A detection method for quercetin and a molybdenum disulfide quantum dot imprinted polymer fluorescence probe used therefor

Abstract

A detection method for quercetin and a molybdenum disulfide quantum dot imprinted polymer fluorescent probe used therein. The detection method includes: establishing a standard working curve of emission fluorescence intensity versus quercetin concentration; Step 2: adding the sample to be tested into a beaker containing ethanol, ultrasonically treating it in a water bath at 8-12 °C, and filtering to obtain an extract; Step 3: taking the extract and diluting it to 1000 mL with ethanol; taking 2 mL of the MoS 2 QDs@SiO2@MIP aqueous solution and adding it to an equal volume of the diluted quercetin solution, incubating at room temperature for 4-6 min, and detecting the fluorescence spectrum intensity; The concentration of the MoS 2 QDs@SiO2@MIP aqueous solution is 0.2 mg / mL; Step 3: obtaining the concentration of the diluted quercetin according to the standard working curve of fluorescence intensity versus quercetin concentration, and calculating the concentration of quercetin in the extract through the dilution multiple relationship, so as to calculate the content of quercetin in onions. The molybdenum disulfide quantum dot imprinted polymer fluorescent probe prepared by the present invention has high selectivity and sensitivity, and the amount of organic solvent used in the synthesis process is small, reducing the secondary pollution to the environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of quercetin detection, and relates to a method for detecting quercetin and a molybdenum disulfide quantum dot imprinted polymer fluorescent probe used therefor. Background Art

[0002] Quercetin is an important class of flavonoid bioactive molecules, which are widely present in various fruits, vegetables, nuts, and Chinese herbal medicines. Quercetin has important value for anti-aging, disease prevention and treatment, and has been attracting much attention in the fields of food chemistry, natural medicine chemistry, biochemistry, and clinical medicine. Accurately detecting and analyzing the content of quercetin is the key to effectively utilizing quercetin compounds and bringing their health benefits into play. The fluorescence method has the characteristics of convenience and high efficiency, and has become an important research direction in the field of quercetin analysis. The primary problem in fluorescence detection is the design and synthesis of fluorescent substances for quercetin determination.

[0003] Currently, the disclosed quercetin fluorescent probes and detection schemes are as follows:

[0004] (1) In 2012, Chen synthesized gold and gold-silver alloy quantum dots and applied them to detect the content of quercetin in human urine and serum (J. Nanopart. Res., 2012, 14: 1264-1272; Plasmonics, 2013, 8: 201-207.).

[0005] (2) In 2013, the research group of Professor Li Songlin at Henan University developed a method for determining quercetin using CdSe / ZnS core-shell quantum dots as fluorescent probes (Anal. Methods., 2014, 6(5): 1442-1447).

[0006] (3) Patent document (Application No. 201910005406.1) Fu Haiyan et al. prepared ZnCdSe quantum dots, and realized the detection of flavonoid compounds with the assistance of the photosensitizer tetra-(4-pyridyl) zinc porphyrin.

[0007] (4) In 2014, Professor Zuo injected glucose solution into P 2 O 5 powder to develop a simple method for preparing carbon quantum dots and applied them to quercetin detection (Microchim. Acta., 2014, 181: 1309-1316).

[0008] (5) In 2019, the research group of Professor Zhao prepared [Tb 4 - based MOF materials and detected quercetin (Anal. Chem. 2019, 91, 2595-2599).

[0009] (6) In 2020, the research group of Professor Dong used 1,2-ethylenediamine (EDA) as a modifier to synthesize carbon quantum dots with a large number of amino groups on the surface and applied them to the detection of quercetin (Talanta, 2020, 206: 120243-120249).

[0010] These methods all directly disperse quantum dots in aqueous solution for detection. During the detection process, the selectivity and repeatability of quantum dots for quercetin detection are poor. Moreover, after detection, due to the difficulty of recycling quantum dots, they are directly discarded, which is extremely easy to cause waste. Especially for quantum dots containing heavy metal elements such as Au, Ag, and Cd, it is easy to cause secondary environmental pollution.

[0011] (7) Xu et al. used the ultrasonic method to synthesize C 3 N 4 quantum dots, and then used the reverse microemulsion method to prepare a molecularly imprinted probe for the detection of quercetin. The ultrasonic method for preparing C 3 N 4 quantum dots is a "top-down" method with high energy consumption, uneven sizes of the prepared quantum dots, and low fluorescence quantum yield. Moreover, a large amount of organic solvents are required to prepare the imprinted polymer by the reverse microemulsion method, resulting in high costs and easy to cause secondary pollution to the environment (Microchimica Acta, 2018, 185(10): 1-9).

[0012] Therefore, how to develop a highly sensitive, selective, low-cost, and environmentally friendly fluorescent probe and its quercetin detection method remains a challenging task.

[0013] Molybdenum disulfide quantum dots (MoS 2 QDs) are a typical zero-dimensional transition metal sulfide nanomaterial. In recent years, due to their high fluorescence performance, strong resistance to light and chemical corrosion, and low biological toxicity, they have received increasing attention in biological and food detection and other fields. Molecular imprinting technique (MIT) is a polymer preparation technique for obtaining a polymer that is completely matched with a certain template molecule in terms of spatial structure and binding sites. This molecularly imprinted polymer (MIP) has a specific recognition function for the template molecule and is widely used in separation, sensing, catalysis, and other aspects. To improve the selectivity and sensitivity of the fluorescent probe for quercetin detection, this patent uses a simple "one-pot" hydrothermal method to synthesize MoS 2 QDs with high fluorescence quantum yield, and uses this as a fluorescent light source, combined with the molecular imprinting technique to prepare a MoS 2 QDs molecular polymer fluorescent probe for the detection of quercetin. Summary of the Invention

[0014] The object of the present invention is to provide a detection method of quercetin and a molybdenum disulfide quantum dot imprinted polymer fluorescence probe used therefor.

[0015] To achieve the above object and other related objects, the technical solution provided by the present invention is: a molybdenum disulfide quantum dot imprinted polymer fluorescence probe, and the preparation method includes the following steps:

[0016] Step 1: Add molybdenum disulfide quantum dots and 3-mercaptopropyltriethoxysilane to ethanol, and stir for reaction;

[0017] Step 2: Add an ethanol solution of quercetin and tetraethyl orthosilicate to the system of Step 1, stir and heat to 55-65 °C, and then add ammonia water for reaction;

[0018] Step 3: After the reaction is completed, centrifuge to remove the supernatant; centrifuge and wash the remaining precipitate product to obtain MoS 2 QDs@SiO 2 @MIP.

[0019] The preferred technical solution is: the molybdenum disulfide quantum dots are an aqueous solution of molybdenum disulfide quantum dots, the ratio between the aqueous solution of molybdenum disulfide quantum dots and 3-mercaptopropyltriethoxysilane is 4.5 mL: 60-64 μL, and the concentration of the aqueous solution of molybdenum disulfide quantum dots is 3-4 mg / ml.

[0020] The preferred technical solution is: the preparation method of the molybdenum disulfide quantum dots includes the following steps:

[0021] Step 1: Add ammonium tetrathiomolybdate to distilled water, ultrasonically dissolve to obtain an ammonium tetrathiomolybdate aqueous solution; add reduced glutathione to the ammonium tetrathiomolybdate aqueous solution, and then add ultrapure water to adjust the pH to 7.9-8.1;

[0022] Step 2: Transfer the solution obtained in Step 1 to a stainless steel hydrothermal reaction kettle with a polytetrafluoroethylene inner liner, and react in a hydrothermal environment at 180-220 °C for 12-18 h; after the reaction is completed, the obtained product is naturally cooled to room temperature, and the crude product is centrifuged at 9000-12000 revolutions per minute for 10-20 minutes, and the supernatant of the crude product is aspirated;

[0023] Step 3: Place the supernatant in a dialysis membrane, and then place it in secondary distilled water for dialysis to obtain a permeate; the permeate is converted into a light yellow powder solid substance through vacuum freeze-drying treatment and stored in a sealed manner.

[0024] The preferred technical solution is as follows: in step 2, the addition amount of the ethanol solution of quercetin is 0.8 - 1.2 mL; the addition amount of tetraethyl orthosilicate is 80 - 120 μL; the addition amount of ammonia water is 320 - 350 μL; the concentration of the ethanol solution of quercetin is 1.8 - 2.2 mg / mL.

[0025] The preferred technical solution is as follows: in step 3, the centrifugation speed is 10,000 - 12,000 revolutions per minute.

[0026] To achieve the above and other related purposes, the technical solution provided by the present invention is: a detection method for quercetin, comprising the following steps:

[0027] Step1: Establish a standard working curve of emission fluorescence intensity and quercetin concentration;

[0028] Step2: Add the sample to be tested into a beaker containing ethanol, ultrasonically treat it in a water bath at 8 - 12 °C, and filter to obtain an extract.

[0029] Step3: Take the extract and dilute it to 1000 mL with ethanol; take 2 mL of MoS 2 QDs@SiO 2 @MIP aqueous solution and add it to an equal - volume dilution of quercetin, incubate at room temperature for 4 - 6 min, and detect its fluorescence spectrum intensity; the concentration of the MoS 2 QDs@SiO 2 @MIP aqueous solution is 0.2 mg / mL;

[0030] Step4: Obtain the concentration of diluted quercetin according to the standard working curve of fluorescence intensity and quercetin concentration, and calculate the concentration of quercetin in the extract through the dilution multiple relationship, so as to calculate the content of quercetin in onions;

[0031] MoS 2 QDs@SiO 2 @MIP is the MoS 2 QDs@SiO 2 @MIP described in any one of claims 1 - 5.

[0032] The preferred technical solution is as follows: establishing a standard working curve of emission fluorescence intensity and quercetin concentration includes: using ethanol with a volume fraction of 80% as a solvent and acetic acid - sodium acetate as a pH regulator to prepare a series of standard - concentration quercetin solutions with a pH value of 4, mixing the MoS 2 QDs@SiO 2 @MIP aqueous solution and the series of standard - concentration quercetin solutions in a volume ratio of 1:1, standing and then detecting its fluorescence spectrum to establish a standard working curve of emission fluorescence intensity and quercetin concentration; the MoS 2QDs@SiO 2 The concentration of the @MIP aqueous solution is 0.2 mg / mL.

[0033] Due to the application of the above technical solution, the advantages of the present invention compared with the prior art are:

[0034] The molybdenum disulfide quantum dot imprinted polymer fluorescent probe prepared by the present invention has high selectivity and sensitivity, and the amount of organic solvent used in the synthesis process is small, reducing the secondary pollution to the environment. Description of the Drawings

[0035] Figure 1 is the absorption spectrum. Among them, a and b are the absorption spectra of MoS 2 QDs and quercetin, and c is the absorption spectrum of MoS 2 QDs @SiO 2 @MIP after adding quercetin.

[0036] Figure 2 The a is the ultraviolet-visible absorption spectrum of quercetin, and b and c are the MoS 2 QDs@SiO 2 @MIP excitation spectrum and emission spectrum.

[0037] Figure 3 is the TEM image of MoS 2 QDs-GSH.

[0038] Figure 4 is the TEM image of MoS 2 QDs-MPTES.

[0039] Figure 5 is the TEM image of MoS 2 QDs@SiO 2 @MIP.

[0040] Figure 6 (a) of is the high-angle annular dark field image (HAADF) of MoS 2 QDs@SiO 2 @MIP; (b) is the local enlarged view of the HAADF of MoS 2 QDs @SiO 2 @MIP.

[0041] Figure 7 is the photoelectron spectrum of MoS 2 QDs-GSH.

[0042] Figure 8 is the photoelectron spectrum of MoS 2 QDs@SiO 2 @MIP.

[0043] Figure 9 For MoS 2 Infrared spectrum of QDs-GSH.

[0044] Figure 10 For MoS 2 QDs@SiO 2 Infrared spectrum of @MIP.

[0045] Figure 11 (a) Absorption spectrum of MoS 2 QDs-GSH; (b) Absorption spectrum of MoS 2 QDs-MPTES; (c) Absorption spectrum of MoS 2 QDs @SiO 2 @MIP.

[0046] Figure 12 (a) Fluorescence spectrum of MoS 2 QDs-GSH, with its relative fluorescence quantum yield (Fluorescence Quantumyield, QY) being 21.8%; (b) Fluorescence spectrum of MoS 2 QDs-MPTES, with its QY being 39.2%; (c) Fluorescence spectrum of MoS 2 QDs@SiO 2 @MIP, with its QY being 16.4%.

[0047] Figure 13 For the same concentration of MoS 2 QDs@SiO 2 @MIP in Example 1 of the present invention at different pH values, fluorescence emission spectra before and after adding the same concentration of quercetin.

[0048] Figure 14 For MoS 2 QDs@SiO 2 @MIP and different concentrations of quercetin (0.0, 2.0, 5.0, 10.0, 20.0, 40.0, 60.0, 80.0, 120.0, 160.0, 200.0, 250.0, 300.0, 400.0, 500.0 ng / mL) after mixing, fluorescence emission spectra. In the figure, each letter in the order from a to o represents the curve in the order from top to bottom in the figure. a represents the fluorescence emission spectrum curve with a quercetin concentration of 0 ng / mL, b represents the fluorescence emission spectrum curve with a quercetin concentration of 2.0 ng / mL, c represents the fluorescence emission spectrum curve with a quercetin concentration of 5.0 ng / mL, and so on.

[0049] Figure 15This is the standard working curve graph in Example 1 of the present invention. The ordinate of the standard working curve graph is the fluorescence intensity ratio: (F 0 -F) / F, and the abscissa is the quercetin concentration (0 - 200 ng / mL).

[0050] Figure 16 This is MoS in Example 1 of the present invention 2 QDs@SiO 2 @MIP when coexisting with organic small molecules with a molecular structure similar to quercetin and the bar graph of the fluorescence intensity ratio of the MoS 2 QDs@SiO 2 @MIP blank sample. Among them, QE, BE, ME, BC, DE, TC, and OC represent quercetin, baicalein, myricetin, bisphenol C, daidzein, tetracycline, and oxytetracycline respectively.

[0051] Figure 17 This is MoS in Example 1 of the present invention 2 QDs@SiO 2 @MIP when coexisting with other ions and the bar graph of the fluorescence intensity ratio of the MoS 2 QDs @SiO 2 @MIP blank sample.

[0052] Figure 18 For the number of recyclable times of MoS 2 QDs@SiO 2 @MIP to detect quercetin molecules. Specific implementation manners

[0053] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0054] Please refer to Figure 1-18 , it should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in the art to understand and read, and are not used to limit the limited conditions under which the present invention can be implemented. Therefore, they do not have practical technical significance. The following embodiments are provided to better understand the present invention, rather than to limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0055] Some of the materials and equipment used in the present invention are as follows:

[0056] I. Reagents: Ammonium tetrathiomolybdate: Xiya Reagent, 99.99%; L-Glutathione: Guoyao Reagent, AR; Quercetin: Exploration Platform, AR; Tetraethyl orthosilicate: Exploration Platform, AR; 3-Aminopropyltriethoxysilane: Exploration Platform, 99%; 3-Mercaptopropyltriethoxysilane: Exploration Platform, 99%; Ammonia water: Exploration Platform, AR; Sodium hydroxide: Exploration Platform, AR; Hydrazine hydrate: Exploration Platform, 80%; Ethanol: Exploration Platform, AR; Acetone: Exploration Platform, AR; Baicalein: Exploration Platform, 99.0%; Tetracycline: Exploration Platform, 97%; Myricetin: Exploration Platform, 98%; Oxytetracycline: Exploration Platform, 98%; Bisphenol C: Exploration Platform, 98%; Daidzein: Exploration Platform, 98%.

[0057] II. Instruments:

[0058] Daskat Precision Constant Temperature Electrothermal Blast Drying Oven (DGT-G) (Hefei Huadeli Equipment Co., Ltd.).

[0059] Example 1: A Detection Method for Quercetin and Its Molybdenum Disulfide Quantum Dot Imprinted Polymer Fluorescent Probe

[0060] (1) Preparation of MoS 2 QDs@SiO 2 @MIP Probe:

[0061] 1) Preparation of MoS 2 QDs

[0062] The specific preparation scheme is as follows:

[0063] ①Accurately weigh 0.13 g (0.5 mmol) of ammonium tetrathiomolybdate and add it to 10 mL of secondary distilled water, and dissolve it by ultrasonic treatment for 30 minutes. Accurately weigh 0.46 g (1.5 mmol) of reduced glutathione and add it to the above ammonium tetrathiomolybdate aqueous solution, then add 20 mL of ultrapure water, and adjust the pH to 8.0 with ammonia water.

[0064] ②Transfer the above solution to a 100 mL stainless steel hydrothermal reaction kettle with a polytetrafluoroethylene liner, and react in a hydrothermal environment at 200 °C for 16 h. After the reaction is completed, the obtained product is naturally cooled to room temperature. The crude product is centrifuged at a high speed (10,000 revolutions per minute) for 15 minutes, and the supernatant of the crude product is aspirated.

[0065] ③Dialyze the solution in secondary distilled water using a dialysis membrane to obtain the permeate. The purified filtrate is transformed into a light yellow powder solid substance by vacuum freeze-drying and stored in a sealed manner.

[0066] 2) Preparation of MoS 2 QDs@SiO 2 @MIP Probe

[0067] ①Accurately weigh 30 mg of quercetin (0.1 mmol) and 58 μL of 3-aminopropyltriethoxysilane, add them to 15 mL of anhydrous ethanol solution to prepare a 2.0 mg / mL quercetin ethanol solution.

[0068] ②Accurately measure 4.5 mL of MoS 2 QDs (3.5 mg / ml) aqueous solution and 15 mL of ethanol and add them to a 100 mL three-necked flask. Under stirring, add 62 μL of 3-mercaptopropyltriethoxysilane and react at room temperature for 30 minutes.

[0069] ③Dropwise add 100 μL of tetraethyl orthosilicate and 338 μL of ammonia water successively to adjust the pH value to about 9. Keep the temperature at 60 °C and react for 1 hour, then add 1 mL of quercetin ethanol solution and continue to react for 2 h.

[0070] ④After the reaction is completed, centrifuge at high speed and remove the supernatant; repeatedly centrifuge and precipitate the above precipitate product with acetone and high-purity water for washing, wash continuously for 3 times, and the product is dried in vacuum and stored sealed away from light.

[0071] (2) Detection of quercetin by MoS 2 QDs@SiO 2 @MIP probe

[0072] 1) Take an appropriate amount of MoS 2 QDs@SiO 2 @MIP and dissolve it in 80% ethanol solution to prepare a 0.2 mg / mL fluorescent probe.

[0073] 2) Use 80% ethanol as the solvent and acetic acid-sodium acetate pH regulator to prepare a series of standard concentration quercetin solutions (0.0 ng / mL, 4.0 ng / mL, 10.0 ng / mL, 20 ng / mL, 40.0 ng / mL, 80.0 ng / mL, 120.0 ng / mL, 160.0 ng / mL, 240.0 ng / mL, 320.0 ng / mL, 400.0 ng / mL, 500.0 ng / mL, 600.0 ng / mL, 800.0 ng / mL, 1000.0 ng / mL) with a pH of 4.

[0074] 3) Take 2 mL of the molecularly imprinted polymer fluorescent probe solution and add it to equal volumes of quercetin solutions with different standard concentrations respectively, incubate for 5 minutes, and measure its fluorescence spectrum.

[0075] 4) Based on the relationship between fluorescence intensity and quercetin concentration, fit and establish a standard concentration-fluorescence intensity working curve.

[0076] (3) MoS 2 QDs@SiO 2Detection of Quercetin Content in Onions by @MIP Probe

[0077] 1) Select an appropriate amount of onion skins or scallions, wash them clean with water. After drying, place them in a crusher and chop them up. Weigh 1 g of onion sample and add it to a beaker containing 20 mL of 80% ethanol. After ultrasonic treatment in a water bath at 10 °C for 5 minutes, filter the extract. Repeat the above ultrasonic - filtration process 3 times. Combine the obtained filtrates to obtain 50 mL of quercetin extract and store it for later use.

[0078] 2) Take 0.5 mL of the quercetin extract and dilute it to 1000 mL with 80% ethanol solution. Take an appropriate amount of MoS 2 QDs@SiO 2 @MIP and dissolve it in 80% ethanol solution to prepare a 0.2 mg / mL fluorescent probe. Take 2 mL of the MoS 2 QDs@SiO 2 @MIP probe solution and add it to an equal - volume diluted solution of quercetin. Incubate at room temperature for 5 minutes and detect its fluorescence spectrum. According to the fluorescence working curve of the MoS 2 QDs@SiO 2 @MIP fluorescent probe for standard - concentration quercetin, obtain the concentration of diluted quercetin. Through the dilution - multiple relationship, calculate the concentration of quercetin in the extract, and thus calculate the quercetin content in onions.

[0079] III. Detection Selectivity Test

[0080] Take 2 mL of MoS 2 QDs@SiO 2 @MIP (0.2 mg / mL) and add it to equal - volume aqueous solutions of quercetin, baicalein, myricetin, bisphenol C, daidzein, tetracycline, and oxytetracycline respectively and mix them. Record the fluorescence intensities of the above - mentioned mixed solutions. The results of the fluorescence intensities are as Figure 13 shown. As Figure 13 can be seen, at the same concentration (200 ng / mL), quercetin has the most significant fluorescence quenching effect on MoS 2 QDs @SiO 2 @MIP, and the fluorescence intensity decreases by 81.9%. The fluorescence quenching effects of other organic small molecules on MoS 2 QDs @SiO 2 @MIP follow the order of myricetin, baicalein, bisphenol C, daidzein, and the fluorescence intensities decrease by 19.0%, 15.8%, 10.3%, and 8.91% respectively. Tetracycline and oxytetracycline have little influence on the fluorescence of MoS 2 QDs@SiO 2 @MIP, which are 4.3% and 2.41% respectively. Obviously, MoS 2 QDs@SiO 2The @MIP fluorescent probe has significant selectivity for quercetin. This may be due to the fact that MoS 2 QDs@SiO 2 @MIP has a cavity structure site for quercetin, which specifically binds to quercetin. However, the structures of other organic molecules do not match well with the cavity, resulting in a low binding rate and poor fluorescence quenching effect.

[0081] The influence of other ions on the fluorescence intensity of MoS 2 QDs@SiO 2 @MIP was also investigated, and the results are as Figure 14 shown:

[0082] As Figure 14 can be seen, Fe 3+ , Br - , I - have a certain quenching effect on the fluorescence intensity of MoS 2 QDs@SiO 2 @MIP, with the fluorescence decreasing by 21.5%, 17.6%, and 26.0% respectively. The influence of the remaining test ions on the fluorescence intensity of MoS 2 QDs@SiO 2 @MIP can be ignored.

[0083] IV. Detection Sensitivity Test

[0084] Using 80% ethanol as the solvent and acetic acid - sodium acetate as the pH regulator, a series of standard concentration quercetin solutions with a pH of 4 were prepared (0.0 ng / mL, 4.0 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 40.0 ng / mL, 80.0 ng / mL, 120.0 ng / mL, 160.0 ng / mL, 240.0 ng / mL, 320.0 ng / mL, 400.0 ng / mL, 500.0 ng / mL, 600.0 ng / mL, 800.0 ng / mL, 1000.0 ng / mL). An appropriate amount of MoS 2 QDs@SiO 2 @MIP was dissolved in 80% ethanol solution to prepare a 0.2 mg / mL fluorescent probe. 2 mL of MIP@MoS 2 QDs@SiO 2 (0.2 mg / mL) solution was added to equal volumes of quercetin solutions with different standard concentrations, incubated for 5 minutes, and its fluorescence spectrum was measured. Through the relationship between fluorescence intensity and quercetin concentration, the working curve equation of quercetin standard concentration - fluorescence intensity was obtained by fitting as y = 0.00631x + 0.00256. The square of the correlation coefficient (R 2 ) was 0.999, and the lowest detection limit (LOD) was 1.2 ng / mL.

[0085] V. Reliability Test of Detection Method

[0086] To verify the reliability of the method for detecting quercetin, this method was used to detect the quercetin content in onions. As shown in Table 1, the quercetin concentration detected by this method was 130.4 ng / mL. According to the calculation, the quercetin content in onions was 52.2 mg / g. Through the sample spiking experiment, the reliability of the detection method was further verified. The experimental results showed that within the spiking concentration range, the recovery rate was 91.2%-104.2%.

[0087] Table 1 Detection and Recovery Experiments of Quercetin in Onions

[0088]

[0089] It is known Figure 1 that quercetin has two absorption peak bands, namely 254 nm (II peak band) and 374 nm (I peak band), and the II peak band has a good overlap with the low-wavelength absorption of MoS 2 QDs@SiO 2 @MIP. After adding MoS 2 QDs@SiO 2 @MIP, the I peak of quercetin was red-shifted by 9 nm. There are o-dihydroxybenzene groups, 3-hydroxy-4-keto groups, and 5-hydroxy-4-keto groups in the quercetin molecule, and these oxygen-containing groups have a strong coordination ability with metal ions. Quercetin is very likely to form a complex with the Mo element in MoS 2 QDs@SiO 2 @MIP, which makes the conjugated system of the whole molecule extended and the wavelength red-shifted.

[0090] It is known Figure 2 that the ultraviolet-visible absorption spectrum of quercetin has partial overlap with the excitation spectrum of MoS 2 QDs@SiO 2 @MIP. Therefore, the quenching of MoS 2 QDs@SiO 2 @MIP fluorescence by quercetin may be caused by energy transfer or inner filter effect.

[0091] Example 2: A Detection Method for Quercetin and a Fluorescent Probe of Molybdenum Disulfide Quantum Dot Imprinted Polymer Used Therein

[0092] A simple method for preparing a fluorescent probe of molybdenum disulfide molecularly imprinted polymer (MoS 2 QDs@SiO 2 @MIP) and its method for detecting quercetin, which improve the selectivity and sensitivity of quercetin detection. Reduce the secondary pollution to the environment during the synthesis process of the quercetin fluorescent probe and reduce the preparation cost.

[0093] A kind of MoS 2 QDs@SiO 2 @MIP preparation and quercetin molecule detection method, comprising the following steps:

[0094] (1) Take appropriate amounts of molybdenum disulfide quantum dots (MoS 2 QDs) and 3-mercaptopropyltriethoxysilane (MPTES) and add them to an ethanol solution, and stir and react at room temperature for a period of time;

[0095] (2) Then add 1 mL of quercetin ethanol solution (2 mg / mL) and 100 μL of tetraethyl orthosilicate (TEOS), stir and heat up to 60 °C, and dropwise add ammonia water and react for a period of time.

[0096] (3) After the reaction is completed, centrifuge at high speed and remove the supernatant; repeatedly centrifuge and wash the above precipitate product with acetone and high-purity water to obtain MoS 2 QDs@SiO 2 @MIP powder;

[0097] (4) Prepare a MoS 2 QDs@SiO 2 @MIP fluorescent probe with the powder in step (3), and establish a method for detecting quercetin with the probe.

[0098] (5) Detect the quercetin content in the sample with the fluorescent probe in step (4).

[0099] Preferably, in step (1), take 4.5 mL of MoS 2 QDs (3.5 mg / ml) aqueous solution and 62 μL of 3-mercaptopropyltriethoxysilane. Stir at room temperature at a speed of 1000 revolutions / min for 30 min.

[0100] Preferably, the preparation steps of MoS 2 QDs are as follows:

[0101] ① Accurately weigh 0.13 grams (0.5 mmol) of ammonium tetrathiomolybdate and add it to 10 mL of secondary distilled water, and dissolve it by ultrasonic treatment for 30 minutes. Accurately weigh 0.46 g (1.5 mmol) of reduced glutathione (GSH) and add it to the above ammonium tetrathiomolybdate aqueous solution, add 20 mL of ultrapure water, and adjust the pH to 8.0 with ammonia water.

[0102] ② Transfer the above solution to a 100 mL stainless steel hydrothermal reaction kettle with a polytetrafluoroethylene lining, and react in a hydrothermal environment at 200 °C for 16 h. After the reaction is completed, the obtained product is naturally cooled to room temperature. The primary product is centrifuged at high speed (10000 revolutions / min) for 15 minutes, and the supernatant of the crude product is aspirated.

[0103] ③Dialysis was carried out using a dialysis membrane in secondary distilled water to obtain a permeate. The purified filtrate was converted into a pale yellow powder solid substance (MoS 2 QDs-GSH) by vacuum freeze-drying and stored in a sealed manner.

[0104] Preferably, 1 mL of quercetin ethanol solution (2 mg / mL) was taken in step (2). The preparation method is as follows: Weigh 30 mg of quercetin (0.1 mmol) and 58 μL of 3-aminopropyltriethoxysilane, add them to 15 mL of anhydrous ethanol solution to prepare a 2.0 mg / mL quercetin ethanol solution.

[0105] Preferably, 100 μL of tetraethyl orthosilicate (TEOS) was taken in step (2), the amount of ammonia water added dropwise was 338 μL, the stirring speed was 1000 r / min, and the reaction time was 2 h.

[0106] Preferably, the high-speed centrifugation speed in step (3) was 10000 r / min;

[0107] The present invention also discloses a MoS prepared by the above method 2 QDs@SiO 2 @MIP fluorescent probe.

[0108] Preferably, MoS was taken in step (4) 2 QDs@SiO 2 @MIP powder was used to prepare an aqueous solution of 0.2 mg / mL. A standard working curve of the emission fluorescence intensity vs. the quercetin concentration was established, including the following steps: Using 80% ethanol as a solvent and acetic acid-sodium acetate as a pH regulator, a series of standard concentration quercetin solutions with a pH of 4 (0.0 ng / mL, 4.0 ng / mL, 10.0 ng / mL, 20 ng / mL, 40.0 ng / mL, 80.0 ng / mL, 120.0 ng / mL, 160.0 ng / mL, 240.0 ng / mL, 320.0 ng / mL, 400.0 ng / mL, 500.0 ng / mL, 600.0 ng / mL, 800.0 ng / mL, 1000.0 ng / mL) were prepared. The MoS 2 QDs @SiO 2 @MIP aqueous solution was mixed with the above different concentration quercetin solutions at a volume ratio of 1:1, allowed to stand for 5 min, and its fluorescence spectrum was measured to establish a standard working curve of the emission fluorescence intensity vs. the quercetin concentration.

[0109] Preferably, the fluorescent probe in step (5) was used to detect quercetin. First, quercetin was extracted from the sample, and the steps were as follows:

[0110] 1) Select an appropriate amount of onion skins or scallion whites, wash them clean with water. After drying, place them in a pulverizer and chop them. Weigh 1 g of the onion sample, add it to a beaker containing 20 mL of 80% ethanol, ultrasonicate it in a water bath at 10 °C for 5 minutes, and then filter the extract. Repeat the above ultrasonic filtration three times. Combine the obtained filtrates to obtain 50 mL of quercetin extract for storage and standby.

[0111] 2) Take 0.5 mL of the above quercetin extract and dilute it to 1000 mL with 80% ethanol solution. Take 2 mL of the MoS 2 QDs@SiO 2 @MIP probe solution and add it to an equal volume of the diluted quercetin solution, incubate at room temperature for 5 minutes, and detect the fluorescence spectrum intensity. According to the MoS 2 QDs@SiO 2 @MIP fluorescence probe, obtain the concentration of the diluted quercetin from the fluorescence working curve of the standard concentration of quercetin. Through the dilution multiple relationship, deduce the concentration of quercetin in the extract, and thus calculate the content of quercetin in the onion.

[0112] Example 3: A method for detecting quercetin and a molybdenum disulfide quantum dot imprinted polymer fluorescence probe used therein

[0113] A molybdenum disulfide quantum dot imprinted polymer fluorescence probe, G, comprises the following steps:

[0114] Step 1: Add molybdenum disulfide quantum dots and 3-mercaptopropyltriethoxysilane to ethanol, and stir to react;

[0115] Step 2: Add an ethanol solution of quercetin and tetraethyl orthosilicate to the system of Step 1, stir and heat to 55 °C, and then add ammonia water to react;

[0116] Step 3: After the reaction is completed, centrifuge to remove the supernatant; centrifuge and wash the remaining precipitate product to obtain MoS 2 QDs@SiO 2 @MIP.

[0117] The preferred technical solution is: The molybdenum disulfide quantum dots are an aqueous solution of molybdenum disulfide quantum dots, and the ratio between the aqueous solution of molybdenum disulfide quantum dots and 3-mercaptopropyltriethoxysilane is 4.5 mL: 60 μL, and the concentration of the aqueous solution of molybdenum disulfide quantum dots is 3 mg / ml.

[0118] The preferred technical solution is: The preparation method of the molybdenum disulfide quantum dots comprises the following steps:

[0119] Step 1: Add ammonium tetrathiomolybdate to distilled water and dissolve it by ultrasound to obtain an ammonium tetrathiomolybdate aqueous solution; add reduced glutathione to the ammonium tetrathiomolybdate aqueous solution, and then add ultrapure water to adjust the pH to 7.9;

[0120] Step 2: Transfer the solution obtained in Step 1 to a stainless-steel hydrothermal reaction kettle lined with polytetrafluoroethylene and react for 12 h under a hydrothermal environment of 180 °C; after the reaction is completed, the obtained product is naturally cooled to room temperature, and the initial product is centrifuged at 9000 revolutions per minute for 10 minutes, and the supernatant of the crude product is aspirated;

[0121] Step 3: Place the supernatant in a dialysis membrane, and then place it in secondary distilled water for dialysis to obtain a permeate; the permeate is converted into a light yellow powder solid substance through vacuum freeze-drying treatment and stored in a sealed manner.

[0122] The preferred embodiment is: in Step 2, the addition amount of the ethanol solution of quercetin is 0.8 mL; the addition amount of tetraethyl orthosilicate is 80 μL; the addition amount of ammonia water is 320 μL; the concentration of the ethanol solution of quercetin is 1.8 mg / mL.

[0123] The preferred embodiment is: in Step 3, the centrifugation speed is 10000 - 12000 revolutions / min.

[0124] The detection method of quercetin includes the following steps:

[0125] Step1: Establish a standard working curve of emission fluorescence intensity and quercetin concentration;

[0126] Step2: Add the sample to be tested into a beaker containing ethanol, ultrasonicate it in a water bath at 8 °C, and filter to obtain an extract.

[0127] Step3: Take the extract and dilute it to 1000 mL with ethanol; take 2 mL of MoS 2 QDs@SiO 2 @MIP aqueous solution and add it to an equal volume of the diluted solution of quercetin, incubate at room temperature for 4 min, and detect its fluorescence spectrum intensity; MoS 2 QDs@SiO 2 @MIP aqueous solution has a concentration of 0.2 mg / mL;

[0128] Step4: Obtain the concentration of diluted quercetin according to the standard working curve of fluorescence intensity and quercetin concentration, and calculate the concentration of quercetin in the extract through the dilution multiple relationship, so as to calculate the content of quercetin in onions;

[0129] Establishing a standard working curve of emission fluorescence intensity and quercetin concentration includes: using ethanol with a volume fraction of 80% as a solvent and acetic acid-sodium acetate as a pH regulator to prepare a series of standard concentration quercetin solutions with a pH value of 4, and mixing the MoS 2 QDs@SiO 2 @MIP aqueous solution with the series of standard concentration quercetin solutions at a volume ratio of 1:1, standing still and then detecting its fluorescence spectrum to establish a standard working curve of emission fluorescence intensity and quercetin concentration; the concentration of the MoS 2 QDs@SiO 2 @MIP aqueous solution is 0.2 mg / mL.

[0130] The above are only preferred embodiments for explaining the present invention and are not intended to limit the present invention in any form. Therefore, any modification or change to the present invention made in the same inventive spirit should still be included within the scope intended to be protected by the present invention.

Claims

1. A molybdenum disulfide quantum dot imprinted polymer fluorescence probe, characterized in that: The preparation method includes the following steps: Step 1: Add molybdenum disulfide quantum dots and 3-mercaptopropyltriethoxysilane to ethanol and stir to react; Step 2: Add an ethanol solution of quercetin and tetraethyl orthosilicate to the system of Step 1, stir and heat up to 55-65 °C, and then add ammonia water to react; Step 3: After the reaction is completed, centrifuge to remove the supernatant; centrifuge and wash the remaining precipitate product to obtain MoS 2 QDs@SiO 2 @MIP; The preparation method of molybdenum disulfide quantum dots includes the following steps: Step 1: Add ammonium tetrathiomolybdate to distilled water and dissolve it by ultrasonic wave to obtain an ammonium tetrathiomolybdate aqueous solution; add reduced glutathione to the ammonium tetrathiomolybdate aqueous solution, and then add ultrapure water to adjust the pH to 7.9-8.1; Step 2: Transfer the solution obtained in Step 1 to a stainless steel hydrothermal reaction kettle lined with polytetrafluoroethylene and react in a hydrothermal environment at 180-220 °C for 12-18 h; after the reaction, the obtained product is naturally cooled to room temperature, and the initial product is centrifuged at 9000-12000 revolutions per minute for 10-20 minutes, and the supernatant of the crude product is aspirated; Step 3: Place the supernatant in a dialysis membrane and then dialyze it in secondary distilled water to obtain a permeate; the permeate is converted into a light yellow powder solid substance by vacuum freeze-drying and stored in a sealed manner.

2. The molybdenum disulfide quantum dot imprinted polymer fluorescence probe according to claim 1, characterized in that: The molybdenum disulfide quantum dot is an aqueous solution of molybdenum disulfide quantum dots, and the ratio between the aqueous solution of molybdenum disulfide quantum dots and 3-mercaptopropyltriethoxysilane is 4.5 mL: 60-64 μL, and the concentration of the aqueous solution of molybdenum disulfide quantum dots is 3-4 mg / ml.

3. The molybdenum disulfide quantum dot imprinted polymer fluorescence probe according to claim 1, characterized in that: In Step 2, the addition amount of the ethanol solution of quercetin is 0.8-1.2 mL; the addition amount of tetraethyl orthosilicate is 80-120 μL; the addition amount of ammonia water is 320-350 μL; the concentration of the ethanol solution of quercetin is 1.8-2.2 mg / mL.

4. The molybdenum disulfide quantum dot imprinted polymer fluorescence probe according to claim 1, characterized in that: In Step 3, the centrifugation speed is 10000-12000 revolutions / min.

5. A detection method for quercetin, characterized in that: includes the following steps: Step1: Establish a standard working curve of emission fluorescence intensity and quercetin concentration; Step2: Add the sample to be tested into a beaker containing ethanol, ultrasonically treat it in a water bath at 8-12 °C, and filter to obtain an extract; Step 3: Take the extraction solution and dilute it to 1000 mL with ethanol; take 2 mL of MoS 2 QDs@SiO 2 @MIP aqueous solution and add it to an equal volume of the diluted quercetin solution, incubate at room temperature for 4 - 6 min, and detect the fluorescence spectral intensity; MoS 2 QDs@SiO 2 The concentration of the @MIP aqueous solution is 0.2 mg / mL; Step4: Obtain the concentration of diluted quercetin according to the standard working curve of fluorescence intensity and quercetin concentration, and calculate the concentration of quercetin in the extract through the dilution multiple relationship, so as to calculate the content of quercetin in onions; MoS 2 QDs@SiO 2 @MIP is the MoS described in any one of claims 1-4 2 QDs@SiO 2 @MIP.

6. The detection method for quercetin according to claim 5, characterized in that: Establishing a standard working curve for the emission fluorescence intensity and quercetin concentration includes: using ethanol with a volume fraction of 80% as the solvent and acetic acid-sodium acetate as the pH regulator to prepare a series of standard concentration quercetin solutions with a pH value of 4, and mixing the MoS 2 QDs@SiO 2 @MIP aqueous solution with the series of standard concentration quercetin solutions at a volume ratio of 1:1, standing still and then detecting its fluorescence spectrum to establish a standard working curve for the emission fluorescence intensity and quercetin concentration; the concentration of the MoS 2 QDs@SiO 2 @MIP aqueous solution is 0.2 mg / mL.

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