Carbon quantum dot nano-enzyme for detecting ascorbic acid as well as preparation method and application of carbon quantum dot nano-enzyme

Abstract

The invention provides a carbon quantum dot nano-enzyme for detecting ascorbic acid as well as a preparation method and application of the carbon quantum dot nano-enzyme, and relates to the technical field of colorimetric detection and analysis. The carbon quantum dot nano-enzyme for detecting ascorbic acid is a carbon quantum dot nano-enzyme prepared from oxalic acid through a hydrothermal reaction. The light-controllable oxide-like enzyme without participation of metal elements is synthesized, the synthesis process of the carbon quantum dots is simple, convenient and rapid, great application potential is shown in the field of ascorbic acid detection, the bottleneck of an existing detection method is expected to be broken through, and a new solution is provided for rapid, efficient and environment-friendly ascorbic acid detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of ascorbic acid detection, and particularly relates to a carbon quantum dot nanometer enzyme for detecting ascorbic acid, a preparation method and application thereof. BACKGROUND

[0002] There are many methods for detecting ascorbic acid (AA), mainly including liquid chromatography-fluorescence method, electrochemical method, chemiluminescence method and the like. These traditional detection methods perform well in sensitivity and accuracy, however, they have some significant limitations, for example, a large amount of chemical reagents are consumed in the experimental process, and they are highly dependent on large-scale precision instruments and professional technical personnel, which makes them difficult to adapt to the demand scenarios of rapid detection. In contrast, colorimetric detection analysis methods based on enzyme sensors are emerging, which have the outstanding advantages of simplicity, speed, low cost and visualization.

[0003] However, colorimetric detection analysis methods are not perfect, and the synthesis conditions of natural enzymes and metal nanometer enzymes are extremely harsh, and there are great environmental toxicity problems. At the same time, most enzyme catalytic mechanisms are concentrated in the field of peroxidase, and additional oxidizing agents are needed to complete the catalytic process. In addition, the enzyme catalytic process is sensitive to oxygen, and the enzyme activity is easily affected by environmental factors and fluctuates, and even excessive coloration phenomenon easily occurs, thereby reducing the detection accuracy.

[0004] Under such a background, photo-controllable oxidase-like enzymes represented by carbon quantum dots have developed rapidly in recent years due to their unique advantage of not needing to add additional oxidizing agents. Compared with traditional oxidases, photo-controllable oxidase-like enzymes can accurately realize the timed coloration of substrates only by specific wavelength light stimulation, greatly simplifying the detection process. At present, most of the synthesized photo-controllable oxidase-like enzymes still need the participation of metal elements.

[0005] CN116660220A discloses a method for detecting ascorbic acid in urine based on nitrogen and boron co-doped fluorescent carbon dots. The invention synthesizes a nitrogen and boron co-doped fluorescent carbon dot solution by using citric acid, ethylenediamine and boric acid as precursors through a one-step hydrothermal method, purifies it, and then dilutes it for subsequent detection of ascorbic acid in urine. However, additional oxidizing agents are needed to complete the catalytic process in this detection process, that is, additional strong oxidizing agent sodium periodate is needed, and the fluorescent carbon dots themselves do not have oxidizing properties, but rely on the fluorescence quenching effect of IO 4- on the fluorescent carbon dots, and ascorbic acid will inhibit IO 4- from quenching fluorescence, thereby realizing quantitative detection of ascorbic acid in urine.

[0006] Therefore, it is urgent to develop a carbon quantum dot nanometer enzyme for detecting ascorbic acid, so as to accurately realize the timed coloration of substrates and greatly simplify the process of detecting ascorbic acid.

[0007] In view of the above, the present application is proposed. SUMMARY

[0008] The present application aims to provide a carbon quantum dot nanoscale enzyme for detecting ascorbic acid, a preparation method and application thereof, which is a light-controllable oxidase-like enzyme without the participation of metal elements, and the synthesis process of carbon quantum dots is simple and fast, which shows great application potential in the field of AA detection, and is expected to break through the bottleneck of the existing detection method, and provide a new solution for fast, efficient and environmentally friendly AA detection.

[0009] In order to achieve the above-mentioned purpose of the present application, the following technical solutions are adopted:

[0010] In a first aspect, the present application provides a carbon quantum dot nanoscale enzyme for detecting ascorbic acid, which is a carbon quantum dot nanoscale enzyme prepared from oxalic acid through a hydrothermal reaction.

[0011] In a second aspect, the present application provides a preparation method of the carbon quantum dot nanoscale enzyme for detecting ascorbic acid according to the first aspect, which comprises:

[0012] After dissolving oxalic acid in water, the hydrothermal reaction is carried out to obtain the carbon quantum dot nanoscale enzyme for detecting ascorbic acid.

[0013] Further, the mass ratio of oxalic acid to water is (1-5):(5-15).

[0014] Further, the temperature of the hydrothermal reaction is 180-220℃.

[0015] Further, the time of the hydrothermal reaction is 8-16h.

[0016] Further, the hydrothermal reaction is carried out in a high-pressure kettle with a polytetrafluoroethylene lining.

[0017] Further, the hydrothermal reaction is carried out in an electric heating air drying oven.

[0018] Further, the hydrothermal reaction further comprises the following post-processing steps:

[0019] The reaction solution obtained by the hydrothermal reaction is cooled, and then washed, dialyzed and concentrated in sequence to obtain the carbon quantum dot nanoscale enzyme for detecting ascorbic acid.

[0020] Further, the cooling comprises cooling the reaction solution to below 35℃.

[0021] Further, the washing comprises washing the cooled reaction solution with ethyl acetate, and collecting the aqueous phase.

[0022] Further, the dialysis comprises: dialysis of the washed water phase by using a dialysis bag with a molecular weight cut-off of 500-1000D.

[0023] Further, the dialysis time is 12-48h.

[0024] In a third aspect, the present application provides a carbon quantum dot nanoscale enzyme for detecting ascorbic acid as a light-controllable pseudo-oxidase.

[0025] In a fourth aspect, the present application provides a carbon quantum dot nanoscale enzyme for detecting ascorbic acid as a light-controllable pseudo-oxidase for detecting ascorbic acid.

[0026] In a fifth aspect, the present application provides a method for detecting ascorbic acid, which comprises:

[0027] Mixing the carbon quantum dot nanoscale enzyme, TMB solution and buffer solution, irradiating under a 365nm ultraviolet lamp, and then carrying out an oxidation reaction, and then mixing with a sample containing ascorbic acid to carry out a reduction reaction, so as to qualitatively and / or quantitatively detect ascorbic acid in the sample;

[0028] The carbon quantum dot nanoscale enzyme comprises the carbon quantum dot nanoscale enzyme for detecting ascorbic acid according to the first aspect, or the carbon quantum dot nanoscale enzyme prepared by the preparation method according to the second aspect.

[0029] Further, the volume ratio of the carbon quantum dot nanoscale enzyme and the TMB solution is (0.8-1.2):(0.8-1.2).

[0030] Further, the concentration of the TMB solution is 0.8-1.0mmol / L.

[0031] Further, the pH of the buffer solution is controlled in the range of 3.5-4.5, and preferably 4.0.

[0032] Further, the buffer solution comprises an acetic acid-sodium acetate buffer.

[0033] Further, the temperature of the oxidation reaction is 5-45℃, and preferably 25℃.

[0034] Further, the time of the oxidation reaction is 1-40min, and preferably 6min.

[0035] Further, the concentration of ascorbic acid in the sample containing ascorbic acid is 0.047μmol / L or more, and preferably 0.14-27μmol / L.

[0036] Further, the temperature of the reduction reaction is 5-45℃.

[0037] Further, the time of the reduction reaction is 30s-5min.

[0038] Further, the qualitative detection of ascorbic acid in the test substance includes:

[0039] The carbon quantum dot nanoscale enzyme and TMB are deep blue after oxidation reaction under irradiation of 365nm ultraviolet lamp; and the solution is light blue after reduction reaction with ascorbic acid in the test substance.

[0040] Further, the quantitative detection of ascorbic acid in the test substance includes:

[0041] The carbon quantum dot nanoscale enzyme, TMB solution and buffer solution are mixed to obtain an oxidation reaction liquid after oxidation reaction under irradiation of 365nm ultraviolet lamp, and the absorbance A0 of the oxidation reaction liquid is measured.

[0042] Different concentrations of ascorbic acid solution are added to the oxidation reaction liquid respectively to obtain a series of reduction reaction liquids after reduction reaction, and the absorbance A of each reduction reaction liquid is measured. n

[0043] The linear relationship between the absorbance change value A0-A n and the concentration of ascorbic acid is constructed, and the unknown concentration of ascorbic acid is detected by using the linear relationship.

[0044] Compared with the prior art, the present application has the following beneficial effects:

[0045] (1) The carbon quantum dot nanoscale enzyme (PO-CD) of the present application is a carbon quantum dot nanoscale enzyme prepared from oxalic acid by hydrothermal reaction, has a light-controllable oxidase-like activity, and exhibits a ladder-like color change behavior, which is consistent with the characteristics that the enzyme-like activity of PO-CD is excited by light and disappears in the dark, thereby fully supporting the light excitation characteristics of the oxidase-like enzyme.

[0046] (2) The carbon quantum dot nanoscale enzyme of the present application is a light-controllable oxidase-like enzyme without the participation of metal elements, has a simple and convenient detection process, high sensitivity, low detection limit, and can realize rapid and sensitive detection of the concentration of ascorbic acid.

[0047] (3) The carbon quantum dot nanoscale enzyme of the present application has strong specificity for probe detection of ascorbic acid, is not significantly interfered by other metal ions and / or amino acids, and has accurate and reliable detection results, and the colorimetric analysis of ascorbic acid based on PO-CD has good selectivity.

[0048] ​(4) The concentration of AA in the detection method of the present application is in the range of 0.14-27 μmol / L, the fluorescence intensity change value has a good linear relationship with the ascorbic acid activity unit concentration, the recovery rate is 110.0-119.0%, and the relative standard deviation is 0.20-3.70%. BRIEF DESCRIPTION OF DRAWINGS

[0049] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0050] Figure 1 The flowchart of the ascorbic acid detection method of the present application.

[0051] Figure 2 The Fourier infrared spectrum of the carbon quantum dot nanoscale enzyme prepared in Example 1.

[0052] Figure 3 The ultraviolet-visible absorption spectrum and excitation and emission spectrum of the carbon quantum dot nanoscale enzyme prepared in Example 1.

[0053] Figure 4 The nuclear magnetic resonance spectrum of oxalic acid and the carbon quantum dot nanoscale enzyme prepared in Example 1.

[0054] Figure 5 The actual photos of PO-CD (light), TMB (light), PO-CD / TMB (dark) and PO-CD / TMB (light irradiation) provided in Test Example 2.

[0055] Figure 6 The activity diagram of the carbon quantum dot nanoscale enzyme prepared in Example 1 provided in Test Example 2.

[0056] Figure 7 The absorbance diagram of the carbon quantum dot nanoscale enzyme prepared in Example 1 under different temperatures provided in Test Example 3.

[0057] Figure 8 The absorbance diagram of the carbon quantum dot nanoscale enzyme prepared in Example 1 under different irradiation times provided in Test Example 4.

[0058] Figure 9 The standard curve diagram provided in Application Example 1.

[0059] Figure 10 The anti-interference test result diagram provided in Test Example 6. DETAILED DESCRIPTION

[0060] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. In this application, the use of "or" means "and / or" unless specifically stated otherwise, e.g., "comprising A or B" means "comprising A or B or both". Also, the use of "comprising" is meant to be non-limiting as the compositions or methods can include more than what is specifically named.

[0061] It should be noted that specific details are set forth in the following description in order to provide a thorough understanding of the application. However, the application can be practiced without many of the details described in this description, many of which are well known in the art. Therefore, the particular implementation described herein is not intended to limit the scope of the application.

[0062] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0063] In order to achieve the above-mentioned purposes of the present application, the following technical solutions are adopted:

[0064] In a first aspect, the present application provides a carbon quantum dot nanoscale enzyme for detecting ascorbic acid, which is a carbon quantum dot nanoscale enzyme prepared by hydrothermal reaction of oxalic acid.

[0065] In the present application, the carbon quantum dot nanoscale enzyme (PO-CD) is a carbon quantum dot nanoscale enzyme prepared by hydrothermal reaction of oxalic acid. The surface of the PO-CD is rich in carboxyl groups (-COOH) and carbonyl groups (C=O). Under the irradiation of ultraviolet light (such as 365 nm), electron transition can be excited, reactive oxygen species (ROS) are generated, and oxidase activity is simulated. The enzyme activity is terminated immediately after the light source is turned off, realizing the controllable start and stop of the detection process. By adjusting the light intensity, the enzyme-like activity can be linearly controlled, which is suitable for the detection requirements of different concentrations of AA. And the PO-CD catalyzes the dissolved oxygen to generate ·OH and O2·- under light, and oxidizes TMB (3,3',5,5'-tetramethylbenzidine) to generate blue oxTMB. AA reduces oxTMB to make the system fade, realizing the purpose of detecting AA, and the catalytic reaction system has high specificity in response to the reducing property of AA.

[0066] In a second aspect, the present application provides a preparation method of the carbon quantum dot nanoscale enzyme for detecting ascorbic acid according to the first aspect, which comprises:

[0067] The carbon quantum dot nanometer enzyme for detecting ascorbic acid is obtained by hydrothermal reaction after oxalic acid is dissolved in water.

[0068] As an optional embodiment, the mass ratio of oxalic acid to water is (1-5):(5-15); wherein, "1-5" may be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.; and wherein, "5-15" may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0069] As a preferred embodiment, the mass ratio of oxalic acid to water is (2-3):10.

[0070] As an optional embodiment, the temperature of the hydrothermal reaction is 180-220℃, for example, 180℃, 182℃, 184℃, 186℃, 188℃, 190℃, 192℃, 194℃, 196℃, 198℃, 200℃, 202℃, 204℃, 206℃, 208℃, 210℃, 212℃, 214℃, 216℃, 218℃, 220℃, etc.

[0071] As an optional embodiment, the time of the hydrothermal reaction is 8-16h, for example, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h, 12h, 12.5h, 13h, 13.5h, 14h, 14.5h, 15h, 15.5h, 16h, etc.

[0072] As an optional embodiment, the hydrothermal reaction is carried out in a high-pressure kettle with a polytetrafluoroethylene lining.

[0073] As an optional embodiment, the hydrothermal reaction is carried out in an electric heating air drying oven.

[0074] As an optional embodiment, the hydrothermal reaction further comprises the following post-processing steps:

[0075] The reaction solution obtained by hydrothermal reaction is cooled, and then washed, dialyzed and concentrated in sequence to obtain the carbon quantum dot nanometer enzyme for detecting ascorbic acid.

[0076] As an optional embodiment, the cooling comprises: cooling the reaction solution to below 35℃, for example, 35℃, 30℃, 25℃, 20℃, 15℃, 10℃, 5℃, etc.

[0077] As an optional embodiment, the washing comprises: washing the cooled reaction solution with ethyl acetate, and collecting the aqueous phase.

[0078] As an optional implementation, the dialysis includes: dialysis of the washed water phase in a dialysis bag with a cut-off molecular weight of 500-1000 D (for example, it can be 500 D, 600 D, 700 D, 800 D, 900 D, 1000 D, etc.).

[0079] As an optional implementation, the dialysis time is 12-48 h (for example, it can be 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, 48 h, etc.).

[0080] As an optional implementation, the carbon quantum dot nanoscale enzyme for detecting ascorbic acid is prepared by the following steps:

[0081] Oxalic acid is dissolved in deionized water to obtain an oxalic acid solution; the oxalic acid solution is transferred to a polytetrafluoroethylene liner, sealed in an autoclave, and then placed in an electric heating air drying oven for hydrothermal reaction; after the reaction is completed, the reaction liquid is cooled to room temperature, the product is washed with ethyl acetate, the water phase is retained, the purified liquid is collected after dialysis, and the carbon quantum dot nanoscale enzyme for detecting ascorbic acid (PO-CD stock solution) is obtained after concentration.

[0082] It should be noted that during the hydrothermal reaction, the oxalic acid first forms a black intermediate polymer, and it is speculated that the temperature continuously rises during the reaction, and a simple polymer is initially formed by polymerization; as the reaction proceeds, the polymer is dissolved and cross-linked to form PO-CD. The obtained PO-CD solution is colorless and transparent under natural light, and emits bright blue fluorescence under 365 nm ultraviolet irradiation.

[0083] In a third aspect, the present application provides a carbon quantum dot nanoscale enzyme for detecting ascorbic acid as described in the first aspect for use as a light-controllable pseudo-oxidase.

[0084] In a fourth aspect, the present application provides a carbon quantum dot nanoscale enzyme for detecting ascorbic acid as described in the first aspect for use as a light-controllable pseudo-oxidase for detecting ascorbic acid.

[0085] In a fifth aspect, the present application provides a method for detecting ascorbic acid, as shown in Figure 1 The method for detecting ascorbic acid includes:

[0086] The carbon quantum dot nanoscale enzyme, TMB solution, and buffer solution are mixed, irradiated by a 365 nm ultraviolet lamp, and then subjected to an oxidation reaction, and then mixed with a sample containing ascorbic acid to perform a reduction reaction, so as to qualitatively and / or quantitatively detect ascorbic acid in the sample;

[0087] The carbon quantum dot nanoscale enzyme includes the carbon quantum dot nanoscale enzyme for detecting ascorbic acid according to the first aspect, or the carbon quantum dot nanoscale enzyme prepared by the preparation method according to the second aspect.

[0088] As an optional embodiment, the volume ratio of the carbon quantum dot nanoscale enzyme and the TMB solution is (0.8-1.2):(0.8-1.2), which can be 0.8:0.8, 0.9:0.8, 1.0:0.8, 1.1:0.8, 1.2:0.8, 0.8:0.9, 1.0:0.9, 1.1:0.9, 1.2:0.9, 0.8:1.0, 0.9:1.0, 1.1:1.0, 1.2:1.0, 0.8:1.1, 0.9:1.1, 1.0:1.1, 1.2:1.1, 0.8:1.2, 0.9:1.2, 1.0:1.2, 1.1:1.2, and the like.

[0089] As an optional embodiment, the concentration of the TMB solution is 0.8-1.0 mmol / L, which can be 0.8 mmol / L, 0.85 mmol / L, 0.9 mmol / L, 0.95 mmol / L, 1.0 mmol / L, and the like.

[0090] As an optional embodiment, the pH of the buffer solution is controlled in the range of 3.5-4.5, which can be 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, and the like.

[0091] As a preferred embodiment, the pH of the buffer solution is 4.0.

[0092] It should be noted that the efficiency of PO-CD catalytic oxidation of TMB is easily affected by pH, and the efficiency is the highest when the pH value of the mixed solution is 4.0.

[0093] As an optional embodiment, the buffer solution includes an acetic acid-sodium acetate buffer.

[0094] As a preferred embodiment, the oxidation reaction is carried out under light irradiation, and the wavelength of the light irradiation is 365 nm.

[0095] As an optional embodiment, the temperature of the oxidation reaction is 5-45°C, which can be 5°C, 7°C, 9°C, 10°C, 11°C, 13°C, 15°C, 17°C, 19°C, 20°C, 21°C, 23°C, 25°C, 27°C, 29°C, 30°C, 31°C, 33°C, 35°C, 37°C, 39°C, 40°C, 41°C, 43°C, 45°C, and the like.

[0096] As a preferred embodiment, the temperature of the oxidation reaction is 25°C.

[0097] It should be noted that with the increase of the reaction temperature, the absorbance of the PO-CD catalytic reaction system at 652 nm gradually increases, reaches the peak at 25℃, and then decreases with the further increase of the temperature. Therefore, the optimal reaction temperature is 25℃.

[0098] As an optional embodiment, the time of the oxidation reaction is 1-40 min, for example, 1 min, 3 min, 5 min, 7 min, 9 min, 11 min, 13 min, 15 min, 17 min, 19 min, 21 min, 23 min, 25 min, 27 min, 29 min, 31 min, 33 min, 35 min, 37 min, 39 min, 40 min, etc.

[0099] As a preferred embodiment, the time of the oxidation reaction is 6 min.

[0100] It should be noted that the change rate of the absorbance of the PO-CD catalytic reaction system is different at different light irradiation times, and the change rate of the absorbance tends to be stable after 6 min of irradiation. Therefore, the optimal irradiation time is 6 min.

[0101] As an optional embodiment, the concentration of ascorbic acid in the ascorbic acid-containing sample to be tested is 0.047 μmol / L or more.

[0102] As an optional embodiment, the concentration of ascorbic acid in the ascorbic acid-containing sample to be tested is 0.14-27 μmol / L, for example, 0.14 μmol / L, 0.34 μmol / L, 0.54 μmol / L, 0.74 μmol / L, 0.94 μmol / L, 1.14 μmol / L, 1.34 μmol / L, 1.54 μmol / L, 1.74 μmol / L, 1.94 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 8 μmol / L, 10 μmol / L, 12 μmol / L, 14 μmol / L, 16 μmol / L, 18 μmol / L, 20 μmol / L, 22 μmol / L, 24 μmol / L, 26 μmol / L, 27 μmol / L, etc.

[0103] As an optional embodiment, the temperature of the reduction reaction is 5-45℃, for example, 5℃, 7℃, 9℃, 10℃, 11℃, 13℃, 15℃, 17℃, 19℃, 20℃, 21℃, 23℃, 25℃, 27℃, 29℃, 30℃, 31℃, 33℃, 35℃, 37℃, 39℃, 40℃, 41℃, 43℃, 45℃, etc.

[0104] As an optional embodiment, the time of the reduction reaction is 30s-5min, for example, it can be 30s, 35s, 40s, 45s, 50s, 55s, 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, etc.

[0105] As an optional embodiment, the qualitative detection of ascorbic acid in the test substance includes:

[0106] The carbon quantum dot nanometer enzyme and TMB are subjected to oxidation reaction under irradiation of a 365nm ultraviolet lamp, and the solution is dark blue; then the ascorbic acid in the test substance is subjected to reduction reaction, and the solution is light blue.

[0107] As an optional embodiment, the quantitative detection of ascorbic acid in the test substance includes:

[0108] The carbon quantum dot nanometer enzyme, TMB solution and buffer solution are mixed, and after oxidation reaction under irradiation of a 365nm ultraviolet lamp, an oxidation reaction liquid is obtained, and the absorbance A0 thereof is measured;

[0109] Different concentrations of ascorbic acid solution are added to the oxidation reaction liquid respectively, and a series of reduction reaction liquids are obtained, and the absorbance A of each reduction reaction liquid is measured respectively. n

[0110] The linear relationship between the absorbance change value A0-A n and the concentration of ascorbic acid is constructed, and the unknown concentration of ascorbic acid is detected by using the linear relationship.

[0111] The application will be further described by examples. Unless otherwise specified, the materials in the examples are prepared according to the existing methods, or directly purchased from the market.

[0112] Example 1

[0113] This example provides a carbon quantum dot nanometer enzyme PO-CD for detecting ascorbic acid, which is prepared by the following steps:

[0114] 2.52g of oxalic acid is dissolved in 10mL of deionized water, and ultrasonic is applied for 5min to make it fully dissolved. The solution is transferred to a 15mL polytetrafluoroethylene liner, and after being packaged into an autoclave, it is placed in an electric heating air drying oven, and reacted at 200℃ for 12h. After the reaction is completed and cooled to room temperature, the product is washed with ethyl acetate, the water phase is reserved, and after dialysis for 24h with a dialysis bag with a molecular weight cutoff of 500D, the purified liquid is collected, concentrated at 30℃ for 10min, and then a PO-CD stock solution (the concentration of PO-CD in the stock solution is 0.252g / mL) is obtained.

[0115] Test Example 1 ​

[0116] Characterization of carbon quantum dot nanozyme PO-CD

[0117] Test sample: carbon quantum dot nanozyme PO-CD provided in Example 1.

[0118] Test methods: Fourier transform infrared, UV-visible absorption, excitation and emission, and nuclear magnetic hydrogen spectroscopy.

[0119] Test results:

[0120] Figure 2 This is the Fourier infrared spectrum of the carbon quantum dot nanozyme prepared in Example 1, as shown in Figure 2 As shown in Example 1, the carbon quantum dot nanozyme prepared was prepared by hydrothermal reaction of oxalic acid, wherein the peak position of v(OH) was 3271 cm -1 The peak position of v(C=O) is 1717cm -1 The peak position of v(C=C,Ar) is 1587 / 1527cm -1 The peak position of δ(Ar) is 880 cm -1 .

[0121] Figure 3 The UV-visible absorption spectrum, excitation spectrum and emission spectrum of the carbon quantum dot nanozyme prepared in Example 1 are as follows: Figure 3 As shown in FIG, the maximum fluorescence excitation wavelength of PO-CD is 269 nm, and the maximum emission wavelength is 336 nm. In addition, there is a characteristic absorption peak at 260 nm in the UV-visible absorption spectrum of PO-CD.

[0122] Figure 4 The nuclear magnetic hydrogen spectrum of oxalic acid and the carbon quantum dot nanozyme prepared in Example 1 is as follows: Figure 4 As shown in the PO-CD spectrum, the characteristic absorption peak of the oxalic acid monomer (δ4.23 ppm) has essentially disappeared, and a new absorption peak signal has emerged in the aromatic ring region (δ5.25-8.00 ppm), indicating that the oxalic acid monomer participated in the polymerization reaction and formed a conjugated aromatic structure. In addition, new absorption peaks have also been observed in the non-aromatic region (δ0-4.00 ppm), indicating that methyl, methylene, or methine groups have also been generated at the mid- or terminal ends of the PO-CD during its formation.

[0123] Test Example 2

[0124] Oxidase activity test

[0125] Test sample: carbon quantum dot nanozyme PO-CD provided in Example 1.

[0126] Test method:

[0127] (1) 0.40 mL PO-CD, 3.20 mL pH 4.0 acetic acid-sodium acetate buffer, 0.40 mL TMB solution were mixed, and the absorbance value at 652 nm was measured after irradiation with 365 nm ultraviolet light for 6 min at 25°C. Four groups of tests were set up to investigate the peroxidase-like activity of PO-CD, including PO-CD (light), TMB (light), PO-CD TMB (dark), and PO-CD TMB (light irradiation).

[0128] (2) Further, an "on-off" type light irradiation cycle test was used, specifically, light irradiation for 2 min, then dark for 2 min, and thus cycled 6 times, to investigate the light-controllable peroxidase-like activity of PO-CD.

[0129] Test results:

[0130] As shown in Figure 5 , the TMB + PO-CD mixed solution turned a deep blue color after being irradiated with 365 nm for 20 min, at which time a strong absorption peak (652 nm) was shown in the absorption spectrum; the same mixed solution but treated in the dark for 20 min only showed a light blue color, indicating that 365 nm light played a key role therein.

[0131] As shown in Figure 6 , PO-CD exhibited a stepwise color development behavior, which was consistent with its enzyme-like activity being excited by light and being eliminated in the dark, fully supporting the light-activated characteristics of the peroxidase-like activity.

[0132] Test Example 3

[0133] Absorbance of carbon quantum dots PO-CD at different temperatures

[0134] Test sample: carbon quantum dots nanoscale enzyme PO-CD provided in Example 1.

[0135] Test method: 0.40 mL PO-CD, 3.20 mL pH 4.0 acetic acid-sodium acetate buffer, 0.40 mL TMB solution were mixed, and the absorbance value at 652 nm was measured after irradiation with 365 nm ultraviolet light for 6 min at 5-45°C, respectively;

[0136] The test results are shown in Table 1 and Figure 7

[0137] Table 1

[0138] Reaction temperature (°C) Absorbance value (a.u.) 5 0.18 10 0.20 25 0.23 45 0.15

[0139] From Table 1 and Figure 7 ​As shown in the figure, as the reaction temperature increases, the absorbance of the PO-CD catalytic reaction system at 652 nm gradually increases and reaches a peak at 25°C. As the temperature continues to rise, the absorbance decreases. Therefore, the optimal reaction temperature is 25°C.

[0140] Test Example 4

[0141] Absorbance of carbon quantum dots PO-CD under different irradiation times

[0142] Test sample: carbon quantum dot nanozyme PO-CD provided in Example 1.

[0143] Test method: Mix 0.40 mL of PO-CD, 3.20 mL of pH 4.0 acetic acid-sodium acetate buffer, and 0.40 mL of TMB solution. Irradiate with 365 nm UV light at 25°C for 1 to 40 minutes, and then measure the rate of change of absorbance at 652 nm.

[0144] The test results are shown in Table 2 and Table 3. Figure 8 As shown:

[0145] Table 2

[0146]

[0147] From Table 2 and Figure 8 As shown in the figure, the absorbance change rate of the PO-CD catalytic reaction system is different at different light irradiation times. After irradiation for 6 minutes, the absorbance change rate tends to be stable, so the optimal irradiation time is selected as 6 minutes.

[0148] Application Example 1

[0149] This application example provides a method for detecting ascorbic acid, which includes:

[0150] (a) 0.40 mL of the carbon quantum dot nanozyme prepared in Example 1, 0.40 mL of TMB solution (concentration of 1.0 μmol / L), and 3.20 mL of pH 4.0 acetic acid-sodium acetate buffer were mixed and irradiated with 365 nm ultraviolet light for 6 min at 25° C. to obtain an oxidation reaction solution, and its absorbance A0 was measured;

[0151] (b) adding ascorbic acid solutions of different concentrations to the oxidation reaction solution and performing reduction reaction at 25°C for 1 min to obtain a series of reduction reaction solutions, and measuring their absorbance A respectively. n ;

[0152] (c) Construct absorbance change value A0-A n The linear relationship between the concentration of ascorbic acid [AA] and the standard curve was obtained.

[0153] like Figure 9 As shown in the figure, when the concentration of AA is between 0.14 and 27 μmol / L, A0-A n It has a positive linear correlation with [AA], where A0 is the absorbance of the system at 652nm when no AA is added, and A n It is the absorbance at 652nm after adding a series of AA concentrations, and the linear correlation coefficient R 2 is 0.9988.

[0154] (d) Adding an unknown AA content to the analyte (Alande ascorbic acid lozenges) in the oxidation reaction solution to perform a reduction reaction at 25° C. for 1 min to obtain a reduction reaction solution, measuring its absorbance, and obtaining an AA concentration of 11.1 μmol / L in the Alande ascorbic acid lozenges based on the standard curve obtained in step (c).

[0155] Test Example 5

[0156] Detection limit and spike recovery test

[0157] (1) Detection limit test method: According to the detection method of Application Example 1, pure PO-CD solution was used as a blank, and the standard deviation was obtained by continuous measurement 11 times, and the detection limit LOD was further calculated;

[0158] Test results: Using pure PO-CD solution as blank, 11 consecutive measurements yielded a standard deviation of 0.0004. The detection limit (LOD) of this method was calculated to be 0.047 μmol / L.

[0159] (2) Spiked recovery test method: According to the detection method of Application Example 1, AA standard solutions of different concentrations (5.00, 10.00, 15.00 μmol / L) were added to the AA lozenges to be tested, and the spiked recovery and relative standard deviation were calculated.

[0160] The test results are shown in Table 3 below:

[0161] Table 3

[0162]

[0163] As shown in Table 3, the AA content of the AA lozenges was determined to be 11.1 μmol / L by AA colorimetric analysis, with a relative error of +11.1%. When AA standard solutions of varying concentrations (5.00, 10.00, and 15.00 μmol / L) were added to the AA lozenges, the spiked recoveries ranged from 110.0% to 119.0%, with relative standard deviations ranging from 0.20% to 3.7%.

[0164] Test Example 6

[0165] Anti-interference test:

[0166] Test item: Considering the influence of common ions and amino acids in health food on ascorbic acid colorimetric determination. Study metal ions Ca 2+ , K + , Mg 2+ , Cu 2+ , Zn 2+ , lysine, phenylalanine, tryptophan, threonine, tyrosine, etc. Amino acids.

[0167] Test method: In the reaction system, add 10 μmol / L of anti-interference substances or 2 μmol / L of ascorbic acid, and place the system at 25°C. Irradiate it with 365nm light for 6min. After the reaction is fully carried out, measure the absorbance of the system at 652nm.

[0168] Test results:

[0169] As Figure 10 shown, 1 represents blank, 2 represents Ca 2+ , 3 represents K + , 4 represents Mg 2+ , 5 represents Cu 2+ , 6 represents Zn 2+ , 7 represents lysine, 8 represents phenylalanine, 9 represents tryptophan, 10 represents threonine, 11 represents tyrosine, and 12 represents ascorbic acid. As shown in Figure 10 , the study metal ions and amino acids have no obvious interference, indicating that the ascorbic acid colorimetric analysis based on PO-CD has good selectivity.

[0170] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A carbon quantum dot nanozyme for detecting ascorbic acid, characterized in that: The carbon quantum dot nanozyme for detecting ascorbic acid is a carbon quantum dot nanozyme prepared by hydrothermal reaction of oxalic acid.

2. A method for preparing a carbon quantum dot nanozyme for detecting ascorbic acid according to claim 1, characterized in that: The preparation method comprises: After dissolving oxalic acid in water, the carbon quantum dot nanozyme for detecting ascorbic acid is obtained through a hydrothermal reaction.

3. The preparation method according to claim 2, characterized in that The mass ratio of the oxalic acid to water is (1-5):(5-15).

4. The preparation method according to claim 2, characterized in that The temperature of the hydrothermal reaction is 180 to 220° C.; the time of the hydrothermal reaction is 8 to 16 hours; Preferably, the hydrothermal reaction is carried out in an autoclave lined with polytetrafluoroethylene.

5. The preparation method according to claim 2, characterized in that The hydrothermal reaction further includes the following post-processing steps: The reaction solution obtained by the hydrothermal reaction is cooled, washed, dialyzed and concentrated in sequence to obtain the carbon quantum dot nanozyme for detecting ascorbic acid.

6. The preparation method according to claim 5, characterized in that The cooling comprises: cooling the reaction solution to below 35° C.; Preferably, the washing comprises: washing the cooled reaction solution with ethyl acetate and collecting the aqueous phase; Preferably, the dialysis comprises: dialyzing the washed aqueous phase using a dialysis bag with a cut-off molecular weight of 500 to 1000D; Preferably, the dialysis time is 12 to 48 hours.

7. Use of the carbon quantum dot nanozyme for detecting ascorbic acid according to claim 1 as a light-controllable oxidase-like enzyme.

8. Use of the carbon quantum dot nanozyme for detecting ascorbic acid according to claim 1 as a light-controllable oxidase-like enzyme for detecting ascorbic acid.

9. A method for detecting ascorbic acid, characterized in that: The detection method of ascorbic acid comprises: The carbon quantum dot nanozyme, TMB solution and buffer solution are mixed and irradiated with a 365nm ultraviolet lamp to produce an oxidation reaction, and then mixed with an analyte containing ascorbic acid to undergo a reduction reaction to qualitatively and / or quantitatively detect ascorbic acid in the analyte; Wherein, the carbon quantum dot nanozyme includes the carbon quantum dot nanozyme for detecting ascorbic acid according to claim 1, or the carbon quantum dot nanozyme prepared by the preparation method according to any one of claims 2 to 6.

10. The method for detecting ascorbic acid according to claim 9, wherein The volume ratio of the carbon quantum dot nanozyme and TMB solution is (0.8-1.2):(0.8-1.2); Preferably, the concentration of the TMB solution is 0.8 to 1.0 mmol / L; Preferably, the pH of the buffer solution is controlled within the range of 3.5 to 4.5, preferably 4.0; Preferably, the buffer solution comprises acetic acid-sodium acetate buffer; Preferably, the temperature of the oxidation reaction is 5 to 45°C, preferably 25°C; Preferably, the oxidation reaction time is 1 to 40 minutes, preferably 6 minutes; Preferably, the concentration of ascorbic acid in the ascorbic acid-containing analyte is 0.047 μmol / L or more, preferably 0.14 to 27 μmol / L; Preferably, the temperature of the reduction reaction is 5 to 45°C; Preferably, the reduction reaction time is 30s to 5min; Preferably, the qualitative detection of ascorbic acid in the analyte comprises: After irradiation with 365nm ultraviolet light, the carbon quantum dot nanozyme and TMB undergo an oxidation reaction, and the solution turns dark blue. After a reduction reaction with ascorbic acid in the analyte, the solution turns light blue. Preferably, the quantitative detection of ascorbic acid in the analyte comprises: The carbon quantum dot nanozyme, TMB solution and buffer solution were mixed and irradiated with a 365nm ultraviolet lamp to undergo an oxidation reaction to obtain an oxidation reaction solution, and its absorbance A0 was measured; Ascorbic acid solutions of different concentrations were added to the oxidation reaction solution to undergo reduction reaction to obtain a series of reduction reaction solutions, and their absorbances A were measured. n ; Construct absorbance change value A0-A n The linear relationship between the concentration of ascorbic acid and the concentration of ascorbic acid was used to detect unknown concentrations of ascorbic acid.

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