CN112710649A

CN112710649A - Method for detecting kanamycin sulfate by using dual-signal-enhanced surface-enhanced Raman spectroscopy

Info

Publication number: CN112710649A
Application number: CN202110056967.1A
Authority: CN
Inventors: 徐志祥; 王汐沫; 陈晨; 徐龙华; 孔非凡
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2021-01-15
Filing date: 2021-01-15
Publication date: 2021-04-27
Anticipated expiration: 2041-01-15
Also published as: CN112710649B

Abstract

The invention discloses a method for detecting kanamycin sulfate by using a dual-signal-enhanced surface-enhanced Raman spectrum, and belongs to the technical field of food safety detection. The invention uses the gold nanorod with the controllable surface plasma resonance peak as a Raman substrate, combines the cascade oxidation-reduction reaction catalyzed by the metal-doped carbon quantum dots and the switch structure controlled by the aptamer, establishes a kanamycin sulfate detection system for doubly enhancing Raman signals, and further improves the sensitivity for detecting kanamycin sulfate. The invention has the advantages of low detection limit, good specificity, simplicity, convenience and rapidness, and is suitable for the ultra-sensitive detection of kanamycin sulfate in food.

Description

Method for detecting kanamycin sulfate by using dual-signal-enhanced surface-enhanced Raman spectroscopy

Technical Field

The invention relates to the technical field of food safety detection, in particular to a method for detecting kanamycin sulfate by using a dual-signal-enhanced surface-enhanced Raman spectrum.

Background

With the improvement of the quality of life of people, the problem of antibiotic medicine residue becomes one of the focus problems concerned by consumers. Kanamycin sulfate is one of high-efficiency aminoglycoside antibiotics widely used for treating animal infectious diseases in the food production and manufacturing industry at present, and the structural formula of the kanamycin sulfate is as follows:

kanamycin sulfate is formed by connecting amino sugar molecules and amino cyclic alcohol through ether bonds, and can quickly kill bacteria mainly by interfering the synthesis of proteins in bacteria. Kanamycin sulfate remained in foods such as meat, egg and milk can be accumulated in a human body, and symptoms such as dizziness, nausea and tinnitus can appear when the foods with kanamycin sulfate exceeding standards are taken for a long time, so that the drug resistance of bacteria in the human body is increased, kidney injury and auditory nerve injury are caused, and great harm is caused to the human body. Therefore, the high-efficiency and rapid detection method for the kanamycin sulfate residue in the food is researched, and the improvement of the sensitivity and the selectivity of the kanamycin sulfate residue is of great significance.

At present, common detection methods aiming at kanamycin sulfate mainly comprise high performance liquid chromatography, chromatography-mass spectrometry, microbiological methods, ELISA, capillary electrophoresis and the like. The high performance liquid chromatography and the chromatography-mass spectrometry are used for detecting the content of kanamycin sulfate in food accurately and reliably, but kanamycin sulfate lacks chromogenic groups, and pre-column derivation or post-column derivation is needed when a fluorescence or ultraviolet detector is used for detection, so that the detection procedure is complex and the requirement on experimenters is high; although the microbiological method is simple to operate, economical and practical, the detection limit is higher and only substances with biological activity can be detected; ELISA has the advantages of high specificity and high sensitivity, but the preparation of the antibody is difficult and the cost is higher; the capillary electrophoresis technology is applied to the kanamycin sulfate detection and analysis, has high speed and small required sample amount, but has poor repeatability and high detection limit, and needs to be combined with technologies such as field amplification, solid phase extraction and the like. Therefore, the detection of kanamycin sulfate in food still has the challenges of low detection limit, high sensitivity, rapidness and reliability.

The Surface Enhanced Raman Spectroscopy (SERS) is an important rapid and lossless inelastic scattering analysis technology, has monomolecular sensitivity, and is expected to become a powerful tool for analyzing kanamycin sulfate in food. The technology utilizes the Raman spectrum technology to measure the sample adsorbed on the surface of the rough noble metal particles, so that the Raman intensity of the sample is greatly improved. However, in actual analysis, the SERS signal acquisition depends not only on the concentration of the analyte, but also on the properties of the nano-roughened metal substrate. Therefore, the Raman substrate which can realize resonance coupling of the plasma resonance peak position and the Raman incident light is screened, and the detection method with high specificity and sensitivity is combined, so that the method has important significance for analyzing kanamycin sulfate in food.

Disclosure of Invention

In view of the prior art, the invention aims to provide a method for detecting kanamycin sulfate by using surface-enhanced Raman spectroscopy with dual signal enhancement. The invention adopts a three-step seed growth method and a chemical dissolution method to prepare gold nanoparticles coupled with Raman incident light resonance as a Raman substrate, combines a cascade oxidation-reduction reaction catalyzed by metal-doped carbon quantum dots and a switch structure controlled by a nucleic acid aptamer, and realizes the sensitive detection of kanamycin sulfate by double enhanced signals of the metal-doped carbon quantum dot catalytic reaction switch controlled by the nucleic acid aptamer and a gold nano substrate with controllable size; the invention has good specificity, simple experimental operation and high sensitivity, and is suitable for trace detection of kanamycin sulfate in food.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a dual signal enhanced surface enhanced raman spectroscopy detection system, comprising: a gold nanorod Raman substrate coupled with Raman incident light in resonance and a metal-doped carbon quantum dot catalytic reaction switch controlled by a nucleic acid aptamer;

the gold nanorod Raman substrate in resonance coupling with Raman incident light is prepared by the following method:

synthesizing large-size gold nanorods by adopting a seed growth method, and then dissolving the synthesized large-size gold nanorods for 5-10 hours by adopting a dissolving solution; the solution contains Cetyl Trimethyl Ammonium Bromide (CTAB) and HAuCl₄·3H₂An aqueous solution of O;

the metal-doped carbon quantum dot catalytic reaction switch controlled by the aptamer is prepared by the following method:

mixing the aptamer and the metal-doped carbon quantum dot, and adding 10-20 mu mol/L HAuCl₄Heating the solution and 0.05-0.2 mol/L glucose solution in a water bath at the temperature of 55-85 ℃ for 20-45 min, and cooling to room temperature; adding 4-mercaptophenylboronic acid (4-MPBA) solution under the action of vortex, and incubating for 1-3 h.

Preferably, the synthesis of the large-size gold nanorod by the seed growth method specifically comprises the following steps: mixing CTAB solution with HAuCl₄Mixing, adding newly prepared NaBH₄Fully stirring the solution, and then standing in a water bath to obtain a seed solution; adding HAuCl₄CTAB and ascorbic acid as a growth solution; and synthesizing the large-size gold nanorod by adopting a three-step seed growth method.

Preferably, the content of CTAB in the solution is 36.4mg/ml, and HAuCl₄·3H₂The O content was 0.66 mg/ml.

Preferably, the synthesized large-size gold nanorods are dissolved by using a dissolving solution until the longitudinal plasma resonance peak is 785cm^-1。

Preferably, the metal-doped carbon quantum dots are zinc-doped carbon quantum dots.

The aptamer is capable of specifically binding to a target substance to be detected.

In a second aspect of the present invention, there is provided the use of the dual signal enhanced surface enhanced raman spectroscopy detection system described above in the detection of antibiotics.

Preferably, the antibiotic is kanamycin sulfate.

In a third aspect of the present invention, a method for detecting kanamycin sulfate based on the above dual signal enhanced surface enhanced raman spectroscopy detection system is provided, which comprises the following steps:

(1) mixing the aptamer, the metal-doped carbon quantum dot and a series of kanamycin sulfate standard solutions with concentration gradients, and then adding 10-20 mu mol/L HAuCl₄Heating the solution and 0.05-0.2 mol/L glucose solution in a water bath at the temperature of 55-85 ℃ for 20-45 min, and cooling to room temperature; in the vortex actionAdding a 4-MPBA solution, incubating for 1-3h, centrifuging, and removing redundant 4-MPBA to obtain gold nanoparticles released by the catalytic oxidation-reduction reaction of the metal-doped carbon quantum dots;

(2) synthesizing a large-size gold nanorod by adopting a seed growth method, and dissolving the synthesized large-size gold nanorod by adopting a dissolving solution until the longitudinal plasma resonance peak is 785cm^-1As a raman substrate;

(3) mixing the gold nanoparticles released by the catalytic oxidation-reduction reaction of the metal-doped carbon quantum dots prepared in the step (1) with a Raman substrate, reacting for 1-3h at 35-40 ℃, and recording 495cm after the reaction is finished^-1(ii) the raman intensity of (d);

(4) the negative logarithm value of the concentration of the kanamycin sulfate standard solution is used as an abscissa, and the concentration is 495cm^-1Drawing a working curve by taking the Raman intensity as a vertical coordinate;

(5) and (3) replacing the kanamycin sulfate standard solution in the step (1) with the solution to be detected, repeating the steps (1) to (3), and detecting the kanamycin sulfate content in the solution to be detected by using the working curve drawn in the step (4).

Preferably, in the step (1), the concentration of the kanamycin sulfate standard solution is 10^-12g/mL、10^-11g/mL、10^-10g/mL、10^-9g/mL、10^-8g/mL、10^-7g/mL、10^-6g/mL、10^-5g/mL。

Preferably, in step (1), the sequence of the aptamer is 5'-TGGGGGTTGAGGCTAAGCC GA-3'; (SEQ ID NO. 1).

Preferably, in the step (5), the solution to be tested is prepared by the following method:

accurately taking 1g of sample into a centrifugal tube, adding deionized water, adding a 5% volume fraction phosphoric acid solution after vortex mixing, performing shake extraction for 5-10 min, then adding a 50% mass fraction trichloroacetic acid solution, and centrifuging after vortex mixing; repeating twice and combining the supernatant, concentrating under reduced pressure, redissolving with deionized water, and filtering with 0.22 μm filter membrane to obtain the solution to be detected.

The invention has the beneficial effects that:

(1) the invention adoptsThe gold nanoparticles which can generate strong resonance coupling with Raman incident light are synthesized and dissolved by a sub-growth method and a chemical method to be used as a Raman substrate, and a metal doped carbon quantum dot catalytic reaction switch structure controlled by a nucleic acid aptamer is used as a main body detection part, so that the effect of dual enhancement of Raman signals can be achieved, and the selectivity and the sensitivity of detection are greatly improved. According to 495cm^-1The Raman intensity of the target is linearly related to the concentration of the target analyte, and a novel sensitive detection means is established. By adopting the method for detecting kanamycin sulfate based on the surface enhanced Raman spectrum of the dual-enhanced signal, the linear range for detecting kanamycin sulfate is 10^-12～10^-5g/mL, minimum detection limit of 3.03X 10^-13g/mL, can meet the trace detection requirement of the actual sample.

(2) The metal-doped carbon quantum dot catalytic switch controlled by the aptamer has good selectivity, and the prepared gold nano substrate with controllable size has high sensitivity and is widely applied to determination of kanamycin sulfate residue in food.

Compared with the existing other methods for detecting kanamycin sulfate, the method disclosed by the invention has the following advantages:

name of method	Detection limit
		The detection method of the invention	3.03×10^-7μg/mL
High performance liquid chromatography	5×10^-2μg/mL
		Liquid chromatography-tandem mass spectrometry	0.5μg/kg
Capillary electrophoresis method	7×10^-3μg/mL

Drawings

FIG. 1: and (3) surface plasma resonance peak spectra of the gold nanorods at different dissolution times.

As can be seen from FIG. 1, the gold nanorods have two plasmon resonance peaks corresponding to the longitudinal plasmon resonance and the transverse plasmon resonance, respectively, indicating that the gold nanorods are in a rod-like structure. With the increase of the dissolution time, the longitudinal plasma resonance peak blue shift is obvious, which indicates that the rod-shaped structure is shortened.

FIG. 2: the longitudinal plasma resonance peak prepared by the embodiment of the invention is a scanning electron microscope image of the gold nanorod with the wavelength of 785 nm; indicating a rod-like structure.

FIG. 3: kanamycin working curve.

As can be seen from FIG. 3, the linear range of kanamycin sulfate detected by the method is 10^-12-10^-5g/mL。

FIG. 4: the invention discloses a selective experimental diagram of a dual-signal enhanced surface enhanced Raman spectrum detection system.

As can be seen from fig. 4, by comparing the responses of kanamycin sulfate (Kanamycinc), Gentamicin (Gentamicin), Amikacin (Amikacin), and Vancomycin (Vancomycin) to the detection system, it can be seen that the raman intensity of two aminoglycoside antibiotics (Gentamicin, Amikacin) and one polypeptide antibiotic (Vancomycin) is much lower than that of kanamycin. The result shows that the surface enhanced Raman spectroscopy detection system has better specificity in detecting kanamycin sulfate.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Description of terms:

the term "room temperature" as used herein means a temperature of 15 to 30 ℃.

As introduced in the background art, the current methods for detecting kanamycin sulfate residues mainly include high performance liquid chromatography, liquid chromatography-mass spectrometry, enzyme linked immunosorbent assay, and the like, and the detection methods used by the methods are complicated or have low sensitivity. Therefore, the research and development of the kanamycin sulfate detection method with high sensitivity and good selectivity has important significance.

Surface Enhanced Raman Spectroscopy (SERS) is an important fast, lossless inelastic scattering analysis technique, gold nanoparticles are a popular raman substrate because of their strong enhancement effect and stability, but most gold nanoparticles are simple in synthesis process and do not control growth, so that the gold nanoparticles are different in size, and limited in shape and color.

In order to realize sensitive and high-selectivity detection of kanamycin sulfate, gold nanoparticles capable of generating strong resonance coupling with Raman incident light are used as a Raman substrate, a metal-doped carbon quantum dot catalytic reaction switch structure controlled by a nucleic acid aptamer is used as a main body detection part, and a method for detecting kanamycin sulfate by using a surface enhanced Raman spectrum based on dual enhanced signals is established.

The invention uses the gold nanorod capable of generating strong resonance coupling with Raman incident light as a Raman substrate. Free electrons on the metal surface will have a frequency when vibrating around a relatively fixed positively charged nucleus, and if the frequency of the incident light is exactly the same as this frequency, surface plasmon resonance will occur, and therefore, it is necessary to construct a raman substrate capable of controlling the position of the plasmon resonance peak. The invention adopts a three-step seed growth method and a chemical method for synthesis and dissolution, can simply and effectively control the position of the plasma resonance peak of the gold nanorod, and opens up a better development path for kanamycin sulfate detection. In the invention, the longitudinal plasma resonance peak of the gold nanorod used as the Raman substrate is controlled to be 785cm^-1785cm for gold as substrate^-1Is the most commonly used wavelength of incident light. The enhancement is best when the raman incident light coincides with the raman substrate frequency and resonance coupling occurs.

Doping metal elements is one of the most effective, most extensive and most sensitive means for controlling the optical and electron transport of carbon quantum dots. As an electron transfer carrier, the catalyst can catalyze the reduction of gold ions into elementary atoms in the presence of a reducing agent. The reason is that the Fermi level of gold is low, and electrons carried by carbon quantum dots tend to migrate to noble metals, so that effective electron-hole separation is realized, effective carrier separation is promoted, and the catalytic efficiency is improved. Meanwhile, due to the load synergistic effect, the gold ions adsorbed on the surfaces of the gold particles are further subjected to catalytic reduction on the surrounding gold ions, so that a cascade redox reaction is generated, more gold nanoparticles are generated, and the effect of dual enhancement of Raman signals can be achieved by combining the gold nanoparticles with a Raman substrate.

Highly catalytic metal-doped carbon quantum dot pair HAuCl₄The oxidation-reduction reaction between the gold nanoparticles and glucose has strong catalytic action, and the gold nanoparticles with high Raman activity are formed. The nucleic acid aptamer is adsorbed on the surface of the carbon quantum dot to inhibit the catalytic activity of the carbon quantum dot, and the Raman intensity is reduced. However, when the target kanamycin sulfate is present, the target kanamycin sulfate can be combined with the aptamer to release free metal-doped carbon quantum dots, and the Raman intensity is increased. 4-MPBA as molecular probe, 495cm^-1The raman intensity at (a) increases with increasing kanamycin sulfate concentration. The linear range of kanamycin sulfate detected by the method is 10^-12～10^-5g/mL, and realizes the ultra-sensitive detection of kanamycin sulfate.

In conclusion, the invention adopts a three-step seed growth method and a chemical method to synthesize and dissolve to 785cm which is the same as the Raman incident light frequency^-1The gold nanorods with the longitudinal plasma resonance peak are combined with the gold nanoparticles released by the catalytic oxidation-reduction reaction of the metal-doped carbon quantum dots, so that the effect of dual enhancement of Raman signals is achieved, and the Raman spectrum analyzer has a wide Raman application prospect.

In one embodiment of the present invention, a method for preparing a size-controllable gold nanorod as a raman substrate is provided, comprising the steps of:

5mL of a 0.2mol/L CTAB solution and 5mL of 0.0005mol/L HAuCl were added₄The solutions were mixed. To the stirred solution was added 0.6mL of freshly prepared 0.01mol/L NaBH₄And (5) obtaining a seed solution.

4.5mL of 2.5X 10^-4mol/L HAuCl₄4.5mL of 0.1mol/L CTAB solution and 0.05mL of 0.1mol/L ascorbic acid solution are mixed to obtain a growth solution.

1mL of the seed solution was added to 9.05mL of the growth solution, and reacted for 4 hours to obtain solution A. 1mL of the solution A was added to 9.05mL of a newly prepared growth solution, and the reaction was carried out for 4 hours to obtain a solution B. And adding 1mL of the solution B into 9.05mL of newly prepared growth solution, and reacting for 12h to obtain the single crystal gold nanorod.

3.64g CTAB and 66mg HAuCl₄·3H₂Dissolving O in 100mL of water to obtain a solution. Adding 1.5mL of the solution into 5mL of the single-crystal gold nanorods, reacting at 38 ℃ for 5-10h, and centrifuging at 5900 Xg for 10min to remove the redundant solution.

In the process of preparing gold nanoparticles with controllable size, HAuCl is added₄·3H₂O and CTAB water solution to control and selectively dissolve the gold nano particles. As the dissolution time increased, the gold nanoparticles decreased in size and the plasmon resonance peak blue shifted (fig. 1). Therefore, the reaction time is accurately controlled, and the position of the gold nanoparticle plasma resonance peak can be adjusted, which is the key to apply the surface enhanced Raman spectroscopy to the detection of trace kanamycin sulfate.

In another embodiment of the invention, a specific preparation method of the aptamer-controlled metal-doped carbon quantum dot catalytic reaction switch is provided, which comprises the following steps:

0.735g of sodium citrate and 0.1704g of zinc chloride were weighed into a beaker, and 25mL of ultrapure water was added thereto to dissolve sufficiently. And transferring the fully dissolved solution into a hydrothermal kettle with the total volume of 50mL, heating the solution to 180-200 ℃ in a constant-temperature air-blast drying oven, and reacting for 2-5 h at the temperature. After cooling the reaction kettle to room temperature, transferring the reaction kettle to a clean test tube, filtering the reaction kettle by a 0.22 mu m filter membrane, and transferring the reaction kettle to the clean test tube to prepare the zinc-doped carbon quantum dots; refrigerating at 4 deg.C for use.

150-250 mu L of 0.03mol/L aptamer is added into a 5mL test tube, and 30 mu L of kanamycin sulfate standard solution and 70 mu L of metal-doped carbon quantum dots are added. After 15min, adding 100 mu L of 10-20 mu mol/L HAuCl₄Heating the solution, 150 mu L of 0.05-0.2 mol/L glucose solution, in a water bath at 55-85 ℃ for 20-45 min, and cooling to room temperature. mu.L of a 0.1mM 4-MPBA solution was added to 1mL of the above mixed solution under vortexing. Incubate at room temperature for 2h, 1475 Xg centrifugation for 10min to remove excess 4-MPBA. The obtained nanoparticles are collected and re-dispersed in 1mL of deionized water to prepare the gold nanoparticles (i.e., the aptamer-controlled metal-doped carbon quantum dot catalytic reaction switch) incubated with 4-MPBA.

The resonance peak of the gold nanoparticles incubated with the 4-MPBA and the longitudinal plasma is 785cm^-1After mixing the Raman substrate of (1), a Raman spectrometer was used to record 495cm^-1And (4) the Raman intensity. The negative logarithm value of the concentration of the kanamycin sulfate standard solution is used as an abscissa, and the concentration is 495cm^-1The raman intensity at position is plotted as the ordinate to draw the working curve. And detecting the content of kanamycin sulfate in the solution to be detected by using the drawn working curve.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. Wherein:

the sequence of the aptamer was 5'-TGGGGGTTGAGGCTAAGCCGA-3'.

Example 1:

1. preparing gold nanoparticles with controllable sizes:

(1) preparing single crystal gold nano particles:

Adding 1mL of seed solution into the growth solution at 28 ℃ to react for 4h (solution A); adding 1mL of new growth solution into the solution A, and reacting for 4h (solution B); adding 1mL of the solution B into a new growth solution, and reacting for 12h to obtain the single-crystal gold nanorod.

(2) Dissolving the single crystal gold nanorods: 3.64g CTAB and 66mg HAuCl₄·3H₂O is dissolved in 100mL of deionized water, 1.5mL of the above solution is added to 5mL of single-crystal gold nanorods, and the reaction is carried out for 7.5h at 38 ℃. After the reaction is finished, the reaction solution is centrifuged at 5900 Xg for 10min to remove the excessive CTAB and HAuCl₄·3H₂O, re-dissolving with 5mL of deionized water to obtain a longitudinal plasma resonance peak of 785cm^-1The average size of the gold nanorods (peaks shown in a in FIG. 1, FIG. 2) was 171.8nm, which was used as a Raman substrate.

2. Preparing the metal-doped carbon quantum dots:

0.735g of sodium citrate and 0.1704g of zinc chloride were weighed into a beaker, and 25mL of ultrapure water was added thereto to dissolve sufficiently. The well-dissolved solution was transferred to a hydrothermal kettle with a total volume of 50mL, heated to 185 ℃ in a constant temperature forced air drying oven and reacted at this temperature for 4 h. After the reaction kettle is cooled to room temperature, the mixture is filtered by a 0.22 mu m filter membrane and then transferred into a clean test tube, and the mixture is refrigerated at 4 ℃ for standby.

3. The establishment of the surface-enhanced Raman spectroscopy analysis method of the metal-doped carbon quantum dot catalytic switch and the gold nanoparticle double-enhanced signal based on the control of the aptamer comprises the following steps:

200. mu.L of 0.03mol/L aptamer was added to a 5mL tube, and 50. mu.L of 10^-12g/mL、10^-11g/mL、10^-10g/mL、10^-9g/mL、10^-8g/mL、10^-7g/mL、10^-6g/mL、10^-5g/mL kanamycin sulfateStandard solution and 70 mul of metallic zinc doped carbon quantum dots. After 15min, 100. mu.L of 15. mu. mol/L HAuCl was added₄The solution, 150. mu.L of a 0.1mol/L glucose solution, was heated in a water bath at 65 ℃ for 30 min. After cooling to room temperature, 10. mu.L of 0.1 mmol/L4-MPBA solution was added to 1mL of gold nanoparticle solution under vortexing. Incubate at room temperature for 2h, 1475 Xg centrifugation for 10min to remove excess 4-MPBA. The resulting nanoparticles were collected and redispersed in 1mL of deionized water to give 4-MPBA incubated gold nanoparticles.

The resonance peak of gold nanoparticles incubated with 4-MPBA and longitudinal plasma is 785cm^-1The Raman substrate was mixed at 38 ℃ and reacted for 2 hours, and then recorded at 495cm using a Raman spectrometer^-1And (4) processing the Raman value.

The negative logarithm of the kanamycin sulfate concentration is used as the abscissa, and the length is 495cm^-1The raman intensity at (a) is plotted on the ordinate, and a working curve is plotted (fig. 3).

The detection specificity of the dual-signal-enhanced surface-enhanced Raman spectrum detection system constructed above is examined, and the results are shown in FIG. 4 by comparing the responses of kanamycin sulfate (Kanamycinc), Gentamicin (Gentamicin), Amikacin (Amikacin) and Vancomycin (Vancomycin) to the detection system. The result shows that the surface enhanced Raman spectroscopy detection system has better specificity in detecting kanamycin sulfate.

Example 2:

the kanamycin sulfate in the milk sample is detected by using the surface-enhanced Raman spectroscopy analysis method based on the metal-doped carbon quantum dot catalytic switch controlled by the aptamer and the gold nanoparticle dual-enhanced signal with controllable size, which is established in the embodiment 1, and the method specifically comprises the following steps:

(1) accurately taking 1g of milk sample, adding 30mL of 5% phosphoric acid solution in volume fraction into a 50mL centrifuge tube, extracting for 10min by shaking, and adding 3mL of 50% trichloroacetic acid solution in mass fraction. Vortex mixed and centrifuged at 944 Xg for 10 min. This procedure was repeated twice and the supernatants were combined. Concentrating the component to be detected under reduced pressure, re-dissolving the component to be detected to 1mL by using water, and filtering the component by using a 0.22 mu m filter membrane to obtain a solution to be detected.

(2) 200. mu.L of 0.03mol/L coreAdding the acid aptamer into a 5mL test tube, and adding 50 mu L of solution to be detected and 70 mu L of metal zinc-doped carbon quantum dots. After 15min, 100. mu.L of 15. mu. mol/L HAuCl was added₄The solution, 150. mu.L of a 0.1mol/L glucose solution, was heated in a water bath at 65 ℃ for 30 min. After cooling to room temperature, 10. mu.L of 0.1 mmol/L4-MPBA solution was added to 1mL of gold nanoparticle solution under vortexing. Incubate at room temperature for 2h, 1475 Xg centrifugation for 10min to remove excess 4-MPBA. The resulting nanoparticles were collected and redispersed in 1mL of deionized water to give 4-MPBA incubated gold nanoparticles.

According to the standard curve drawn in example 1, the kanamycin sulfate content in the milk sample was found to be 94.5. mu.g/kg.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> method for detecting kanamycin sulfate by using surface enhanced Raman spectroscopy with double signal enhancement

<130> 2021

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence

<400> 1

tgggggttga ggctaagccg a 21

Claims

1. A dual signal enhanced surface enhanced raman spectroscopy detection system comprising: a gold nanorod Raman substrate coupled with Raman incident light in resonance and a metal-doped carbon quantum dot catalytic reaction switch controlled by a nucleic acid aptamer;

synthesizing large-size gold nanorods by adopting a seed growth method, and then dissolving the synthesized large-size gold nanorods for 5-10 hours by adopting a dissolving solution; the solution contains CTAB and HAuCl₄·3H₂An aqueous solution of O;

mixing the aptamer and the metal-doped carbon quantum dot, and adding 10-20 mu mol/L HAuCl₄Heating the solution and 0.05-0.2 mol/L glucose solution in a water bath at the temperature of 55-85 ℃ for 20-45 min, and cooling to room temperature; adding 4-MPBA solution under the action of vortex, and incubating for 1-3 h.

2. The dual-signal-enhanced surface-enhanced Raman spectroscopy detection system according to claim 1, wherein the synthesis of the large-size gold nanorods by the seed growth method specifically comprises: mixing CTAB solution with HAuCl₄Mixing, adding newly prepared NaBH₄Fully stirring the solution, and then standing in a water bath to obtain a seed solution; adding HAuCl₄CTAB and ascorbic acid as a growth solution; and synthesizing the large-size gold nanorod by adopting a three-step seed growth method.

3. The dual signal enhanced surface-enhanced Raman spectroscopy detection system according to claim 1, wherein the amount of CTAB in the dissolution solution is 36.4mg/ml and HAuCl in the dissolution solution₄·3H₂The O content was 0.66 mg/ml.

4. The dual signal enhanced surface-enhanced Raman spectroscopy detection system of claim 1, wherein the synthesized large-sized gold nanorods are dissolved to longitudinal plasma with a dissolving solutionThe volume resonance peak is 785cm^-1。

5. The dual signal enhanced surface-enhanced raman spectroscopy detection system of claim 1, wherein the metal-doped carbon quantum dots are zinc-doped carbon quantum dots.

6. Use of the dual signal enhanced surface enhanced raman spectroscopy detection system of any one of claims 1 to 5 in the detection of antibiotics.

7. A method for detecting kanamycin sulfate based on the dual signal enhanced surface enhanced Raman spectroscopy detection system of any one of claims 1-5, comprising the following steps:

(1) mixing the aptamer, the metal-doped carbon quantum dot and a series of kanamycin sulfate standard solutions with concentration gradients, and then adding 10-20 mu mol/L HAuCl₄Heating the solution and 0.05-0.2 mol/L glucose solution in a water bath at the temperature of 55-85 ℃ for 20-45 min, and cooling to room temperature; adding a 4-MPBA solution under the action of vortex, incubating for 1-3h, centrifuging, and removing redundant 4-MPBA to obtain gold nanoparticles released by the catalytic oxidation-reduction reaction of the metal-doped carbon quantum dots;

8. The method according to claim 7, wherein in the step (1), the concentrations of the kanamycin sulfate standard solutions are 10 respectively^-12g/mL、10^-11g/mL、10^-10g/mL、10^-9g/mL、10^-8g/mL、10^-7g/mL、10^-6g/mL、10^-5g/mL。

9. The method according to claim 7, wherein the sequence of the aptamer in step (1) is 5'-TGGGGGTTGAGGCTAAGCCGA-3'.

10. The method according to claim 7, wherein in the step (5), the solution to be tested is prepared by the following method: