CN109115711B - Method for colorimetric detection of kanamycin based on Au @ Ag core-shell structure - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Abstract
The invention discloses a colorimetric detection method for kanamycin based on an Au @ Ag core-shell structure, which comprises the following steps: (1) designing and synthesizing nucleic acid aptamer fragments; (2) preparing Au @ Ag core-shell structure nanoparticles; (3) taking a centrifuge tube, adding an aptamer with the concentration of 10 nM-50 nM and NaCl with the concentration of 10 nM-50 nM into 1500 muL of PBS buffer solution, adding a sample to be tested, mixing, stabilizing at room temperature for 20min, adding 500 muL of nano solution into the centrifuge tube, and scanning the ultraviolet visible absorption spectrum; (4) kanamycin concentration was determined according to the linear equation: y is 0.00011x + 0.39901; the method can realize simple, rapid and high-sensitivity detection of the kanamycin in the food, and can meet the detection requirement of an actual sample on the kanamycin.
Description
Technical Field
The invention relates to a method for detecting kanamycin, in particular to a method for detecting kanamycin based on Au @ Ag core-shell structure colorimetric detection, and belongs to the technical field of food safety.
Background
Kanamycin is a broad-spectrum antibiotic, belongs to aminoglycosides, can be combined with ribosome to achieve the effect of inhibiting protein biosynthesis, and is a widely applied veterinary drug in China due to obvious effect and low price. However, aminoglycoside antibiotics have strong side effects and toxicity, and are easy to cause hearing loss and kidney damage after long-term contact, and even can penetrate placenta barrier to cause fetal damage. Kanamycin can remain in animal food and is transmitted to human beings through the food chain enrichment effect, so that the health of the human beings is damaged. With the improvement of the living quality level of people, the requirements on food safety are stricter and stricter, and the highest residual quantity of the product in livestock is also regulated in China, so the problem of the detection limit of the product is more and more emphasized by people and becomes a hotspot of research.
Currently, methods for detecting kanamycin are classified into a microbiological method, a high performance liquid chromatography, a spectrophotometric method, an electrochemical method, and the like. Although the result of the method is accurate, the operation of the microbiological method is complex, time-consuming, poor in specificity and sensitivity, and the high performance liquid chromatography needs large instruments and high cost, so that the method is generally difficult to be used for on-site rapid detection, and a novel detection method is urgently needed to be developed on the basis.
Disclosure of Invention
The invention aims to provide a colorimetric detection method for kanamycin based on an Au @ Ag core-shell structure, which can realize simple, rapid and high-sensitivity detection of kanamycin in food (not limited to food).
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
a colorimetric kanamycin detection method based on an Au @ Ag core-shell structure comprises the following steps:
(1) designing and synthesizing nucleic acid aptamer fragments;
designing a DNA fragment capable of binding to kanamycin with specific affinity, and preparing a sequence by a DNA synthesizer, wherein the DNA: 5'-AGA TGG GGG TTG AGG CTA AGC CGA-3'
(2) The preparation method of the Au @ Ag core-shell structure nano particle comprises the following steps:
1) and reducing silver nitrate by trisodium citrate to obtain gold nanoparticles, wherein the particle size of the gold nanoparticles is 25nm, and the maximum absorption peak is 523 nm.
2) And concentrating the prepared gold nanoparticles by 10 times, cooling to room temperature, and storing at 4 ℃ for later use.
3) Mixing 500 mu L0.1M phosphate buffer solution, 100 mu L of 1% polyvinylpyrrolidone solution and 50 mu L0.1M ascorbic acid, adding concentrated nanogold, adding 1mM silver nitrate solution, and uniformly mixing to obtain the Au @ Ag core-shell structure nano-particle.
(3) Taking a centrifuge tube, adding the aptamer with the concentration of 10 nM-50 nM and the NaCl with the concentration of 10 nM-50 nM into 1500 muL of PBS buffer solution, adding the sample to be tested, mixing, stabilizing at room temperature for 20min, adding 500 muL of nano solution into the centrifuge tube, and scanning the ultraviolet visible absorption spectrum.
Since the amount of aptamer and NaCl directly affect the results of the assay, to select the optimal amount, 6 2mL centrifuge tubes were taken, 1500. mu.L of phosphate buffer (0.2M PBS, pH 7.4) was added, NaCl was added to each tube to give final concentrations of 0,10, 20, 30, 40 and 50nM, respectively, and 500. mu.L of the nano-solution was added thereto. According to the ultraviolet-visible spectrum, when the concentration of NaCl is 30nM, the absorption value is minimum, the agglomeration degree of the nano particles is maximum, and the optimal concentration of NaCl is 30 nM.
And then taking 6 centrifuge tubes, adding 1500 mu L of PBS buffer solution and NaCl with the final concentration of 30nM into the centrifuge tubes, adding the aptamer into each centrifuge tube to enable the final concentration to be 0,10, 20, 30, 40 and 50nM respectively, mixing, stabilizing at room temperature for 20min, then adding 500 mu L of nano solution into each centrifuge tube, and obtaining the optimal concentration to be 30nM according to the detection result.
Therefore, 30nM was selected as the optimum reaction condition for both NaCl and aptamer concentration.
(4) Determining the concentration of kanamycin in the sample according to a linear equation:
y＝0.00011x+0.39901，y(y＝A519/A410) The ultraviolet absorption value ratio under different kanamycin concentrations, x is the corresponding kanamycin concentration, and the linear correlation>0.995, a linear range of 80-5000nmol/L, a detection limit of 60 nM.
Establishing a composite reaction and a standard curve:
taking 10 centrifuge tubes, adding aptamer with the concentration of 30nM and NaCl with the concentration of 30nM into 1500 uL PBS buffer solution, adding kanamycin to make the final concentration respectively 0nM, 10nM, 100nM, 500nM, 1000nM, 2000nM, 4000nM, 5000nM, 6000nM and 7000nM, mixing and stabilizing at room temperature for 20min, then adding 500 uL nanometer solution into each centrifuge tube, scanning the ultraviolet visible absorption spectrum, drawing the standard curve of kanamycin concentration according to the absorption value measured under different kanamycin concentrations, the linear range of the sensor is 80-5000nmol/L, the detection limit is 60nM, the linear equation of the standard curve is y 0.00011x +0.39901, y (y is A) is519/A410) The ultraviolet absorption value ratio under different kanamycin concentrations, x is the corresponding kanamycin concentration, and the linear correlation>0.995。
Determination of kanamycin specificity:
taking 5 centrifuge tubes, adding aptamer with the concentration of 30nM and NaCl with the concentration of 30nM into 1500 uL PBS buffer solution, adding analogs of ampicillin, neostreptothricin, gentamicin, tetracycline and kanamycin into each centrifuge tube respectively to enable the final concentration of the added analogs to be 3000nM, mixing and reacting at room temperature for 20 minutes, then adding 500 uL of nano solution into each centrifuge tube, and measuring the visible light absorption value after mixing and reacting.
The aptamer is a short-chain DNA or RNA sequence which is obtained by an exponential enrichment technology and has specific affinity to a target, can be selectively bound to a target such as a drug, a protein, a small molecule and the like, and has high specific binding force. The Au @ Ag core-shell structure nano particle has unique optical performance, can be applied to colorimetric detection of DNA, and is more stable than the ordinary nano particle in property. The Au @ Ag core-shell structure nano particle is yellow, and when a proper salt solution is added into the Au @ Ag core-shell structure nano particle, the nano particle is induced to aggregate, so that the color of the solution is changed, and finally the solution is red. Based on the functional DNA principle, development of biosensors by combining nanomaterials is a hot point of research.
The action of kanamycin and nucleic acid and the characteristic analysis of the nanoparticles show that: the dispersion state can affect the optical property of the Au @ Ag core-shell structure nano-particle, when single-stranded DNA is attached to the surface of the nano-particle, the single-stranded DNA has a protection effect on the nano-particle to disperse the nano-particle, the nano-particle loses the protection of nucleic acid along with the combination of kanamycin and the single-stranded DNA, and the nano-particle is agglomerated into larger particles under a certain salt concentration, so that the surface plasmon resonance absorption of the nano-particle is affected, and the intensity of ultraviolet absorption peaks at different wavelengths is weakened or enhanced and corresponding color changes are caused.
The method can realize simple, rapid and high-sensitivity detection of the kanamycin in the food, and can meet the detection requirement of an actual sample on the kanamycin.
Drawings
FIG. 1 is an absorption spectrum diagram.
Fig. 2 is a standard graph.
FIG. 3 is a diagram of specificity analysis.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
(1) Designing and synthesizing the corresponding aptamer fragment:
a DNA fragment capable of binding to kanamycin with specific affinity was designed, and the sequence was prepared by a DNA synthesizer.
DNA：5’-AGA TGG GGG TTG AGG CTA AGC CGA-3’
(2) Preparing Au @ Ag core-shell structure nanoparticles:
reducing silver nitrate with trisodium citrate to obtain gold nanoparticles with the particle size of about 25nm and the maximum absorption peak of about 523nm, concentrating the prepared gold nanoparticles by 10 times, cooling to room temperature, and storing at 4 ℃ for later use. Mixing 500 mu L0.1M phosphate buffer solution, 100 mu L of 1% polyvinylpyrrolidone solution and 50 mu L0.1M ascorbic acid, adding concentrated nanogold, adding 1mM silver nitrate solution, and uniformly mixing to obtain the Au @ Ag core-shell structure nano-particle.
(3) And (3) optimizing conditions:
in view of the direct effect of the amount of aptamer and NaCl on the assay, to optimize the experimental conditions, 6 2mL centrifuge tubes were taken, 1500. mu.L of phosphate buffer (0.2M PBS, pH 7.4) was added, NaCl was added to each tube to give final concentrations of 0,10, 20, 30, 40 and 50nM, respectively, and 500. mu.L of the nanosol was added. According to the ultraviolet-visible spectrum, when the concentration of NaCl is 30nM, the absorption value is minimum, the agglomeration degree of the nano particles is maximum, and the optimal concentration of NaCl is 30 nM. And then taking 6 centrifuge tubes, adding 1500 mu L of PBS buffer solution and NaCl with the final concentration of 30nM into the centrifuge tubes, adding the aptamer into each centrifuge tube to enable the final concentration to be 0,10, 20, 30, 40 and 50nM respectively, mixing, stabilizing at room temperature for 20min, then adding 500 mu L of nano solution into each centrifuge tube, and obtaining the optimal concentration to be 30nM according to the detection result. Therefore, in the following experiments, 30nM of NaCl and aptamer were used as the optimal reaction conditions for measuring kanamycin in the solution.
(4) Establishing a composite reaction and a standard curve:
10 centrifuge tubes were taken, aptamer concentration (30nM) and NaCl (30nM) were added to 1500. mu.L of PBS buffer, kanamycin was added to give final concentrations of 0,10,100,500, 1000,2000,4000,5000,6000,7000nM, respectively, mixed and stabilized at room temperature for 20min, 500. mu.L of the nano solution was added to each of the centrifuge tubes, and the UV-visible absorption spectrum was scanned, and the spectrum is shown in FIG. 1, and a standard curve of kanamycin concentration was plotted based on the absorption values measured at different kanamycin concentrations, as shown in FIG. 2. The linear range of the sensor is 80-5000nmol/L, the detection limit is 60nM, the linear equation of the standard curve is y ═ 0.00011x +0.39901, and y (y ═ A-519/A410) The ultraviolet absorption value ratio under different kanamycin concentrations, x is the corresponding kanamycin concentration, and the linear correlation>0.995。
(5) Determination of kanamycin specificity: ampicillin, neostreptothricin, gentamicin, tetracycline
Taking 5 centrifuge tubes, adding aptamer (30nM) and NaCl (30nM) into 1500. mu.L of PBS buffer, adding ampicillin, neostreptothricin, gentamicin, tetracycline and kanamycin into each centrifuge tube respectively to make the final concentration of the added analogues to be 3000nM, mixing and reacting at room temperature for 20 minutes, then adding 500. mu.L of nano solution into each centrifuge tube, and measuring the visible light absorption value after mixing and reacting. From the experimental results, as shown in fig. 3, it is known that the kanamycin detection sensor based on the colorimetric detection technique has high specificity.
(6) Determination of the actual addition sample: kanamycin was added to tap water samples at 200, 400, 1000 and 2000nM, respectively.
4 centrifuge tubes were taken, aptamer concentration (30nM) and NaCl (30nM) were added to 1500. mu.L of PBS buffer, and then the above tap water samples were added, respectively, mixed and stabilized at room temperature for 20min, and then 500. mu.L of the nano solution was added to each centrifuge tube, and its UV-VIS absorption spectrum was scanned to determine kanamycin concentration in the sample according to the above linear equation, as shown in Table 1 below.
As can be seen from Table 1, the addition recovery rate of kanamycin in an actual sample determined by the Au @ Ag core-shell structure based colorimetric kanamycin detection method is between 98.25% and 104.40%, and the kanamycin detection requirement of the actual sample can be met.
TABLE 1 determination of kanamycin water samples
The above embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation fall within the protection scope of the present invention.
<110> institute of technology and technology
<120> method for colorimetric detection of kanamycin based on Au @ Ag core-shell structure
<130> 20180710
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213> Artificial Synthesis
<400> 1
agatgggggt tgaggctaag ccga
Claims (4)
1. A colorimetric kanamycin detection method based on an Au @ Ag core-shell structure is characterized by comprising the following steps:
(1) designing and synthesizing nucleic acid aptamer fragments;
designing a DNA fragment capable of binding to kanamycin with specific affinity, and preparing a sequence by a DNA synthesizer, wherein the DNA: 5'-AGA TGG GGG TTG AGG CTA AGC CGA-3'
(2) Preparing Au @ Ag core-shell structure nanoparticles;
(3) taking a centrifuge tube, adding an aptamer with the concentration of 10 nM-50 nM and NaCl with the concentration of 10 nM-50 nM into 1500 muL of PBS buffer solution, adding a sample to be detected, mixing and stabilizing at room temperature, then adding 500 muL of Au @ Ag core-shell structure nano solution into the centrifuge tube, and scanning the ultraviolet visible absorption spectrum;
(4) determining the concentration of kanamycin in the sample according to a linear equation: y =0.00011x +0.39901, y is the ratio of the UV absorbance at different kanamycin concentrations, x is the corresponding kanamycin concentration, the linear correlation is >0.995, the linear range is 80-5000nmol/L, the detection limit is 60 nM.
2. The method for colorimetric detection of kanamycin based on Au @ Ag nucleocapsid structure according to claim 1, characterized in that in the step (3), the concentration of aptamer is 30nM, and the concentration of NaCl is 30 nM.
3. The method for colorimetric detection of kanamycin based on the Au @ Ag core-shell structure according to claim 1, characterized in that a standard curve corresponding to the linear equation is established: 10 centrifuge tubes were taken, aptamer at a concentration of 30nM and NaCl at a concentration of 30nM were added to 1500. mu.L of PBS buffer, adding kanamycin to give final concentrations of 0nM, 10nM, 100nM, 500nM, 1000nM, 2000nM, 4000nM, 5000nM, 6000nM, 7000nM, mixing, stabilizing at room temperature for 20min, then 500 mu L of Au @ Ag core-shell structure nano solution is added into each centrifugal tube, the ultraviolet visible absorption spectrum of the nano solution is scanned, a standard curve of kanamycin concentration was plotted based on the absorbance measured at different kanamycin concentrations, with a linear range of 80-5000nmol/L and a detection limit of 60nM, and the linear equation of the standard curve was y =0.00011x +0.39901, y is the ratio of the UV absorbance at different kanamycin concentrations, x is the corresponding kanamycin concentration, and the linear correlation was > 0.995.
4. The method for colorimetric detection of kanamycin based on Au @ Ag core-shell structure according to claim 1, characterized in that the kanamycin specificity determination method comprises the following steps: taking 5 centrifuge tubes, adding aptamer with the concentration of 30nM and NaCl with the concentration of 30nM into 1500 muL PBS buffer solution, adding analogs of ampicillin, neostreptothricin, gentamicin, tetracycline and kanamycin into each centrifuge tube respectively to enable the final concentration of the added analogs to be 3000nM, mixing and reacting for 20 minutes at room temperature, then adding 500 muL of Au @ Ag core-shell structure nano solution into each centrifuge tube, and measuring the visible light absorption value after mixing and reacting.
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