CN114958365A - Preparation method and application of periostracum cicadae carbon quantum dots - Google Patents
Preparation method and application of periostracum cicadae carbon quantum dots Download PDFInfo
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Abstract
The invention relates to a preparation method and application of periostracum cicada carbon quantum dots. According to the invention, the periostracum cicada carbon quantum dots (CT-CQDs) are prepared by using biomass periostracum cicada as a precursor material and combining a one-step hydrothermal synthesis method. The preparation method of the cicada slough carbon quantum dots comprises the following steps: (1) pulverizing periostracum Cicadae, and dissolving in water to obtain solution; (2) heating the solution obtained in the step (1); (3) and (3) cooling and centrifuging the product obtained after heating in the step (2), collecting supernatant, filtering, and dialyzing to obtain cicada slough carbon quantum dots, namely CT-CQDs. The carbon quantum dots of the cicada slough synthesized by the method can be used as a carbon quantum dot fluorescent probe, and can sensitively and selectively detect enrofloxacin in aquatic products. The invention has the advantages of no pollution, wide and cheap raw material source, simple and convenient operation and the like.
Description
Technical Field
The invention belongs to the technical field of preparation of carbon quantum dot fluorescent materials, and particularly relates to a preparation method and application of periostracum cicada carbon quantum dots.
Background
Enrofloxacin (Enrofloxacin, ENR), also known as ethylciprofloxacin, is a fluoroquinolone antibacterial (Fluoroquinolones, FQs). Since ENR has the advantages of a wide antimicrobial spectrum, a strong antimicrobial effect, and wide distribution, ENR is applied to the industries such as animal husbandry, aquaculture, poultry farming, and the like. However, the antibiotic is a double-edged sword, which has many hazards to human body and ecological environment, and is mainly expressed as follows: bacterial drug resistance, adverse reactions, superinfection, threat to food safety, harm to soil microorganisms and aquatic organisms and the like.
The current methods for detecting enrofloxacin mainly comprise: fluorescence spectrophotometry, enzyme-linked method, high performance liquid chromatography and mass spectrometry. Although the methods have good accuracy and sensitivity, the methods have the defects of expensive instruments, time-consuming analysis, complex operation, troublesome data processing and the like. And because animal products contain a large amount of protein and fat, the matrix is complex, and the samples are usually subjected to pretreatment such as precipitation, centrifugation, enrichment, nitrogen drying, redissolution and the like before detection and analysis. A large amount of toxic organic reagent is used in the treatment process, so that the safety and the environmental protection are reduced.
In view of the numerous hazards, in many countries and regions, in order to ensure the edible safety of aquatic products, the maximum residual limit standard in aquatic products has been established for ENR, for example, the maximum residual limit of ENR in crustacean aquatic products is 0.1 μ g/kg as specified in korea, and the maximum residual limit of ENR in finfish muscles is 100 μ g/kg as specified in the european union. At present, most of enrofloxacin residue detection is concentrated in livestock and poultry tissues and vegetables, and no report is provided about residue detection of enrofloxacin residue in aquatic products.
The method for detecting the enrofloxacin has the problems of low accuracy, incapability of detecting the enrofloxacin with lower concentration and the like. Therefore, in order to ensure the safety of human bodies and protect the ecological environment, it is necessary to develop a simple, easy-to-operate, green and environment-friendly method for monitoring the antibiotic residues in aquatic products.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a preparation method and application of a cicada slough carbon quantum dot. The cicada slough carbon quantum dots (CT-CQDs) provided by the invention have the advantages of higher fluorescence intensity, good accuracy, high sensitivity, wide and cheap raw material sources, and green and pollution-free synthesis process. The CT-CQDs are used as fluorescent probes, and the established fluorescence analysis method can sensitively and selectively detect ENR in aquatic products.
The technical scheme for solving the technical problems is as follows:
the invention provides a preparation method of periostracum cicadae carbon quantum dots, which comprises the following steps:
(1) pulverizing periostracum Cicadae, and dissolving in water to obtain solution;
(2) heating the solution obtained in the step (1);
(3) and (3) cooling and centrifuging the product obtained after heating in the step (2), collecting supernatant, filtering, and dialyzing to obtain cicada slough carbon quantum dots, namely CT-CQDs.
The beneficial effects of adopting the above scheme include: the preparation method provided by the invention takes the biomass cicada slough as a precursor material and combines a one-step hydrothermal method to synthesize the cicada slough carbon quantum dots (CT-CQDs). The periostracum cicada carbon quantum dots prepared by the method have the advantages of good dispersity, uniform particle size distribution and the like.
The invention adopts biomass precursor materials, has the advantages of reproducibility, rich content, easy obtainment and low price, avoids introducing other components in the synthesis process, and the like. Can be used as a cicada slough carbon quantum dot fluorescent probe (CT-CQDs fluorescent probe) and can be used for detecting the concentration of enrofloxacin in aquatic products. The invention adopts a one-step hydrothermal method, and has the advantages of cheap instrument, simple and convenient operation, environmental protection, rapidness and the like.
The periostracum cicadae carbon quantum dots can be used as a fluorescent probe, are novel fluorescent carbon nano materials, have the size of less than 20nm, and have the advantages of good accuracy, good selectivity to ENR, good linear relation, low detection limit (0.069 mu M), good water solubility, low price and the like. Compared with the traditional semiconductor quantum dot, the CQDs prepared based on the biomass periostracum cicada have the advantages of low toxicity, environmental protection, simple synthesis, good water solubility and the like, and can be widely applied to the fields of fluorescence sensing, biological imaging, drug/gene transfer, photoelectrocatalysis, food safety and the like.
Further, in the step (1), the feeding ratio of the powder of the crushed cicada slough to water is 1.0g:50mL, and the water is purified water.
The beneficial effects of adopting the above scheme include: the adoption of the feeding ratio is favorable for further improving the fluorescence quantum yield of the CT-CQDs.
Further, in the step (2), heating is carried out in a reaction kettle under the condition of 180 ℃ for 4 hours.
The beneficial effects of adopting the above scheme include: the temperature in the reaction in the step (2) is 180 ℃, the duration is 4 hours, the heating in the reaction kettle is used for selecting the optimal fluorescence intensity, and the fluorescence intensity is weakened due to too high or too low temperature. The heating time is 4h, and the higher fluorescence quantum yield of the CT-CQDs can be obtained. Therefore, the appropriate reaction temperature and time are favorable for obtaining the condition of higher fluorescence intensity, and further higher fluorescence quantum yield of the CT-CQDs is obtained.
Further, in the step (3), the product obtained by heating in the step (2) is cooled to room temperature and then is subjected to 4500 r.min -1 Centrifuging for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, and dialyzing for 24 hr.
Adopt above-mentioned scheme's beneficial effect to include: at 4500r min -1 Centrifuging for 10min, filtering with 0.22 μm microporous membrane, and dialyzing to remove macromolecular impurities.
Further, the method also comprises a step of freeze-drying the carbon quantum dots of the periostracum cicadae obtained after dialysis.
The beneficial effects of adopting the above scheme include: after freeze-drying, the storage, the use and the like are convenient.
The invention provides a periostracum cicadae carbon quantum dot fluorescent probe which is obtained by adopting the preparation method.
Adopt above-mentioned scheme's beneficial effect to include: the method has the advantages of good dispersity, uniform particle size, good accuracy, good selectivity to ENR, good linear relation, low detection limit (0.069 mu M), good water solubility, low price and the like.
The invention provides a reagent or a kit for detecting enrofloxacin, which comprises the periostracum cicada carbon quantum dot fluorescent probe.
The beneficial effects of adopting the above scheme include: the method has the advantages of accurate detection result, good selectivity to ENR, good linear relation, low detection limit (0.069 mu M), good water solubility, low price and the like.
The invention provides application of the periostracum cicada carbon quantum dots prepared by the preparation method in detection of enrofloxacin.
For example: the invention provides application of the periostracum cicadae carbon quantum dot CT-CQDs fluorescent probe prepared by the preparation method in detecting enrofloxacin content in aquatic products.
The beneficial effects of adopting the above scheme include: when in application, the periostracum cicada carbon quantum dot provided by the invention has the advantages of low chemical cost, good stability, environmental friendliness, rapidness and accuracy in detection, simplicity in operation, wide linear range, low detection limit (0.069 mu M) and the like.
The CT-CQDs provided by the invention have good selectivity on enrofloxacin in a certain range, and the influence of other antibiotics and common ions in aquatic products on the fluorescence intensity of the CT-CQDs is extremely small. Therefore, the effective detection of enrofloxacin in aquatic products can be realized through the CT-CQDs.
The invention provides a method for detecting enrofloxacin, which comprises the following steps:
s1, plotting a standard curve for ENR concentration: drawing a standard curve about the enrofloxacin concentration by taking the relative change value of the fluorescence intensity of a standard solution containing periostracum cicadae carbon quantum dots as a vertical coordinate and the enrofloxacin concentration as a horizontal coordinate to obtain a standard curve graph;
s2, detecting the enrofloxacin concentration in the unknown solution: mixing the periostracum cicadae carbon quantum dot solution with an unknown solution, diluting with a buffer solution, carrying out fluorescence detection after reaction, detecting the fluorescence intensity of a solution to be detected, and combining a standard curve graph to obtain the concentration of enrofloxacin in the unknown solution.
Further, the reaction mixture was diluted with a buffer solution having a pH of 3.6, and the fluorescence detection was carried out after 5min of the reaction.
The beneficial effects of adopting the above scheme include: the fluorescence intensity of CT-CQDs solutions in the presence of ENR varied with pH, reaching a maximum when the pH was 3.6. Therefore, the optimum solution pH was selected to be 3.6 for the test. Better fluorescence intensity can be obtained by detecting the fluorescence intensity of the solution to be detected after 5 min. The detection method has the advantages of simplicity in operation, greenness, no pollution, rapidness in detection, wider linear range and the like.
The CT-CQDs can be prepared by the above preparation method.
The method can be adopted to detect the enrofloxacin content in the aquatic products.
For example: the CT-CQDs fluorescent probe is used for detecting the enrofloxacin concentration in aquatic products, and the method comprises the following steps:
s1, plotting a standard curve for ENR concentration: stepwise diluting ENR stock solution (10mM) to obtain standard solution with ENR concentration range of 0.13-16.67 μ M, and respectively adding 400 μ L ENR standard solution and CT-CQDs solution (0.3 mg/mL) -1 ) mu.L of the mixture was diluted with PBS buffer solution having a pH of 3.6, and fluorescence detection was performed after 5min, and the fluorescence spectrum of each solution was recorded. Then linearly fitting the relative change value of the fluorescence intensity of the CT-CQDs at 355nm with the ENR concentration to obtain a standard curve graph;
s2, detecting the ENR concentration in the aquatic product: accurately weighing 5.0g of the homogenized sample tissue, placing in 10mL centrifuge tube, homogenizing at high speed for 1min with 4.0mL acetonitrile and 0.04mL 1.0% acetic acid as extractive solution, ultrasonic extracting for 15min at 4500 r.min -1 Centrifuging for 15min, collecting supernatant, extracting residue for 1 time, and mixing the two supernatants. 400. mu.L of CT-CQDs solution, 400. mu.L of low, medium and high concentration ENR standard solution (0.67. mu.M, 3.33. mu.M, 13.33. mu.M) and 400. mu.L of actual sample solution were taken, diluted to 3mL with PBS buffer solution having pH of 3.6, mixed well and transferred to a series of 4mL cuvettes. And after 5min, detecting the fluorescence intensity of the solution to be detected, and combining a standard curve graph to obtain the concentration of ENR in the unknown solution.
Drawings
FIG. 1 is a schematic diagram of a detection principle of a periostracum cicada carbon quantum dot fluorescent probe for detecting enrofloxacin in an aquatic product.
FIG. 2 is a perspective electron micrograph of CT-CQDs.
FIG. 3 is a histogram of the particle size distribution of CT-CQDs.
FIG. 4 is a graph showing fluorescence emission spectra of CQDs synthesized from different precursor materials.
FIG. 5 is a graph comparing the effect of different ions and antibiotics on the fluorescence intensity of CT-CQDs.
FIG. 6 shows fluorescence emission spectra of CT-CQDs at different ENR concentrations.
FIG. 7 is a graph showing the relationship between different ENR concentrations and the relative change value of the fluorescence intensity of CT-CQDs.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
The invention provides a preparation method of a periostracum cicadae carbon quantum dot fluorescent probe, which comprises the following steps:
(1) pulverizing periostracum Cicadae, and dissolving in purified water to obtain solution;
(2) transferring the solution obtained in the step (1) into a reaction kettle, and putting the reaction kettle into an oven for heating;
(3) and (3) cooling and centrifuging the product obtained after heating in the step (2), collecting supernatant, filtering, performing primary dialysis, and dialyzing for 24h to obtain periostracum cicada carbon quantum dots, namely CT-CQDs, freeze-drying the sample for later use, wherein the CT-CQDs can be used as periostracum cicada carbon quantum dot fluorescent probes (CT-CQDs fluorescent probes).
Further, the adding ratio of the cicada slough powder and the purified water in the step (1) is 1.0g to 50 mL; specifically, step (1) may take the following actions: crushing periostracum cicadae, weighing 1.0g of crushed periostracum cicadae powder, and mixing with 50mL of purified water to obtain a solution;
further, the temperature of the reaction in the step (2) is 180 ℃, and the reaction time is 4 h. Wherein the heating in the reaction vessel is performed for the purpose of selecting an optimum fluorescence intensity, and too high or too low results in a decrease in fluorescence intensity. The heating time is 4h, and the higher fluorescence quantum yield of the CT-CQDs can be obtained. Therefore, the appropriate reaction temperature and time are favorable for obtaining the condition of higher fluorescence intensity, and further higher fluorescence quantum yield of the CT-CQDs is obtained.
Further, in the step (3), the product obtained after heating in the step 2) is cooled to room temperature and then is subjected to 4500 r.min -1 And centrifuging for 10 min. The supernatant was collected and filtered through a 0.22 μm filter. And (5) carrying out primary dialysis for 24 hours to remove macromolecular impurities in the solution.
Further, the periostracum cicada carbon quantum dot fluorescent probe can be prepared by the preparation method.
The cicada slough carbon quantum dot fluorescent probe can be assembled into products such as reagents or kits.
The CT-CQDs fluorescent probe can be applied to the determination of enrofloxacin in aquatic products, such as the determination of the content (concentration and the like) of enrofloxacin in aquatic products. But are not limited to, determining enrofloxacin in the aquatic product.
The application of the periostracum cicadae carbon quantum dot fluorescent probe can adopt the following method when being used for detecting the concentration of enrofloxacin in an aquatic product, and comprises the following steps:
s1, drawing a standard curve related to the ENR concentration by taking the relative change value of the fluorescence intensity of the standard solution containing CT-CQDs as a vertical coordinate and the ENR concentration as a horizontal coordinate to obtain a standard curve graph;
s2, detecting the ENR concentration in the unknown solution: mixing the CT-CQDs solution with the unknown solution, diluting with PBS buffer solution with pH of 3.6, performing fluorescence detection after 5min, detecting the fluorescence intensity of the solution to be detected, and combining a standard curve graph to obtain the concentration of ENR in the unknown solution.
For example, the following steps may be employed:
s1, plotting a standard curve for ENR concentration: sequentially diluting ENR stock solution (10mM) to obtain standard solution with ENR concentration range of 0.13-16.67 μ M, and respectively adding 400 μ L of ENR standard solution and CT-CQDs solution (concentration of 0.3 mg/mL) -1 ) mu.L of the mixture was diluted with PBS buffer solution having a pH of 3.6, and fluorescence detection was performed after 5min, and the fluorescence spectrum of each solution was recorded. Then linearly fitting the relative change value of the fluorescence intensity of the CT-CQDs at 355nm with the ENR concentration to obtain a standard curve graph;
s2, detecting the ENR concentration in the aquatic product: accurately weighing 5.0g of homogenized sample tissue, placing in 10mL centrifuge tube, adding 4.0mL acetonitrile and 0.04mL 1.0% acetic acid as extractive solution, homogenizing at high speed for 1min, ultrasonic extracting for 15min at 4500 r.min -1 Centrifuging for 15min, collecting supernatant, extracting residue for 1 time, and mixing the two supernatants. Taking 400 mu L of CT-CQDs solution, and respectively taking the solution as low as400. mu.L of medium and high concentration ENR standard solutions (0.67, 3.33, 13.33. mu.M) and 400. mu.L of the actual sample solution, which were diluted to 3mL with a PBS buffer solution having a pH of 3.6 and thoroughly mixed, were transferred to a series of 4mL cuvettes. And after 5min, detecting the fluorescence intensity of the solution to be detected, and combining a standard curve graph to obtain the concentration of ENR in the unknown solution.
Because CT-CQDs have higher fluorescence quantum yield, the CT-CQDs solution turns light blue under an ultraviolet lamp with the wavelength of 365 nm. The periostracum cicada carbon quantum dot fluorescent probe and the preparation method thereof and the application thereof in detecting the concentration of enrofloxacin are provided based on the characteristics, the fluorescent probe has good selectivity and sensitivity to enrofloxacin in a certain range, and other metal ions or antibiotics have little interference on the detection system. Therefore, the effective detection of enrofloxacin can be realized through the CT-CQDs, and the method has good application value and application prospect in the fields of food safety and the like.
Compared with the prior art, the invention has the following advantages:
(1) the carbon quantum dot fluorescent probe prepared by the invention has wide raw material sources, is cheap and is easy to obtain.
(2) The carbon quantum dot fluorescent probe prepared by the invention has the advantages of simple and convenient preparation process, easy operation and environmental protection.
(3) The fluorescent probe shows stronger selectivity to enrofloxacin and can effectively reduce Mg 2+ ，Ca 2+ ，Na + ，K + ，Zn 2+ The interference of metal ions or other antibiotics on the detection results have higher reliability.
(4) The relative change value of the fluorescence intensity of the CT-CQDs fluorescent probe has a good linear relation with the ENR concentration within a certain range.
(5) The CT-CQDs fluorescent probe can realize effective detection of enrofloxacin in aquatic products, and has good application value and application prospect in the fields of food safety and the like.
The materials used in the examples of the present invention, unless otherwise specified, are all conventional experimental materials in the art, and may be prepared by conventional methods or commercially available.
The methods used in the examples of the present invention are all routine experimental methods in the art unless otherwise specified.
In the examples, Enrofloxacin (ENR), Ciprofloxacin (CIP), Norfloxacin (NFX), Dexamethasone (DEX), Gentamicin (GEN) were purchased from Dudet Biotechnology Ltd, NaCl solution, KCl solution, ZnCl 2 Solution, CaCl 2 Solution, MgCl 2 The solutions were purchased from Tianjin Bailun Biochemical Co., Ltd.
Example 1
The detection principle of the periostracum cicada carbon quantum dot fluorescent probe for detecting enrofloxacin in aquatic products is shown in figure 1.
Preparing water-soluble CT-CQDs, comprising the following steps: the preparation method comprises the steps of crushing the cicada slough, weighing 1.0g of crushed cicada slough powder, mixing with 50mL of purified water, transferring to a 100mL reaction kettle, and heating the reaction kettle in an oven at 180 ℃ for 4 hours. After the reaction is finished, the reaction kettle is naturally cooled to room temperature. At 4500r min -1 After centrifugation for 10min, the supernatant of CT-CQDs is collected, filtered by a 0.22 μm filter membrane, dialyzed once, and the sample is freeze-dried after dialysis for 24h for further characterization and application.
Performing transmission electron microscope characterization on the CT-CQDs, wherein the characterization result is as follows:
the transmission electron microscope characterization result is shown in FIG. 2, and it can be observed from FIG. 2 that CT-CQDs have better dispersibility and more uniform particle size distribution. When the particle size distribution of CT-CQDs was examined, the mean particle size of CT-CQDs was 19.6. + -. 0.2nm as shown in FIG. 3.
In order to obtain carbon quantum dots with high fluorescence intensity, the preparation method selects cicada slough, mosquito incense ash, a drawn filter tip, an undrawn filter tip and soot as precursor materials, and different CQDs are prepared by the operation according to the preparation method. A400. mu.L solution of CQDs was diluted to 3mL with water and subjected to fluorescence intensity detection at λ ex/λ em-355/436 nm, and the fluorescence spectrum of each CQDs was recorded. As shown in fig. 4, the curves from top to bottom are cicada slough, filter tip not drawn, soot and mosquito incense ash, respectively, and the results show that the fluorescence intensity of the carbon quantum dots synthesized by using the biomass of the cicada slough as the precursor material is higher, and the CT-CQDs synthesized by using the cicada slough as the precursor material have better effect than the CQDs synthesized by selecting other precursor materials.
Example 2
Detecting the influence of different ions and antibiotics on the fluorescence intensity of the CT-CQDs solution. The method comprises the following steps:
a blank control (blank) group and a test group were set.
Test groups: taking 400 μ L of standard solution or chloride ion solution (concentration is 10mM), adding into 5mL centrifuge tube, adding into CT-CQDs solution (0.3 mg. mL) -1 )400 μ L was diluted to 3mL with PBS buffer pH 3.6, mixed well and transferred to a 4mL cuvette. After 5min, the fluorescence was detected at 355nm and the fluorescence spectrum recorded for each solution.
The standard substances are respectively: enrofloxacin (ENR), Ciprofloxacin (CIP), Norfloxacin (NFX), Dexamethasone (DEX), Gentamicin (GEN). The ionic solutions of chloride were respectively: NaCl solution, KCl solution, ZnCl 2 Solution, CaCl 2 Solution, MgCl 2 And (3) solution. The concentration is 10mM, and the preparation method comprises the following steps: the standard substance or the chloride is precisely weighed and respectively placed in a 10mL measuring flask, dissolved by methanol, and diluted by water to a constant volume to prepare a 10mM stock solution.
Blank control (blank): adding 400 μ L of purified water into 5mL centrifuge tube, adding CT-CQDs solution (0.3 mg. mL) into the centrifuge tube -1 )400 μ L was diluted to 3mL with PBS buffer pH 3.6, mixed well and transferred to a 4mL cuvette. After 5min, fluorescence detection was performed at 355nm and the fluorescence spectrum was recorded.
The selectivity result is shown in fig. 5, compared with the ENR fluoroquinolone antibiotics, the fluorescence intensity of the CT-CQDs added with other antibiotics or ions (10mM) is not changed greatly, which indicates that the influence of the other antibiotics and the ions commonly found in aquatic products on the fluorescence intensity of the CT-CQDs is negligible, namely the CT-CQDs as a fluorescent probe can realize the selective detection of the ENR fluoroquinolone antibiotics in the aquatic products.
Example 3
Detecting fluorescence emission spectra of CT-CQDs at different ENR concentrations, comprising the following steps:
a plurality of 5mL centrifuge tubes were used to gradually dilute the ENR stock solution (10mM) to 0.13. mu.M, 0.67. mu.M, 1.67. mu.M, 3.33. mu.M, 6.67. mu.M, 13.33. mu.M, and 16.67. mu.M, and 400. mu.L of ENR standard solution and CT-CQDs solution (0.3 mg. mL) were used -1 ) After mixing 400 μ L, it was diluted to 3mL with PBS buffer pH 3.6 and mixed well before transferring to a series of 4mL cuvettes. After 5min, the fluorescence was detected at 355 nm.
As shown in FIG. 6, the results of the measurements are plotted from top to bottom for ENR concentrations of 16.67. mu.M, 13.33. mu.M, 6.67. mu.M, 3.33. mu.M, 1.67. mu.M, 0.67. mu.M, 0.13. mu.M, and 0. mu.M, respectively, and it can be seen that the relative change value of the fluorescence intensity of CT-CQDs increases with the concentration when the concentration of the standard solution of ENR is in the range of 0.13 to 16.67. mu.M.
The results of linear fitting of the ENR solution concentration and the relative change value of fluorescence intensity are shown in FIG. 7, and it can be seen that when the concentration of the ENR standard substance solution is in the range of 0.13 to 16.67. mu.M, there is a good linear relationship (R) between the concentration and the relative change value of fluorescence intensity 2 ＝0.9979)。
The limit of detection (LOD) was calculated to be 0.069. mu.M according to the formula 3. sigma./s (σ is the variance of the detection data of 11 blank samples, and s is the slope of the standard curve).
Example 4
Taking aquatic products as an example, the CT-CQDs are utilized to detect ENR in a sample. The feasibility and the applicability of measuring ENR in aquatic products by CT-CQDs are detected by adopting the method of the embodiment 3.
ENR detection was performed on 4 kinds of aquatic products (carp, freshwater shrimp, scallop, and fresh oyster), respectively.
(1) Pretreating a sample to be detected: homogenizing the sample to be detected with an electric homogenizer, accurately weighing 5.0g of the homogenized sample tissue, placing in a 10mL centrifuge tube, adding 4.0mL acetonitrile and 0.04mL 1.0% acetic acid as extractive solutions, homogenizing at high speed for 1min, and ultrasonically extractingTaking 15min at 4500 r.min -1 Centrifuging for 15min, collecting supernatant, extracting residue for 1 time, and mixing the two supernatants to obtain sample solution to be detected.
(2) Detecting ENR in a sample using CT-CQDs comprising the steps of: taking CT-CQDs solution (0.3 mg. mL) -1 )400 μ L of ENR standard solution with low (0.67 μ M), medium (3.33 μ M) and high (13.33 μ M) concentration and 400 μ L of sample solution to be detected are respectively taken, the sample solution to be detected and the sample of the standard solution are analyzed by the same procedure and conditions as in example 3, and the concentration of ENR in the sample solution to be detected and the sample solution to be standard solution is calculated. The spiked solution sample refers to the sample solution to which the ENR standard solution was added for further calculation of spiked recovery.
When a blank real sample (referring to a sample without adding ENR standard solution) is added, the fluorescence intensity of the CT-CQDs is not changed significantly, which indicates that the blank real sample does not contain or contains ENR with the quantity lower than the detection limit.
The recovery rates for ENR were further calculated and, as shown in table 1, the recovery rates for ENR in the samples ranged from 88.6% to 104.7% with RSD ≦ 5.4% (n ═ 3). The results show that the method can accurately determine the ENR content in the aquatic product. Meanwhile, compared with the existing chromatographic analysis method and liquid chromatography mass spectrometry combined technology, the method for measuring ENR parameters has wider linear range; the detection limit is lower compared with the titration method and SERS technology. Therefore, the method has the advantages of simple operation, no pollution, quick detection, wider linear range and the like. Has good application value and application prospect in the fields of food safety and the like.
TABLE 1 results of recovery of ENR from four aquatic products with spiked standard
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be apparent to those skilled in the art that various modifications, equivalents, and the like can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make modifications and alterations without departing from the scope of the present invention.
Claims (10)
1. A preparation method of periostracum cicadae carbon quantum dots is characterized by comprising the following steps:
(1) pulverizing periostracum Cicadae, and dissolving in water to obtain solution;
(2) heating the solution obtained in the step (1);
(3) and (3) cooling and centrifuging the product obtained after heating in the step (2), collecting supernatant, filtering, and dialyzing to obtain cicada slough carbon quantum dots, namely CT-CQDs.
2. The preparation method according to claim 1, wherein in the step (1), the ratio of the powder of the pulverized periostracum cicada to water is 1.0g to 50mL, and the water is purified water.
3. The method according to claim 1, wherein the heating in step (2) is carried out in a reaction vessel at 180 ℃ for 4 hours.
4. The process according to claim 1, wherein in the step (3), the product obtained by heating in the step (2) is cooled to room temperature and then subjected to temperature reduction at 4500 r-min -1 Centrifuging for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, and dialyzing for 24 hr.
5. The preparation method according to any one of claims 1 to 4, further comprising a step of freeze-drying the carbon quantum dots of the periostracum cicadae obtained after dialysis.
6. A periostracum cicadae carbon quantum dot fluorescent probe is characterized by being obtained by the preparation method of any one of claims 1-5.
7. A reagent or a kit for detecting enrofloxacin, which is characterized by comprising the periostracum cicadae carbon quantum dot fluorescent probe of claim 6.
8. Application of the periostracum cicada carbon quantum dots prepared by the preparation method of any one of claims 1-5 in detection of enrofloxacin.
9. The method for detecting enrofloxacin is characterized by comprising the following steps:
s1, drawing a standard curve about enrofloxacin concentration by taking the relative change value of fluorescence intensity of a standard solution containing periostracum cicadae carbon quantum dots as a vertical coordinate and the enrofloxacin concentration as a horizontal coordinate to obtain a standard curve graph;
s2, detecting the enrofloxacin concentration in the unknown solution: mixing the periostracum cicadae carbon quantum dot solution with an unknown solution, diluting with a buffer solution, carrying out fluorescence detection after reaction, detecting the fluorescence intensity of a solution to be detected, and combining a standard curve graph to obtain the concentration of enrofloxacin in the unknown solution.
10. The detection method according to claim 9, wherein in S2, the pH of the buffer solution is 3.6, and the reaction time is 5 min.
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| CN107490565A (en) * | 2017-06-27 | 2017-12-19 | 昆明理工大学 | A kind of method of nitrogen-doped carbon quantum dot fluorescence enhanced sensitivity detection Ciprofloxacin |
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| CN113134347A (en) * | 2021-03-23 | 2021-07-20 | 西安理工大学 | Preparation method and application of heteroatom porous carbon |
| CN113721024A (en) * | 2021-09-16 | 2021-11-30 | 天津温阳生物技术有限公司 | Fluorescence immunoassay rapid detection kit and detection method for enrofloxacin carbon quantum dots in animal derived food |
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| CN107490565A (en) * | 2017-06-27 | 2017-12-19 | 昆明理工大学 | A kind of method of nitrogen-doped carbon quantum dot fluorescence enhanced sensitivity detection Ciprofloxacin |
| CN107515206A (en) * | 2017-06-27 | 2017-12-26 | 昆明理工大学 | A kind of method of sulfur doping carbon quantum dot fluorescence sensitivity detection Norfloxacin |
| CN112852420A (en) * | 2021-02-04 | 2021-05-28 | 四川农业大学 | Carbon quantum dot fluorescent probe and method for detecting thiamphenicol content |
| CN113134347A (en) * | 2021-03-23 | 2021-07-20 | 西安理工大学 | Preparation method and application of heteroatom porous carbon |
| CN113721024A (en) * | 2021-09-16 | 2021-11-30 | 天津温阳生物技术有限公司 | Fluorescence immunoassay rapid detection kit and detection method for enrofloxacin carbon quantum dots in animal derived food |
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