CN111380857A - Method for detecting ciprofloxacin in milk - Google Patents
Method for detecting ciprofloxacin in milk Download PDFInfo
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- CN111380857A CN111380857A CN201811653955.1A CN201811653955A CN111380857A CN 111380857 A CN111380857 A CN 111380857A CN 201811653955 A CN201811653955 A CN 201811653955A CN 111380857 A CN111380857 A CN 111380857A
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
Abstract
The invention discloses a method for detecting ciprofloxacin in milk, which comprises the following steps: deproteinizing a milk sample to obtain a solution to be detected; placing a surface enhancing reagent in the container; placing the liquid to be detected in a container; placing a flocculant in a container; uniformly mixing the solution in the container to obtain a mixed solution; performing Raman spectrum scanning on the mixed solution to obtain a Raman spectrum of the mixed solution, and determining whether ciprofloxacin exists in the milk based on a characteristic peak of the obtained Raman spectrum; the method adopts the surface enhancing reagent and the flocculating agent as SERS enhancing reagents, can enhance the SERS effect of the ciprofloxacin in the milk, obtain stronger ciprofloxacin Raman signals, greatly improve the detection sensitivity of the ciprofloxacin in the milk, and has lower detection lower limit of the ciprofloxacin.
Description
Technical Field
At least one embodiment of the present disclosure relates to a method for detecting ciprofloxacin in milk, and more particularly, to a method for detecting ciprofloxacin in milk.
Background
Ciprofloxacin is a quinolone veterinary drug and is often added into feed of food livestock and poultry animals to reduce the incidence of animal diseases. Excessive ciprofloxacin in the feed can remain in downstream food, and the human health can be threatened by eating a large amount of food with high residual ciprofloxacin. Therefore, the establishment of the method for detecting ciprofloxacin in food is very important for controlling the residual quantity of ciprofloxacin and protecting the health of human bodies.
Milk is a food possibly containing ciprofloxacin residues, and ciprofloxacin detection methods aiming at milk matrixes are divided into a laboratory method and a quick detection method. The laboratory method mainly comprises a liquid chromatogram-tandem mass spectrometry method, and is detailed in GB/T22985-2008 milk and milk powder for determining the residual quantity of enrofloxacin, danofloxacin, ciprofloxacin, sarafloxacin, orbifloxacin, difloxacin and marbofloxacin.
The fast detection method mainly comprises a colloidal gold-based method and a Surface Enhanced Raman Spectroscopy (SERS) -based method, wherein the colloidal gold method has the characteristic of high sensitivity, but has relatively high cost and cannot quantitatively detect fluoroquinolone drug residues in milk by colloidal gold immunochromatography (wanyuping, Zhao Zheng Miao, Tiantian sweet, Wankunlong, Konjingpeng. 2012 (12): 50-54.). And the common method based on the Surface Enhanced Raman Spectroscopy (SERS) has the defects of low detection sensitivity and incapability of detecting ciprofloxacin with low content in milk.
Disclosure of Invention
An object of the present disclosure is to solve at least one aspect of the above problems and disadvantages in the related art.
At least one embodiment of the disclosure provides a method for detecting ciprofloxacin in milk, which can detect ciprofloxacin with low residual quantity and greatly improve the detection sensitivity of ciprofloxacin in milk.
At least one embodiment of the present disclosure provides a method of detecting ciprofloxacin in milk, comprising the steps of: deproteinizing a milk sample to obtain a solution to be detected; placing a surface enhancing reagent in the container; placing the liquid to be detected in a container; placing a flocculant in a container; uniformly mixing the solution in the container to obtain a mixed solution; and performing Raman spectrum scanning on the mixed solution to obtain a Raman spectrum of the mixed solution, and determining whether the ciprofloxacin exists in the milk based on characteristic peaks of the obtained Raman spectrum.
According to an embodiment of one aspect of the present disclosure, the surface enhancing agent includes at least one of a nanogold sol agent and a nanosilver sol agent.
According to an embodiment of one aspect of the present disclosure, the flocculant includes an electrolyte solution containing halogen ions.
According to an embodiment of the present disclosure, the halide ions include at least one of fluoride, chloride, bromide, and iodide.
According to an embodiment of the present disclosure, the concentration of the halide ion ranges from 1 mmol.L-1-100mmol·L-1。
According to one embodiment of the present disclosure, the volume ratio of the surface enhancing reagent to the liquid to be tested ranges from 10: 1 to 50: 1, and the volume ratio of the electrolyte solution containing halogen ions to the liquid to be tested ranges from 10: 1 to 20: 1.
According to another embodiment of the present disclosure, the volume of the solution to be measured ranges from 10 μ L to 100 μ L, the volume of the surface enhancing reagent ranges from 300 μ L to 700 μ L, and the volume of the electrolyte solution containing halogen ions ranges from 10 μ L to 100 μ L.
According to an embodiment of the present disclosure, the deproteinizing a milk sample to obtain a test solution includes: placing a milk sample in a centrifuge tube; placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution; centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant; taking the supernatant as the solution to be detected.
According to another embodiment of the present disclosure, the deproteinizing a milk sample to obtain a test solution comprises: placing a milk sample in a centrifuge tube; placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution; putting the alcohol organic matters into a centrifugal tube to precipitate protein in the milk sample; centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant; taking the supernatant in the centrifuge tube as the liquid to be tested.
According to another embodiment of the present disclosure, a method for deproteinizing a milk sample to obtain a test solution comprises: placing a milk sample in a centrifuge tube; placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution; putting the alcohol organic matters into a centrifugal tube to precipitate protein in the milk sample; placing an organic acid solution in a centrifuge tube to precipitate proteins in the milk sample; centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant; taking the supernatant in the centrifuge tube as the liquid to be tested.
According to one embodiment of the present disclosure, the acidic solution is an organic acid solution such as glacial acetic acid or formic acid, or an inorganic acid aqueous solution such as hydrochloric acid, sulfuric acid, or nitric acid having a pH of 1 to 2.
According to an embodiment of the present disclosure, the alcohol organic is ethanol or propanol.
According to one embodiment of the present disclosure, the organic acid solution is trichloroacetic acid or trifluoroacetic acid.
According to one embodiment of the present disclosure, the concentration of the organic acid solution ranges from 1% to 10%.
According to one embodiment of the present disclosure, the volume ratio of the milk sample to the acidic solution ranges from 10: 1 to 20: 1.
According to an embodiment of the present disclosure, a volume ratio of the milk sample to the alcoholic organic substance is 5: 1.
According to one embodiment of the present disclosure, the volume ratio of the milk sample to the organic acid solution is 5: 1.
According to an embodiment of one aspect of the present disclosure, centrifuging a solution in a centrifuge tube comprises: the solution in the centrifuge tube was centrifuged for 2 minutes at 10000 rpm in a centrifuge.
According to one embodiment of the present disclosure, determining whether ciprofloxacin is present in milk based on characteristic peaks of the obtained raman spectrum comprises: there is a Raman shift of 1266 + -5 cm in the obtained Raman spectrum-1Determining that the ciprofloxacin exists in the milk when the characteristic peak is reached.
The various embodiments of the disclosure provide a method for detecting ciprofloxacin in milk, and a surface enhancing reagent and a flocculating agent are adopted to enhance the SERS effect of ciprofloxacin in milk, so that a stronger annular ciprofloxacin Raman signal can be obtained, ciprofloxacin with lower content in milk can be detected, and the detection sensitivity of ciprofloxacin in milk is greatly improved.
Other objects and advantages of the present disclosure will become apparent from the following description of the disclosure, which is made with reference to the accompanying drawings, and can assist in a comprehensive understanding of the disclosure.
Drawings
FIG. 1 shows a schematic flow diagram of a method of detecting ciprofloxacin in milk, in accordance with embodiments of the present disclosure;
FIG. 2 shows a schematic flow diagram of a deproteinization operation on a milk sample to obtain a liquid to be tested according to an embodiment of the present disclosure;
FIG. 3 shows Raman spectra of milk with gradually increasing ciprofloxacin spiking concentrations and a ciprofloxacin spiking solution, according to embodiments of the present disclosure;
fig. 4 shows a comparison graph of the detection results of the method of detecting ciprofloxacin in milk and the conventional method according to an embodiment of the present disclosure.
Detailed Description
The technical solution of the present disclosure is further specifically described below by way of examples and with reference to the accompanying drawings. In the specification, the same or similar reference numerals denote the same or similar components. The following description of the embodiments of the present disclosure with reference to the accompanying drawings is intended to explain the general inventive concept of the present disclosure and should not be construed as limiting the present disclosure.
Furthermore, in the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the disclosure. It may be evident, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are shown in schematic form in order to simplify the drawing.
According to the general inventive concept of the present disclosure, a method of detecting ciprofloxacin in milk includes the steps of: deproteinizing a milk sample to obtain a solution to be detected; placing a surface enhancing reagent in a container (e.g., a sample vial); placing the liquid to be detected in a container; placing a flocculant in a container; uniformly mixing the solution in the container to obtain a mixed solution; and performing Raman spectrum scanning on the mixed solution to obtain a Raman spectrum of the mixed solution, and determining whether the ciprofloxacin exists in the milk based on characteristic peaks of the obtained Raman spectrum.
FIG. 1 shows a schematic flow diagram of a method of detecting ciprofloxacin in milk, in accordance with embodiments of the present disclosure; FIG. 2 shows a schematic flow diagram of a deproteinization operation performed on a milk sample to obtain a solution to be tested according to an embodiment of the present disclosure.
In an exemplary embodiment of the present disclosure, as shown in fig. 1, a method of detecting ciprofloxacin in milk comprises the steps of: deproteinizing a milk sample to obtain a solution to be detected; placing a surface enhancing reagent in the container; placing the liquid to be detected in a container; placing a flocculant in a container; uniformly mixing the solution in the container to obtain a mixed solution; and performing Raman spectrum scanning on the mixed solution to obtain a Raman spectrum of the mixed solution, and determining whether the ciprofloxacin exists in the milk based on characteristic peaks of the obtained Raman spectrum. The container may be a vial. The detection method of the present disclosure is not limited to ciprofloxacin, and is also applicable to detection of illegally added substances in milk like ciprofloxacin and the like.
In one embodiment, the surface enhancing agent comprises at least one of a nanogold sol agent and a nanosilver sol agent. In one embodiment, the surface enhancing agent is a nanogold sol agent. In another embodiment, the surface enhancing agent is a nanosilver sol agent. In yet another embodiment, the surface enhancing agent is a mixture of a nanogold sol agent and a nanosilver sol agent. In the previous examples, the nanogold sol reagent has the best effect on enhancing the SERS phenomenon of ciprofloxacin in milk. The surface enhancing agent may also be a solution containing other nanoparticles.
In one embodiment, the flocculating agent comprises at least one of an electrolyte solution or an acidic solution containing halide ions. In one embodiment, the flocculant is an electrolyte solution containing halide ions. According to an aspect of an embodiment of the present disclosure, the halide ion includes at least one of fluoride ion, chloride ion, bromide ion, and iodide ion. The electrolyte solution containing halogen ions may contain only one of fluorine ions, chlorine ions, bromine ions, and iodine ions, or may contain at least two of the halogen ions. In one embodiment, when the flocculant is an electrolyte solution containing halogen ions, the concentration of the halogen ions is in the range of 1 mmol.L-1-100mmol·L-1. In another embodiment, the flocculant is an acidic solution comprising at least one of nitric acid, hydrochloric acid.
According to an aspect of an embodiment of the present disclosure, when the flocculant is an electrolyte solution containing halogen ions, a volume ratio of the surface enhancing reagent to the liquid to be tested ranges from 10: 1 to 50: 1, and a volume ratio of the electrolyte solution containing halogen ions to the liquid to be tested ranges from 10: 1 to 20: 1.
According to another aspect of an embodiment of the present disclosure, the volume of the solution to be measured ranges from 10 μ L to 100 μ L, the volume of the surface enhancing reagent ranges from 300 μ L to 700 μ L, and the volume of the electrolyte solution containing halogen ions ranges from 10 μ L to 100 μ L.
In one embodiment, the order in which the test solution, surface enhancing reagent, and flocculating agent are placed in the vessel is: firstly adding a surface enhancing reagent, then adding the liquid to be detected, and finally adding a flocculating agent. In this embodiment, after sequentially placing the surface enhancing reagent and the test solution into the container, the container may be shaken manually or vortexed using a vortexer for a period of time (e.g., 10 seconds) to mix the surface enhancing reagent and the test solution uniformly. After the surface enhancing reagent and the liquid to be tested are mixed uniformly, the flocculating agent is added, and similarly, the container may be shaken manually or vortexed using a vortexer for a period of time (e.g., 10 seconds) to uniformly mix the flocculating agent with the solution already in the container. According to the method, the surface enhancing reagent and the flocculating agent are adopted to treat the liquid to be detected, the SERS effect of the ciprofloxacin in the milk can be enhanced, so that a strong ciprofloxacin Raman signal can be obtained when the mixed solution is subjected to Raman spectrum scanning, even the ciprofloxacin with low content can be detected, and the detection sensitivity of the ciprofloxacin in the milk is greatly improved.
In another embodiment, the order in which the test solution, surface enhancing reagent, and flocculating agent are placed in the vessel is: the liquid to be tested is added firstly, then the surface enhancing reagent is added, and finally the flocculating agent is added. In this embodiment, after the liquid to be tested and the surface enhancing reagent are sequentially placed in the container, the container may be shaken manually or vortexed using a vortexer for a period of time (e.g., 10 seconds) to uniformly mix the liquid to be tested and the surface enhancing reagent. After the liquid to be tested and the surface enhancing reagent are mixed uniformly, the flocculant is placed into the container, and similarly, the container can be shaken manually or vortexed by a vortexing instrument for a period of time (for example, 10 seconds) to thoroughly and uniformly mix the flocculant with the existing solution in the container. The embodiment can also achieve the effect of enhancing the SERS effect of the ciprofloxacin in the milk, so that a stronger ciprofloxacin Raman signal is obtained, the detection sensitivity of the ciprofloxacin in the milk is improved, and the lower detection limit of the ciprofloxacin is reduced.
In the two embodiments, it can be seen that the surface enhancing reagent of the solution containing the gold nanoparticles or silver nanoparticles is mixed with the liquid to be detected, and then the flocculating agent is added to the mixed solution, and the two embodiments have better enhancing effect on the raman signal. The enhancement principle is as follows: the premise of triggering the SERS phenomenon is that ciprofloxacin molecules in the liquid to be detected are fully adsorbed on the surfaces of the nanoparticles, and the solution containing the nanoparticles and the liquid to be detected are mixed firstly, namely, the ciprofloxacin molecules are fully adsorbed on the nanoparticles in the process of mixing the solution containing the nanoparticles and the liquid to be detected. And then, a flocculating agent is added, so that clustering effect can be generated on the nanoparticles, the distance between the nanoparticles is remarkably shortened, and when the distance between the nanoparticles is reduced to a nanoscale, the SERS phenomenon of the ciprofloxacin can be remarkably enhanced, so that a reinforced ciprofloxacin Raman signal is obtained.
In an embodiment, the liquid to be detected and the flocculant are mixed first, and then the surface enhancing reagent is added, the method of the embodiment also has a certain enhancing effect on the signal of the raman signal, but the adsorption process of the ciprofloxacin and the clustering process of the nanoparticles are carried out simultaneously, so that the ciprofloxacin cannot be completely adsorbed on the nanoparticles, and the enhancing effect of the raman signal of the ciprofloxacin of the embodiment is not as good as that of the two embodiments of adding the surface enhancing reagent and then the flocculant. In another embodiment, when the flocculant is added into the liquid to be detected first and then the liquid to be detected is added finally, the method of this embodiment will also have a certain enhancement effect on the signal of the raman signal, but since the nanoparticles will cluster and settle first, the ciprofloxacin molecules of milk cannot be completely adsorbed onto the surface of the nanoparticles before clustering, so that the raman signal of ciprofloxacin in this embodiment is also inferior to the enhancement effect of the two embodiments of adding the flocculant after adding the surface enhancing agent first. In yet another embodiment, the liquid to be tested, the surface enhancing reagent and the flocculating agent are placed in the container at the same time, and the method of this embodiment will also have a certain enhancement effect on the raman signal, but the method of this embodiment will also have a less enhancement effect on the raman signal of ciprofloxacin than the two embodiments in which the surface enhancing reagent is added and then the flocculating agent is added.
In one embodiment, a milk sample is deproteinized to obtain a test solution comprising: placing a milk sample in a centrifuge tube; placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution; centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant; taking the supernatant as the solution to be detected. Milk contains a large amount of protein impurities, and the protein causes strong Raman signal interference on ciprofloxacin, so that the protein in the milk needs to be efficiently removed on the premise of retaining the ciprofloxacin. In the method of the embodiment, the acidic solution can precipitate the protein in the milk, and the addition of the acidic solution can also adjust the pH value of the milk sample to promote the dissolution of the ciprofloxacin in the acidic aqueous solution because the ciprofloxacin has the best solubility in the acidic aqueous solution environment. The solution in the centrifuge tube can be centrifuged for 2 minutes by a centrifuge with a rotation speed of 10000 rpm or by standing. After the centrifugation is completed, the protein is precipitated to the lower layer, and the supernatant is the ciprofloxacin-containing solution. According to an aspect of an embodiment of the present disclosure, the acidic solution is an organic acid solution of glacial acetic acid, formic acid, or the like, or an inorganic acid aqueous solution of hydrochloric acid, sulfuric acid, nitric acid, or the like having a pH of 1 to 2.
In another embodiment, a milk sample is deproteinized to obtain a test solution comprising: placing a milk sample in a centrifuge tube; placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution; putting the alcohol organic matters into a centrifugal tube to precipitate protein in the milk sample; centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant; taking the supernatant in the centrifuge tube as the liquid to be tested. In the method of this example, the protein was precipitated simultaneously by adding an acidic solution to facilitate the dissolution of ciprofloxacin in the acidic aqueous solution. The acidic aqueous solution can only precipitate a part of protein, and the function of adding the alcohol organic substance is to further precipitate the protein which is not precipitated, and simultaneously leave the ciprofloxacin in the milk in the acidic aqueous solution, thereby achieving the effect of efficiently removing the protein and simultaneously keeping the ciprofloxacin, and obviously reducing the interference of the protein on the raman signal caused by the ciprofloxacin. In the method of this embodiment, the step of mixing the milk sample with the acidic solution must precede the step of mixing the milk sample with the alcoholic organic substance. On the contrary, if the step of adding the acidic solution into the milk sample is omitted or the sequence of the step and the step of adding the alcohol organic matter into the milk sample is changed, ciprofloxacin is co-precipitated with the protein in the process of protein precipitation, and further, when the raman detection is performed on the mixed solution, the raman signal of ciprofloxacin cannot be obtained. The order of adding the milk sample and the acidic solution to the centrifuge tube can be changed. In the method of this embodiment, the solution in the centrifuge tube can be mixed by manually shaking the centrifuge tube or vortexing the centrifuge tube using a vortexer for a period of time (e.g., 10 seconds) until the solution in the centrifuge tube is thoroughly mixed. According to an aspect of an embodiment of the present disclosure, the acidic solution is an organic acid solution of glacial acetic acid, formic acid, or the like, or an inorganic acid aqueous solution of hydrochloric acid, sulfuric acid, nitric acid, or the like having a pH of 1 to 2. In another aspect of an embodiment of the disclosure, the alcoholic organic is ethanol or propanol. Ethanol is relatively low in cost and is most commonly used. Similarly, the solution in the centrifuge tube may be centrifuged for 2 minutes by a centrifuge at 10000 rpm to precipitate the protein, and the conventional standing method may be used.
In yet another embodiment, a method of deproteinizing a milk sample to obtain a test solution comprises: placing a milk sample in a centrifuge tube; placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution; putting the alcohol organic matters into a centrifugal tube to precipitate protein in the milk sample; placing an organic acid solution in a centrifuge tube to precipitate proteins in the milk sample; centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant; taking the supernatant in the centrifuge tube as the liquid to be tested. In the method of this example, the PH of the milk sample was adjusted by adding an acidic solution to promote ciprofloxacin to dissolve in the acidic aqueous solution while precipitating a portion of the protein, and then adding an alcoholic organic to precipitate another portion of the protein in the milk sample. Because some proteins in the milk cannot be precipitated under the action of the acidic aqueous solution and the alcohol organic substance, in the embodiment, after the alcohol organic substance is added, the organic acid solution is continuously added, and the proteins which cannot be precipitated by the acidic aqueous solution and the alcohol organic substance are precipitated by the organic acid solution, so that the proteins in the milk sample are more thoroughly precipitated. According to the method, the protein is precipitated by adopting the acidic solution, the alcohol organic substance and the organic acid solution, so that the protein in the milk sample can be removed more comprehensively, and the signal interference of the protein on the ciprofloxacin in the subsequent Raman detection is avoided, so that the sensitivity is further improved. In this embodiment, the solution in the centrifuge tube can be mixed by manually shaking the centrifuge tube or vortexing the centrifuge tube using a vortexer for a period of time (e.g., 10 seconds or 30 seconds) until the solution in the centrifuge tube is thoroughly mixed. According to an aspect of an embodiment of the present disclosure, the acidic solution is an organic acid solution of glacial acetic acid, formic acid, or the like, or an inorganic acid aqueous solution of hydrochloric acid, sulfuric acid, nitric acid, or the like having a pH of 1 to 2. In another aspect of an embodiment of the disclosure, the alcoholic organic is ethanol or propanol. In yet another aspect of embodiments of the present disclosure, the organic acid solution is trichloroacetic acid or trifluoroacetic acid at a concentration of 1% to 10%. Similarly, the solution in the centrifuge tube may be centrifuged for 2 minutes by a centrifuge at 10000 rpm or more to precipitate the protein. In one embodiment, a milk sample of 1 ml volume is used, hydrochloric acid of 50 μ l volume is used as the acidic solution, ethanol of 200 μ l volume is used as the alcoholic organic substance, and trichloroacetic acid of 5% concentration is used as the organic acid solution of 200 μ l volume.
According to one aspect of the embodiments of the present disclosure, the placing of the milk sample in a centrifuge tube and the placing of the acidic solution in the centrifuge tube to precipitate the proteins in the milk sample while dissolving the ciprofloxacin in the milk sample in the acidic solution specifically includes: placing the milk sample and the acid solution in a volume ratio of 10: 1-20: 1 into a centrifuge tube. According to another aspect of the embodiments of the present disclosure, the step of placing the alcoholic organic substance in the centrifugal tube to precipitate the protein in the milk sample specifically includes: the alcohol organics with the volume of 1/5 of the milk sample were placed in a centrifuge tube. According to yet another aspect of embodiments of the present disclosure, placing an organic acid solution in a centrifuge tube to precipitate proteins in a milk sample specifically includes: the organic acid solution with the volume of 1/5 milk sample was placed in a centrifuge tube.
In one embodiment, the raman spectroscopy scanning of the mixed solution is specifically: and performing Raman spectrum scanning on the mixed solution by using a laser source Raman spectrometer. In one embodiment, determining whether ciprofloxacin is present in the milk based on the characteristic peaks of the obtained raman spectrum comprises: there is a Raman shift of 1266 + -5 cm in the obtained Raman spectrum-1Determining that the ciprofloxacin exists in the milk when the characteristic peak is reached. That is, the Raman shift is 1266. + -.5 cm-1The characteristic peak of (2) is used for qualitative determination of ciprofloxacin, and the intensity of the characteristic peak is used for quantitative determination of ciprofloxacin. In another embodiment, the laser source raman spectroscopy instrument is a model RT5000 or RT6000 785nm laser source raman spectroscopy instrument.
Fig. 3 shows SERS raman spectra of milk with progressively increasing ciprofloxacin spiking concentrations and ciprofloxacin spiking solutions according to embodiments of the present disclosure; fig. 4 shows a comparison graph of the detection results of the method of detecting ciprofloxacin in milk and the conventional method according to an embodiment of the present disclosure.
As shown in FIG. 3, the standard ciprofloxacin solution, that is, the aqueous solution containing only ciprofloxacin, was found to have a Raman spectrum of 1266. + -.5 cm on the abscissa-1The characteristic peak of ciprofloxacin appeared in the range of (2), and the Raman shift was found to be 1266. + -.5 cm-1The characteristic peak of (A) is reasonable for the ciprofloxacin to be qualitatively determined.
Referring to the 6 raman lines above the cyprohexate star liquid line in fig. 3, it is shown that milk containing ciprofloxacin has its corresponding raman lines as the concentration of ciprofloxacin therein increases. According to the change rule of the 6 raman spectral lines, it can be seen that when the ciprofloxacin content in the milk is low, due to the interference of raman signals of impurities such as proteins, in the raman spectral line of the milk, the characteristic peak corresponding to the ciprofloxacin is almost invisible, that is, the raman signal of the ciprofloxacin in the milk is weak, the characteristic peak of the ciprofloxacin gradually appears along with the increase of the concentration of the ciprofloxacin in the milk, and when the concentration value reaches a certain degree, the raman spectral line of the milk containing the ciprofloxacin is very similar to the shape of the spectral line of the ciprofloxacin standard solution.
Referring to fig. 4, a graph showing a comparison of the detection results of the method for detecting ciprofloxacin in milk according to the embodiment of the present disclosure and the conventional method is shown. The conventional method adopts 1 mol.L-1Hydrochloric acid is used as a milk protein precipitator, only nano-silver colloid solution is used as an SERS enhancing reagent, and the peak position is 1384cm-1The method adopts the steps that hydrochloric acid, ethanol and trichloroacetic acid are sequentially added to serve as a milk protein precipitator, and a surface enhancing reagent of a nano gold colloid reagent and a flocculating agent of an electrolyte solution containing halogen ions are used as SERS enhancing reagents.
In fig. 4 there are 4 raman lines, which we name as a first raman line, a second raman line, a third raman line and a fourth raman line in order from top to bottom. The first raman line represents the raman spectrum of the SERS enhancing agent of this patent obtained by performing raman spectrum scanning on the SERS enhancing agent (nanogold sol agent and electrolyte solution containing halogen ions) used in the method according to the embodiment of the present disclosure, and the two peaks on the right side in the first raman line correspond to the characteristic peaks of the SERS enhancing agent (i.e. surface enhancing agent and flocculating agent). And the second raman spectral line represents the raman spectral scanning of the SERS enhancing reagent (nano silver colloid solution) adopted by the conventional method, and the two peaks on the left side in the second raman spectral line correspond to the characteristic peaks of the SERS enhancing reagent. The third raman line represents a raman line of the ciprofloxacin standard solution measured by the method of the embodiment of the present disclosure (using the nanogold sol reagent and the electrolyte solution containing the halogen ion as the SERS enhancing reagent). The fourth raman line represents the raman line of the ciprofloxacin standard solution measured by a conventional method (using a nano silver colloid solution as a SERS enhancing reagent).
Comparing the second raman line with the fourth raman line, when the SERS enhancing reagent (nano silver colloid solution) used in the conventional method is used to test the raman signal of the ciprofloxacin standard solution, in the fourth raman line, comparing the second raman line, the characteristic peak corresponding to the nano silver colloid solution still exists, and the intensity of the characteristic peak is almost the same as that of the characteristic peak in the second raman line, meanwhile, the intensity of the characteristic peak corresponding to the ring shaped sand star is very small, and the intensity of the characteristic peak of the ring shaped sand star is much smaller than that of the nano silver colloid solution, so that it can be seen that the detected nano silver raman signal is strong, and the detected raman signal of ciprofloxacin is weak or almost none, so that i can think that the enhancing reagent used in the conventional method hardly has the effect of enhancing the ciprofloxacin, but rather has good intensity as the background signal, and may adversely affect or interfere with the raman signal of the detected ciprofloxacin.
Comparing the first raman line and the third raman line, when the raman signal of the ciprofloxacin standard solution is tested by using the surface enhancing reagent and the flocculant (the nano gold sol reagent and the electrolyte solution containing halogen ions) used in the method of the embodiment of the disclosure, the characteristic peaks corresponding to the surface enhancing reagent and the flocculant (the nano gold sol reagent and the electrolyte solution containing halogen ions) in the third raman line are very little, while the characteristic peak intensity corresponding to the ciprofloxacin is particularly large, it can be seen that background signals of the nano gold particles and the halogen ions serving as the surface enhancing reagent and the flocculant are greatly attenuated or even not attenuated, while the signal of the ciprofloxacin signal is greatly increased.
According to the comparison of the raman spectral lines, the surface enhancement reagent of the nanogold sol reagent and the flocculant of the electrolyte solution containing the halogen ions adopted in the method disclosed by the embodiment of the disclosure can obviously enhance the raman signal of the ciprofloxacin signal, and the background signals of the surface enhancement reagent and the flocculant are very weak, so that the raman signal of the ciprofloxacin cannot be influenced or interfered, the sensitivity of the detection method disclosed by the patent is very high, and meanwhile, the lower limit of the ciprofloxacin which can be detected is also greatly reduced.
It will be appreciated by those skilled in the art that the embodiments described above are exemplary and can be modified by those skilled in the art, and that the structures described in the various embodiments can be freely combined without conflict in structure or principle.
While the present disclosure has been described in connection with the accompanying drawings, the embodiments disclosed in the drawings are intended to be illustrative of the preferred embodiments of the disclosure, and should not be construed as limiting the disclosure.
Although a few embodiments of the present general inventive concept have been shown and described, it will be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the general inventive concept, the scope of which is defined in the appended claims and their equivalents.
It should be noted that the word "comprising" does not exclude other elements or steps, and the words "a" or "an" do not exclude a plurality. Additionally, any element numbers of the claims should not be construed as limiting the scope of the disclosure.
Claims (19)
1. A method of detecting ciprofloxacin in milk, comprising:
deproteinizing a milk sample to obtain a solution to be detected;
placing a surface enhancing reagent in the container;
placing the liquid to be detected in a container;
placing a flocculant in a container;
uniformly mixing the solution in the container to obtain a mixed solution;
and performing Raman spectrum scanning on the mixed solution to obtain a Raman spectrum of the mixed solution, and determining whether the ciprofloxacin exists in the milk based on characteristic peaks of the obtained Raman spectrum.
2. The method for detecting ciprofloxacin in milk according to claim 1, wherein the surface enhancing reagent comprises at least one of a nano gold sol reagent and a nano silver sol reagent.
3. The method for detecting ciprofloxacin in milk according to claim 1, wherein the flocculating agent comprises an electrolyte solution containing halogen ions.
4. The method for detecting ciprofloxacin in milk according to claim 3, wherein the halogen ion comprises at least one of fluoride ion, chloride ion, bromide ion and iodide ion.
5. Method for detecting ciprofloxacin in milk according to claim 3, wherein the concentration of the halogen ion is in the range of 1 mmol-L-1-100mmol·L-1。
6. The method for detecting ciprofloxacin in milk according to claim 3, wherein the volume ratio of the surface enhancing reagent to the liquid to be detected is in the range of 10: 1 to 50: 1, and the volume ratio of the electrolyte solution containing halogen ions to the liquid to be detected is in the range of 10: 1 to 20: 1.
7. The method for detecting ciprofloxacin in milk according to claim 3, wherein the volume of the liquid to be detected is in a range of 10 μ L to 100 μ L, the volume of the surface enhancing reagent is in a range of 300 μ L to 700 μ L, and the volume of the electrolyte solution containing halogen ions is in a range of 10 μ L to 100 μ L.
8. The method for detecting ciprofloxacin in milk according to claim 1, wherein the deproteinizing the milk sample to obtain the liquid to be detected comprises:
placing a milk sample in a centrifuge tube;
placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution;
centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant;
taking the supernatant as the solution to be detected.
9. The method for detecting ciprofloxacin in milk according to claim 1, wherein the deproteinizing the milk sample to obtain the liquid to be detected comprises:
placing a milk sample in a centrifuge tube;
placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution;
putting the alcohol organic matters into a centrifugal tube to precipitate protein in the milk sample;
centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant;
taking the supernatant in the centrifuge tube as the liquid to be tested.
10. The method for detecting ciprofloxacin in milk according to claim 1, wherein the deproteinizing the milk sample to obtain the liquid to be detected comprises:
placing a milk sample in a centrifuge tube;
placing the acidic solution in a centrifuge tube to precipitate the proteins in the milk sample and simultaneously dissolve the ciprofloxacin in the milk sample in the acidic solution;
putting the alcohol organic matters into a centrifugal tube to precipitate protein in the milk sample;
placing an organic acid solution in a centrifuge tube to precipitate proteins in the milk sample;
centrifuging the solution in the centrifuge tube to obtain a solution comprising a supernatant;
taking the supernatant in the centrifuge tube as the liquid to be tested.
11. Method for detecting ciprofloxacin in milk according to any one of claims 8 to 10, characterized in that the acidic solution is an organic acid solution such as glacial acetic acid or formic acid or an inorganic acid aqueous solution such as hydrochloric acid, sulfuric acid or nitric acid having a pH of 1 to 2.
12. The method for detecting ciprofloxacin in milk according to claim 9 or 10, wherein the alcohol organic substance is ethanol or propanol.
13. The method for detecting ciprofloxacin in milk according to claim 10, wherein the organic acid solution is trichloroacetic acid or trifluoroacetic acid.
14. The method for detecting ciprofloxacin in milk according to claim 10, wherein the concentration of the organic acid solution is in the range of 1% -10%.
15. Method for detecting ciprofloxacin in milk according to any one of claims 8 to 10, characterized in that the volume ratio of the milk sample to the acidic solution ranges from 10: 1 to 20: 1.
16. The method for detecting ciprofloxacin in milk according to claim 9 or 10, wherein the volume ratio of the milk sample to the alcoholic organic substance is 5: 1.
17. The method for detecting ciprofloxacin in milk according to claim 10, wherein the volume ratio of the milk sample to the organic acid solution is 5: 1.
18. Method for detecting ciprofloxacin in milk according to any one of claims 8 to 10, wherein centrifuging the solution in a centrifuge tube comprises: the solution in the centrifuge tube was centrifuged for 2 minutes at 10000 rpm in a centrifuge.
19. The method for detecting ciprofloxacin in milk according to claim 1, wherein determining whether ciprofloxacin in the milk is present based on the characteristic peaks of the obtained Raman spectrum comprises:
there is a Raman shift of 1266 + -5 cm in the obtained Raman spectrum-1Determining that the ciprofloxacin exists in the milk when the characteristic peak is reached.
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