A method for detecting food additives using bluefish stone fluorescent carbon quantum dots

Abstract

The invention relates to the technical field of food detection, and in particular to a method for detecting food additives by using bluefish stone fluorescent carbon quantum dots. The food additive detection method constructed based on the fluorescence quenching phenomenon of bluefish stone fluorescent carbon quantum dots has good selectivity and anti-interference for amaranth and sunset yellow. Under optimal experimental conditions, the fluorescence intensity of the carbon quantum dots has a good linear relationship with the concentrations of sunset yellow and amaranth, and the detection limit is low, which is 1.513 μM (amaranth) and 2.044 μM (sunset yellow), respectively. The precision is high and the relative standard deviation is small. The average recovery rate of actual sample detection is high, which is 100.36% (amaranth) and 98.40% (sunset yellow), respectively. The method can meet the requirements for detecting additives in food, provide a new and reliable method for detecting food additives, and has broad application prospects in the field of food safety.

Description

Technical Field

[0001] The invention relates to the technical field of food detection, and in particular to a method for detecting food additives by utilizing bluefish stone fluorescent carbon quantum dots. Background Art

[0002] Food additives are widely used in the food industry, and they play an important role in extending the shelf life of food, improving food quality and taste. However, if some food additives are used excessively or illegally, they may cause potential harm to human health, such as organ toxicity, carcinogenicity, mutagenicity, etc., and may also cause environmental pollution. Therefore, accurate and rapid detection of the content of food additives is crucial to ensure food safety and human health.

[0003] At present, conventional methods for detecting food additives include electrochemical methods, spectroscopy, chromatography, etc. Although these methods are constantly developing and improving, they still have some disadvantages, such as long time consumption, high sample requirements, reliance on large equipment, expensive equipment, and the need for professional operators. With the development of nanomaterials and nanotechnology, carbon quantum dots (CDs) have shown great application potential in the field of food testing due to their unique optical properties, chemical stability, anti-bleaching and photobleaching properties, low toxicity, good biocompatibility and other excellent properties. However, the traditional carbon quantum dot synthesis method has limitations such as long processing time, harsh reaction conditions, expensive materials and equipment, and the need to add irritating reagents.

[0004] Therefore, according to the above-mentioned related technologies, it is urgent to develop a method for detecting food additives using bluefish stone fluorescent carbon quantum dots. Summary of the invention

[0005] In view of this, the purpose of the present invention is to propose a method for detecting food additives using bluefish stone fluorescent carbon quantum dots, so as to provide a green, efficient and sensitive method for detecting food additives using carbon quantum dots.

[0006] Based on the above purpose, the present invention provides a method for detecting food additives using bluefish stone fluorescent carbon quantum dots.

[0007] A method for detecting food additives using bluefish stone fluorescent carbon quantum dots comprises the following steps:

[0008] Step S1. Preparation of carbon quantum dots;

[0009] Step S2. drawing a standard curve;

[0010] Step S3. Sample testing;

[0011] The carbon quantum dots in step S1 are prepared from blackfish stone and blackfish stone modified by plant extracts;

[0012] The average particle size of the carbon quantum dots in step S1 is 3.2-3.8 nm.

[0013] The preparation of the carbon quantum dots in step S1 comprises the following steps:

[0014] Step S101. Take blackfish stone, clean it and grind it into powder to obtain blackfish stone powder;

[0015] Step S102. Weigh the blackfish stone powder, add it to deionized water, then add the plant extract, stir magnetically for 28-32 minutes to fully disperse the blackfish stone powder in the water, and then perform ultrasonic treatment for 4-6 minutes to obtain a mixed solution 1;

[0016] Step S103. The mixed solution 1 is transferred to a polytetrafluoroethylene-lined autoclave, sealed and placed in an oven for reaction to obtain a mixed solution 2;

[0017] Step S104. After the reaction is completed, the mixed solution 2 is naturally cooled to room temperature, and then transferred to a centrifuge tube, centrifuged and the supernatant is collected;

[0018] Step S105. Filter the supernatant with a 0.22 μm microporous filter membrane to obtain a clear light yellow carbon quantum dot solution;

[0019] Step S106. placing the obtained carbon quantum dot solution in a dialysis bag with a molecular weight of 500 Da, and dialyzing it in deionized water for 8-10 h;

[0020] Step S107. Finally, the dialyzed solution is freeze-dried to obtain a yellow-brown solid product, which is dispersed with deionized water to obtain a transparent suspension with a concentration of 5-6 mg / mL, thereby obtaining the bluefish stone fluorescent carbon quantum dots for detecting food additives.

[0021] Preferably, the usage ratio of the blackfish stone powder, deionized water and plant extract in step S102 is 0.18-0.22 g: 9-12 mL: 0.018-0.022 g.

[0022] The plant extract in step S102 is any one of green tea extract and grape seed extract.

[0023] Preferably, the reaction temperature in step S103 is 178-182° C., and the reaction time is 10-12 h.

[0024] Preferably, the centrifugal speed in step S104 is 4100-4200 rpm, and the centrifugal time is 5-7 min.

[0025] Preferably, the standard curve drawing in step S2 comprises the following steps:

[0026] Step S201. Prepare standard solutions of food additives with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM respectively;

[0027] Step S202. Adding food additive standard solutions with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM and 60 μM into different colorimetric tubes, respectively adding carbon quantum dot solutions into each colorimetric tube, and mixing thoroughly to obtain a carbon quantum dot-food additive system;

[0028] Step S203. Under a fluorescence excitation wavelength of 373 nm, the fluorescence intensity of each carbon quantum dot-food additive system is measured using a fluorescence spectrophotometer;

[0029] Step S204. Draw a standard curve with the concentration of the food additive as the horizontal axis and the fluorescence quenching rate as the vertical axis to obtain the corresponding linear regression equation.

[0030] Preferably, it is characterized in that the dosage ratio of the food additive solution and the carbon quantum dot solution in step S202 is 4.4-4.6mL:0.4-0.6mL.

[0031] Preferably, it is characterized in that the formula of the fluorescence quenching rate in step S204 is: ,in is the fluorescence intensity of the carbon quantum dot solution without adding food additives, and F is the fluorescence intensity of the carbon quantum dot solution after adding food additives.

[0032] Preferably, it is characterized in that the sample detection in step S3 comprises the following steps:

[0033] Step S301. Dilute the sample to be tested 50 times with PBS buffer solution to obtain a mixed solvent;

[0034] Step S302. Adding the food additive to the mixed solvent to prepare 5 μM, 30 μM and 50 μM food additive solutions respectively;

[0035] Step S303. Take 0.4-0.6 mL of carbon quantum dot solution in a colorimetric tube, add 4.4-4.6 mL of food additive solution, and mix thoroughly to obtain a sample detection system;

[0036] Step S304. Using a fluorescence spectrophotometer to measure the fluorescence intensity of the sample detection system at a fluorescence excitation wavelength of 373 nm;

[0037] Step S305. Calculate the concentration of the food additive in the sample based on the measured fluorescence intensity and the linear regression equation of the standard curve;

[0038] Step S306. Calculate the recovery rate through the spike recovery experiment to evaluate the accuracy of the detection method;

[0039] The pH of the PBS buffer solution in step S301 is 7.

[0040] Beneficial effects of the present invention:

[0041] The present invention provides a method for detecting food additives by using fluorescent carbon quantum dots from blackfish stone. The present invention uses blackfish stone as a raw material to prepare carbon quantum dots. Blackfish stone is a waste. The resource utilization of waste is realized, which conforms to the concept of green chemistry and reduces costs at the same time.

[0042] The present invention adopts a hydrothermal synthesis method to prepare carbon quantum dots. The method has simple operation, mild reaction conditions, does not require complex equipment and expensive reagents, and has good repeatability and large-scale production potential.

[0043] The bluefish stone fluorescent carbon quantum dots prepared by the invention have good stability, high fluorescence quantum yield, good biocompatibility and good water solubility. Under different pH values ​​and ionic strength conditions, the fluorescence intensity thereof remains basically stable, which is conducive to detection in complex food systems.

[0044] The food additive detection method constructed based on the fluorescence quenching phenomenon of bluefish stone fluorescent carbon quantum dots has good selectivity and anti-interference for amaranth and sunset yellow. Under the optimal experimental conditions, the fluorescence intensity of the carbon quantum dots has a good linear relationship with the concentrations of sunset yellow and amaranth, and the detection limit is low, which is 1.513 μM (amaranth) and 2.044 μM (sunset yellow), respectively. The precision is high and the relative standard deviation is small. The average recovery rate of actual sample detection is high, which is 100.36% (amaranth) and 98.40% (sunset yellow), respectively. The method can meet the requirements for the detection of additives in food, provide a new and reliable method for the detection of food additives, and has broad application prospects in the field of food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 TEM images of carbon quantum dots at different magnifications in the present invention;

[0047] Figure 2 It is a statistical diagram of the particle size distribution of carbon quantum dots in the present invention;

[0048] Figure 3 is the XRD image of the carbon quantum dots in the present invention;

[0049] Figure 4 The XPS measurement spectrum of the carbon quantum dots in the present invention;

[0050] Figure 5 is the high-resolution C1s peak and fitting curve in the present invention;

[0051] Figure 6 It is the high-resolution N1s peak and fitting curve in the present invention;

[0052] Figure 7 It is the high-resolution O1s peak and fitting curve in the present invention;

[0053] Figure 8 It is the high-resolution Sp2 peak and fitting curve in the present invention;

[0054] Fig. 9 FT-IR spectrum of carbon quantum dots in the present invention;

[0055] Fig.10 The ultraviolet absorption spectra of carbon quantum dots CDs, amaranth and sunset yellow, the excitation spectrum and emission spectrum of carbon quantum dots CDs in the present invention, and the illustrations are CDs solutions under natural light and CDs solutions under ultraviolet light;

[0056] Fig.11 is the emission spectrum of carbon quantum dots CDs at different excitation wavelengths;

[0057] Fig.12 is the change of fluorescence intensity under different pH conditions;

[0058] Fig.13 is the change of fluorescence intensity under different concentrations of NaCl;

[0059] Fig.14 is the linear relationship between fluorescence intensity and time;

[0060] Fig.15 The selectivity of carbon quantum dots CDs to amaranth;

[0061] Fig.16 is the selectivity of carbon quantum dots CDs to sunset yellow;

[0062] Fig.17 The anti-interference of carbon quantum dots CDs to amaranth red;

[0063] Fig.18 The anti-interference property of carbon quantum dots CDs to sunset yellow;

[0064] Fig.19 is the linearity test of sunset yellow;

[0065] Fig. 20 is the linearity test of amaranth;

[0066] Fig.21 Cell survival rate at different carbon quantum dots CDs concentrations. DETAILED DESCRIPTION

[0067] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0068] The sources and properties of some of the raw materials used in the present invention are as follows:

[0069] Deionized water was from the laboratory (Shenyang, China);

[0070] Oriental leaf and green plum green tea were purchased from a local market (Shenyang, China);

[0071] Food additives Amaranth, sunset yellow, acesulfame potassium, sodium citrate, malic acid, citric acid, calcium carbonate, titanium dioxide, potassium chloride, and vitamin C were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China);

[0072] NaOH and HCl were purchased from Li’an Long Bohua Pharmaceutical Chemical Co., Ltd. (Tianjin, China);

[0073] NaCl was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd. (Tianjin, China);

[0074] PBS buffer solution was purchased from Shanghai Ruichu Biotechnology Co., Ltd. (Shanghai, China);

[0075] Green tea extract is commercially available green tea extract;

[0076] The grape seed extract is commercially available grape seed extract.

[0077] The instruments and equipment used in the present invention are as follows:

[0078] F97 fluorescence spectrophotometer (Shanghai Lingguang Technology Co., Ltd., China);

[0079] Thermo Fisher EscaLab 250Xi X-ray photoelectron spectrometer (Thermo Fisher Scientific, USA);

[0080] Thermo Fisher Nicolet IS50 FT-IR spectrometer (Thermo FisherScientific, USA);

[0081] PHS-3C pH meter (INESA Scientific Instrument Co., Ltd., Shanghai, China);

[0082] UV-5500 PC spectrophotometer (Metasch Instrument Co., Ltd., Shanghai, China);

[0083] FEI talos F200S high-resolution transmission electron microscope (Thermo Fisher Scientific, USA);

[0084] D8 ADVANCE X-ray diffractometer (Bruker, Germany);

[0085] TD5G desktop low-speed centrifuge (Kate Experimental Instrument Co., Ltd., Yancheng City, China);

[0086] KQ-250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China);

[0087] HJ-6B digital display constant temperature and speed measurement magnetic stirrer (Changzhou Su Rui Instrument Co., Ltd.).

[0088] Embodiment 1: A method for preparing carbon quantum dots, comprising the following steps:

[0089] S1. Take the black fish stone, clean it and grind it into powder to obtain black fish stone powder;

[0090] S2. Weigh 0.18 g of herringbone powder, add it to 9 mL of deionized water, then add 0.018 g of green tea extract, stir magnetically for 28 min to fully disperse the herringbone powder in water, and then perform ultrasonic treatment for 4 min to obtain a mixed solution 1;

[0091] S3. The mixed solution 1 was transferred to a polytetrafluoroethylene-lined autoclave, sealed, and placed in an oven for reaction at 178° C. for 10 h to obtain a mixed solution 2;

[0092] S4. After the reaction, the mixture 2 was naturally cooled to room temperature, then transferred to a centrifuge tube, centrifuged at 4100 rpm for 5 min and the supernatant was collected;

[0093] S5. The supernatant was filtered through a 0.22 μm microporous filter membrane to obtain a clear light yellow carbon quantum dot solution;

[0094] S6. The obtained carbon quantum dot solution was placed in a dialysis bag with a molecular weight of 500 Da and dialyzed in deionized water for 8 h;

[0095] S7. Finally, the dialyzed solution is freeze-dried to obtain a yellow-brown solid product, which is dispersed with deionized water to prepare a transparent suspension with a concentration of 5 mg / mL, thereby obtaining the bluefish stone fluorescent carbon quantum dots for detecting food additives, wherein the particle size of the carbon quantum dots is 3.2 nm.

[0096] Embodiment 2: A method for preparing carbon quantum dots, comprising the following steps:

[0097] S1. Take the black fish stone, clean it and grind it into powder to obtain black fish stone powder;

[0098] S2. Weigh 0.2 g of herringbone powder, add it to 10.5 mL of deionized water, then add 0.02 g of green tea extract, stir magnetically for 30 min to fully disperse the herringbone powder in water, and then perform ultrasonic treatment for 5 min to obtain a mixed solution 1;

[0099] S3. The mixed solution 1 was transferred to a polytetrafluoroethylene-lined autoclave, sealed and placed in an oven, and reacted at 180° C. for 11 h to obtain a mixed solution 2;

[0100] S4. After the reaction, the mixture 2 was naturally cooled to room temperature, then transferred to a centrifuge tube, centrifuged at 4150 rpm for 6 min and the supernatant was collected;

[0101] S5. The supernatant was filtered through a 0.22 μm microporous filter membrane to obtain a clear light yellow carbon quantum dot solution;

[0102] S6. The obtained carbon quantum dot solution was placed in a dialysis bag with a molecular weight of 500 Da and dialyzed in deionized water for 9 h;

[0103] S7. Finally, the dialyzed solution is freeze-dried to obtain a yellow-brown solid product, which is dispersed with deionized water to prepare a transparent suspension with a concentration of 5.5 mg / mL, that is, the bluefish stone fluorescent carbon quantum dots for detecting food additives are obtained, wherein the particle size of the carbon quantum dots is 3.5 nm.

[0104] Embodiment 3: A method for preparing carbon quantum dots, comprising the following steps:

[0105] S1. Take the black fish stone, clean it and grind it into powder to obtain black fish stone powder;

[0106] S2. Weigh 0.22 g of herringite powder, add it to 12 mL of deionized water, then add 0.022 g of grape seed extract, stir magnetically for 32 min to fully disperse the herringite powder in water, and then perform ultrasonic treatment for 6 min to obtain a mixed solution 1;

[0107] S3. The mixed solution 1 was transferred to a polytetrafluoroethylene-lined autoclave, sealed and placed in an oven, and reacted at 182° C. for 12 h to obtain a mixed solution 2;

[0108] S4. After the reaction, the mixture 2 was naturally cooled to room temperature, then transferred to a centrifuge tube, centrifuged at 4200 rpm for 7 min and the supernatant was collected;

[0109] S5. The supernatant was filtered through a 0.22 μm microporous filter membrane to obtain a clear light yellow carbon quantum dot solution;

[0110] S6. The obtained carbon quantum dot solution was placed in a dialysis bag with a molecular weight of 500 Da and dialyzed in deionized water for 10 h;

[0111] S7. Finally, the dialyzed solution is freeze-dried to obtain a yellow-brown solid product, which is dispersed with deionized water to prepare a transparent suspension with a concentration of 6 mg / mL, thereby obtaining the bluefish stone fluorescent carbon quantum dots for detecting food additives, wherein the particle size of the carbon quantum dots is 3.8 nm.

[0112] Example 4: Standard curve drawing, comprising the following steps:

[0113] S1. Prepare amaranth standard solutions with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM respectively;

[0114] S2. 4.4 mL of amaranth standard solution with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM and 60 μM were added to different colorimetric tubes, and 0.4 mL of carbon quantum dot solution was added to each colorimetric tube, and the carbon quantum dot-amaranth system was obtained after thorough mixing;

[0115] S3. The fluorescence intensity of each carbon quantum dot-amaranth system was measured using a fluorescence spectrophotometer at a fluorescence excitation wavelength of 373 nm;

[0116] S4. With the concentration of amaranth red as the horizontal axis and the fluorescence quenching rate as the vertical axis, the formula for the fluorescence quenching rate is: ,in is the fluorescence intensity of the carbon quantum dot solution without adding food additives, F is the fluorescence intensity of the carbon quantum dot solution after adding food additives, draw a standard curve, and get the corresponding linear regression equation.

[0117] Example 5: Standard curve drawing, comprising the following steps:

[0118] S1. Prepare amaranth standard solutions with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM respectively;

[0119] S2. 4.5 mL of amaranth standard solution with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM and 60 μM were added to different colorimetric tubes, and 0.5 mL of carbon quantum dot solution was added to each colorimetric tube, and the carbon quantum dot-amaranth system was obtained after thorough mixing;

[0120] S3. The fluorescence intensity of each carbon quantum dot-amaranth system was measured using a fluorescence spectrophotometer at a fluorescence excitation wavelength of 373 nm;

[0121] S4. With the concentration of amaranth red as the horizontal axis and the fluorescence quenching rate as the vertical axis, the formula for the fluorescence quenching rate is: ,in is the fluorescence intensity of the carbon quantum dot solution without adding food additives, F is the fluorescence intensity of the carbon quantum dot solution after adding food additives, draw a standard curve, and get the corresponding linear regression equation.

[0122] Example 6: Standard curve drawing, comprising the following steps:

[0123] S1. Prepare sunset yellow standard solutions with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM respectively;

[0124] S2. Add 4.6 mL of sunset yellow standard solution with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM and 60 μM into different colorimetric tubes, add 0.6 mL of carbon quantum dot solution into each colorimetric tube, mix thoroughly to obtain a carbon quantum dot-sunset yellow system;

[0125] S3. The fluorescence intensity of each carbon quantum dot-sunset yellow system was measured using a fluorescence spectrophotometer at a fluorescence excitation wavelength of 373 nm;

[0126] S4. The concentration of sunset yellow is taken as the horizontal axis, and the fluorescence quenching rate is taken as the vertical axis. The formula of fluorescence quenching rate is: ,in is the fluorescence intensity of the carbon quantum dot solution without adding food additives, F is the fluorescence intensity of the carbon quantum dot solution after adding food additives, draw a standard curve, and get the corresponding linear regression equation.

[0127] Example 7: Sample detection, comprising the following steps:

[0128] S1. The sample to be tested was diluted 50 times with a PBS buffer solution having a pH of 7 to obtain a mixed solvent;

[0129] S2. Adding amaranth to the mixed solvent to prepare 5 μM, 30 μM and 50 μM amaranth solutions respectively;

[0130] S3. Take 0.4 mL of carbon quantum dot solution in a colorimetric tube, add 4.4 mL of amaranth solution, and mix thoroughly to obtain a sample detection system;

[0131] S4. The fluorescence intensity of the sample detection system was measured using a fluorescence spectrophotometer at a fluorescence excitation wavelength of 373 nm;

[0132] S5. Calculate the concentration of amaranth in the sample based on the measured fluorescence intensity and the linear regression equation of the standard curve;

[0133] S6. Calculate the recovery rate through spike recovery experiment to evaluate the accuracy of the detection method.

[0134] Example 8: Sample detection, comprising the following steps:

[0135] S1. The sample to be tested was diluted 50 times with a PBS buffer solution having a pH of 7 to obtain a mixed solvent;

[0136] S2. Adding amaranth to the mixed solvent to prepare 5 μM, 30 μM and 50 μM amaranth solutions respectively;

[0137] S3. Take 0.5 mL of carbon quantum dot solution in a colorimetric tube, add 4.5 mL of amaranth solution, and mix thoroughly to obtain a sample detection system;

[0138] S4. The fluorescence intensity of the sample detection system was measured using a fluorescence spectrophotometer at a fluorescence excitation wavelength of 373 nm;

[0139] S5. Calculate the concentration of amaranth in the sample based on the measured fluorescence intensity and the linear regression equation of the standard curve;

[0140] S6. Calculate the recovery rate through spike recovery experiment to evaluate the accuracy of the detection method.

[0141] Example 9: Sample detection, comprising the following steps:

[0142] S1. The sample to be tested was diluted 50 times with a PBS buffer solution having a pH of 7 to obtain a mixed solvent;

[0143] S2. adding sunset yellow to the mixed solvent to prepare 5 μM, 30 μM and 50 μM sunset yellow solutions respectively;

[0144] S3. Take 0.6 mL of carbon quantum dot solution in a colorimetric tube, add 4.6 mL of sunset yellow solution, and mix thoroughly to obtain a sample detection system;

[0145] S4. The fluorescence intensity of the sample detection system was measured using a fluorescence spectrophotometer at a fluorescence excitation wavelength of 373 nm;

[0146] S5. Calculate the concentration of sunset yellow in the sample based on the measured fluorescence intensity and the linear regression equation of the standard curve;

[0147] S6. Calculate the recovery rate through spike recovery experiment to evaluate the accuracy of the detection method.

[0148] Comparative Example 1:

[0149] Compared with Example 1, green tea extract was not added during the preparation of carbon quantum dots in this comparative example, and the remaining steps and parameters were the same, which will not be repeated in this comparative example, and carbon quantum dots were finally obtained.

[0150] Comparative Example 2:

[0151] Compared with Example 1, this comparative example only adjusts the amount of "green tea extract" from "0.018g" to "0.05g", and the remaining steps and parameters are the same, which will not be repeated in this comparative example, and finally carbon quantum dots are obtained.

[0152] Performance Testing:

[0153] Stability test of carbon quantum dots CDs:

[0154] This study investigated the effects of solution pH, ionic strength and fluorescence intensity.

[0155] Effect of solution pH on the fluorescence intensity of CDs: CDs (0.1 mg / mL) were adjusted to different pH values ​​(1-14) using NaOH and HCl, and their fluorescence intensity was measured.

[0156] Effect of ionic strength on the fluorescence intensity of CDs: NaCl solutions of different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mol / L) were added to CDs (0.1 mg / mL) solution and mixed thoroughly before measuring the fluorescence intensity.

[0157] Determination of fluorescence quantum yield (QY):

[0158] The fluorescence quantum yield of CDs was calculated by dissolving quinine sulfate in 0.1 mol·L -1 H 2 SO 4 The quantum yield (QY=54%) in is used as the reference standard. In order to minimize the influence of the internal filter, the CDs sample is diluted to keep its UV absorption value between 0.05-0.1. The quantum yield is calculated according to formula (2-1):

[0159]

[0160] Where I is the integrated emission intensity, A is the optical density, and n is the refractive index. The subscript R refers to the reference sample of quinine sulfate.

[0161] Study on the selectivity and anti-interference of Amaranth and Sunset Yellow:

[0162] The fluorescence quenching rate F / F 0 As indicators, the selectivity of CDs for a series of food additives such as amaranth, sunset yellow, acesulfame potassium, sodium citrate, and DL-malic acid was evaluated, and the fluorescence intensity of the system with different food additives was detected. In addition, whether the presence of other food additives would affect the selectivity of CDs for amaranth and sunset yellow was studied. Different food additives (acesulfame potassium, sodium citrate, DL-malic acid, citric acid, etc.) were added to the CDs-Amaranth (AR) / Sunset Yellow (SY) system as interfering substances, and the fluorescence intensity changes of the system with interfering substances were detected at a fluorescence excitation wavelength of 373 nm.

[0163] Linearity detection of Amaranth and Sunset Yellow by CDs:

[0164] To 0.5 ml CDs solution, 4.5 ml of amaranth or sunset yellow solution of different concentrations (0-150 μM) was added in sequence and mixed thoroughly to obtain the CDs-AR / SY system. The fluorescence intensity of the CDs-AR / SY system was measured at a fluorescence excitation wavelength of 373 nm, and the linear relationship between the quenching degree and the concentration of amaranth or sunset yellow was investigated.

[0165] Actual sample analysis:

[0166] According to the National Food Safety Standard for Food Additives, colorants such as amaranth and sunset yellow are prohibited from being added to tea beverages. Therefore, the probe was used to detect amaranth in oriental leaves and sunset yellow in green plum green tea. The two beverage samples were purchased from a local convenience store. The two beverages were diluted 50 times with PBS buffer solution (pH=7), and the diluted solution was used as a solvent to prepare amaranth and sunset yellow solutions at high, medium and low concentrations. The concentration values ​​of amaranth and sunset yellow in real complex samples were determined by fluorescence spectroscopy. Finally, the recovery rate was calculated based on the degree of fluorescence enhancement and the linear regression equation of the day.

[0167] Cytotoxicity:

[0168] The CCK-8 assay was used to detect the cytotoxicity of CDs and the survival rate of RAW 264.7 (mouse mononuclear macrophage leukemia cells) in the presence of CDs was determined. RAW 264.7 cells were seeded into 96-well plates with approximately 10,000 cells per well and incubated at 37°C and 5% CO 2The cells were cultured in a cell culture incubator for 24 h, and then different concentrations of CDs solution (6.25-100 μg / ml) were added for 24 h, with 6 replicate wells for each concentration. After the incubation, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. Finally, the absorbance of each well was measured at 450 nm using an ELISA reader. The cell survival rate was calculated according to the following formula:

[0169] 00%

[0170] Among them, As is the absorbance of the experimental well, Ac is the absorbance of the control well, and Ab is the absorbance of the blank well.

[0171] Results and Analysis:

[0172] Characterization of CDs:

[0173] Structural characterization of CDs:

[0174] The morphology and elements of CDs were characterized by using TEM and XPS. The TEM images showed the relationship between the morphology and particle size of CDs (e.g. Figure 1 As shown in Figure 2, CDs are granular, well dispersed, and evenly distributed in the range of 1.5nm-5.5nm, with an average particle size of 3.5nm (as shown in Figure 2). Figure 2 ). Figure 3 The X-ray diffraction pattern of CDs. The figure shows a strong diffraction peak at about 20.06°2θ, corresponding to the (002) plane. Subsequently, the elemental composition, content and chemical bond information of CDs were obtained by XPS. Figure 4 In the study, XPS was used to perform a full elemental composition scan analysis of CDs. The scan results showed that CDs mainly contained carbon, nitrogen, oxygen, and sulfur, which were represented by four peaks at 284.80eV, 400.49eV, 532.11eV, and 169.39eV, respectively, with element contents of 59.91%, 12.92%, 20.70%, and 2.94%, respectively. At the same time, there were no other elements in the spectrum, indicating that the carbon skeleton in CDs was modified by nitrogen, oxygen, sulfur, and other groups. In the high-resolution XPS spectrum of C1s ( Figure 5 ), three characteristic peaks were recorded at 284.8eV, 286eV, and 288.5eV, representing CC / C=C, CO, and C=O, respectively; in the high-resolution XPS spectrum of N1s ( Figure 6 ), two characteristic peaks were recorded at 399.90eV and 401.81eV, representing NC and NO respectively; the high-resolution XPS spectrum of O1s ( Figure 7 ) recorded a characteristic peak at 531.75 eV, indicating the presence of -CO-; the high-resolution XPS spectrum of Sp2 ( Figure 8) Two characteristic peaks were recorded at 168.92eV and 163.38eV, which are respectively related to S 6+ Sulfate structure 、 Thiophene structure.

[0175] Optical Characterization of CDs: FT-IR Characterization

[0176] In order to confirm the type of functional groups contained in N,S-CQDs, FT-IR was used for identification, such as Fig. 9 As shown, 3228cm -1 The absorption peak at 1633 cm is the stretching vibration peak of the NH bond. -1 The stretching vibration of C=O bond is 1415cm -1 The CH absorption peak is caused by the carbon atom structure of CDs. This indicates that oxygen-containing groups, nitrogen-containing groups and unsaturated carbon structures are enriched on the surface CDs, and the synthesized CDs have good hydrophilicity.

[0177] UV Characterization: CDs were further optically characterized by using fluorescence spectrophotometer and UV-visible spectroscopy. Fig.10 As shown in the figure, under natural light, the carbon dot solution is light yellow, while under ultraviolet light, the carbon dot solution exhibits blue fluorescence. In the ultraviolet absorption spectrum, there is a strong absorption peak at 210nm, which is attributed to the π-π * transition, indicating the existence of a conjugated skeleton in the carbon dots. Fig.11 It was shown that within the excitation wavelength range of 310nm-460nm, the emission peak was positively correlated with the excitation wavelength and red-shifted, with the maximum emission peak occurring at 375nm. After multiple experiments, it was determined that the optimal excitation wavelength was 373nm, under which the emission wavelength was 447nm.

[0178] Stability test: Effect of pH on the fluorescence properties of CDs

[0179] NaOH and HCl were added to the CDs solution with the same concentration (0.1 mg / mL) to adjust to different pH values ​​( Fig.12 ), the effect of pH on fluorescence was evaluated by the change in fluorescence intensity to examine the fluorescence properties of carbon quantum dots under different pH environments. When the pH value varies in the range of 2-9, the fluorescence intensity remains basically unchanged. When the pH value is too low or too high, the fluorescence intensity decreases. This obvious phenomenon is due to the deprotonation and protonation of the carboxyl and hydroxyl groups on the surface of CDs in extreme acid-base environments. This shows that under normal pH conditions, even if the pH changes partially, the fluorescence intensity of CDs will not be affected.

[0180] Effect of NaCl on the fluorescence properties of CDs: Different concentrations of NaCl were added to CDs (0.1 mg / mL) solution, and the effect of ionic strength on CDs fluorescence was further evaluated by the change in fluorescence intensity. Fig.13 As shown in the figure, the fluorescence intensity did not change significantly with the increase of NaCl concentration, which indicates that CDs have excellent tolerance to high ionic strength.

[0181] Fluorescence lifetime and quantum yield: After diluting the CDs solution, the fluorescence intensity change was measured under ultraviolet light using a fluorescence spectrophotometer, the process and time of fluorescence quenching were observed and recorded, and the linear relationship between the fluorescence intensity change and time was analyzed. The detection data was then processed and a fluorescence intensity-time relationship curve was drawn ( Fig.14 ). It can be seen from the curve that after about 1μs, the detected fluorescence intensity is lower than 0.001 au and tends to be stable, indicating that after about 1μs, the fluorescence has been completely quenched. The average fluorescence lifetime is calculated to be 0.01969μs. In addition, the fluorescence quantum yield was measured under the ultraviolet wavelength of 360nm, and the result showed that it was only 1.59%, indicating that the luminescence efficiency of the CDs is low and needs to be carried out at a higher concentration when used as a fluorescent probe. However, since the raw materials for synthesizing the CDs are abundant and the preparation process is simple, the detection conditions are relatively easy to meet.

[0182] Study on the selectivity and anti-interference of CDs: In order to understand the selectivity of CDs for amaranth and sunset yellow, the fluorescence intensity ratio F / F 0 As indicators, the selective quenching of CDs by amaranth and sunset yellow was studied in comparison with some common food additives, including acesulfame potassium, sodium citrate, malic acid, citric acid, calcium carbonate, titanium dioxide, potassium chloride, and vitamin C. Fig.15 and Fig.16 It can be seen that compared with other common food additives, amaranth and sunset yellow have significant fluorescence quenching effects on CDs. This result proves that compared with other common food additives, CDs has better selectivity for amaranth and sunset yellow.

[0183] In addition, in order to find out whether the fluorescence quenching degree of CDs by amaranth and sunset yellow will be affected in the presence of interference, we added the above food additives to amaranth and sunset yellow and conducted anti-interference tests. Fig.17 and Fig.18 As shown in the figure, in the presence of other interfering substances, neither of them had a significant effect on the fluorescence quenching degree of CDs. This result shows that CDs has good anti-interference ability in detecting the two.

[0184] Linearity test of CDs: According to the experimental method, the fluorescence spectra of CDs in different concentrations of sunset yellow and amaranth were measured. Under the optimal experimental conditions, the concentrations of sunset yellow and amaranth were in the range of 10-60μM and 5-60μM respectively. 0 -F) / F 0 All showed good linear relationships. The detection limit of sunset yellow was 2.044μM, and the solution containing sunset yellow was measured 10 times continuously, with a relative standard deviation of 0.007086; the detection limit of amaranth red was 1.513μM, and the solution containing amaranth red was measured 10 times continuously, with a relative standard deviation of 0.00583, indicating that the method has high sensitivity and precision.

[0185] Analysis of actual samples: In order to evaluate the applicability of this detection method in the detection of real samples, CDs were used to detect amaranth in oriental leaves (tea beverages) and sunset yellow in green plum green tea (tea beverages) by the spike recovery method. The data in Table 1 show that the average recovery rate of sunset yellow is 98.40%, and the average recovery rate of amaranth is 100.36%. This shows that the detection method of this experiment has high accuracy.

[0186] Table 1 Actual sample analysis

[0187]

[0188] Cytotoxicity: RAW 264.7 (mouse mononuclear macrophage leukemia cells) were cultured in CDs, and the toxicity of CDs on cells was evaluated by measuring the cell survival rate. The relative viability of RAW 264.7 cells in a series of CDs solutions at concentrations ranging from 0 to 100 μg / ml was studied using the CCK-8 assay. Fig.21 It can be seen that after 24 hours of incubation in CDs, at the maximum concentration of 100 μg / ml, the cell survival rate is still more than 90%, which shows that high concentrations of CDs in this experiment are unlikely to have a significant impact on cell activity. Therefore, the CDs synthesized in this experiment have ideal application value in the biomedical field.

[0189] XPS and FT-IR spectra confirmed that the carbon dots are rich in amino and carbonyl groups, so the carbon dots are rich in heteroatom doping defects, which cause CDs to emit blue fluorescence under ultraviolet light. The ultraviolet absorption spectrum and fluorescence emission spectrum confirmed that the carbon dots used in this experiment have a maximum emission wavelength of 447nm under the condition of 373nm excitation wavelength, which has obvious overlap with the absorption spectra of amaranth and sunset yellow. According to the inner filter effect / fluorescence resonance energy transfer, that is, when the absorption spectrum of the detected substance overlaps significantly with the excitation spectrum or emission spectrum of the fluorescent probe, the emission light or incident light of the fluorescent substance is absorbed by the quencher, and the fluorescence lifetime remains unchanged / shortened, which greatly weakens or even quenches the fluorescence of the fluorescent probe. Therefore, amaranth and sunset yellow can significantly quench the fluorescence of CDs through the inner filter effect.

[0190] In summary, the carbon dots (CDs) prepared by hydrothermal synthesis using herring stone as raw material and modified herring stone by plant extracts have good stability, high fluorescence quantum yield and good biocompatibility. The plant extracts are rich in bioactive ingredients, such as polyphenols, flavonoids, alkaloids, etc. These ingredients can interact with the components in the herring stone during the preparation of herring stone carbon quantum dots, and can hydrogen bond or redox reaction with the groups on the surface of the carbon quantum dots, thereby changing the surface chemical environment of the carbon quantum dots, forming a coating layer on the surface of the carbon quantum dots, or intermixing with the internal structure of the carbon quantum dots, thereby improving its fluorescence performance and stability. However, in Comparative Example 2, when the amount of plant extract added is large, although the biocompatibility of the prepared carbon quantum dots is improved, the fluorescence intensity decreases. In addition, the experimental and characterization results show that the oxygen-containing groups in the CDs improve their solubility and fluorescence performance. Under the optimal experimental conditions, the fluorescence intensity of CDs has a good linear relationship with the concentration of sunset yellow and amaranth, and the experimental method has good precision and high accuracy. This study fully illustrates the feasibility of synthesizing carbon quantum dots under the concept of waste utilization. The experimental method can meet the requirements for the determination of sunset yellow and amaranth pigments in tea beverages, so it is expected to be used in food quality testing and medical quality testing.

[0191] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0192] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting food additives using bluefish stone fluorescent carbon quantum dots, characterized in that: The following steps are involved: Step S1. Preparation of carbon quantum dots; The preparation of the carbon quantum dots in step S1 comprises the following steps: Step S101. Take blackfish stone, clean it and grind it into powder to obtain blackfish stone powder; Step S102. Weigh the blackfish stone powder, add it to deionized water, then add the plant extract, stir magnetically for 28-32 minutes to fully disperse the blackfish stone powder in the water, and then perform ultrasonic treatment for 4-6 minutes to obtain a mixed solution 1; Step S103. The mixed solution 1 is transferred to a polytetrafluoroethylene-lined autoclave, sealed and placed in an oven for reaction to obtain a mixed solution 2; Step S104. After the reaction is completed, the mixed solution 2 is naturally cooled to room temperature, and then transferred to a centrifuge tube, centrifuged and the supernatant is collected; Step S105. Filter the supernatant with a 0.22 μm microporous filter membrane to obtain a clear light yellow carbon quantum dot solution; Step S106. placing the obtained carbon quantum dot solution in a dialysis bag with a molecular weight of 500 Da, and dialyzing it in deionized water for 8-10 h; Step S107. Finally, the dialyzed solution is freeze-dried to obtain a yellow-brown solid product, which is dispersed with deionized water to obtain a transparent suspension with a concentration of 5-6 mg / mL, that is, the bluefish stone fluorescent carbon quantum dots for detecting food additives are obtained; Step S2. drawing a standard curve; The standard curve drawing in step S2 comprises the following steps: Step S201. Prepare standard solutions of food additives with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM respectively; Step S202. Adding food additive standard solutions with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM and 60 μM into different colorimetric tubes, respectively adding carbon quantum dot solutions into each colorimetric tube, and mixing thoroughly to obtain a carbon quantum dot-food additive system; Step S203. Under a fluorescence excitation wavelength of 373 nm, the fluorescence intensity of each carbon quantum dot-food additive system is measured using a fluorescence spectrophotometer; Step S204. Draw a standard curve with the concentration of the food additive as the abscissa and the fluorescence quenching rate as the ordinate to obtain a corresponding linear regression equation; Step S3. Sample testing; The sample detection in step S3 includes the following steps: Step S301. Dilute the sample to be tested 50 times with PBS buffer solution to obtain a mixed solvent; Step S302. Adding the food additive to the mixed solvent to prepare 5 μM, 30 μM and 50 μM food additive solutions respectively; Step S303. Take 0.4-0.6 mL of carbon quantum dot solution in a colorimetric tube, add 4.4-4.6 mL of food additive solution, and mix thoroughly to obtain a sample detection system; Step S304. Using a fluorescence spectrophotometer to measure the fluorescence intensity of the sample detection system at a fluorescence excitation wavelength of 373 nm; Step S305. Calculate the concentration of the food additive in the sample based on the measured fluorescence intensity and the linear regression equation of the standard curve; Step S306. Calculate the recovery rate through the spike recovery experiment to evaluate the accuracy of the detection method; The pH of the PBS buffer solution in step S301 is 7; The carbon quantum dots in step S1 are prepared from blackfish stone and blackfish stone modified by plant extracts; The average particle size of the carbon quantum dots in step S1 is 3.2-3.8 nm.

2. The method for detecting food additives using bluefish stone fluorescent carbon quantum dots according to claim 1, characterized in that: The usage ratio of the blackfish stone powder, deionized water and plant extract in step S102 is 0.18-0.22 g: 9-12 mL: 0.018-0.022 g.

3. The method for detecting food additives using bluefish stone fluorescent carbon quantum dots according to claim 1, characterized in that: The plant extract in step S102 is any one of green tea extract and grape seed extract.

4. The method for detecting food additives using bluefish stone fluorescent carbon quantum dots according to claim 1, characterized in that: The reaction temperature in step S103 is 178-182° C., and the reaction time is 10-12 h.

5. The method for detecting food additives using bluefish stone fluorescent carbon quantum dots according to claim 1, characterized in that: The centrifugal speed in step S104 is 4100-4200 rpm, and the centrifugal time is 5-7 min.

6. The method for detecting food additives using bluefish stone fluorescent carbon quantum dots according to claim 1, characterized in that: The dosage ratio of the food additive solution and the carbon quantum dot solution in step S202 is 4.4-4.6 mL: 0.4-0.6 mL.

7. The method for detecting food additives using bluefish stone fluorescent carbon quantum dots according to claim 1, characterized in that: The formula for the fluorescence quenching rate in step S204 is: ,in is the fluorescence intensity of the carbon quantum dot solution without adding food additives, and F is the fluorescence intensity of the carbon quantum dot solution after adding food additives.

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