0.2 M within 8 h. After continued slow stirring at room temperature for 24 h, the obtained three-dimensional DNA walker (i.e., AuNP-DNAs) was collected and centrifuged in a centrifuge tube (12500 rpm, 30 min). The unbound DNA was washed three times with 1× Tris-HCl buffer, and finally the obtained AuNP-DNAs were resuspended in the buffer.

3. The construction method according to claim 2, characterized in that: The volume of the sodium citrate solution in step 1) is 0.3 to 2 mL, preferably 1.6 mL.

4. According to claim 2, a method for constructing a rapid kanamycin detection technology based on dual amplification of three-dimensional DNA walker operation and polymerase nicking reaction, characterized in that: The molar ratio of the walking chain W to the closed chain B in step 2) is 1:1 to 1:7, preferably 1:

5.

5. According to claim 2, a method for constructing a rapid kanamycin detection technology based on dual amplification of three-dimensional DNA walker operation and polymerase nicking reaction, characterized in that: The molar ratio of AuNPs, closed walking chain W, and hairpin track chain in step 2) is 1:30:150 to 1:10:

150.

6. The method for constructing a rapid kanamycin detection technology based on dual amplification of three-dimensional DNA walker operation and polymerase nicking reaction according to claim 2, characterized in that: In the preparation method, the reagents are all analytically pure; the solutions are all prepared in water unless otherwise specified.

7. A technique for rapid determination of kanamycin based on a three-dimensional DNA walker constructed according to any one of claims 1 to 6, characterized in that: Used for rapid testing of kanamycin content in animal-derived food, the test method is fluorescence analysis.

8. The use of a technique for rapid determination of kanamycin based on a three-dimensional DNA walker according to claim 7, characterized in that: The fluorescence analysis method is: 1) Establishment of standard curve: At room temperature, the AuNP-DNAs solution prepared above was added to 50 μL of Tris-HCl buffer containing 3 UNt.BbvCI endonuclease and 1.5 U KF polymerase, and then kanamycin was added in a concentration gradient ranging from 5 pM to 500 nM. The mixture was incubated at 37 ºC for 3 h, and the absorbance values ​​were measured at a wavelength of 520 nm to establish a concentration-fluorescence intensity standard curve and obtain the standard curve equation. 2) Detection of test samples: Pretreat the test samples to obtain dilutions, add AuNP-DNAs, 3 UNt.BbvCI endonuclease and 1.5 U KF polymerase to the test dilutions, incubate at 37 ºC for 3 h, measure the absorbance at a wavelength of 520 nm, and then calculate the concentration of kanamycin contained in the sample based on the concentration-fluorescence intensity standard curve and the standard curve equation.

9. The application of the kanamycin rapid detection technology based on dual amplification of three-dimensional DNA walker operation and polymerase nicking reaction according to claim 8, characterized in that: The Tris-HCl buffer solution in step 1) has a pH of 7.4 and a concentration of 140 mM NaCl, 20 mM Tris, and 5.0 mM KCl; Step 2) Test sample: Test the test sample: if it is a solid sample, the cells need to be ultrasonically crushed before soaking in clean water, soak in 3 times the volume of clean water for 24-36 hours, take the soaking supernatant and dilute it 2-6 times with clean water to obtain a dilution; if it is a liquid sample, it is directly diluted 5-12 times with clean water to obtain a dilution.

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