A method for detecting acrylamide based on copper nanocluster ratio fluorescence
By using double-modal emission copper nanoclusters synthesized by bovine serum albumin and ascorbic acid in food detection, and combining carboxymethyl chitosan, the problem of unstable fluorescence performance of copper nanoclusters in the prior art and the use of toxic reagents during the synthesis process is solved, and the food detection effect with high stability and low background interference is achieved.
Patent Information
- Application Number
- CN202311051373.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-21
- Publication Date
- 2025-03-25
- Estimated Expiration
- 2043-08-21
AI Technical Summary
In the prior art, the fluorescence performance of copper nanoclusters is susceptible to environmental interference, Stokes displacement is small, and toxic reagents are often used during synthesis, which affects health and safety.
Bovine serum albumin as ligand and ascorbic acid as reducing agent were used to synthesize bimodal emitted copper nanoclusters under water bath conditions, and carboxymethyl chitosan was used to improve its fluorescence intensity and storage stability.
The obtained copper nanoclusters have good fluorescence stability and have a large Stokes displacement, which reduces background interference during the detection process, enhances the stability of the detection results, and avoids the risk of using toxic reagents.
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Figure CN117186880B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for detecting acrylamide based on copper nanocluster ratio fluorescence, and belongs to the technical field of food safety and rapid detection. Background Art
[0002] Acrylamide is a water-soluble small molecule with potential neurotoxicity, reproductive toxicity, genotoxicity and carcinogenicity, and is a Class 2A carcinogen. Acrylamide in food mainly comes from the side effects of the Maillard reaction and is commonly found in foods that have been processed at high temperatures such as frying and baking. According to EU regulations, the maximum allowable concentration of acrylamide in food is 750μg / kg, and foods processed at high temperatures will inevitably produce more or less acrylamide, which poses a threat to people's health.
[0003] The detection methods of acrylamide in food include liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, surface enhanced Raman method, electrochemical method, etc. The above methods have achieved good detection results, but have the disadvantages of expensive instruments, complex operation, long detection time, and low repeatability.
[0004] In recent years, fluorescence detection methods have attracted much attention due to their high sensitivity, strong selectivity, simple operation, and rapid detection. However, most reports are single signal responses of fluorescence enhancement or quenching, which are easily interfered by the environment. Therefore, the ratiometric fluorescence detection system provides a fast and accurate detection method for acrylamide.
[0005] Copper nanoclusters are composed of several to hundreds of copper atoms, and their size is close to the Fermi wavelength. They have the characteristics of strong fluorescence stability, good biocompatibility, and large Stokes shift. Compared with precious metal nanoclusters, they have significant economic advantages and have been used in the detection of various substances.
[0006] The synthesis of copper nanoclusters in the prior art includes hydrothermal reduction, electrochemical deposition, etching, etc. The hydrothermal reduction method has attracted much attention due to its simple operation. The ligands of copper nanoclusters are also diverse, mainly including proteins, thiol small molecules, DNA, dendritic polymers, etc. Most of the copper nanoclusters synthesized now emit blue or green light, with small Stokes shift and are easily disturbed by the background environment. The near-infrared emitting copper nanoclusters synthesized with bovine serum albumin as ligand use strong reducing agents such as sodium borohydride and hydrazine hydrate, which are easy to threaten human health. The synthesis process of near-infrared emitting copper nanoclusters with thiol small molecules as ligands involves toxic reagents or their fluorescence properties are easily affected by temperature and pH.
[0007] Therefore, there is an urgent need for a copper nanocluster with stable fluorescence properties, large Stokes shift, and green synthesis for food detection. Summary of the invention
[0008] In view of the defects and shortcomings of the prior art, the present invention provides a method for detecting acrylamide by using ratio fluorescence of copper nanoclusters. In the method, copper nanoclusters with double-peak emission are synthesized by using bovine serum albumin as a ligand and ascorbic acid as a reducing agent. The obtained copper nanoclusters have good fluorescence stability and a large Stokes shift. Meanwhile, carboxymethyl chitosan is used to further improve the fluorescence intensity and storage stability of the copper nanoclusters with double-peak emission. The copper nanoclusters are used as fluorescent probes and ratio fluorescence is used to reduce background interference in the detection process and enhance the stability of the detection results.
[0009] The first object of the present invention is to provide a method for preparing a copper nanocluster fluorescent probe with double peak emission, the method comprising the following steps:
[0010] (1) At room temperature, carboxymethyl chitosan is added to a bovine serum albumin solution and stirred thoroughly; then a copper sulfate solution is added and stirred continuously to form a milky white mixed solution;
[0011] (2) Add ascorbic acid solution to the milky white mixed solution of step (1), and adjust the pH value of the system to 10-13; then place in a water bath at 50-55° C. and stir thoroughly until the solution eventually turns brown-yellow. Collect the reaction solution, dialyze, and collect the dialyzate to obtain the product.
[0012] In one embodiment, the concentration of the bovine serum albumin solution in step (1) is 10 to 80 mg / mL.
[0013] In one embodiment, the mass ratio of the carboxymethyl chitosan to bovine serum albumin in step (1) is 1-6:1-8.
[0014] In one embodiment, the concentration of copper sulfate in step (1) is 2 to 10 mmol / L.
[0015] In one embodiment, the mass ratio of the carboxymethyl chitosan to bovine serum albumin and copper sulfate in step (1) is 1-3:1-3:0.02-0.09.
[0016] In one embodiment, the concentration of the ascorbic acid solution in step (2) is 0.2-1.2 mol / L.
[0017] In one embodiment, the sufficient stirring time in step (2) is 1 to 4 hours.
[0018] In one embodiment, the dialysis bag used for dialysis in step (2) is 3500Da; and the dialysis time is 10 to 40 hours.
[0019] The second object of the present invention is to provide a copper nanocluster prepared by the above method.
[0020] The third object of the present invention is to provide an application of the copper nanoclusters described above in the field of food detection.
[0021] A fourth object of the present invention is to provide a method for detecting acrylamide based on copper nanocluster ratio fluorescence, the method comprising the following steps:
[0022] (1) Constructing a quantitative relationship model
[0023] A series of acrylamide standard solutions with different concentrations are prepared, and the acrylamide standard solutions with different concentrations are mixed with a glutathione solution, a tri(2-carboxyethyl)phosphine solution and a phosphate buffer with a pH value of 9 to 10 to form a reaction solution; the reaction solution is then mixed with a phosphate buffer with a pH value of 7 and the copper nanoclusters prepared above, and the reaction is continued. After the reaction is completed, a fluorescence spectrometer is used to record the fluorescence spectrum at 370-770 nm;
[0024] The fluorescence intensity at 650nm and 395nm in the fluorescence spectrum was selected to establish the relationship between acrylamide concentration and F 650 / F 395 The quantitative relationship between the concentration of acrylamide and 650 / F 395 The value is the vertical axis, and the quantitative relationship model is constructed;
[0025] (2) Determination of acrylamide content in the sample to be tested
[0026] The sample to be tested is mixed with a glutathione solution, a tri(2-carboxyethyl)phosphine solution and a phosphate buffer solution with a pH value of 9 to 10 to form a reaction solution; the reaction solution is then mixed with a phosphate buffer solution with a pH value of 7 and the copper nanoclusters prepared above, and the reaction is continued. After the reaction is completed, a fluorescence spectrometer is used to record the fluorescence spectrum at 370-770 nm; the fluorescence intensity at 650 nm and 395 nm of the detection system is selected to calculate F 650 / F 395 The value of is obtained, and based on the quantitative relationship constructed in step (1), the content of acrylamide in the sample to be tested is calculated.
[0027] In one embodiment, the concentration of glutathione is 200-360 μmol / L, preferably 320 μmol / L.
[0028] In one embodiment, the concentration of the tri(2-carboxyethyl)phosphine solution is 20-30 μmol / L, preferably 21 μmol / L.
[0029] In one embodiment, the reaction is continued for 2 to 8 minutes, preferably 5 minutes.
[0030] In one embodiment, the detection system is formed by mixing 500 μL of reaction solution with 10 μL of copper nanoclusters and 490 μL of phosphate buffer (100 mmol / L, PH=7); the reaction solution is formed by mixing 100 μL of standard acrylamide solution or sample solution to be tested with 100 μL of glutathione (320 μmol / L) and 100 μL of tris(2-carboxyethyl)phosphine (21 μmol / L) solution and 200 μL of phosphate buffer (10 mmol / L, PH=9).
[0031] The fifth object of the present invention is to provide an application of the above-mentioned method for detecting acrylamide based on copper nanocluster ratio fluorescence in the field of food detection.
[0032] Beneficial effects of the present invention:
[0033] (1) The present invention synthesizes copper nanoclusters with double peak emission for the first time using bovine serum albumin as a ligand and ascorbic acid as a reducing agent. The preparation method is simple and takes a short time. The obtained copper nanoclusters have good fluorescence stability and a large Stokes shift. At the same time, carboxymethyl chitosan is used to further improve the fluorescence intensity and storage stability of the copper nanoclusters.
[0034] (2) The acrylamide detection method established in the present invention uses a proportional fluorescence method, which reduces background interference in the detection process and enhances the stability of the detection results; and effectively avoids complicated pre-treatment steps and the use of large instruments. It has a wide linear range (0-300 μmol / L), a low detection limit (0.15 μmol / L) and good selectivity, and has application prospects in the detection of actual samples. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 This is a transmission electron microscopy image of the copper nanoclusters prepared in Example 1 of the present invention;
[0036] Figure 2 The excitation and emission spectra of the copper nanoclusters prepared in Example 1 of the present invention;
[0037] Figure 3 This is an infrared spectrum of the copper nanoclusters prepared in Example 1 of the present invention;
[0038] Figure 4 This is an X-ray photoelectron spectrum of the copper nanoclusters prepared in Example 1 of the present invention;
[0039] Figure 5 The fluorescence spectra of acrylamide at different concentrations in Example 2 of the present invention are shown;
[0040] Figure 6 This is a quantitative relationship model diagram constructed in Example 2 of the present invention. DETAILED DESCRIPTION
[0041] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not intended to limit the present invention.
[0042] Example 1 Synthesis and Characterization of Copper Nanoclusters
[0043] The method for preparing copper nanoclusters comprises the following steps:
[0044] (1) Under stirring conditions at room temperature, 0.3 g of carboxymethyl chitosan was added to 10 mL of bovine serum albumin solution (30 mg / mL), and the mixture was stirred for 10 min to form a mixed solution;
[0045] (2) 10 mL of copper sulfate solution (5 mmol / L) was added dropwise to the mixed solution of step (1), and the mixture was stirred for 2 min until the mixture turned milky white. Then 500 μL of ascorbic acid solution (1 mol / L) was added, and the pH was adjusted to 11 using sodium hydroxide solution. The mixture was then placed in a 55°C water bath and stirred for 2 h. The solution eventually turned brown-yellow, and the reaction solution was collected.
[0046] (3) The reaction solution obtained in step (2) was dialyzed using a 3500Da dialysis bag for 12 h, the liquid in the dialysis bag was collected, and sealed and stored in a 4°C refrigerator to obtain copper nanoclusters.
[0047] Characterization of copper nanoclusters
[0048] The performance of the copper nanoclusters prepared in Example 1 was characterized:
[0049] Transmission electron microscopy of copper nanoclusters Figure 1 As shown, the particles are dispersed in a spherical shape with an average particle size of 2.16 nm.
[0050] The excitation and emission spectra of copper nanoclusters are shown in Figure 2. Figure 2 As shown, it can be seen from the figure that the optimal excitation wavelength of the copper nanoclusters is 310nm, and the optimal emission wavelengths are 395nm and 650nm.
[0051] The infrared spectrum of copper nanoclusters is shown in Figure 3 As shown, it can be seen from the figure that copper nanoclusters contain functional groups such as hydroxyl, carboxyl, and amino, which are consistent with bovine serum albumin.
[0052] X-ray electron spectrum of copper nanoclusters Figure 4 As shown, it can be seen from the figure that copper nanoclusters are mainly composed of elements such as copper, carbon, nitrogen, oxygen, and sulfur.
[0053] Example 2
[0054] A method for constructing a fluorescent probe for detecting acrylamide based on the copper nanoclusters prepared in Example 1, the method specifically comprising the following steps:
[0055] (1) Construction of quantitative relationship model
[0056] Prepare acrylamide standard solutions of different concentrations (0-300 μmol / L), then take 100 μL of acrylamide solutions of different concentrations, mix them with 100 μL of glutathione (320 μmol / L), 100 μL of tris(2-carboxyethyl)phosphine (21 μmol / L) solution and 200 μL of phosphate buffer (10 mmol / L, PH=9), and react at 25°C for 30 min to form a reaction solution; then mix 10 μL of copper nanoclusters (copper nanocluster stock solution prepared in Example 1), 490 μL of phosphate buffer (100 mmol / L, PH=7) and 500 μL of the reaction solution for 5 min. After the reaction, take the solution and place it in a quartz cuvette, and place it in an RF-6000 fluorescence spectrometer to record the fluorescence spectrum of 370-770 nm;
[0057] Select the fluorescence intensity at 650nm and 395nm of the detection system to establish the relationship between acrylamide concentration and F 650 / F 395 (Evaluation of the fluorescence on-time efficiency of acrylamide on copper nanocluster system), with acrylamide concentration as the horizontal axis; F 650 / F 395 The value is the vertical axis, and the quantitative relationship model is constructed;
[0058] (2) Determination of acrylamide content in the sample to be tested
[0059] The sample to be tested is pretreated to form a test solution, 100 μL of the test solution is mixed with 100 μL of glutathione solution (320 μmol / L), 100 μL of tris(2-carboxyethyl)phosphine solution (21 μmol / L) and 200 μL of phosphate buffer (10 mmol / L, PH=9), and reacted at 25°C for 30 minutes to form a reaction solution; then 10 μL of copper nanoclusters, 490 μL of phosphate buffer (100 mmol / L, PH=7) and 500 μL of the reaction solution are mixed and reacted for 5 minutes. After the reaction is completed, the solution is placed in a quartz cuvette, and placed in an RF-6000 fluorescence spectrometer to record the fluorescence spectrum of 370-770 nm; the fluorescence intensity at 650 nm and 395 nm of the detection system is selected to calculate F 650 / F 395 The value of is obtained, and based on the quantitative relationship constructed in step (1), the content of acrylamide in the sample to be tested is calculated.
[0060] The fluorescence spectra of different concentrations of acrylamide in step (1) are as follows: Figure 5 As shown, it can be seen from the figure that acrylamide can restore the fluorescence intensity of copper nanoclusters at 650nm.
[0061] The quantitative relationship constructed is as follows Figure 6 As shown, the acrylamide concentration and F 650 / F 395 There is a significant linear correlation (R 2 =0.99843); the regression equation is F 650 / F 395 =0.00694C(μmol / L)+1.15464, the detection limit LOD is 0.15μmol / L (LOD=3s / k, where s is the standard deviation of the blank solution and k is the slope of the standard curve).
[0062] Example 3 Selectivity and recovery of acrylamide
[0063] Referring to the detection method of Example 2, a selective experiment was performed: taking 300 μmol / L of the test substance as an example, acrylamide was replaced with other analogs under the same conditions, and the fluorescence spectrum was measured and the F 650 / F 395 ; The test results are shown in Table 1:
[0064] Table 1. Selectivity of different analytes
[0065]
[0066] From the results in Table 1, it can be seen that the detection method of the present invention has a significantly better corresponding intensity for acrylamide than other analogs, showing specific selectivity.
[0067] Addition recovery of actual samples
[0068] Take 1g of bread and grind it, mix it with acrylamide standard solution (0, 10, 50, 100μmol / L) respectively, add 5mL of hexane, ultrasonicate for 15min, add 5mL of mixed extractant consisting of water and acetonitrile (volume ratio 1:1), vortex oscillate for 10min; then centrifuge at 12000rpm for 20min, take the supernatant, add QuECHERS salt, evaporate the acetonitrile phase under nitrogen blowing at 40℃, pass through a 0.22μm hydrophilic membrane to obtain the test solution.
[0069] Referring to the detection method of Example 2, the content of acrylamide in the bread samples was detected; all the measurements were repeated three times, and the test results are shown in Table 2. The recovery rate of acrylamide in the toast bread samples was between 90.29% and 101.30%, indicating that the method has good accuracy and can be used for the detection of actual samples.
[0070] Table 2. Results of acrylamide spike experiment in toast samples
[0071]
[0072] Example 4
[0073] 1. Optimization of pH conditions of the detection system
[0074] 10 μL of copper nanoclusters, 100 μL of glutathione solution (320 μmol / L), and 100 μL of tris(2-carboxyethyl)phosphine (21 μmol / L) solution were mixed with 790 μL of phosphate buffer (100 mmol / L) at pH 6, 7, 8, 9, or 10, respectively, and reacted for 5 min. The fluorescence spectra were measured using RF-6000 and the F 650 / F 395 To evaluate the quenching efficiency of glutathione on copper nanoclusters, the results are shown in Table 3:
[0075] Table 3. Quenching efficiency of copper nanoclusters by glutathione at different pH values
[0076]
[0077] From the results in Table 3, we can see that with the increase of system pH, F 650 / F 395 It shows a trend of first decreasing and then increasing, and is lowest when the pH reaches 7, so the optimal pH of the system is 7.
[0078] 2. Optimization of glutathione concentration in the detection system
[0079] 10 μL of copper nanoclusters, 100 μL of tris(2-carboxyethyl)phosphine (21 μmol / L) solution, and 790 μL of phosphate buffer (100 mmol / L, pH=7) were mixed with 100 μL of glutathione solution with concentrations of 200, 240, 280, 320, and 400 μmol / L, respectively, and reacted for 5 min. The fluorescence spectrum was measured using RF-6000 and the F 650 / F 395 To evaluate the quenching efficiency of glutathione on copper nanoclusters, the results are shown in Table 4:
[0080] Table 4. Quenching efficiency of copper nanoclusters at different concentrations of glutathione
[0081]
[0082] From the results in Table 4, we can see that with the increase of glutathione concentration, F 650 / F 395 It showed a downward trend and remained unchanged after the glutathione concentration reached 320μmol / L, so the optimal glutathione concentration was 320μmol / L.
[0083] 3. Time optimization of detection system
[0084] 10 μL of copper nanoclusters, 100 μL of tris(2-carboxyethyl)phosphine (21 μmol / L) solution, 100 μL of glutathione solution (320 μmol / L) and 790 μL of phosphate buffer (100 mmol / L, pH=7) were mixed and reacted for 2, 3, 5, 8 and 10 min, respectively. The fluorescence spectra were measured using RF-6000 and the F 650 / F 395 To evaluate the quenching efficiency of glutathione on copper nanoclusters, the results are shown in Table 5:
[0085] Table 5. Quenching efficiency of copper nanoclusters by glutathione at different times
[0086]
[0087] From the results in Table 5, we can see that as the system reaction proceeds, F 650 / F 395 It shows a downward trend and remains unchanged after 5 minutes of reaction, so the optimal reaction time is 5 minutes.
[0088] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.
Claims
1. A method for detecting acrylamide based on copper nanocluster ratio fluorescence, characterized in that: The method comprises the following steps: (1) Constructing a quantitative relationship model A series of acrylamide standard solutions with different concentrations are prepared, and the acrylamide standard solutions with different concentrations are mixed with a glutathione solution, a tri(2-carboxyethyl)phosphine solution and a phosphate buffer with a pH value of 9 to 10 to form a reaction solution; the reaction solution is then mixed with a phosphate buffer with a pH value of 7 and copper nanoclusters, and the reaction is continued. After the reaction is completed, a fluorescence spectrometer is used to record the fluorescence spectrum at 370-770 nm; The fluorescence intensity at 650nm and 395nm in the fluorescence spectrum was selected to establish the relationship between acrylamide concentration and F 650 / F 395 The quantitative relationship between the concentration of acrylamide and 650 / F 395 The value is the vertical coordinate, and the quantitative relationship model is constructed; (2) Determination of acrylamide content in the sample to be tested The sample to be tested is mixed with a glutathione solution, a tri(2-carboxyethyl)phosphine solution and a phosphate buffer solution with a pH value of 9 to 10 to form a reaction solution; the reaction solution is then mixed with a phosphate buffer solution with a pH value of 7 and copper nanoclusters to continue the reaction. After the reaction is completed, a fluorescence spectrometer is used to record the fluorescence spectrum at 370-770 nm; Select the fluorescence intensity at 650nm and 395nm of the detection system and calculate F 650 / F 395 The value of, and according to the quantitative relationship constructed in step (1), calculate the content of acrylamide in the sample to be tested; The method for preparing the copper nanoclusters described in step (1) and step (2) comprises the following steps: 1) At room temperature, add carboxymethyl chitosan to the bovine serum albumin solution and stir thoroughly; then add the copper sulfate solution and continue stirring to form a milky white mixed solution; 2) Add ascorbic acid solution to the milky white mixed solution of step 1) and adjust the pH value of the system to 10-13; then place it in a water bath at 50-55° C. and stir it thoroughly until the solution eventually turns brown-yellow. Collect the reaction solution, dialyze it, and collect the dialyzate to obtain the product.
2. The method according to claim 1, characterized in that In step 1), the mass ratio of the carboxymethyl chitosan to bovine serum albumin is 1-6:1-8.
3. The method according to claim 1, characterized in that In step 1), the mass ratio of the carboxymethyl chitosan to bovine serum albumin and copper sulfate is 1-3:1-3:0.02-0.
09.
4. The method according to claim 1, characterized in that: Step 2) The concentration of the ascorbic acid solution is 0.2-1.2 mol / L.
5. The method according to claim 1, characterized in that The concentration of the glutathione is 200-360 μmol / L.
6. The method according to claim 1, characterized in that The reaction time is 2 to 8 minutes.
7. Application of the method for detecting acrylamide based on copper nanocluster ratio fluorescence according to any one of claims 1 to 6 in the field of food detection.