CuCDs, preparation method thereof and application of CuCDs in detection of NADH (Nicotinamide Adenine Dinucleotide)

Abstract

The invention provides CuCDs, a preparation method of the CuCDs and application of the CuCDs in detection of NADH (Nicotinamide Adenine Dinucleotide), and belongs to the technical field of NADH detection. The CuCDs provided by the invention are obtained by carrying out a reaction on citric acid monohydrate, urea and CuCl2. 2H2O. The CuCDs provided by the invention have double-enzyme activity, not only catalyzes the NADH to generate NAD +, but also oxidizes the TMB to generate oxTMB in the presence of H2O2, and the NADH can inhibit the CuCDs from peroxidizing the TMB, the invention provides a colorimetric detection method of the NADH with the detection limit as low as 3.6 mu M by virtue of the double-enzyme activity of the CuCDs, and the method has the advantages of simplicity in operation, low cost, visualization and the like. According to the electrode CuCDs / CS / GCE and the method for electrochemically detecting the NADH, the quantitative detection of the NADH can be accurately and sensitively carried out, and the detection limit is as low as 0.51 mu M.

Description

Technical Field

[0001] The invention belongs to the technical field of NADH detection, and in particular relates to CuCDs and a preparation method thereof and application in detecting NADH. Background Art

[0002] β-Nicotinamide adenine dinucleotide (NADH) is naturally formed in cells through food supplementation and is the most important coenzyme found in living organisms. This may be related to the formation of ATP (adenosine triphosphate) molecules, which are responsible for storing energy in their chemical structure. NADH deficiency can lead to insufficient energy at the cellular level due to the lack of ATP production. On the other hand, an increase in intracellular NADH may also lead to an excess supply of energy, resulting in the adverse effects of overstimulation. Cytoplasmic NADH is produced by lactate dehydrogenase (LDH), which catalyzes the reversible conversion between lactate and pyruvate. Intracellular NADH has different sensitivities to different stimuli, and the total NADH in the cytoplasm and nucleus is susceptible to changes in the levels of intracellular nutrients, such as glucose, lactate, pyruvate, etc. NADH is an important coenzyme in the human body and is involved in hundreds of enzymatic reactions involving more than 300 dehydrogenases. Accurate, convenient and rapid quantification of NADH plays an important role. NADH is closely related to the metabolism of organisms, and the life state of organisms can be reflected to a certain extent by the NADH content. Detecting the content of NADH in organisms is of great significance for evaluating the growth status of organisms. The existing methods for detecting NADH in the art include optical, liquid chromatography, electrophoresis and chemiluminescence techniques, but these techniques are expensive, require a lot of preparation, and have low response time and sensitivity. Summary of the invention

[0003] In view of this, one of the purposes of the present invention is to provide a CuCDs with dual enzyme (TMB (tetramethylbenzidine) and NADH) activity, and a method for colorimetric detection of NADH is designed based on the principle that NADH can inhibit CuCDs from peroxidizing TMB.

[0004] The second object of the present invention is to provide an electrode CuCDs / CS / GCE, and a method for electrochemically detecting NADH is provided by using the electrode CuCDs / CS / GCE.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides a method for preparing CuCDs capable of catalytically oxidizing TMB and NADH, comprising the following steps: dissolving monohydrated citric acid, urea and CuCl2·2H2O in water, reacting at 180-220°C for 8-12 hours, centrifuging and discarding precipitates, filtering and dialysis the filtrate, collecting products, and freeze-drying to obtain CuCDs.

[0006] Preferably, the mass volume ratio of citric acid monohydrate, urea and water is (0.5~2) g: (0.5~2) g: 20 mL; and the final concentration of the CuCl2·2H2O is 1~4 mM.

[0007] The present invention also provides CuCDs capable of catalytically oxidizing TMB and NADH, which is prepared by the above method.

[0008] The present invention also provides the use of the CuCDs in detecting NADH or in preparing a product for detecting NADH.

[0009] The present invention also provides a method for colorimetric detection of NADH, comprising the following steps: mixing a sample to be tested with a reaction solution, reacting at room temperature for 15 minutes, measuring the absorbance at 652 nm to obtain ΔA652 nm, substituting into the formula ΔA652 nm=0.0081[NADH]+0.0326, to obtain the concentration of NADH in the sample to be tested; the ΔA652 nm value is the A652 nm value when the sample to be tested is not added minus the A652 nm value after the sample to be tested is added; the reaction solution is composed of HAc-NaAc (acetic acid-sodium acetate buffer) with a pH of 4-6, 0.05-0.4 mM TMB, 0.025-0.2 mM H2O2 and 5-20 μg / mL of the above-mentioned CuCDs.

[0010] The present invention also provides a method for preparing an electrode CuCDs / CS / GCE, comprising the following steps: mixing a solution containing the CuCDs with CS to obtain a mixed solution; and drop-coating the mixed solution on a surface of GCE to obtain an electrode CuCDs / CS / GCE.

[0011] Preferably, the concentration of the solution containing the above-mentioned CuCDs is 3-10 mg / mL, and the final mass volume fraction of CS in the mixed solution is 0.05%-0.5%.

[0012] The present invention also provides an electrode CuCDs / CS / GCE, which is prepared by the above method.

[0013] The present invention also provides a method for electrochemically detecting NADH, comprising the following steps: placing the above-mentioned electrode CuCDs / CS / GCE in PBS, scanning it, setting the potential to 0.5-0.8 V, adding a sample to be tested after the current is stable, obtaining a current increase ΔI, substituting into the formula ΔI=0.01757[NADH]+0.00174, and obtaining the concentration of NADH in the sample to be tested; the current increase ΔI is I after adding the sample to be tested minus the initial I when no sample to be tested is added.

[0014] Preferably, the sample to be tested includes bacteria and / or cells.

[0015] Beneficial effects of the present invention: The preparation method provided by the present invention can prepare CuCDs with dual enzyme activity, which can not only catalyze NADH to generate NAD in the presence of H2O2, but also + , and can oxidize TMB to generate oxTMB, and NADH can inhibit CuCDs from peroxidizing TMB. The present invention provides a method for colorimetric detection of NADH by means of the dual enzyme activity of CuCDs, which has the advantages of simple operation, low cost, visualization, etc., and has a wider range of practical applications. The detection limit of the colorimetric detection method of NADH provided by the present invention is as low as 3.6 μM.

[0016] The electrode CuCDs / CS / GCE and the method for electrochemically detecting NADH provided by the present invention can accurately and sensitively perform quantitative detection of NADH in a sample to be tested, with a detection limit as low as 0.51 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the study on the NADH peroxidase activity of CuCDs obtained in Example 1, wherein A is the effect of different pH on the peroxidation of NADH in CuCDs, B is the effect of different temperatures on the peroxidation of NADH in CuCDs, C is the effect of different concentrations of NADH on the NADH peroxidase activity of CuCDs, and D is the effect of different concentrations of H2O2 on the NADH peroxidase activity of CuCDs; Figure 2 The results of the study on the TMB peroxidase activity of CuCDs obtained in Example 1, wherein A is the effect of different pH on the catalysis of TMB by CuCDs, B is the effect of different temperatures on the catalysis of TMB by CuCDs, C is the effect of different concentrations of TMB on the catalysis of TMB by CuCDs, and D is the effect of different concentrations of H2O2 on the catalysis of TMB by CuCDs; Figure 3 The effect of NADH on TMB peroxidation by CuCDs; Figure 4 To further study the effect of NADH on the peroxidation of TMB by CuCDs, A is the monitoring result of the peroxidation reaction of TMB by CuCDs with or without NADH, B is the color change and absorbance change results of adding different concentrations of NADH to the reaction system of CuCDs, H2O2 and TMB, and C is the relationship curve between NADH concentration and ΔA652 nm. Figure 5 The effect of different concentrations of CuCDs solution on the electrocatalytic NADH of electrode CuCDs / CS / GCE; Figure 6The effect of different potentials on the electrocatalytic NADH of CuCDs / CS / GCE electrode; Figure 7 The results of Example 12, wherein A is the response current result of the electrode CuCDs / CS / GCE at different NADH concentrations, and B is the relationship curve between NADH concentration and current increment ΔI; Figure 8 The results of the colorimetric method and the electrochemical method for detecting NADH in Escherichia coli of the present invention, wherein A is the result of the electrochemical method for detecting NADH in Escherichia coli of different concentrations, B is the result of the colorimetric method for detecting NADH in Escherichia coli of different concentrations, C is the result of the colorimetric method and the electrochemical method for calculating NADH in Escherichia coli of different weights; D is the result of the electrochemical method for detecting NADH in Escherichia coli of the same weight at different culture times, E is the result of the colorimetric method for detecting NADH in Escherichia coli of the same weight at different culture times, and F is the result of further calculating the NADH content in Escherichia coli of the same weight at different times based on the current and color changes; Fig. 9 The results of the colorimetric and electrochemical methods for detecting NADH in mouse fibroblast L929 of the present invention, wherein A is the result of the electrochemical method for detecting NADH in different numbers of L929 cells, B is the result of the colorimetric method for detecting NADH in different numbers of L929 cells, C is the result of the colorimetric and electrochemical methods for calculating NADH in different numbers of L929 cells; D is the result of the electrochemical method for detecting NADH in the same number of L929 cells at different culture times, E is the result of the colorimetric method for detecting NADH in the same number of L929 cells at different culture times, and F is the result of further calculating the NADH content in the same number of L929 cells at different culture times based on the current and color changes; Fig.10 The electrocatalytic effects of two different electrodes on NADH; Fig.11 The electrocatalytic effects of three different electrodes on NADH. DETAILED DESCRIPTION

[0018] The invention provides a method for preparing CuCDs capable of catalytically oxidizing TMB and NADH, comprising the following steps: dissolving monohydrated citric acid, urea and CuCl2·2H2O in water, reacting at 180-220°C for 8-12 hours, centrifuging and discarding precipitates, filtering and dialysis the filtrate, collecting products, and freeze-drying to obtain CuCDs.

[0019] The present invention introduces Cu into carbon dots (CDs) so that the prepared CuCDs have electrocatalytic properties in solution and on electrodes. In the present invention, the mass volume ratio of citric acid monohydrate, urea and water is preferably (0.5~2) g: (0.5~2) g: 20 mL, more preferably (0.8~1.8) g: (0.7~1.5) g: 20 mL, and the final concentration of the CuCl2·2H2O is preferably 1~4 mM, more preferably 2~3 mM. In the present invention, the temperature of the reaction is preferably 190~210°C, more preferably 200~208°C, and the reaction time is preferably 9~11 h, more preferably 10~10.5 h. After the reaction is completed, centrifugation is performed, and the centrifugation condition is preferably 10000 rpm for 10 min. In the present invention, the filtration is preferably performed using a membrane filter, and the pore size of the membrane is preferably 0.22 μm. In the present invention, the dialysis molecular weight is preferably 500 molecular weight, and the dialysis time is preferably 48 hours.

[0020] The present invention also provides CuCDs capable of catalytically oxidizing TMB and NADH, which is prepared by the above method.

[0021] The present invention also provides the use of the CuCDs in detecting NADH or in preparing a product for detecting NADH.

[0022] The present invention also provides a method for colorimetric detection of NADH, comprising the following steps: mixing a sample to be tested with a reaction solution, reacting at room temperature for 15 minutes, measuring the absorbance at 652 nm to obtain ΔA652 nm, substituting into the formula ΔA652 nm=0.0081[NADH]+0.0326, and obtaining the concentration of NADH in the sample to be tested; the ΔA652 nm value is the A652 nm value when the sample to be tested is not added minus the A652 nm value after the sample to be tested is added; the reaction solution is composed of HAc-NaAc with a pH of 4-6, 0.05-0.4 mM TMB, 0.025-0.2 mM H2O2, and 5-20 μg / mL of the above-mentioned CuCDs.

[0023] The present invention is based on the fact that the prepared CuCDs have dual enzyme activity, and NADH can inhibit CuCDs from peroxidizing TMB. Therefore, the present invention uses the natural enzyme simulation activity of CuCDs to convert the colorless substrate into a colored substrate to design the above-mentioned colorimetric detection method of NADH. In the colorimetric detection method of NADH provided by the present invention, the room temperature is preferably 20°C~25°C, more preferably 21°C~24°C, and further preferably 22°C~23°C. In the reaction solution of the present invention, the pH of HAc-NaAc is preferably 5; the concentration of TMB is preferably 0.1~0.3 mM, more preferably 0.15~0.25 mM; the concentration of H2O2 is preferably 0.05~0.15 mM, more preferably 0.1~0.12 mM; the concentration of CuCDs is preferably 8~15 μg / mL, more preferably 9~12 μg / mL. In the present invention, the sample to be tested preferably includes bacteria and / or cells, the bacteria preferably include Escherichia coli, and the cells preferably include mouse epithelial fibroblasts L929.

[0024] The present invention also provides a method for preparing an electrode CuCDs / CS / GCE, comprising the following steps: mixing a solution containing the above-mentioned CuCDs with chitosan (CS) to obtain a mixed solution; and dropwise coating the mixed solution on the surface of a glassy carbon electrode (GCE) to obtain an electrode CuCDs / CS / GCE.

[0025] The present invention does not specifically limit the specific sources of CS and GCE. In the preparation method of the present invention, the concentration of the CuCDs solution is preferably 3-10 mg / mL, more preferably 5-8 mg / mL; the final mass volume fraction of CS in the mixed solution is preferably 0.05%-0.5%, more preferably 0.1%-0.4%. In the present invention, GCE is preferably polished, cleaned, and then dried under N2 before use. When the mixed solution is drop-coated on the surface of GCE, the amount of the mixed solution drop-coated is preferably 5 μL. After the drop-coating is completed, it is preferably dried naturally at room temperature to obtain the electrode CuCDs / CS / GCE.

[0026] The present invention also provides an electrode CuCDs / CS / GCE, which is prepared by the above method.

[0027] The present invention also provides a method for electrochemically detecting NADH, comprising the following steps: placing the above-mentioned electrode CuCDs / CS / GCE in PBS, scanning it, setting the potential to 0.5-0.8 V, adding a sample to be tested after the current is stable, obtaining a current increase ΔI, substituting into the formula ΔI=0.01757[NADH]+0.00174, and obtaining the concentration of NADH in the sample to be tested; the current increase ΔI is I after adding the sample to be tested minus the initial I when no sample to be tested is added.

[0028] In the electrochemical detection method of NADH of the present invention, the concentration of PBS is preferably 0.1 M, the scanning potential setting is preferably 0.6-0.7 V, and the scanning rate is preferably 10 mV / s. In the electrochemical detection method of NADH of the present invention, the sample to be tested preferably includes bacteria and / or cells, the bacteria preferably include Escherichia coli, and the cells preferably include mouse epithelial fibroblast L929.

[0029] The present invention provides a colorimetric and electrochemical dual-mode NADH detection method based on CuCDs, which can achieve more accurate and effective NADH detection. The dual-mode NADH detection method provided by the present invention can accurately and quantitatively analyze NADH in bacteria and cells, and has great practical application potential.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] Example 1 A CuCDs capable of catalytically oxidizing TMB and NADH, wherein the preparation method of the CuCDs is: First, 1.051 g of citric acid monohydrate and 0.9 g of urea were dissolved in 20 mL of water, and 2 mM CuCl2·2H2O was added. After ultrasonic dissolution, the mixture was transferred to a high-pressure reactor and reacted at 200°C for 10 h. Then, the mixture was centrifuged at 10,000 rpm for 10 min, the precipitate was discarded, and the mixture was filtered using a 0.22 μm filter membrane. Finally, the filtrate was dialyzed for 48 h (the molecular weight of the dialyzed product was 500), the product was collected, and solid powder was obtained after freeze-drying, which was CuCDs.

[0034] Example 2 A CuCDs capable of catalytically oxidizing TMB and NADH, wherein the preparation method of the CuCDs is: First, 0.5 g of citric acid monohydrate and 0.8 g of urea were dissolved in 20 mL of water, and 1 mM CuCl2·2H2O was added. After ultrasonic dissolution, the mixture was transferred to a high-pressure reactor and reacted at 180°C for 9 h. Then, the mixture was centrifuged at 10,000 rpm for 10 min, the precipitate was discarded, and the mixture was filtered using a 0.22 μm filter membrane. Finally, the filtrate was dialyzed for 48 h (the molecular weight of the dialyzed product was 500), the product was collected, and solid powder was obtained after freeze-drying, which was CuCDs.

[0035] Example 3 A CuCDs capable of catalytically oxidizing TMB and NADH, wherein the preparation method of the CuCDs is: First, 2 g of citric acid monohydrate and 2 g of urea were dissolved in 20 mL of water, and 4 mM CuCl2·2H2O was added. After ultrasonic dissolution, the mixture was transferred to a high-pressure reactor and reacted at 210°C for 12 h. Then, the mixture was centrifuged at 10,000 rpm for 10 min, the precipitate was discarded, and the mixture was filtered using a 0.22 μm filter membrane. Finally, the filtrate was dialyzed for 48 h (the molecular weight of the dialyzed product was 500), the product was collected, and solid powder was obtained after freeze-drying, which was CuCDs.

[0036] Example 4 Example 1 Study on the Enzyme Activity of CuCDs Obtained 4.1 NADH peroxidase activity 50 μg / mL CDs or CuCDs prepared in Example 1 were reacted with 0.2 mM NADH and 2.0 mM H2O2 in 1 mL PBS at pH 5 at 50°C for 15 min, and the absorbance at 340 nm was measured. The NADH peroxidase activity of CuCDs was analyzed based on the decrease in absorbance at 340 nm (ΔA340 nm = A340 nm before reaction - A340 nm after reaction).

[0037] 50 μg / mL of the CuCDs prepared in Example 1 was reacted with 0.2 mM NADH and 2.0 mM H2O2 in 1 mL PBS at pH 3, pH 4, pH 5, pH 6, pH 7, pH 8 and 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 15 min, and the absorbance at 340 nm was measured. The effect of different pH and temperature on the peroxidation of NADH by CuCDs was analyzed based on the decrease in absorbance at 340 nm (ΔA340 nm).

[0038] 0.04 mM, 0.08 mM, 0.12 mM, 0.16 mM, and 0.2 mM NADH were reacted with 50 μg / mL CuCDs prepared in Example 1 in 1 mL PBS (pH 5) containing 2.0 mM H2O2 for 15 min, and the absorbance at 340 nm was measured. The reaction rate v was calculated based on ΔA340 nm, and the relationship between 1 / c(NADH) and 1 / v was plotted by double reciprocal to calculate K m (NADH).

[0039] 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, and 1.2 mM H2O2 were reacted with 50 μg / mL CuCDs in 1 mL PBS (pH 5) containing 0.2 mM NADH for 15 min, and the absorbance change at 340 nm was measured. The reaction rate v was calculated based on ΔA340 nm, and the relationship between 1 / c(H2O2) and 1 / v was plotted using a double reciprocal graph. Calculation K m (H2O2).

[0040] Results: In the presence of H2O2, CuCDs oxidized NADH, which means that CuCDs had NADH peroxidase-like activity. Since CDs had no obvious peroxidation effect on NADH, this was related to the presence of Cu in CuCDs.

[0041] The NADH peroxidase-like activity of CuCDs was evaluated at different pH and temperature. Figure 1 As shown in A and B in FIG, it was found that the activity of CuCDs decreased with increasing pH in the pH range of 3 to 8, and increased with increasing temperature in the range of 25-70°C.

[0042] Depend on Figure 1 As can be seen from C and D in Figure 1, the reaction rate v increases with the increase of [NADH] and [H2O2]. As the active sites of CuCDs tend to be saturated, the reaction rate increases slowly. Plot the double reciprocal of the reaction rate v and the substrate concentration [NADH] or [H2O2] ( Figure 1 According to the Michaelis-Menten calculation, CuCDs have a significant influence on NADH and H2O2. K m The values ​​are 0.12 mM and 0.16 mM respectively.

[0043] 4.2 TMB peroxidase activity In HAc-NaAc (pH 5) containing 1 mM TMB and 1 mM H2O2, 10 μg / mL of CDs or CuCDs prepared in Example 1 were added respectively, with a total reaction system of 1 mL. The mixture was thoroughly mixed and reacted at 50°C for 5 min. The color change was observed and photographed in time. The absorbance at 652 nm was measured, and the peroxidation activity of CuCDs on TMB was analyzed based on ΔA652 nm (ΔA652 nm = A652 nm after reaction - A652 nm before reaction).

[0044] 10 μg / mL of CuCDs obtained in Example 1 was added to 1 mL of HAc-NaAc containing 1 mM H2O2 and 1 mM TMB, respectively, at pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8 and 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and the reaction time was 5 min, and the color change was recorded. The absorbance at 652 nm was measured, and the effects of different pH and temperature on CuCDs peroxidation of TMB were analyzed based on ΔA652nm.

[0045] 10 μg / mL of CuCDs obtained in Example 1 was dissolved in 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, and 1.0 mM TMB in HAc-NaAc containing 1.0 mM H2O2 at pH 5, and reacted at 50°C for 5 min. The reaction rate v was calculated based on ΔA652 nm, and the relationship between 1 / c(TMB) and 1 / v was plotted by double reciprocal plotting to calculate K m (TMB).

[0046] 10 μg / mL of CuCDs obtained in Example 1 was dissolved in 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, and 1.0 mM H2O2 in HAc-NaAc at pH 5 containing 1.0 mM TMB, and reacted at 50°C for 5 min. The reaction rate v was calculated based on ΔA652nm, and the relationship between 1 / c(H2O2) and 1 / v was plotted by double reciprocal plotting to calculate K m (H2O2).

[0047] Results: CuCDs has peroxidase-like activity towards TMB. The effects of pH and temperature on the activity of CuCDs were further studied. Figure 2 From A and B in Figure 3, we can see that acidic environment and increased temperature can promote the activity of CuCDs, reaching the highest level at pH 5 and 60℃. Figure 2 Figures C and D show the reaction rate v of different concentrations of TMB and H2O2 on the catalytic color development of TMB by CuCDs. As [TMB] and [H2O2] increase, the reaction rate v increases, and the final reaction rate increases slowly, which is due to the saturation of the activity of CuCDs. The reaction rate v and substrate concentration [TMB] or [H2O2] were plotted by double reciprocal plots (see Figure 2The insets in C and D of Figure 1 show that the CuCDs have a strong affinity for TMB and H2O2. K m The values ​​are 0.40 mM and 0.14 mM, respectively.

[0048] Example 5 Experimental group (CuCDs+TMB+H2O2+NADH): 10 μg / mL of CuCDs obtained in Example 1 was added to 1 mL of HAc-NaAc at pH 5 containing 0.2 mM TMB, 0.1 mM H2O2 and 0.1 mM NADH.

[0049] Control group (CuCDs+TMB+H2O2): 10 μg / mL of CuCDs obtained in Example 1 was added to 1 mL of HAc-NaAc at pH 5 containing 0.2 mM TMB and 0.1 mM H2O2.

[0050] The experimental group and the control group were reacted at room temperature (the room temperature was 20℃~22℃) for 1 h, the color change of the reaction system was recorded, the absorbance at 652 nm was measured, and the effect of NADH on CuCDs oxidation of TMB was analyzed based on ΔA652 nm. Figure 3 As shown, compared with the reaction system without adding 0.1 mM NADH, in the reaction system where NADH and TMB coexist, it is found that the blue color of oxTMB becomes weaker, indicating that the peroxidation process of TMB by CuCDs is inhibited, and this inhibition comes from NADH.

[0051] Example 6 The inhibitory effect of NADH on CuCDs oxidation of TMB was further studied.

[0052] 10 μg / mL of CuCDs prepared in Example 1 was reacted with 0.2 mM TMB, 0.1 mM H2O2 and 0.1 mM NADH in 1 mL of HAc-NaAc at pH 5 at room temperature (the room temperature is 21°C to 23°C) for 75 min. The reaction without adding 0.1 mM NADH was used as the control group. A652 nm was monitored every 30 s. The effect of NADH on CuCDs catalysis of TMB was analyzed based on A652 nm. The results are shown in Figure 4 As shown in A, ΔA652 nm reached its maximum value at 15 min, indicating that the inhibitory effect was most obvious at 15 min.

[0053] 0.2 mM TMB, 0.1 mM H2O2, and 10 μg / mL of CuCDs obtained in Example 1 were added to HAc-NaAc at pH 5, wherein different concentrations of NADH (1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM) were added respectively, with a total reaction system of 1 mL. The mixture was mixed thoroughly, reacted at room temperature (the room temperature is 21°C to 23°C) for 15 min, and the absorbance at 652 nm was measured. The results are as follows: Figure 4 As shown in B, as the NADH concentration increases, the inhibitory effect becomes stronger and stronger, and the color also changes regularly.

[0054] Calculate the absorbance change ΔA652 nm at A652 nm (ΔA652 nm is the difference between A652 nm without NADH and A652 nm with different concentrations of NADH added), and draw a curve of [NADH] and ΔA652 nm. The results are as follows: Figure 4 As shown in C, the fitting results show that: ΔA652 nm = 0.0081 [NADH] + 0.0326, and there is a linear relationship between [NADH] and ΔA652 nm in the range of 1-100 μM. A detection limit of 3.6 μM can be calculated.

[0055] Example 7 A method for colorimetric detection of NADH, the specific steps are as follows: mixing a sample to be tested with a reaction solution, reacting at room temperature (the room temperature is 21°C~23°C) for 15 minutes, measuring the absorbance at 652 nm to obtain ΔA652 nm, substituting into the formula ΔA652 nm=0.0081[NADH]+0.0326, and calculating the concentration of NADH in the sample to be tested.

[0056] The ΔA652 nm value is the A652 nm value when no sample to be tested is added minus the A652 nm value after the sample to be tested is added; The reaction solution consisted of HAc-NaAc at pH 5, 0.2 mM TMB, 0.1 mM H2O2 and 10 μg / mL of CuCDs obtained in Example 1.

[0057] Example 8 A method for colorimetric detection of NADH, the specific steps are as follows: mixing a sample to be tested with a reaction solution, reacting at room temperature (the room temperature is 20°C~25°C) for 15 minutes, measuring the absorbance at 652 nm to obtain ΔA652 nm, substituting it into the formula ΔA652 nm=0.0081[NADH]+0.0326, and calculating the concentration of NADH in the sample to be tested.

[0058] The ΔA652 nm value is the A652 nm value when no sample to be tested is added minus the A652 nm value after the sample to be tested is added; The reaction solution consisted of HAc-NaAc at pH 6, 0.1 mM TMB, 0.2 mM H2O2 and 15 μg / mL of CuCDs obtained in Example 2.

[0059] Example 9 An electrode CuCDs / CS / GCE, the preparation method is as follows: A 7.5 mg / mL CuCDs solution (the CuCDs was prepared in Example 1, and the solvent of the CuCDs solution was water) was mixed with chitosan (CS) so that the final mass volume fraction of CS was 0.1%, thereby obtaining a mixed solution; a glassy carbon electrode (GCE) was polished, cleaned, and then dried under N2, 5 μL of the mixed solution was dropwise applied to the surface of the GCE electrode, and naturally dried at room temperature to obtain an electrode CuCDs / CS / GCE.

[0060] Example 10 CuCDs solutions of different concentrations (2.5 mg / mL, 5.0 mg / mL, 7.5 mg / mL, 10 mg / mL) (the CuCDs were prepared in Example 1) were mixed with CS to obtain a final mass volume fraction of CS of 0.1% to obtain a mixed solution; 5 μL of the mixed solution was drop-coated on the surface of the polished and cleaned GCE electrode and dried naturally at room temperature to obtain different electrodes CuCDs / CS / GCE. The electrode without CuCDs was used as the control group, recorded as CS / GCE.

[0061] The obtained different electrodes were placed in 0.1 M PBS containing 1 mM NADH, and the CV was scanned (scan it), with parameter settings: 0-1 V, scan rate 10 mV / s. The results are shown in Figure 5As shown. Except that the peak current of NADH oxidation by 2.5 mg / mL CuCDs is lower than that by CS / GCE, the peak current increases with the increase of concentration, and the peak current of 7.5 mg / mL CuCDs reaches the maximum. However, when the concentration continues to increase to 10.0 mg / mL, the peak current of NADH by CuCDs decreases. The results show that too high or too low concentration will reduce the oxidation current performance of NADH on GCE. On the whole, 7.5 mg / mL CuCDs achieves the best balance between the mass transfer of NADH and the electron transfer in the electrocatalytic oxidation process, and shows a relatively strong promoting effect on the electrocatalytic oxidation of NADH.

[0062] Embodiment 11 The CuCDs / CS / GCE prepared in Example 9 was placed in 0.1 M PBS and scanned. The potential was set to 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, and 0.8 V. After the current stabilized, 1 mM NADH was added. The effect of different oxidation potentials on the electrocatalytic NADH of CuCDs / CS / GCE was determined based on the increase in oxidation peak current. The results are shown in Figure 2. Figure 6 As shown in the figure, after the current stabilizes, NADH is added and the current increases. However, the current response of NADH on CuCDs / CS / GCE is different at different potentials. At fixed potentials of 0.3 V and 0.4 V, the current change amplitude is small, and the current change amplitude increases significantly at 0.5 V. When the potential increases to 0.6 V, the current increase reaches the maximum. When the potential is further increased to 0.7 V, the current increase does not continue to increase, and the current increase even decreases at 0.8 V. The results show that a fixed potential of 0.6 V can make the current response of NADH on CuCDs / CS / GCE reach the highest level, that is, when it is fixed at a potential of 0.6 V, CuCDs / CS / GCE can achieve the highest sensitivity in detecting NADH.

[0063] Example 12 The CuCDs / CS / GCE prepared in Example 9 was placed in 0.1 M PBS and scanned. The oxidation potential was set to 0.6 V. After the current stabilized, different concentrations of NADH (0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM) were added in sequence. The next concentration of NADH was added after the current stabilized for 10 s each time, and the process was repeated in sequence. A linear curve was drawn based on the current increase Δi and the NADH concentration, and the relationship between Δi and c(NADH) was obtained by fitting.

[0064] The results are as follows Figure 7As shown in the figure, in 0.1 M PBS, NADH was added every 10 s. As the NADH concentration increased, the current increased. Figure 7 It can be observed in A that the response current is stable at low NADH concentrations, and the current fluctuation increases with increasing concentrations. This is because excessive NADH concentrations cause a large disturbance in the charge balance on the CuCDs / CS / GCE surface, resulting in large current fluctuations. By fitting the data between the NADH concentration [NADH] and the current increment ΔI (ΔI is the I minus the initial I after adding different NADH), it was found that there is a linear relationship in the range of 0.25-120 μM (see Figure 7 B), ΔI=0.01757[NADH]+0.00174, linear correlation coefficient R 2 =0.9965, the sensitivity is 17.57 μA / mM, and according to the formula LOD=3σ / k, the detection limit LOD=0.51 μM can be calculated.

[0065] Example 13 A method for electrochemically detecting NADH, the specific method is as follows: The electrode CuCDs / CS / GCE prepared in Example 9 was placed in 0.1 M PBS, and it was scanned. The potential was set to 0.6 V. After the current stabilized, the sample to be tested was added to obtain the current increase ΔI. Substituting the current into the formula ΔI=0.01757[NADH]+0.00174, the concentration of NADH in the sample to be tested was calculated; the current increase ΔI was I after adding the sample to be tested minus the initial I when no sample to be tested was added.

[0066] Embodiment 14 Detection of bacteria E. coli NADH, Escherichia coli ( E. coli ) was activated in LB medium at a 1‰ inoculum volume and cultured for 12 h before being passed on to the next generation. After three generations of activation, E. coli When the growth state is stable, re-inoculate into new culture medium. Cultivate for 4 h, 8 h, 12 h, 24 h, and 48 h respectively, centrifuge to obtain bacterial cells and weigh them. Then resuspend the bacterial cells in 5 mL PBS, ultrasonically disrupt them, centrifuge to obtain the supernatant, and use the colorimetric method of Example 7 and the electrochemical method of Example 13 to detect the E. coli NADH in the solution. The liquid without E. coli was used as the control group. Three parallel tests were performed at the same time.

[0067] The colorimetric method of Example 7 and the electrochemical method of Example 13 were used to detect NADH of Escherichia coli of different concentrations and weights. The results are as follows: Figure 8As shown in A to C, as the number of bacteria increases, the NADH content increases, the TMB color development is inhibited and the color becomes lighter, and the current signal increases. The NADH concentrations of 25 mg, 50 mg, and 100 mg of E. coli are 9.73±0.22, 15.16±0.12, and 22.50±0.59 nmol (colorimetric method), and 9.42±0.40, 15.41±0.61, and 22.13±0.86 nmol (electrochemical method).

[0068] The NADH in the bacteria with the same weight at different culture times was detected. The results are as follows Figure 8 As shown in D~F in the figure. From 4 h to 12 h, the color inhibition becomes stronger and the current increase becomes larger. This is because the bacteria are in a proliferating state, the bacterial metabolism is faster, and the NADH content is increasing. At 12 h, the bacterial growth state reaches a plateau, the overall bacterial metabolism reaches the fastest, the NADH content reaches the highest, the color inhibition is the greatest, and the current increase is the greatest. At 24 h and 48 h, due to nutrient deficiency, metabolism slows down, the NADH in the bacteria gradually decreases, the color inhibition becomes smaller, and the current increase decreases. The NADH content in the same mass of bacteria at different times was further calculated based on the color and current changes, as shown in the figure. Figure 8 As shown in F, the NADH contents in the same weight of bacteria at 4 h, 8 h, 12 h, 24 h, and 48 h detected by colorimetric and electrochemical methods were 5.87±0.05, 6.65±0.02, 7.72±0.04, 5.48±0.37, and 3.95±0.19 nmol (colorimetric method) and 5.85±0.24, 6.71±0.13, 7.70±1.11, 6.08±1.00, and 3.75±0.64 nmol (electrochemical method), respectively.

[0069] Embodiment 15 Mouse fibroblast L929 was revived and cultured in DMEM containing 10% fetal bovine serum. After the cell growth state stabilized, the cells were re-passaged and cultured for 24 h, 48 h, and 72 h, respectively. The cells were digested with trypsin, centrifuged, resuspended in PBS, counted, ultrasonically disrupted, and centrifuged to obtain the supernatant. The NADH in L929 was detected by the colorimetric method of Example 7 and the electrochemical method of Example 13, respectively. The liquid without L929 was used as the control group. Three parallel tests were performed at the same time.

[0070] Result analysis: Different numbers of cells cultured for 24 h were taken to detect NADH in L929 using the colorimetric method of Example 7 and the electrochemical method of Example 13, respectively. The results are as follows: Fig. 9As shown in A to C, as the number of cells increases, the color reaction is more strongly inhibited and the current signal increases, indicating that NADH increases with the increase in cell number. 6 pcs, 2×10 6 pcs, 3×10 6 The NADH contents of each cell were 4.07±0.06 nmol, 6.89±0.20 nmol, 9.65±0.36 nmol (colorimetric method), 4.08±0.09 nmol, 7.06±0.20 nmol, 9.63±0.36 nmol (electrochemical method) (see Fig. 9 C in.

[0071] The NADH content in cells at 24 h, 48 h, and 72 h was detected by colorimetric and electrochemical methods, respectively. Fig. 9 As shown in D-F in Figure 2, the cells at 24 h had a good growth state and a fast metabolism, and the color inhibition was the strongest and the current signal increased by a large margin. At 48 h and 72 h, due to insufficient nutrition, the cell metabolism slowed down and turned into a senescent state, the NADH content decreased, the color inhibition weakened, and the current signal increased by a small margin. It was further determined that every 1×10 6 The NADH contents of each cell were 4.41±0.59 nmol, 3.93±0.59 nmol, 3.67±0.52 nmol (colorimetric method), 4.99±0.48 nmol, 4.30±0.60 nmol, 3.80±0.86 nmol (electrochemical method) (see Fig. 9 F in.

[0072] Comparative Example 1 An electrode CDs / CS / GCE, which is different from Example 9 in that the CuCDs in Example 9 is replaced by CDs, and the rest is the same as Example 9.

[0073] The CuCDs / CS / GCE obtained in Example 9 was compared with the CDs / CS / GCE obtained in this comparative example to explore the electrocatalytic effects of different electrodes on NADH.

[0074] The two different electrodes were placed in 0.1 M PBS containing 1 mM NADH and scanned with the following parameters: 0-1 V and a scan rate of 10 mV / s.

[0075] The results are as follows Fig.10As shown in the figure, the oxidation potentials of CDs / CS / GCE and CuCDs / CS / GCE for NADH are 0.579 V and 0.533 V, respectively. From the peak potential, CuCDs / CS / GCE is more likely to oxidize NADH, indicating that the presence of Cu can promote the electrocatalysis of NADH. In addition, the peak current of NADH on CuCDs / CS / GCE is larger than that on CDs / CS / GCE, indicating that the presence of CuCDs can provide a larger effective surface area than CDs, allowing more NADH to be oxidized on the electrode surface and generate a larger current.

[0076] Comparative Example 2 An electrode CS / GCE, which is different from Example 9 in that CuCDs are not added, and the rest is the same as Example 9.

[0077] CS / GCE, CDs / CS / GCE (obtained in Comparative Example 1), and CuCDs / CS / GCE (obtained in Example 9) were placed in 0.1 M PBS, respectively, and the potential was set to 0.6 V. After the current stabilized, different concentrations of NADH were added, and the electrocatalytic effects of CS / GCE, CDs / CS / GCE, and CuCDs / CS / GCE on NADH were determined based on the increase in oxidation peak current.

[0078] The results are as follows Fig.11 As shown in the figure, the response currents of CS / GCE, CDs / CS / GCE and CuCDs / CS / GCE to NADH all increase with the increase of concentration, but the response amplitude of CDs / CS / GCE to NADH is smaller than that of CS / GCE, indicating that CDs has a certain inhibitory effect on the oxidation of NADH on the electrode. The response of CuCDs / CS / GCE to NADH is significantly improved compared with CS / GCE and CDs / CS / GCE. This is due to the electrocatalytic oxidation activity of CuCDs to NADH, which makes the response current more sensitive, and this also confirms the previous CV results, further confirming that CuCDs has a promoting effect on the electrocatalytic oxidation of NADH.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing CuCDs capable of catalytic oxidation of TMB and NADH, characterized in that: The method comprises the following steps: dissolving monohydrated citric acid, urea and CuCl2·2H2O in water, reacting at 180-220°C for 8-12 hours, centrifuging and discarding precipitate, filtering and dialyzing the filtrate, collecting the product, and freeze-drying to obtain CuCDs.

2. The method according to claim 1, characterized in that The mass volume ratio of citric acid monohydrate, urea and water is (0.5~2) g: (0.5~2) g: 20 mL; the final concentration of the CuCl2·2H2O is 1~4 mM.

3. A CuCDs capable of catalytic oxidation of TMB and NADH, characterized in that: Prepared by the method of claim 1 or 2.

4. Use of the CuCDs according to claim 3 in detecting NADH or in preparing a product for detecting NADH.

5. A method for colorimetric detection of NADH, characterized in that: The method comprises the following steps: mixing a sample to be tested with a reaction solution, reacting at room temperature for 15 minutes, measuring the absorbance A at 652 nm to obtain ΔA652 nm, substituting the absorbance A at 652 nm into the formula ΔA652 nm=0.0081[NADH]+0.0326, and calculating the concentration of NADH in the sample to be tested; the ΔA652 nm value is the A652 nm value when the sample to be tested is not added minus the A652 nm value after the sample to be tested is added; the reaction solution is composed of HAc-NaAc with a pH of 4 to 6, 0.05 to 0.4 mM TMB, 0.025 to 0.2 mM H2O2, and 5 to 20 μg / mL of the CuCDs described in claim 3.

6. A method for preparing an electrode CuCDs / CS / GCE, characterized in that: The method comprises the following steps: mixing the solution containing the CuCDs according to claim 3 with CS to obtain a mixed solution; and drop-coating the mixed solution on the surface of GCE to obtain an electrode CuCDs / CS / GCE.

7. The method according to claim 6, characterized in that The concentration of the solution containing the CuCDs according to claim 3 is 3-10 mg / mL, and the final mass volume fraction of CS in the mixed solution is 0.05%-0.5%.

8. An electrode CuCDs / CS / GCE, characterized in that Prepared by the method of claim 6 or 7.

9. A method for electrochemical detection of NADH, characterized in that: The method comprises the following steps: placing the electrode CuCDs / CS / GCE according to claim 8 in PBS, scanning it, setting the potential to 0.5-0.8 V, adding the sample to be tested after the current is stable, obtaining the current increase ΔI, substituting the current into the formula ΔI=0.01757[NADH]+0.00174, and calculating the concentration of NADH in the sample to be tested; the current increase ΔI is I after adding the sample to be tested minus the initial I when no sample to be tested is added.

10. The method according to claim 5 or claim 9, characterized in that: The sample to be tested includes bacteria and / or cells.

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