A fluorescence detection method for total antioxidant capacity based on infrared emission carbon dots

Abstract

This invention provides a method for detecting total antioxidant capacity. Near-infrared emitting carbon dot nanoparticles are treated with hydrazine hydrate to obtain aminated carbon dots (NH2-CDs). The surface of NH2-CDs is rich in hydroxyl and amino groups, resulting in enhanced fluorescence intensity, which helps NH2-CDs specifically recognize Fe. 3+ And Fe 3+ It can form strong coordination and electrostatic interactions with NH2-CDs, thus significantly quenching the fluorescence emission of NH2-CDs at 652 nm. Ascorbic acid (AA) has strong reducing properties and can convert Fe... 3+ Reduced to Fe 2+ Because NH2-CDs affect Fe 3+ Specific recognition, when Fe 3+ Reduced to Fe 2+ Afterwards, the fluorescence of NH2-CDs at 652 nm will recover. Therefore, this invention first utilizes Fe... 3+ Fluorescence quenching of NH2-CDs yields Fe with extremely weak fluorescence intensity. 3+ @NH2-CDs, then use AA to remove Fe 3+ Reduced to Fe 2+ Thus, Fe 3+ The fluorescence intensity of @NH2-CDs increased. This was observed by measuring Fe... 3+ By enhancing the fluorescence intensity of NH2-CDs, this invention can quantitatively detect the concentration of AA in solution, i.e., detect TAC. This invention is not only easy to operate, but also has advantages such as high sensitivity, rapid response, and low detection limit.

Description

Technical Field

[0001] This invention relates to a method for detecting total antioxidant capacity, belonging to the field of total antioxidant capacity detection. Background Technology

[0002] Antioxidants, as the body's metabolic defense system, protect cells and organs from oxidative damage. However, most antioxidant molecules must be obtained from exogenous nutrients, including fruits and vegetables, because the human body cannot produce antioxidants itself. To examine the cumulative activity of antioxidants in various foods, the concept of Total Antioxidant Capacity (TAC) has been proposed as a comprehensive antioxidant indicator. Furthermore, due to its high predictive value, TAC can be considered an essential antioxidant indicator for characterizing several high-risk diseases. Therefore, obtaining quantitative information on TAC in food is crucial, as it can guide the rational selection of diets to regulate oxidative stress. Currently, various techniques such as chromatography, spectrometry, and electrochemical techniques have been reported for TAC detection, but these methods suffer from drawbacks such as complex operation and low detection sensitivity.

[0003] Fluorescence methods offer advantages such as high sensitivity and ease of operation for TAC detection. Furthermore, although the fluorescence properties of carbon quantum dots (CDs) are rarely used for TAC detection in food, CDs that emit blue light have been used to detect ascorbic acid (AA) in food. AA is a typical antioxidant, and TAC can be equivalently described by its AA content. Summary of the Invention

[0004] This invention provides a method for detecting total antioxidant capacity. Near-infrared emitting carbon nanoparticles are treated with hydrazine hydrate to obtain aminated carbon nanoparticles (NH2-CDs). The fluorescence emission peak of NH2-CDs at 680 nm shifts to 652 nm. Compared to unmodified CDs, the hydrazine-modified CDs show a significant increase in the content of hydroxyl and amino groups on their surface, resulting in enhanced fluorescence intensity. The abundant hydroxyl and amino groups on the surface of NH2-CDs facilitate their specific recognition of Fe. 3+ Fe 3+ It can form strong coordination and electrostatic interactions with NH2-CDs, thus significantly quenching the fluorescence emission peak of NH2-CDs at 652 nm. Ascorbic acid (AA) has strong reducing properties and can convert Fe... 3+ Reduced to Fe 2+ Because NH2-CDs affect Fe 3+ Specific recognition, when Fe 3+ Reduced to Fe 2+ Afterwards, the fluorescence of NH2-CDs at 652 nm will recover. Therefore, we can use Fe 3+First, the fluorescence of NH2-CDs is quenched to obtain Fe with extremely weak fluorescence intensity. 3+ @NH2-CDs, then utilize the reducing property of AA to reduce Fe 3+ Reduced to Fe 2+ Thus, Fe 3+ The fluorescence intensity of @NH2-CDs increased. This was observed by measuring Fe... 3+ By observing changes in the fluorescence intensity of @NH2-CDs, we can quantitatively detect the concentration of AA in the solution, i.e., detect TAC. This invention is not only easy to operate, but also has the advantages of high sensitivity, rapid response, and low detection limit.

[0005] This invention provides a method for detecting total antioxidant capacity, comprising the following steps:

[0006] (1) Preparation of near-infrared emitting carbon dot nanoparticles (CDs);

[0007] (2) Prepare aminated near-infrared emitting carbon dot nanoparticles NH2-CDs using the CDs described in step (1) as raw materials;

[0008] (3) Prepare an NH2-CDs carbon quantum dot solution by preparing the aminated near-infrared emitting carbon nanoparticles NH2-CDs described in step (2). Titrate the NH2-CDs carbon quantum dot solution with a FeCl3 solution of a certain concentration, calculate the amount of FeCl3 solution required to completely quench the NH2-CDs carbon quantum dot solution, and incubate the NH2-CDs carbon quantum dot solution with the same concentration and volume of FeCl3 solution to obtain Fe 3+ @NH2-CDs solution; repeat the above steps to obtain multiple portions of Fe 3+ @NH2-CDs solution, for later use;

[0009] (4) Take Fe from step (3) 3+ @NH2-CDs solution and its fluorescence intensity at 652 nm was measured and recorded as F0; another Fe from step (3) was taken 3+ The NH2-CDs solution was titrated with ascorbic acid solutions of different concentrations to obtain a standard sample system, and its fluorescence intensity at 652 nm was measured and denoted as F. a Then establish (F) a -F0) and ascorbic acid concentration C AA The linear relationship between them;

[0010] (5) Prepare an ascorbic acid test solution of unknown concentration, and determine Fe according to the methods in steps (3) and (4). 3+ The fluorescence intensity F0 of the NH2-CDs solution at 652 nm and the fluorescence intensity F of the sample after titration with ascorbic acid solution at 652 nm are also considered. aAccording to step (4) (F) a -F0) and ascorbic acid concentration C AA The concentration of the ascorbic acid test solution can be calculated by establishing a linear relationship between the two, thereby obtaining the total antioxidant capacity.

[0011] in:

[0012] Preferably, in step (1), the near-infrared emitting carbon dot nanoparticles (CDs) are prepared by taking 0.1-1g of glutathione and 10-20g of formamide solution, mixing them evenly, and reacting them in a high-pressure reactor at 100-200℃ for 5-15h. The reaction solution is then filtered, dialyzed, and freeze-dried to obtain the final product.

[0013] Preferably, in step (2), the preparation method of the aminated near-infrared emitting carbon dot nanoparticles NH2-CDs is as follows: weigh 5-20 mg of near-infrared emitting carbon dot nanoparticles CDs prepared in step (1), dilute with 10-30 mL of deionized water, add 10-100 μL of hydrazine hydrate solution, mix evenly, and then reflux magnetically in an oil bath at 30-100℃ for 1-24 h. Filter, dialyze, and freeze-dry the reaction solution after the reaction to obtain the final product.

[0014] Preferably, in step (3), the NH2-CDs carbon quantum dot solution is prepared by weighing 1-15 mg of the aminated near-infrared emitting carbon dot nanoparticles NH2-CDs prepared in step (2), adding deionized water to dilute, and obtaining an NH2-CDs carbon quantum dot solution with a concentration of 1-30 μg / mL.

[0015] Preferably, in step (3), the FeCl3 solution is prepared by dissolving a certain mass of FeCl3 in 1-10 mL of deionized water to obtain a FeCl3 solution with a concentration of 10 mM.

[0016] Preferably, in step (3), the incubation reaction time is 1-10 min;

[0017] Preferably, in step (4), the titration reaction time is 1-5 min;

[0018] Preferably, in steps (4) and (5), the conditions for fluorescence measurement are: excitation wavelength of 420 nm and excitation and emission slits of 5 nm.

[0019] The present invention also provides a method for detecting total antioxidant capacity and its use in detecting ascorbic acid concentration in food or fruit.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] First, the aminated near-infrared emitting carbon dot nanoparticles (CDs) prepared in this invention contain a large number of amino and hydroxyl groups on their surface, which can react with Fe. 3+ Strong coordination and electrostatic interactions occur between them, enabling them to specifically recognize Fe. 3+ Therefore, titrating an aminated near-infrared emitting NH2-CDs carbon quantum dot solution with FeCl3 solution can quench the fluorescence of the carbon dots. Secondly, ascorbic acid AA has strong reducing properties and can quench the fluorescence of Fe... 3+ Reduced to Fe 2+ Because NH2-CDs affect Fe 3+ Specific recognition, when Fe 3+ Reduced to Fe 2+ Subsequently, the fluorescence of NH2-CDs at 652 nm gradually increases, and the concentration of ascorbic acid AA can be quantitatively detected by the intensity of the fluorescence enhancement of this carbon dot, that is, TAC is measured.

[0022] The method of this invention is simple and convenient to use, highly sensitive, fast in response and has a low detection limit. The detection limit of the method disclosed in this invention for ascorbic acid is 1.89 nmol / L, which is 1-2 orders of magnitude lower than that of other fluorescence assay methods. Attached Figure Description

[0023] Figure 1 The fluorescence spectrum of the standard sample in Example 1 was obtained at an excitation wavelength of 420 nm.

[0024] Figure 2 In Example 1, the ascorbic acid concentration is used as the x-axis, and (F) a The standard curve is plotted using -F0 as the ordinate.

[0025] Figure 3 The fluorescence spectrum of the standard sample in Example 2 was obtained at an excitation wavelength of 420 nm.

[0026] Figure 4 In Example 2, ascorbic acid concentration is used as the x-axis, (F a The standard curve is plotted using -F0 as the ordinate.

[0027] Figure 5 The bar chart shows the TAC (Total Acrylic Acid) bars for the standard and test samples of different foods in Example 3.

[0028] Figure 6 This is a bar chart of the vitamin C content of different fruits in Example 4. Detailed Implementation Plan

[0029] The present invention will be described in more detail below through specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0030] Example 1:

[0031] A method for detecting total antioxidant capacity specifically includes the following steps:

[0032] 1. Preparation of near-infrared emitting carbon dot nanoparticles (CDs):

[0033] Weigh 0.6g of glutathione and 19.4g of formamide solution, mix them ultrasonically for 30min, and then put them into a 50mL polytetrafluoroethylene high-pressure reactor. React at 160℃ for 10h. After natural cooling, filter with a 0.22-micron microporous membrane, and then dialyze with a dialysis bag with a molecular weight of 1000 for two days. Finally, freeze the product and freeze-dry it in a freeze dryer for two days to obtain near-infrared emitting carbon dot nanoparticles (CDs).

[0034] 2. Preparation of amination-modified near-infrared emitting carbon dot nanoparticles NH2-CDs:

[0035] Weigh 10 mg of the near-infrared emitting carbon dot nanoparticles (CDs) prepared in step 1, dissolve them in 20 mL of deionized water to obtain a carbon dot solution, then add 25 μL of hydrazine hydrate to the above carbon dot solution, mix well, and then add the mixture to a 50 mL round-bottom flask. Stir and reflux magnetically in an oil bath at 60 °C for 6 h. After the reaction is complete, allow the reaction solution to cool naturally, then filter it through a 0.22 μm microporous membrane. Finally, freeze the product and place it in a freeze dryer to freeze dry for two days to obtain amination-modified near-infrared emitting carbon dot nanoparticles (NH2-CDs).

[0036] 3. Preparation of Fe 3+ @NH2-CDs

[0037] Weigh 10 mg of the NH2-CDs prepared in step 2 and dilute with deionized water to prepare a carbon quantum dot solution with a concentration of 20 μg / mL. Take 2 mL of the above carbon quantum dot solution and titrate it with a 10 mM FeCl3 solution. Calculations show that the amount of FeCl3 solution required to completely quench the fluorescence of NH2-CDs at 652 nm is 10 μL. Take another 2 mL of the above carbon quantum dot solution, add 10 μL of 10 mM FeCl3 solution, and incubate for 5 min to obtain Fe... 3+ @NH2-CDs solution; repeat the above steps multiple times to obtain multiple portions of Fe 3+ @NH2-CDs solution, for later use;

[0038] 4. Prepare ascorbic acid solutions (AA) with concentrations of 4 μM, 8 μM, 12 μM, 16 μM, 20 μM, 24 μM, and 28 μM, and react them with the Fe obtained in step 3 at 25°C. 3+ The standard sample system was obtained by reacting the NH2-CDs solution for 2 min.

[0039] 5. Using an RF-6000 fluorescence spectrophotometer, with the excitation wavelength set to 420 nm and both the excitation and emission slits at 5 nm, measure the Fe obtained in step 3. 3+ The fluorescence intensity of the NH2-CDs solution was measured and denoted as F0. Using an RF-6000 fluorescence spectrophotometer, with the excitation wavelength set to 420 nm and both the excitation and emission slits at 5 nm, the fluorescence intensity of the standard sample from step 4 was detected at 652 nm, and F0 was recorded. a ;

[0040] 6. Obtain the fluorescence spectrum of the standard sample system at an excitation wavelength of 420 nm, as shown below. Figure 1 As shown. Then, with the concentration of ascorbic acid as the x-axis, the intensity of carbon dot fluorescence enhancement (F) is... a Plot a standard curve using -F0) as the ordinate, such as Figure 2 As shown, we obtain (F) a -F0) and C AA It exhibits a good linear relationship, and the linear equation is F. a -F0 = 2075.73C AA +958.38, R 2 =0.992;

[0041] 7. Prepare a test solution of ascorbic acid with an unknown concentration, and determine the fluorescence intensity F according to steps 5-6. a And F0, according to (F a -F0) and C AA The concentration of the ascorbic acid test solution can be calculated by establishing a linear relationship between the two.

[0042] Example 2:

[0043] A method for detecting total antioxidant capacity specifically includes the following steps:

[0044] 1. Preparation of near-infrared emitting carbon dot nanoparticles (CDs): Same as in Example 1;

[0045] 2. Preparation of amination-modified near-infrared emitting carbon dot nanoparticles NH2-CDs:

[0046] Weigh 10 mg of the near-infrared emitting carbon dot nanoparticles (CDs) prepared in step 1, dissolve them in 20 mL of deionized water, and add 50 μL of hydrazine hydrate to the above carbon dot solution. After mixing evenly, add the mixture to a 50 mL round-bottom flask and reflux with magnetic stirring in an oil bath at 60 °C for 9 h. After the reaction is complete, allow the refluxed reaction solution to cool naturally, then filter it through a 0.22 μm microporous membrane. Finally, freeze the product and place it in a freeze dryer to freeze dry for two days to obtain the final product.

[0047] 3. Preparation of Fe 3+ @NH2-CDs

[0048] Weigh 10 mg of the aminated near-infrared emitting carbon dot nanoparticles NH2-CDs prepared in step 2, dissolve them in 2 mL of deionized water to prepare a carbon quantum dot solution with a concentration of 5 mg / mL. Then dilute the carbon quantum dot solution with deionized water to 20 μg / mL, with a volume of 2 mL for subsequent testing. Prepare a 10 mM FeCl3 solution; titration shows that adding 10 μL of 10 mM FeCl3 solution completely quenches the fluorescence of NH2-CDs at 652 nm. Take another 2 mL of the above carbon quantum dot solution, add 10 μL of 10 mM FeCl3 solution, and incubate for 5 min to obtain Fe... 3+ @NH2-CDs solution; repeat the above steps multiple times to obtain multiple portions of Fe 3+ @NH2-CDs solution, for later use;

[0049] 4. Prepare ascorbic acid solutions (AA) with concentrations of 2 μM, 6 μM, 10 μM, 14 μM, 18 μM, 22 μM, 26 μM, and 30 μM, and react them with the Fe obtained in step 3 at 25°C. 3+ The standard sample system was obtained by reacting the NH2-CDs solution for 2 min.

[0050] 5. Using an RF-6000 fluorescence spectrophotometer, with the excitation wavelength set to 420 nm and both the excitation and emission slits at 5 nm, measure the Fe obtained in step 3. 3+ The fluorescence intensity of the NH2-CDs solution was measured and denoted as F0. Using an RF-6000 fluorescence spectrophotometer, with the excitation wavelength set to 420 nm and both the excitation and emission slits at 5 nm, the fluorescence intensity of the standard sample from step 4 was detected at 652 nm, and F0 was recorded. a ;

[0051] 6. Obtain the fluorescence spectrum of the standard sample system at an excitation wavelength of 420 nm, as shown below. Figure 3 As shown, the intensity of carbon dot fluorescence enhancement (F) is plotted on the x-axis with the concentration of ascorbic acid. a A standard curve is plotted with -F0) as the ordinate, such as Figure 4 As shown, we obtain (F) a -F0) and C AA It exhibits a good linear relationship, and the linear equation is F. a -F0 = 2098.10C AA +848.82, R 2 =0.995;

[0052] 7. Prepare a test solution of ascorbic acid with an unknown concentration, and determine the fluorescence intensity F according to steps 5-6. a And F0, according to (F a -F0) and C AA The concentration of the ascorbic acid test solution can be calculated by establishing a linear relationship between the two.

[0053] The detection limit of ascorbic acid using the method of this embodiment is 1.89 nmol / L, which is 1-2 orders of magnitude lower than that of other fluorescence assay methods, as shown in Table 1.

[0054] Table 1 Comparison of detection results between Example 2 of the present invention and the references

[0055]

[0056]

[0057] Example 3:

[0058] Four types of beverages containing vitamin C and vitamin C tablets were purchased. A standard sample system was obtained according to the test method described in Example 1, and F was obtained. a and F0, then according to (F a -F0) and C AA The linear relationship between these two factors allows for the calculation of the concentration of ascorbic acid in the test solution of different foods. For example... Figure 5 As shown, the calculated concentration of vitamin C in the food is comparable to the actual concentration of vitamin C in the food.

[0059] Example 4:

[0060] Five fruits containing vitamin C were purchased: kiwi, strawberry, tomato, orange, and apple. A standard sample system was obtained according to the method described in Example 1, and F was obtained. a and F0, then according to (F a -F0) and C AA The linear relationship between these two factors allows for the calculation of the concentration of ascorbic acid in the test solution of different foods. For example... Figure 6 As shown, the calculated concentration of vitamin C in the fruit is consistent with the actual situation.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

[0062] References:

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Claims

1. A method for detecting ascorbic acid concentration, comprising the following steps: (1) Preparation of near-infrared emitting carbon dot nanoparticles (CDs); Take 0.1-1g of glutathione and 10-20g of formamide solution, mix them evenly, and react them in a high-pressure reactor at 100-200℃ for 5-15 hours. After the reaction is completed, filter, dialyze, and freeze dry to obtain the product. (2) Prepare aminated near-infrared emitting carbon dot nanoparticles NH2-CDs using CDs as raw materials in step (1); weigh 5-20 mg of near-infrared emitting carbon dot nanoparticles CDs prepared in step (1), dilute with 10-30 mL of deionized water, add 10-100 μL of hydrazine hydrate solution, mix evenly, and reflux magnetically in an oil bath at 30-100℃ for 1-24 h. Filter, dialyze, and freeze dry the reaction solution after the reaction to obtain the final product. (3) Prepare an NH2-CDs carbon quantum dot solution by preparing the amination-modified near-infrared emitting carbon nanoparticles NH2-CDs described in step (2). Titrate the NH2-CDs carbon quantum dot solution with a FeCl3 solution of a certain concentration, calculate the amount of FeCl3 solution required to completely quench the NH2-CDs carbon quantum dot solution, and incubate the NH2-CDs carbon quantum dot solution with the same concentration and volume of FeCl3 solution to obtain Fe 3+ @NH2-CDs solution; repeat the above steps to obtain multiple portions of Fe 3+ @NH2-CDs solution, for later use; (4) Take Fe from step (3) 3+ @NH2-CDs solution and its fluorescence intensity at 652 nm was measured and recorded as F0; another Fe from step (3) was taken 3+ The NH2-CDs solution was titrated with ascorbic acid solutions of different concentrations to obtain a standard sample system, and its fluorescence intensity at 652 nm was measured and denoted as F. a Then establish (F) a -F0) and ascorbic acid concentration C AA The linear relationship between them; (5) Prepare an ascorbic acid test solution of unknown concentration, and determine Fe according to the methods in steps (3) and (4). 3+ The fluorescence intensity F0 of the NH2-CDs solution at 652 nm and the fluorescence intensity F of the sample after titration with ascorbic acid solution at 652 nm are also considered. a According to step (4) (F) a -F0) and ascorbic acid concentration C AA The concentration of the ascorbic acid test solution can be calculated by establishing a linear relationship between the two.

2. The method for detecting ascorbic acid concentration as described in claim 1, characterized in that, In step (3), the NH2-CDs carbon quantum dot solution is prepared by weighing 1-15 mg of the aminated near-infrared emitting carbon nanoparticles NH2-CDs prepared in step (2), adding deionized water to dilute, and obtaining a carbon quantum dot solution with a concentration of 1-30 μg / mL.

3. The method for detecting ascorbic acid concentration as described in claim 1, characterized in that, In step (3), the FeCl3 solution is prepared by dissolving a certain mass of FeCl3 in 1-10 mL of deionized water to obtain a FeCl3 solution with a concentration of 10 mM.

4. The method for detecting ascorbic acid concentration as described in claim 1, characterized in that, In step (3), the incubation reaction time is 1-10 min.

5. The method for detecting ascorbic acid concentration as described in claim 1, characterized in that, In step (4), the titration reaction time is 1-5 min.

6. The method for detecting ascorbic acid concentration as described in claim 1, characterized in that, In steps (4) and (5), the conditions for fluorescence measurement are: excitation wavelength of 420 nm and excitation and emission slits of 5 nm.

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