Quantum dot fluorescence-based method for detecting peroxide value in animal fat

Abstract

The present application relates to the technical field of food detection, in particular to a method for detecting peroxide value in animal fat based on quantum dot fluorescence, comprising: dissolving the animal fat to be detected with an organic solvent to obtain a clear solution; refluxing a specific proportion of sulfur compound, cadmium source and selenium source in water phase to prepare cadmium selenide quantum dot fluorescence probe with high quantum yield; mixing the quantitative probe and standard hydrogen peroxide solution with known concentration in buffer to obtain a gradient solution, measuring the initial fluorescence intensity under a specific excitation wavelength, measuring the intensity after reaction at a preset temperature for a certain period of time; calculating the intensity difference of each concentration point, taking the difference as the vertical axis and the concentration as the horizontal axis to draw a standard curve fitting equation, and obtaining the peroxide value of the sample based on the equation. The method realizes rapid and accurate detection of peroxide value in animal fat by synthesizing high-activity probe in water phase and quantifying the fluorescence intensity difference before and after reaction.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a method for detecting peroxide value in animal fats based on quantum dot fluorescence. Background Technology

[0002] Peroxide value of animal fats is a key indicator for measuring the degree of oxidative rancidity. Current detection technologies mainly rely on chemical methods such as iodometric titration and sodium thiosulfate titration, or indirect detection methods based on organic fluorescent dyes. Chemical methods require multiple titrations, large reagent volumes, are cumbersome, and are easily affected by environmental interference. Organic fluorescent dye probes are often synthesized using toxic organic solvents, have low quantum yields, and their fluorescence emission is often located in the ultraviolet region. Detection requires light-protected operation and suffers from poor stability, making it difficult to meet the needs for rapid and accurate detection. Existing quantitative fluorescence strategies often use fluorescence quenching rate or a single intensity value correlated with target analyte concentration, which is easily affected by instrument baseline drift and background fluorescence fluctuations, leading to quantitative deviations.

[0003] There is a need to overcome the limitations of existing fluorescent probe synthesis and the defects of quantitative methods. Current probe synthesis often relies on high-temperature organic phase injection, which is complex and produces products with poor water solubility; furthermore, it fails to fully utilize the absolute change in fluorescence signal before and after the reaction for quantification. This invention aims to solve the problems of existing fluorescent probes, such as difficulties in aqueous phase synthesis, insufficient quantum yield, incompatibility of emission bands with visible light detection, and the limitation of accuracy due to quantification dependence on relative changes. It aims to prepare high-performance probes through a specific aqueous phase synthesis pathway and establish a novel quantitative mechanism based on the absolute difference in fluorescence intensity before and after the reaction. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and propose a method for detecting peroxide value in animal fats based on quantum dot fluorescence.

[0005] To achieve the above objectives, the present invention employs the following technical solution: a method for detecting peroxide value in animal fats based on quantum dot fluorescence, comprising: Take the animal fat sample to be tested, dissolve it with an organic solvent, and obtain a clear fat solution to be tested; A cadmium selenide quantum dot fluorescent probe solution with high quantum yield was prepared by reacting a sulfur-based compound with a cadmium source and a selenium source in an aqueous phase under reflux heating. The fluorescence emission wavelength of the cadmium selenide quantum dot fluorescent probe solution is in a specific visible light band. A quantitative amount of the cadmium selenide quantum dot fluorescent probe solution was mixed with a hydrogen peroxide standard solution of known concentration in a buffer solution to form a series of standard reaction solutions with concentration gradients. The initial fluorescence intensity value of each standard reaction solution at a specific excitation wavelength was measured. After placing the standard reaction solutions of the series of concentration gradients at a preset temperature and reacting for a preset time, the fluorescence intensity value of each standard reaction solution after the reaction is measured at the same specific excitation wavelength. Calculate the difference between the initial fluorescence intensity value and the fluorescence intensity value after the reaction corresponding to each concentration point, plot the difference as the ordinate and the concentration of the corresponding hydrogen peroxide standard solution as the abscissa to obtain a standard working curve, and fit the curve to obtain a linear regression equation. Based on the linear regression equation, the peroxide value of the animal fat sample to be tested is obtained.

[0006] As a further aspect of the present invention, the method of preparing a cadmium selenide quantum dot fluorescent probe solution with high quantum yield by reacting a sulfur-based compound with a cadmium source and a selenium source in an aqueous phase under reflux heating comprises: Weigh out sodium citrate and cadmium chloride in a predetermined molar ratio, dissolve them in deionized water, and obtain a clear cadmium-containing precursor solution. Under inert gas protection, the cadmium-containing precursor solution was transferred to a three-necked flask and heated to a preset first reaction temperature; A sodium selenite solution of a predetermined concentration is mixed with a sulfur-based compound solution of a predetermined concentration using a syringe to form a mixed solution of selenium source and sulfur-based ligand, wherein the sulfur-based compound is one of mercaptoacetic acid, mercaptopropionic acid or glutathione. The mixed solution of the selenium source and the sulfur-based ligand is rapidly injected into the cadmium-containing precursor solution that has reached the first reaction temperature. Then, the reaction system is heated to a preset second reaction temperature for reflux reaction. During the reflux reaction process, the growth of cadmium selenide quantum dots was monitored by taking samples at regular intervals and measuring their ultraviolet-visible absorption and fluorescence emission spectra. When the peak wavelength of the fluorescence emission spectrum of the cadmium selenide quantum dots reaches the specific visible light band, heating is stopped and the mixture is allowed to cool naturally to room temperature. The cooled reaction mixture was purified to remove unreacted ions and small molecule ligands. The purified cadmium selenide quantum dots were then redispersed in a buffer solution with a specified pH to obtain the cadmium selenide quantum dot fluorescent probe solution.

[0007] As a further aspect of the present invention, the purification treatment of the cooled reaction mixture to remove unreacted ions and small molecule ligands includes: An equal volume of polar organic solvent is added to the cooled reaction mixture to promote the precipitation of the cadmium selenide quantum dots. The reaction mixture was centrifuged at high speed, and the supernatant containing free ions and small molecule ligands was discarded. Redissolve the quantum dot precipitate at the bottom of the centrifuge tube in an appropriate amount of deionized water; Repeat the steps of adding polar organic solvent, high-speed centrifugation, and redissolving at least twice; The resulting quantum dot aqueous solution was filtered and separated by an ultrafiltration centrifuge tube with a specific molecular weight cutoff, and the retentate was collected. The retentate was diluted with deionized water and then subjected to ultrafiltration centrifugation again. The dilution and ultrafiltration steps were repeated several times until the conductivity of the filtrate in the lower layer of the centrifuge tube after ultrafiltration was lower than a preset threshold.

[0008] As a further aspect of the present invention, the measured amount of the cadmium selenide quantum dot fluorescent probe solution is mixed with a hydrogen peroxide standard solution of known concentration in a buffer solution to form a series of standard reaction solutions with concentration gradients, including: A fixed volume of the cadmium selenide quantum dot fluorescent probe solution was accurately measured using a micropipette and added to multiple identical transparent reaction containers. Add the same volume of buffer solution, which is either a phosphate buffer solution or a borate buffer solution, to each of the transparent reaction vessels. The pH value of the buffer solution is pre-adjusted to a preset range. A set of hydrogen peroxide standard working solutions with progressively increasing concentrations and covering the expected detection range were prepared using the gradient dilution method. Different volumes of the hydrogen peroxide standard working solution were added to each transparent reaction vessel containing the cadmium selenide quantum dot fluorescent probe solution and the buffer solution, and the buffer solution was added to ensure that the total volume of the solution in each transparent reaction vessel was equal. The mixed solution in each transparent reaction vessel is thoroughly shaken to form a series of standard reaction solutions with different concentration gradients for which the fluorescence intensity is to be measured.

[0009] As a further aspect of the present invention, the calculation of the difference between the initial fluorescence intensity value and the fluorescence intensity value after the reaction corresponding to each concentration point includes: Record the number of each standard reaction solution and the concentration of its corresponding hydrogen peroxide standard solution; The initial fluorescence intensity value of each standard reaction solution measured before the reaction is retrieved from the storage unit of the fluorescence spectrometer. The initial fluorescence intensity value is the fluorescence intensity value read at the peak wavelength of the fluorescence emission spectrum of the cadmium selenide quantum dot at the specific excitation wavelength. The fluorescence intensity value after the reaction of each standard reaction solution is retrieved from the storage unit of the fluorescence spectrometer. The fluorescence intensity value after the reaction is obtained under the same instrument parameters as the initial fluorescence intensity value. For standard reaction solutions with the same number, the fluorescence intensity value after the reaction is subtracted from the initial fluorescence intensity value to obtain a fluorescence intensity difference value, which corresponds to the hydrogen peroxide concentration of the standard reaction solutions with the same number.

[0010] As a further aspect of the present invention, a standard working curve is plotted with the difference as the ordinate and the corresponding concentration of the hydrogen peroxide standard solution as the abscissa, and a linear regression equation is obtained by fitting the curve, including: Establish a two-dimensional coordinate system and use the fluorescence intensity difference calculated for each standard reaction solution as the ordinate value of the data point; The concentration of the hydrogen peroxide standard solution corresponding to each standard reaction solution is used as the x-axis value of the same data point; Plot all data points in the two-dimensional coordinate system; The linear least squares method is used to fit all data points in the two-dimensional coordinate system to obtain a straight line characterizing the relationship between the fluorescence intensity difference and the hydrogen peroxide concentration, which is the standard working curve. Obtain the slope and intercept parameters of the straight line to form a mathematical expression with hydrogen peroxide concentration as the independent variable and fluorescence intensity difference as the dependent variable, i.e., the linear regression equation.

[0011] As a further aspect of the present invention, the peroxide value of the animal fat sample to be tested is obtained based on the linear regression equation, including: Take the same amount of the cadmium selenide quantum dot fluorescent probe solution used to construct the standard curve, and mix it with the clarified test oil solution in the same buffer solution to form the test sample reaction solution; The initial fluorescence intensity of the test sample reaction solution at the specific excitation wavelength was measured. After reacting the test sample solution at the preset temperature for the preset time, the fluorescence intensity value of the test sample after reaction is measured at the specific excitation wavelength. Calculate the difference between the initial fluorescence intensity value of the sample to be tested and the fluorescence intensity value of the sample to be tested after the reaction, substitute the difference into the linear regression equation, and calculate the concentration of hydrogen peroxide equivalent in the reaction solution of the sample to be tested; Based on the dilution factor and sample volume of the oil solution to be tested, the concentration of the hydrogen peroxide equivalent is converted into the peroxide value of the animal oil sample to be tested. The step of taking the same amount of the cadmium selenide quantum dot fluorescent probe solution used to construct the standard curve and mixing it with the clarified test oil solution in the same buffer solution to form the test sample reaction solution specifically includes: Using the same micropipette as when measuring the cadmium selenide quantum dot fluorescent probe solution, measure the exact same volume of the cadmium selenide quantum dot fluorescent probe solution as when constructing the standard curve, and add it to a new transparent reaction vessel. Add the same volume and type of buffer solution as used when constructing the standard curve to the transparent reaction vessel; Using another micropipette, accurately measure a predetermined volume of the clear test oil solution and add it to the transparent reaction vessel; The buffer solution is added to the transparent reaction vessel so that the total volume of the solution in the transparent reaction vessel is exactly equal to the total volume of the standard reaction solution used to construct the standard curve; The mixed solution in the transparent reaction vessel is thoroughly shaken to form the test sample reaction solution.

[0012] As a further aspect of the present invention, measuring the initial fluorescence intensity value of the test sample reaction solution at the specific excitation wavelength includes: Place the transparent reaction container containing the reaction solution of the sample to be tested into the sample chamber of the fluorescence spectrometer; Set the excitation wavelength of the fluorescence spectrometer to the specific excitation wavelength, and set the scanning range of the emission wavelength to cover the band where the peak wavelength of the fluorescence emission spectrum of the cadmium selenide quantum dots is located; Start the scanning program of the fluorescence spectrometer to obtain the complete fluorescence emission spectrum of the reaction solution of the sample to be tested; From the obtained complete fluorescence emission spectrum, locate the position corresponding to the peak wavelength of the fluorescence emission spectrum of the cadmium selenide quantum dot, read the fluorescence intensity value at the position, and record the fluorescence intensity value as the initial fluorescence intensity value of the sample to be tested.

[0013] As a further aspect of the present invention, after reacting the test sample solution at the preset temperature for the preset time, the fluorescence intensity value of the test sample after reaction at the specific excitation wavelength is measured, including: Remove the transparent reaction vessel from the sample chamber of the fluorescence spectrometer after the initial fluorescence intensity value of the sample to be tested has been determined; The transparent reaction vessel is transferred to a constant temperature water bath or constant temperature oscillator that has been preheated to the preset temperature, and the reaction is started on a timer. When the reaction time reaches the preset duration, the transparent reaction vessel is removed from the temperature control device and allowed to cool naturally to room temperature. After cooling, the transparent reaction vessel is placed back into the sample chamber of the fluorescence spectrometer. Without changing any instrument parameters, the scanning program of the fluorescence spectrometer is restarted to obtain the complete fluorescence emission spectrum of the reaction solution of the sample to be tested after the reaction. From the complete fluorescence emission spectrum obtained after the reaction, at the same wavelength position as the initial fluorescence intensity value of the sample to be tested, the fluorescence intensity value is read and recorded as the fluorescence intensity value of the sample to be tested after the reaction.

[0014] As a further aspect of the present invention, the concentration of the hydrogen peroxide equivalent is converted into the peroxide value of the animal fat sample to be tested, based on the dilution factor and sample volume of the oil solution to be tested, including: The concentration value calculated from the linear regression equation is the molar concentration of hydrogen peroxide equivalent in the reaction solution of the test sample. Calculate the amount of hydrogen peroxide equivalent contained in the volume of the clarified oil solution added when preparing the test sample reaction solution. Calculate the mass of the original animal fat per unit volume in the clarified test fat solution based on the volume of the organic solvent used to initially dissolve the animal fat sample and the mass of the animal fat sample weighed. By combining the mass of the original animal fat per unit volume in the clarified test oil solution and the amount of hydrogen peroxide equivalent in the test sample reaction solution, the number of millimoles of active oxygen per kilogram of the original animal fat sample is calculated according to the standard definition of peroxide value, thus obtaining the peroxide value of the test animal fat sample in millimoles per kilogram.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are as follows: Cadmium selenide quantum dot fluorescent probes were prepared by reacting a sulfur-based compound with cadmium and selenium sources in an aqueous phase under reflux. The sulfur-based compound provides surface coordinating groups, ensuring stable dispersion of the quantum dots in the aqueous phase and avoiding the toxicity and complexity associated with organic phase synthesis. Optimizing the molar ratio of cadmium, selenium, and sulfur-based compounds promotes uniform growth of quantum dot nuclei, resulting in a higher quantum yield than products synthesized using conventional organic phases. The fluorescence emission wavelength of the generated quantum dots is modulated to the visible light band, allowing direct observation using a conventional fluorescence spectrophotometer without the need for a special light source. This technique enables the probe to possess high sensitivity, good water solubility, and operational safety, overcoming the limitations of existing probes, such as difficult synthesis, poor stability, and inconvenient detection.

[0016] The quantitative probe solution and hydrogen peroxide standard solution are mixed in a buffer solution, and the initial fluorescence intensity is measured at a specific excitation wavelength. After reacting at a preset temperature and time, the fluorescence intensity after the reaction is measured again at the same excitation wavelength, and the difference between the two is calculated as the quantitative signal to plot a standard curve. This method eliminates the indirect correlation between fluorescence quenching rate or a single intensity value, directly using the absolute change in fluorescence intensity before and after the reaction to reflect the hydrogen peroxide content, reducing instrument drift and background interference. Preset reaction conditions ensure that the reaction process between the probe and hydrogen peroxide is controllable, making the standard curve more linear and stable. This technology improves the accuracy and reproducibility of peroxide value quantification, avoiding the drawbacks of existing fluorescence quantitative methods that are easily affected by external factors. Attached Figure Description

[0017] Figure 1 This is a flowchart of the method for detecting peroxide value in animal fats based on quantum dot fluorescence as described in this invention; Figure 2 Flowchart for the purification process of the reaction mixture; 3 Figure 3 This is a flowchart for calculating the fluorescence intensity difference. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] In the description of this invention, it should be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, in the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0020] See Figure 1The present invention provides a method for determining peroxide value in animal fats based on quantum dot fluorescence. Its core implementation path includes four main steps: sample pretreatment, probe preparation, standard curve establishment, and sample measurement. The animal fat sample to be tested is taken and dissolved in an organic solvent such as n-hexane, isopropanol, or chloroform to obtain a clear solution. A cadmium selenide quantum dot fluorescent probe solution with high quantum yield and fluorescence emission wavelength in a specific visible light band is prepared by aqueous synthesis, using a specific ratio of thiol compounds as stabilizers, in a reaction with cadmium and selenium sources under reflux conditions. A quantitative amount of this probe solution is mixed with a series of hydrogen peroxide standard solutions of known concentrations in a buffer system to form a concentration gradient standard reaction solution. The initial fluorescence intensity of each standard reaction solution is measured at a specific excitation wavelength. After the reaction system is placed under a preset isothermal condition for a preset time, the fluorescence intensity of each tube after the reaction is measured again at the same excitation wavelength. The fluorescence intensity difference corresponding to each concentration point was calculated. Using this difference as the ordinate and the corresponding hydrogen peroxide concentration as the abscissa, a standard working curve was plotted and a linear regression equation was fitted. Finally, the treated oil sample to be tested was reacted with an equal volume of probe solution under the same conditions, and the fluorescence intensity change was measured. The calculated fluorescence intensity difference was substituted into the linear regression equation to calculate the concentration of hydrogen peroxide equivalent in the sample. Based on the sample dilution factor and weight, the peroxide value of the animal oil sample was finally calculated in millimoles per kilogram.

[0021] In one embodiment of the invention, sodium citrate and cadmium chloride are accurately weighed in a molar ratio of 2.5:1, for example, 2.50 mmol of sodium citrate and 1.00 mmol of cadmium chloride are weighed and transferred together to a beaker containing 50 mL of deionized water. The mixture is stirred at room temperature using a magnetic stirrer until the solid is completely dissolved, obtaining a clear and transparent cadmium-containing precursor solution. In a specific implementation, the cadmium-containing precursor solution is transferred to a three-necked flask equipped with a reflux condenser, a thermometer, and a nitrogen inlet tube. The magnetic stirrer and heating device are turned on, and high-purity nitrogen is continuously introduced into the reaction system to maintain an inert protective atmosphere. The oil bath temperature is controlled to allow the reaction solution temperature to rise uniformly to a first reaction temperature of 90 degrees Celsius.

[0022] In the specific implementation, 0.173 g of sodium selenite powder was accurately weighed using an analytical balance and dissolved in 10 mL of deionized water to prepare a sodium selenite solution with a concentration of 0.1 mol / L. In the specific implementation, 0.21 mL of mercaptopropionic acid stock solution was measured using a pipette and transferred to a 10 mL volumetric flask. The solution was then diluted to the mark with deionized water and shaken well to prepare a mercaptopropionic acid solution with a concentration of 0.2 mol / L. In the specific implementation, two clean syringes were used to draw 5 mL of the prepared sodium selenite solution and 5 mL of the prepared mercaptopropionic acid solution, respectively. The needles of both syringes were simultaneously inserted into an empty mixing bottle, and the stopcock was pushed to inject both solutions simultaneously, forming a mixed solution of selenium source and mercaptopropionic acid ligand.

[0023] In the specific implementation, a new syringe was used to draw up the entire mixture of selenium source and thiol ligand. The syringe needle was quickly inserted into the feeding port of a three-necked flask, and under continuous nitrogen purging and stirring, the mixture was rapidly injected in one go into the cadmium-containing precursor solution, which had reached 90 degrees Celsius. Immediately after injection, the oil bath temperature was raised from 90 degrees Celsius to a second reaction temperature of 110 degrees Celsius, and maintained at this temperature under reflux conditions. During the reflux reaction, approximately 0.5 ml of the reaction mixture was drawn from the reaction system every 10 minutes using another syringe. The drawn sample was immediately injected into a centrifuge tube containing 2 ml of an ice-water mixture for rapid cooling to terminate the reaction. The cooled sample solution was then transferred to a quartz cuvette. In practice, a UV-Vis spectrophotometer is used to scan the UV-Vis absorption spectrum of the sample, and a fluorescence spectrophotometer is used to scan the fluorescence emission spectrum of the sample at an excitation wavelength of 380 nm. The growth of cadmium selenide quantum dots is tracked by monitoring the changes in the position of the first exciton absorption peak in the absorption spectrum and the peak wavelength of the fluorescence emission spectrum.

[0024] In practice, when monitoring data shows that the peak wavelength of the fluorescence emission spectrum reaches a specific band of 560 nm, for example, when the peak wavelength of the sample's fluorescence emission stabilizes at 560 nm after 50 minutes of reaction, the heating device is immediately turned off and the three-necked flask is removed from the oil bath. In practice, the reaction mixture, along with the flask, is placed at room temperature and allowed to cool naturally to 25 degrees Celsius under continuous stirring and nitrogen protection. The reaction mixture, cooled to room temperature, is then purified to remove unreacted ions and small molecule ligands. In practice, an equal volume of isopropanol is added to the reaction mixture, and after thorough stirring, it is allowed to stand for 10 minutes to promote the precipitation of cadmium selenide quantum dots. In practice, the mixture is transferred to centrifuge tubes and centrifuged at 12,000 rpm for 15 minutes using a high-speed centrifuge. After centrifugation, the supernatant containing free ions and small molecule ligands is carefully discarded. In practice, the cadmium selenide quantum dot precipitate at the bottom of the centrifuge tube is redissolved in 20 ml of deionized water, and a vortex mixer is used to aid dissolution to obtain a homogeneous quantum dot aqueous solution.

[0025] In the specific implementation, the purified cadmium selenide quantum dot aqueous solution was transferred to a dialysis bag. Using a dialysis bag with a molecular weight cutoff of 8000 Daltons, the bag was placed in a phosphate buffer solution with a pH of 8.0 for dialysis under continuous stirring. The external dialysis solution was replaced every 4 hours, and dialysis was performed continuously for 24 hours. After dialysis, the cadmium selenide quantum dot solution in the dialysis bag was transferred to a sample vial, and the solution volume was adjusted to 50 mL using a phosphate buffer solution with a pH of 8.0. This resulted in a homogeneous and stable cadmium selenide quantum dot fluorescent probe solution with a fluorescence emission spectrum peak wavelength of 560 nm.

[0026] See Figure 2In one embodiment of the present invention, 50 ml of the prepared cadmium selenide quantum dot fluorescent probe solution was taken, and an equal volume of 50 ml of isopropanol was added. The mixture was stirred with a glass rod until homogeneous. The mixture was then left to stand at 4 degrees Celsius for 10 minutes. Slight turbidity was observed in the solution, indicating that cadmium selenide quantum dots had precipitated. In a specific implementation, the mixture was dispensed into multiple clean 50 ml polypropylene centrifuge tubes, ensuring that the liquid volume in each tube did not exceed three-quarters of its maximum capacity. The centrifuge tubes were symmetrically placed in the rotor of a high-speed centrifuge. The centrifuge speed was set to 12,000 rpm, and the centrifugation time was set to 10 minutes. The centrifuge was then started for high-speed centrifugation. In a specific implementation, after the centrifugation program was completed, the centrifuge tubes were carefully removed. Orange-red precipitate was observed to have accumulated at the bottom of the tubes. The centrifuge tubes were kept upright, and the supernatant containing free ions and small molecule ligands was discarded using a pipette or by pouring, ensuring that the cadmium selenide quantum dot precipitate at the bottom of the centrifuge tube was not disturbed. In practice, 10 ml of deionized water is added to each centrifuge tube, and the centrifuge tube is shaken vigorously using a vortex mixer to completely redissolve the cadmium selenide quantum dot precipitate at the bottom of the centrifuge tube in the deionized water, thereby obtaining a uniformly dispersed cadmium selenide quantum dot aqueous solution.

[0027] In the specific implementation, the steps of adding polar organic solvent, high-speed centrifugation, and redissolution are repeated twice. The first repetition involves adding an equal volume of isopropanol to each centrifuge tube (10 mL of isopropanol to each tube containing 10 mL of cadmium selenide quantum dot aqueous solution), stirring, allowing to stand for 5 minutes, and then centrifuging again at 12000 rpm for 10 minutes. After discarding the supernatant, 10 mL of deionized water is added to each centrifuge tube, and the mixture is vortexed to completely redissolve the cadmium selenide quantum dot precipitate. The second repetition involves repeating the above steps, adding an equal volume of isopropanol, centrifuging again, discarding the supernatant, and then redissolving the cadmium selenide quantum dot precipitate with 10 mL of deionized water, completing three complete purification cycles.

[0028] In practice, the cadmium selenide quantum dot aqueous solutions obtained after three purification processes are combined and filtered using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10,000 Daltons. The combined cadmium selenide quantum dot aqueous solution is transferred to the upper sample well of the ultrafiltration centrifuge tube, ensuring that the maximum calibration volume is not exceeded. The ultrafiltration centrifuge tube is placed in a centrifuge and centrifuged at 5000 rpm for 20 minutes. The centrifugal force drives the solution through the ultrafiltration membrane, and the retentate retained in the upper sample well of the ultrafiltration centrifuge tube is collected. In practice, the retentate is diluted with deionized water. Deionized water is added to the retentate in the upper layer of the ultrafiltration centrifuge tube to restore the total volume to the initial volume, and the tube is centrifuged again at 5000 rpm for 20 minutes. This dilution and ultrafiltration centrifugation process is repeated five times. In practice, after each ultrafiltration centrifugation, the conductivity of the filtrate collected from the bottom of the ultrafiltration centrifuge tube is measured using a conductivity meter. When the conductivity of the filtrate collected after the fifth ultrafiltration centrifugation is less than 10 microsiemens per centimeter, the repeated operation is stopped and the purification process ends.

[0029] In the specific implementation, a series of standard reaction solutions with varying concentrations are prepared. Using calibrated micropipettes with a volume range of 20 μL to 200 μL, the pipette tip is immersed below the surface of the prepared cadmium selenide quantum dot fluorescent probe solution. 200 μL of the cadmium selenide quantum dot fluorescent probe solution is then drawn and poured into the bottom of a clean, dry quartz cuvette. In the specific implementation, 2.0 mL of pre-prepared phosphate buffer solution with a pH of 7.4 is added to the quartz cuvette containing the cadmium selenide quantum dot fluorescent probe solution. In the specific implementation, a series of hydrogen peroxide standard working solutions with progressively increasing concentrations, covering the expected detection range, are prepared using a gradient dilution method. In practice, a hydrogen peroxide standard stock solution with a concentration of 1.0 mmol / L is used as the stock solution, and phosphate buffer solution is used as the diluent. A series of dilutions are performed to prepare hydrogen peroxide standard working solutions with concentrations of 0 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L, and 80 μmol / L.

[0030] In the specific implementation, using another clean micropipette, 100 μL of hydrogen peroxide standard working solution with a concentration of 0 μmol / L was added to the first quartz cuvette that already contained cadmium selenide quantum dot fluorescent probe solution and phosphate buffer solution. Following the same procedure, 100 μL of hydrogen peroxide standard working solution with concentrations of 10 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L, and 80 μmol / L were added sequentially to the second through sixth quartz cuvettes. In the specific implementation, phosphate buffer solution was added to each quartz cuvette, the volume of which was calculated based on the total volume requirement, ensuring that the total volume of solution in each quartz cuvette was equal and 2.5 mL. In practice, the opening of each quartz cuvette is sealed with sealing film. The cuvette is then placed on a vortex mixer and vortexed at a moderate speed for 30 seconds to ensure the mixed solution inside is thoroughly homogenized, forming a series of standard reaction solutions with varying concentration gradients for fluorescence intensity measurement. In practice, the final concentrations of hydrogen peroxide in the series of standard reaction solutions are 0 μmol / L, 4 μmol / L, 8 μmol / L, 16 μmol / L, 24 μmol / L, and 32 μmol / L, respectively. The formula for calculating the final concentration is as follows: in: This represents the final concentration of hydrogen peroxide in the standard reaction solution. This represents the concentration of the added hydrogen peroxide standard working solution. This represents the volume of hydrogen peroxide standard working solution added. This represents the total volume of the standard reaction solution.

[0031] See Figure 3, in one embodiment of the present invention, a unique number is assigned to each standard reaction solution. Each number S1 to S6 is associated and recorded with the corresponding concentration of the known hydrogen peroxide standard solution. The recording method is to record in the data recording table that the concentration corresponding to number S1 is 0 micromoles per liter, the concentration corresponding to number S2 is 4 micromoles per liter, the concentration corresponding to number S3 is 8 micromoles per liter, the concentration corresponding to number S4 is 16 micromoles per liter, the concentration corresponding to number S5 is 24 micromoles per liter, and the concentration corresponding to number S6 is 32 micromoles per liter. In a specific implementation, operate the fluorescence spectrometer and retrieve the initial fluorescence intensity value measured before the reaction of the standard reaction solution numbered S1 from the data storage unit of the data processing software supporting the fluorescence spectrometer. The initial fluorescence intensity value of the standard reaction solution numbered S1 is the fluorescence intensity value measured by the fluorescence spectrometer under the conditions of an excitation wavelength of 380 nanometers and an emission wavelength of 560 nanometers. In a specific implementation, sequentially retrieve from the data storage unit of the fluorescence spectrometer the initial fluorescence intensity values measured before the reaction of the standard reaction solutions numbered S2, S3, S4, S5, and S6. The initial fluorescence intensity values of the standard reaction solutions numbered S2, S3, S4, S5, and S6 are all measured under the same conditions of an excitation wavelength of 380 nanometers and an emission wavelength of 560 nanometers.

[0032] In a specific implementation, retrieve from the data storage unit of the fluorescence spectrometer the fluorescence intensity value after the reaction measured after the standard reaction solution numbered S1 reacts for a preset duration at a preset temperature. The fluorescence intensity value after the reaction of the standard reaction solution numbered S1 is obtained under exactly the same instrument parameters as those for measuring the initial fluorescence intensity value. The instrument parameters include the same excitation wavelength of 380 nanometers, the same emission wavelength of 560 nanometers, the same instrument slit width, and the same detector gain. In a specific implementation, sequentially retrieve from the data storage unit of the fluorescence spectrometer the fluorescence intensity values after the reaction measured after the standard reaction solutions numbered S2, S3, S4, S5, and S6 have completed the reaction. The fluorescence intensity values after the reaction of the standard reaction solutions numbered S2, S3, S4, S5, and S6 are all obtained under the conditions of the same excitation wavelength of 380 nanometers, emission wavelength of 560 nanometers, instrument slit width, and detector gain.

[0033] In the specific implementation, for the standard reaction solution numbered S1, the initial fluorescence intensity value of the standard reaction solution numbered S1 is retrieved from the data recording table, and the post-reaction fluorescence intensity value of the standard reaction solution numbered S1 is retrieved from the data recording table. The post-reaction fluorescence intensity value of the standard reaction solution numbered S1 is subtracted from its initial fluorescence intensity value to obtain a specific fluorescence intensity difference value. The fluorescence intensity difference value of the standard reaction solution numbered S1 corresponds to a hydrogen peroxide concentration of 0 μmol / L for the standard reaction solution numbered S1. In the specific implementation, for the standard reaction solution numbered S2, a subtraction operation is performed, and the post-reaction fluorescence intensity value of the standard reaction solution numbered S2 is subtracted from its initial fluorescence intensity value to obtain the fluorescence intensity difference value of the standard reaction solution numbered S2. The fluorescence intensity difference value of the standard reaction solution numbered S2 corresponds to a hydrogen peroxide concentration of 4 μmol / L for the standard reaction solution numbered S2. In practice, the same subtraction operation was performed sequentially on the standard reaction solutions numbered S3, S4, S5, and S6 to obtain the fluorescence intensity differences of the standard reaction solutions numbered S3, S4, S5, and S6, respectively. The fluorescence intensity difference of the standard reaction solution numbered S3 corresponds to a hydrogen peroxide concentration of 8 μmol / L, the fluorescence intensity difference of the standard reaction solution numbered S4 corresponds to a hydrogen peroxide concentration of 16 μmol / L, the fluorescence intensity difference of the standard reaction solution numbered S5 corresponds to a hydrogen peroxide concentration of 24 μmol / L, and the fluorescence intensity difference of the standard reaction solution numbered S6 corresponds to a hydrogen peroxide concentration of 32 μmol / L.

[0034] In practical implementation, a system based on the difference in fluorescence intensity is established. The vertical axis represents the concentration of hydrogen peroxide, and the horizontal axis represents the concentration of hydrogen peroxide. This is a two-dimensional rectangular coordinate system with the x-axis as the abscissa. In the specific implementation, the fluorescence intensity difference calculated from the standard reaction solution numbered S1 is used as the ordinate value of the first data point, and the known hydrogen peroxide concentration of 0 μmol / L corresponding to the standard reaction solution numbered S1 is used as the abscissa value of the first data point. In the specific implementation, the fluorescence intensity difference calculated from the standard reaction solution numbered S2 is used as the ordinate value of the second data point, and the known hydrogen peroxide concentration of 4 μmol / L corresponding to the standard reaction solution numbered S2 is used as the abscissa value of the second data point. In the specific implementation, the fluorescence intensity differences calculated from the standard reaction solutions numbered S3, S4, S5, and S6 are used as the ordinate values ​​of the third, fourth, fifth, and sixth data points, respectively, and the known hydrogen peroxide concentrations of 8 μmol / L, 16 μmol / L, 24 μmol / L, and 32 μmol / L corresponding to the standard reaction solutions numbered S3, S4, S5, and S6 are used as the abscissa values ​​of the corresponding data points.

[0035] In practice, data plotting software is used to plot the six data points. , , , , , The data is plotted in a two-dimensional Cartesian coordinate system. In practice, the linear least squares method is used to fit all data points in the two-dimensional Cartesian coordinate system. The linear least squares method finds the best-fitting line by minimizing the sum of the squares of the perpendicular distances from each data point to the fitted line. In practice, the linear least squares method fits a straight line characterizing the relationship between the fluorescence intensity difference and the hydrogen peroxide concentration; this straight line is the standard working curve. In practice, the slope parameter of the standard working curve is obtained from data plotting software or calculation programs. With intercept parameter The slope parameter of the standard working curve The intercept parameter of the standard working curve represents the rate of change of fluorescence intensity difference with hydrogen peroxide concentration. This represents the theoretical fluorescence intensity difference when the hydrogen peroxide concentration is zero. In practical implementation, a value is formed based on the hydrogen peroxide concentration... As the independent variable, the difference in fluorescence intensity is used as the value. The dependent variable is expressed mathematically as a linear regression equation: in: Represents hydrogen peroxide concentration. Represents the difference in fluorescence intensity. The slope parameter representing the standard working curve. The intercept parameter represents the standard working curve.

[0036] In one embodiment of the present invention, 0.500 g of the lard sample to be tested is placed in a 10 mL volumetric flask. Hexane solvent is added to the volumetric flask, and the lard sample is completely dissolved using a vortex mixer. The solution is then diluted to the mark with hexane solvent and shaken well to obtain a clear lard sample solution. In a specific implementation, a 20 μL to 200 μL micropipette of the same type as used for measuring the cadmium selenide quantum dot fluorescent probe solution is used to pipette 200 μL of the cadmium selenide quantum dot fluorescent probe solution. The pipette is then transferred to the bottom of a new, clean, and dry quartz cuvette. In another specific implementation, 2.0 mL of phosphate buffer solution with a pH of 7.4 is added to the quartz cuvette containing the cadmium selenide quantum dot fluorescent probe solution. In the specific implementation, using another micropipette with a volume range of 100 μL to 1000 μL, 100 μL of clear lard sample solution is drawn up and added to a quartz cuvette containing cadmium selenide quantum dot fluorescent probe solution and phosphate buffer solution. In the specific implementation, phosphate buffer solution is added to the quartz cuvette using a micropipette, with a volume of 200 μL, ensuring that the total volume of the solution in the quartz cuvette is strictly equal to the total volume of the standard reaction solution, both being 2.5 mL. In the specific implementation, the quartz cuvette is sealed with sealing film and placed on a vortex mixer, oscillated at a moderate speed for 60 seconds to ensure thorough homogenization of the mixture within the quartz cuvette, forming the sample reaction solution.

[0037] In the specific implementation, open the sample chamber door of the fluorescence spectrometer and place the quartz cuvette containing the reaction solution of the sample to be tested inside the sample chamber of the fluorescence spectrometer, ensuring that the smooth and transparent side of the quartz cuvette is aligned with the light path direction. In the specific implementation, set the excitation wavelength parameter in the control software interface of the fluorescence spectrometer, setting the excitation wavelength to 380 nm. In the specific implementation, set the scanning range of the emission wavelength of the fluorescence spectrometer, setting the starting scan point of the emission wavelength to 500 nm, the ending scan point of the emission wavelength to 650 nm, and the scanning step size of the emission wavelength to 1 nm. In the specific implementation, start the emission spectrum scanning program of the fluorescence spectrometer. The fluorescence spectrometer scans the fluorescence emission intensity of the reaction solution of the sample to be tested in the wavelength range of 500 nm to 650 nm at an excitation wavelength of 380 nm. After the scanning program is completed, obtain the complete fluorescence emission spectrum of the reaction solution of the sample to be tested. In the specific implementation, view the obtained complete fluorescence emission spectrum in the data analysis interface of the fluorescence spectrometer, and find the wavelength position corresponding to the maximum fluorescence emission intensity in the complete fluorescence emission spectrum. This wavelength position is the peak wavelength of the fluorescence emission spectrum of cadmium selenide quantum dots. In practice, the fluorescence intensity value at a wavelength of 560 nm is read from the obtained complete fluorescence emission spectrum. This fluorescence intensity value at 560 nm is recorded as the initial fluorescence intensity value of the sample to be tested, and the initial fluorescence intensity value of the sample to be tested can be recorded in any unit. Refer to Table 1, which shows the key wavelength points extracted from the complete fluorescence emission spectrum of the sample reaction solution and their corresponding fluorescence intensity values: Table 1: Key Wavelengths and Corresponding Fluorescence Intensity Values In the specific implementation, the quartz cuvette, after the initial fluorescence intensity value of the sample to be tested has been measured, is removed from the sample chamber of the fluorescence spectrometer, ensuring the exterior of the quartz cuvette is clean. The quartz cuvette is then transferred to a preheated water bath at 40°C (the water bath temperature display reads 40.0°C). The quartz cuvette is placed in the water bath, and the reaction time is started. When the preset reaction time of 15 minutes has been reached, the quartz cuvette is removed from the 40°C water bath. The removed quartz cuvette is placed on a room temperature experimental platform to allow the sample reaction solution inside to cool naturally to room temperature (25°C). Finally, the cooled quartz cuvette is placed back into the sample chamber of the fluorescence spectrometer, maintaining the same position and orientation as during the first measurement. In the specific implementation, the emission spectrum scanning program of the fluorescence spectrometer is restarted without changing any instrument parameters. The instrument parameters include the same excitation wavelength of 380 nm, emission wavelength scanning range of 500 nm to 650 nm, scanning step size of 1 nm, instrument slit width, and detector gain as the first measurement. After the scanning program is completed, the complete fluorescence emission spectrum of the reaction solution of the test sample is obtained. From the complete fluorescence emission spectrum obtained after the reaction, the fluorescence intensity value is read at a wavelength of 560 nm, the same as the initial fluorescence intensity value of the test sample. This fluorescence intensity value read at 560 nm is recorded as the fluorescence intensity value of the test sample after the reaction, where the fluorescence intensity value after the reaction is 135 arbitrary units. The difference between the initial fluorescence intensity value and the fluorescence intensity value after the reaction is calculated. The initial fluorescence intensity value is 158 arbitrary units, and the fluorescence intensity value after the reaction is 135 arbitrary units. The calculation formula is: in: Represents the difference in fluorescence intensity. This represents the initial fluorescence intensity value of the sample to be tested. The fluorescence intensity value represents the fluorescence intensity value of the sample after the reaction, and the fluorescence intensity difference is calculated. Any unit.

[0038] In one embodiment of the present invention, based on a linear regression equation The slope parameter in the linear regression equation The value is 0.75 per micromoles per liter, the intercept parameter in the linear regression equation. The value is 1.2 in any unit. In specific implementation, the fluorescence intensity difference calculated in Example 4 will be used. Substituting any unit into the linear regression equation Substituting the values, we get the equation. ,right The molar concentration of hydrogen peroxide equivalent in the reaction solution of the test sample is obtained by solving the problem. Micromoles per liter. In practice, the experimental records confirm that the volume of the clarified lard sample solution added during the preparation of the test sample reaction solution was 100 microliters, i.e. Microliters. In practice, this is determined based on the molar concentration of hydrogen peroxide equivalent in the reaction solution of the sample to be tested. The volume of the clarified lard sample solution added per micromolar per liter Calculate the amount of hydrogen peroxide equivalent in the added volume of the clarified lard sample solution (µL). The calculation formula is: , This represents the amount of hydrogen peroxide equivalent contained in the added volume of the clarified lard sample solution. This represents the molar concentration of hydrogen peroxide equivalents in the reaction solution of the sample to be tested. The volume of the clarified lard sample solution added during the preparation of the test sample reaction solution is used to calculate the volume of the solution. .

[0039] In practice, the sample pretreatment process was traced back, and the volume of organic solvent used when initially dissolving the lard sample was verified from the experimental records. 0.500 g of lard sample was placed in a 10 mL volumetric flask, dissolved in n-hexane, and diluted to the 10 mL mark. The volume of organic solvent was... Milliliters. In practice, the mass of the weighed original lard sample is verified from the experimental records. Grams. In specific implementation, the mass of raw animal fat contained per unit volume in the clarified lard sample solution to be tested is calculated. The calculation formula is: , This represents the mass of the original animal fat contained per unit volume in the clarified lard sample solution. This represents the mass of the original lard sample taken. The volume of organic solvent used when initially dissolving the lard sample was calculated. .

[0040] In practice, this is combined with the mass of the original animal fat contained per unit volume in the clarified lard sample solution to be tested. The amount of hydrogen peroxide equivalent per milliliter of the clarified lard sample solution added, in grams per milliliter. Micromoles (µmol) are the number of millimoles of active oxygen per kilogram of raw lard sample, calculated according to the standard definition of peroxide value. The standard definition of peroxide value is the number of millimoles of active oxygen per kilogram of fat. The calculation formula is: , Peroxide value is expressed in millimoles per kilogram. The amount of hydrogen peroxide equivalent contained in the added volume of the clarified lard sample solution is expressed in micromoles. This represents the volume of clear lard sample solution added during the preparation of the test sample reaction solution, in milliliters. This represents the mass of raw animal fat per unit volume in a clarified lard sample solution, expressed in grams per milliliter. The calculation yields... .

[0041] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for detecting peroxide value in animal fats based on quantum dot fluorescence, characterized in that, The method includes: Take the animal fat sample to be tested, dissolve it with an organic solvent, and obtain a clear fat solution to be tested; Sodium citrate and cadmium chloride were accurately weighed according to a molar ratio of 2.5:1 and dissolved in deionized water to obtain a clear cadmium-containing precursor solution. Under inert gas protection, the cadmium-containing precursor solution was transferred to a three-necked flask and heated to a first reaction temperature of 90 degrees Celsius. A sodium selenite solution with a concentration of 0.1 mol / L and a sulfur-based compound solution with a concentration of 0.2 mol / L are mixed using a syringe to form a mixed solution of selenium source and sulfur-based ligand, wherein the sulfur-based compound is one of mercaptoacetic acid, mercaptopropionic acid or glutathione. The mixed solution of the selenium source and the sulfur-based ligand is rapidly injected into the cadmium-containing precursor solution that has reached the first reaction temperature. Then, the reaction system is heated to the second reaction temperature of 110 degrees Celsius for reflux reaction. During the reflux reaction, samples were taken every 10 minutes and their ultraviolet-visible absorption and fluorescence emission spectra were measured to monitor the growth of cadmium selenide quantum dots. When the peak wavelength of the fluorescence emission spectrum of the cadmium selenide quantum dots reaches a specific visible light band of 560 nanometers, heating is stopped and the mixture is allowed to cool naturally to room temperature. The cooled reaction mixture was purified to remove unreacted ions and small molecule ligands. The purified cadmium selenide quantum dots were then redispersed in a phosphate buffer solution with a pH of 8.0 to obtain a cadmium selenide quantum dot fluorescent probe solution. A quantitative amount of the cadmium selenide quantum dot fluorescent probe solution was mixed with a hydrogen peroxide standard solution of known concentration in a buffer solution to form a series of standard reaction solutions with concentration gradients. The initial fluorescence intensity value of each standard reaction solution at an excitation wavelength of 380 nm was measured. After the series of standard reaction solutions with concentration gradients are placed at a preset temperature and reacted for a preset time, the fluorescence intensity value of each standard reaction solution after the reaction is measured at the same 380 nm excitation wavelength. Calculate the difference between the initial fluorescence intensity value and the fluorescence intensity value after the reaction corresponding to each concentration point, plot the difference as the ordinate and the concentration of the corresponding hydrogen peroxide standard solution as the abscissa to obtain a standard working curve, and fit the curve to obtain a linear regression equation. Based on the linear regression equation, the peroxide value of the animal fat sample to be tested is obtained.

2. The method for detecting peroxide value in animal fats based on quantum dot fluorescence according to claim 1, characterized in that, The purification process of the cooled reaction mixture to remove unreacted ions and small molecule ligands includes: Add an equal volume of isopropanol to the cooled reaction mixture, stir thoroughly, and let stand for 10 minutes to promote the precipitation of the cadmium selenide quantum dots. The reaction mixture was centrifuged at 12,000 rpm for 15 minutes, and the supernatant containing free ions and small molecule ligands was discarded. The cadmium selenide quantum dot precipitate at the bottom of the centrifuge tube was redissolved in 20 ml of deionized water, and a vortex mixer was used to aid dissolution. Repeat the steps of adding isopropanol, high-speed centrifugation, and redissolving at least twice; The resulting quantum dot aqueous solution was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10,000 Daltons and centrifuged at 5,000 rpm for 20 minutes for filtration separation. The retentate was then collected. The retentate was diluted with deionized water and then subjected to ultrafiltration centrifugation again. The dilution and ultrafiltration steps were repeated five times until the conductivity of the filtrate in the lower layer of the centrifuge tube after ultrafiltration was less than 10 microsiemens per centimeter.

3. The method for detecting peroxide value in animal fats based on quantum dot fluorescence according to claim 2, characterized in that, The measured amount of the cadmium selenide quantum dot fluorescent probe solution is mixed with a hydrogen peroxide standard solution of known concentration in a buffer solution to form a series of standard reaction solutions with concentration gradients, including: Accurately measure 200 μL of the cadmium selenide quantum dot fluorescent probe solution using a micropipette and add it to multiple identical quartz cuvettes. Add 2.0 mL of buffer solution, which is a phosphate buffer solution with a pH of 7.4, to each of the quartz cuvettes. Hydrogen peroxide standard working solutions with concentrations of 0 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L and 80 μmol / L were prepared using a gradient dilution method. Add 100 μL of hydrogen peroxide standard working solution of different concentrations to each quartz cuvette containing the cadmium selenide quantum dot fluorescent probe solution and buffer solution, and replenish with phosphate buffer solution to ensure that the total volume of the solution in each quartz cuvette is 2.5 mL. After sealing the mixed solution in each quartz cuvette with sealing film, place it on a vortex shaker and shake at a moderate speed for 30 seconds to form a series of standard reaction solutions with the fluorescence intensity to be measured. The final concentrations of hydrogen peroxide in the standard reaction solutions of the series of concentration gradients were 0 μmol / L, 4 μmol / L, 8 μmol / L, 16 μmol / L, 24 μmol / L, and 32 μmol / L, respectively.

4. The method for detecting peroxide value in animal fats based on quantum dot fluorescence according to claim 3, characterized in that, The calculation of the difference between the initial fluorescence intensity value and the fluorescence intensity value after the reaction corresponding to each concentration point includes: Record the number of each standard reaction solution and the concentration of its corresponding hydrogen peroxide standard solution; The initial fluorescence intensity value of each standard reaction solution measured before the reaction is retrieved from the storage unit of the fluorescence spectrometer. The initial fluorescence intensity value is the fluorescence intensity value read at the peak wavelength of the fluorescence emission spectrum of the cadmium selenide quantum dots at 560 nm under an excitation wavelength of 380 nm. The fluorescence intensity value after reaction of each standard reaction solution is retrieved from the storage unit of the fluorescence spectrometer. The fluorescence intensity value after reaction is obtained under the same instrument parameters as the initial fluorescence intensity value, including the same excitation wavelength of 380 nm, the same emission wavelength of 560 nm, the same instrument slit width, and the same detector gain. For standard reaction solutions with the same number, the fluorescence intensity value after the reaction is subtracted from the initial fluorescence intensity value to obtain a fluorescence intensity difference value, which corresponds to the hydrogen peroxide concentration of the standard reaction solutions with the same number.

5. The method for detecting peroxide value in animal fats based on quantum dot fluorescence according to claim 4, characterized in that, A standard working curve was plotted with the difference as the ordinate and the corresponding concentration of the hydrogen peroxide standard solution as the abscissa, and a linear regression equation was obtained by fitting the curve, including: Establish a system based on the difference in fluorescence intensity The vertical axis represents the concentration of hydrogen peroxide, and the horizontal axis represents the concentration of hydrogen peroxide. A two-dimensional rectangular coordinate system with the x-axis as the coordinate. The fluorescence intensity difference calculated for each standard reaction solution is used as the ordinate value of the data point; The concentration of the hydrogen peroxide standard solution corresponding to each standard reaction solution is used as the x-axis value of the same data point; Plot all data points in the two-dimensional Cartesian coordinate system. The linear least squares method is used to fit all data points in the two-dimensional rectangular coordinate system to obtain a straight line characterizing the relationship between the fluorescence intensity difference and the hydrogen peroxide concentration, which is the standard working curve. Obtain the slope parameter k and intercept parameter b of the straight line, and form a line with hydrogen peroxide concentration. As the independent variable, the difference in fluorescence intensity is used as the value. The mathematical expression for the dependent variable is the linear regression equation.

6. The method for detecting peroxide value in animal fats based on quantum dot fluorescence according to claim 5, characterized in that, Based on the linear regression equation, the peroxide value of the animal fat sample to be tested is obtained, including: Take the same amount of the cadmium selenide quantum dot fluorescent probe solution used to construct the standard curve, and mix it with the clarified test oil solution in the same buffer solution to form the test sample reaction solution; The initial fluorescence intensity of the test sample reaction solution was measured at the excitation wavelength of 380 nm. After reacting the test sample solution at the preset temperature for the preset time, the fluorescence intensity value of the test sample after reaction is measured at the excitation wavelength of 380 nm. Calculate the difference between the initial fluorescence intensity value of the sample to be tested and the fluorescence intensity value of the sample to be tested after the reaction, substitute the difference into the linear regression equation, and calculate the concentration of hydrogen peroxide equivalent in the reaction solution of the sample to be tested; Based on the dilution factor and sample volume of the oil solution to be tested, the concentration of the hydrogen peroxide equivalent is converted into the peroxide value of the animal oil sample to be tested. The step of taking the same amount of the cadmium selenide quantum dot fluorescent probe solution used to construct the standard curve and mixing it with the clarified test oil solution in the same buffer solution to form the test sample reaction solution specifically includes: Using a 20-µL to 200-µL micropipette of the same type as used for measuring the cadmium selenide quantum dot fluorescent probe solution, aspirate 200 µL of the cadmium selenide quantum dot fluorescent probe solution and transfer it into a new quartz cuvette. Add 2.0 mL of phosphate buffer solution with a pH of 7.4 to the quartz cuvette; Using another micropipette with a volume range of 100 μL to 1000 μL, accurately measure 100 μL of the clear oil solution to be tested and add it to the quartz cuvette. Add 200 μL of phosphate buffer solution to the quartz cuvette so that the total volume of the solution in the quartz cuvette is 2.5 mL, which is exactly equal to the total volume of the standard reaction solution used to construct the standard curve. The quartz cuvette is sealed with a sealing film and placed on a vortex shaker and oscillated at a moderate speed for 60 seconds to form the reaction solution of the sample to be tested.

7. The method for detecting peroxide value in animal fats based on quantum dot fluorescence according to claim 6, characterized in that, The determination of the initial fluorescence intensity value of the test sample reaction solution at the excitation wavelength of 380 nm includes: Place the transparent reaction container containing the reaction solution of the sample to be tested into the sample chamber of the fluorescence spectrometer; Set the excitation wavelength of the fluorescence spectrometer to the 380 nm excitation wavelength, and set the scanning range of the emission wavelength to cover the band where the peak wavelength of the fluorescence emission spectrum of the cadmium selenide quantum dots is located; Start the scanning program of the fluorescence spectrometer to obtain the complete fluorescence emission spectrum of the reaction solution of the sample to be tested; From the obtained complete fluorescence emission spectrum, locate the position at 560 nm corresponding to the peak wavelength of the fluorescence emission spectrum of the cadmium selenide quantum dot, and read the fluorescence intensity value at the position. Record the fluorescence intensity value as the initial fluorescence intensity value of the sample to be tested.

8. The method for detecting peroxide value in animal fats based on quantum dot fluorescence according to claim 7, characterized in that, After reacting the test sample solution at the preset temperature for the preset time, the fluorescence intensity value of the test sample after reaction at the excitation wavelength of 380 nm is measured, including: Remove the quartz cuvette, from the sample chamber of the fluorescence spectrometer, after the initial fluorescence intensity value of the sample to be tested has been determined. The quartz cuvette was transferred to a preheated water bath at 40 degrees Celsius, and the reaction was started. When the reaction time reaches 15 minutes, remove the quartz cuvette from the constant temperature water bath and allow it to cool naturally to room temperature. After cooling, the quartz cuvette was placed back into the sample chamber of the fluorescence spectrometer. Without changing any instrument parameters, the emission spectrum scanning program of the fluorescence spectrometer was restarted to obtain the complete fluorescence emission spectrum of the reaction solution of the sample to be tested after the reaction. From the complete fluorescence emission spectrum obtained after the reaction, the fluorescence intensity value is read at the same wavelength position of 560 nm as the initial fluorescence intensity value of the sample to be tested, and the fluorescence intensity value is recorded as the fluorescence intensity value of the sample to be tested after the reaction.

9. The method for detecting peroxide value in animal fats based on quantum dot fluorescence according to claim 8, characterized in that, Based on the dilution factor and sample volume of the oil solution to be tested, the concentration of the hydrogen peroxide equivalent is converted into the peroxide value of the animal oil sample to be tested, including: The concentration value calculated from the linear regression equation is the molar concentration of hydrogen peroxide equivalent in the reaction solution of the test sample. Calculate the amount of hydrogen peroxide equivalent contained in the volume of the clarified oil solution added when preparing the test sample reaction solution. Calculate the mass of the original animal fat per unit volume in the clarified test fat solution based on the volume of the organic solvent used to initially dissolve the animal fat sample and the mass of the animal fat sample weighed. By combining the mass of the original animal fat per unit volume in the clarified test oil solution and the amount of hydrogen peroxide equivalent in the test sample reaction solution, the number of millimoles of active oxygen per kilogram of the original animal fat sample is calculated according to the standard definition of peroxide value, thus obtaining the peroxide value of the test animal fat sample in millimoles per kilogram.

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