CuCDs, a preparation method thereof and application thereof in detection of nadh

Abstract

The application provides a CuCDs, a preparation method thereof and application thereof in detection of NADH, and belongs to the technical field of NADH detection. The CuCDs provided by the application is obtained by reaction of monohydrate citric acid, urea and CuCl2.2H2O. The CuCDs provided by the application has double enzyme activity, can not only catalyze NADH to generate NAD + in the presence of H2O2, but also can oxidize TMB to generate oxTMB, and NADH can inhibit CuCDs from peroxidizing TMB. The application provides a method for colorimetric detection of NADH with a detection limit of 3.6 mu M by means of the double enzyme activity of CuCDs, and has the advantages of simple operation, low cost and visualization. The electrode CuCDs / CS / GCE and the method for electrochemical detection of NADH can accurately and sensitively detect NADH quantitatively, and the detection limit is as low as 0.51 mu M.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of NADH detection, and particularly relates to CuCDs, a preparation method thereof and application of the CuCDs in NADH detection. BACKGROUND

[0002] Beta-nicotinamide adenine dinucleotide (NADH) is naturally formed in cells by supplementing food, and is the most important coenzyme found in organisms. This may be related to the formation of ATP (adenosine triphosphate) molecules, which are responsible for storing energy in their chemical structure. Due to the lack of ATP production, the lack of NADH can lead to insufficient energy at the cellular level. On the other hand, an increase in NADH in cells can also lead to an excess of energy supply, resulting in adverse effects of excessive stimulation. Cytosolic NADH is produced by lactate dehydrogenase (LDH), which catalyzes the reversible conversion between lactic acid and pyruvic acid. The NADH in cells has different sensitivities to different stimuli, and the total NADH in the cytoplasm and nucleus is susceptible to changes in the levels of nutrients in the cells, such as glucose, lactic acid, pyruvic acid, 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 is important. NADH is closely related to the metabolism of organisms, and to some extent, the content of NADH can reflect the life state of organisms. Detecting the content of NADH in organisms is of great significance to evaluate the growth status of organisms. The methods for detecting NADH currently existing in the art include optical, liquid chromatography, electrophoresis and chemiluminescence techniques, but these techniques are expensive, require a large amount of preparation, and have low response time and sensitivity. SUMMARY

[0003] Therefore, one of the purposes of the present application is to provide a CuCDs with dual enzyme (TMB (tetramethylbenzidine) and NADH) activity, and a colorimetric method for detecting NADH is designed by virtue of the principle that NADH can inhibit the peroxidation of TMB by CuCDs.

[0004] The second purpose of the present application is to provide an electrode CuCDs / CS / GCE, and an electrochemical method for detecting NADH is provided by using the electrode CuCDs / CS / GCE.

[0005] In order to achieve the above-mentioned purposes of the application, the present application provides the following technical solutions:

[0006] The application provides a method for preparing CuCDs capable of catalyzing oxidation of TMB and NADH, comprising the following steps: dissolving citric acid monohydrate, urea and CuCl2.2H2O in water, reacting at 180-220 DEG C for 8-12 h, centrifuging to discard the precipitate, filtering to take the filtrate, dialyzing, collecting the product, and freeze-drying to obtain CuCDs.

[0007] 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 CuCl2.2H2O is 1-4 mM.

[0008] The application further provides CuCDs capable of catalyzing oxidation of TMB and NADH, which are prepared by the above method.

[0009] The application further provides application of the above CuCDs in detection of NADH or in preparation of a product for detecting NADH.

[0010] The application further provides a method for colorimetric detection of NADH, comprising the following steps: mixing a sample to be detected with a reaction solution, reacting at room temperature for 15 min, measuring the absorbance at 652 nm to obtain ΔA652 nm, and substituting the ΔA652 nm into the formula ΔA652 nm=0.0081[NADH]+0.0326 to obtain the concentration of NADH in the sample to be detected; the ΔA652 nm value is the A652 nm value without the sample to be detected minus the A652 nm value after the sample to be detected is added; and the reaction solution is composed of HAc-NaAc (acetic acid-sodium acetate buffer solution) 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 CuCDs.

[0011] The application further provides a method for preparing an electrode CuCDs / CS / GCE, comprising the following steps: mixing a solution containing the above CuCDs with CS to obtain a mixed solution; and dropping the mixed solution on the surface of a GCE to obtain the electrode CuCDs / CS / GCE.

[0012] Preferably, the concentration of the solution containing the above CuCDs is 3-10 mg / mL, and the final mass-volume fraction of CS in the mixed solution is 0.05%-0.5%.

[0013] The application further provides an electrode CuCDs / CS / GCE, which is prepared by the above method.

[0014] The application further provides a method for electrochemically detecting NADH, comprising the following steps: placing the electrode CuCDs / CS / GCE in PBS, scanning i-t, setting the potential to 0.5-0.8 V, adding a sample to be detected after the current is stable, obtaining the current increment DI, and substituting DI into the formula DI=0.01757[NADH]+0.00174 to obtain the concentration of NADH in the sample to be detected; the current increment DI is the initial current I before the sample to be detected is added.

[0015] Preferably, the sample to be detected comprises bacteria and / or cells.

[0016] The application has the following beneficial effects:

[0017] The preparation method provided by the application can prepare CuCDs with double enzyme activities, which can not only catalyze NADH to generate NAD + in the presence of H2O2, but also can oxidize TMB to generate oxTMB, and NADH can inhibit CuCDs from peroxidizing TMB, and the application provides a colorimetric method for detecting NADH by means of the double enzyme activities of CuCDs, which has the advantages of simple operation, low cost, visualization and the like, and is more widely applied in practice, and the detection limit of the colorimetric method for detecting NADH provided by the application is as low as 3.6 μM.

[0018] The electrode CuCDs / CS / GCE and the method for electrochemically detecting NADH provided by the application can accurately and sensitively quantitatively detect NADH in a sample to be detected, and the detection limit is as low as 0.51 μM. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The NADH peroxidase activity research results of CuCDs obtained in Example 1 are shown in the table, wherein A is the influence of different pH on the peroxidation of NADH by CuCDs, B is the influence of different temperatures on the peroxidation of NADH by CuCDs, C is the influence of different concentrations of NADH on the NADH peroxidase activity of CuCDs, and D is the influence of different concentrations of H2O2 on the NADH peroxidase activity of CuCDs;

[0020] Figure 2 The TMB peroxidase activity research results of CuCDs obtained in Example 1 are shown in the table, wherein A is the influence of different pH on the catalysis of TMB by CuCDs, B is the influence of different temperatures on the catalysis of TMB by CuCDs, C is the influence of different concentrations of TMB on the catalysis of TMB by CuCDs, and D is the influence of different concentrations of H2O2 on the catalysis of TMB by CuCDs;

[0021] Figure 3 The influence of NADH on the peroxidation of TMB by CuCDs is shown in the table.

[0022] Figure 4 For further study on the effect of NADH on CuCDs peroxidation of TMB, A is the monitoring result of CuCDs peroxidation of TMB with or without NADH, B is the color change and absorbance change result of adding different concentrations of NADH in the reaction system of CuCDs, H2O2 and TMB, and C is the relationship curve between NADH concentration and ΔA652 nm;

[0023] Figure 5 For the effect of different concentrations of CuCDs solution on the electrocatalysis of NADH on the electrode CuCDs / CS / GCE;

[0024] Figure 6 For the effect of different potentials on the electrocatalysis of NADH on the electrode CuCDs / CS / GCE;

[0025] Figure 7 For the results of Example 12, A is the response current result of the electrode CuCDs / CS / GCE with different concentrations of NADH, B is the relationship curve between NADH concentration and current increment ΔI;

[0026] Figure 8 For the results of NADH detected by colorimetric method and electrochemical method of E. coli according to the application, A is the result of detecting NADH in different concentrations of E. coli by electrochemical method, B is the result of detecting NADH in different concentrations of E. coli by colorimetric method, C is the result of NADH in different weights of E. coli calculated by colorimetric method and electrochemical method; D is the result of detecting NADH in different culture times and same weights of E. coli by electrochemical method, E is the result of detecting NADH in different culture times and same weights of E. coli by colorimetric method, and F is the result of further calculating NADH content in different culture times and same weights of E. coli according to current and color change;

[0027] Figure 9 For the results of NADH detected by colorimetric method and electrochemical method of mouse fibroblast L929 according to the application, A is the result of detecting NADH in different numbers of L929 cells by electrochemical method, B is the result of detecting NADH in different numbers of L929 cells by colorimetric method, C is the result of NADH in different numbers of L929 cells calculated by colorimetric method and electrochemical method; D is the result of detecting NADH in different culture times and same numbers of L929 cells by electrochemical method, E is the result of detecting NADH in different culture times and same numbers of L929 cells by colorimetric method, and F is the result of further calculating NADH content in different culture times and same numbers of L929 cells according to current and color change;

[0028] Figure 10 For the electrocatalysis effect of two different electrodes on NADH;

[0029] Figure 11 The electrocatalytic effects of three different electrodes on NADH. DETAILED DESCRIPTION

[0030] The application provides a method for preparing CuCDs capable of catalyzing oxidation of TMB and NADH, comprising the following steps: dissolving citric acid monohydrate, urea and CuCl2.2H2O in water, reacting at 180-220 DEG C for 8-12 h, centrifuging to discard the precipitate, filtering to take the filtrate, dialyzing, collecting the product and freeze-drying to obtain CuCDs.

[0031] The application introduces Cu into carbon dots (CDs), so that the obtained CuCDs have electrocatalytic properties in solution and on electrodes. In the application, 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 CuCl2.2H2O is preferably 1-4 mM, more preferably 2-3 mM. In the application, the reaction temperature is preferably 190-210 DEG C, more preferably 200-208 DEG 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 conditions are preferably 10000 rpm for 10 min. In the application, the filtration is preferably performed using a filter membrane, and the pore size of the filter membrane is preferably 0.22 μm. In the application, the molecular weight of the dialysis is preferably 500, and the dialysis time is preferably 48 h.

[0032] The application also provides CuCDs capable of catalyzing oxidation of TMB and NADH, which are prepared by the above method.

[0033] The application also provides application of the above CuCDs in detection of NADH or in preparation of a product for detecting NADH.

[0034] The application also provides a method for colorimetric detection of NADH, comprising the following steps: mixing a sample to be detected with a reaction solution, reacting at room temperature for 15 min, measuring the absorbance at 652 nm to obtain ΔA652 nm, and substituting into the formula ΔA652 nm=0.0081[NADH]+0.0326 to obtain the concentration of NADH in the sample to be detected; the ΔA652 nm value is the A652 nm value without the sample to be detected minus the A652 nm value after the sample to be detected is added; and the reaction solution is composed of HAc-NaAc with pH 4-6, 0.05-0.4 mM TMB, 0.025-0.2 mM H2O2 and 5-20 μg / mL of the above CuCDs.

[0035] The application designs the above-mentioned colorimetric detection method of NADH by virtue of the natural enzyme simulation activity of CuCDs, which can convert colorless substrates into colored substrates, according to the prepared CuCDs having double enzyme activity and the NADH can inhibit the CuCDs from peroxidizing TMB. In the colorimetric detection method of NADH provided by the application, the room temperature is preferably 20-25 DEG C, more preferably 21-24 DEG C, and further preferably 22-23 DEG C. In the reaction solution, 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; and the concentration of CuCDs is preferably 8-15 μg / mL, more preferably 9-12 μg / mL. In the application, the sample to be detected preferably comprises bacteria and / or cells, the bacteria preferably comprise Escherichia coli, and the cells preferably comprise mouse epithelial fibroblasts L929.

[0036] The application further 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 dropping the mixed solution on the surface of a glassy carbon electrode (GCE) to obtain the electrode CuCDs / CS / GCE.

[0037] The application does not have special limitations on the specific sources of CS and GCE. In the preparation method of the application, the concentration of the CuCDs solution is preferably 3-10 mg / mL, more preferably 5-8 mg / mL; and 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 application, the GCE is preferably polished, cleaned and then dried under N2 before use. When the mixed solution is dropped on the surface of the GCE, the amount of the dropped mixed solution is preferably 5 μL, and after the dropping is completed, the electrode CuCDs / CS / GCE is obtained by natural drying at room temperature.

[0038] The application further provides an electrode CuCDs / CS / GCE, which is prepared by the above-mentioned method.

[0039] The application further provides an electrochemical method for detecting NADH, comprising the following steps: placing the above-mentioned electrode CuCDs / CS / GCE in PBS, scanning i-t, setting the potential to 0.5-0.8 V, adding a sample to be detected after the current is stable, obtaining the current increment ΔI, and substituting the current increment ΔI into the formula ΔI=0.01757[NADH]+0.00174 to obtain the concentration of NADH in the sample to be detected; the current increment ΔI is the initial current I when the sample to be detected is not added.

[0040] In the method for electrochemically detecting NADH, the concentration of the PBS is preferably 0.1 M, the set potential for scanning is preferably 0.6-0.7 V, and the scanning rate is preferably 10 mV / s. In the method for electrochemically detecting NADH, the sample to be detected preferably comprises bacteria and / or cells, the bacteria preferably comprise Escherichia coli, and the cells preferably comprise mouse epithelial fibroblasts L929.

[0041] The application provides a colorimetric and electrochemical dual-mode NADH detection method based on CuCDs, which can realize more accurate and effective NADH detection, and the dual-mode NADH detection method can accurately quantify NADH in bacteria and cells, and has great practical application potential.

[0042] The technical solutions provided by the application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the protection scope of the application.

[0043] In the following examples, all are conventional methods unless otherwise specified.

[0044] In the following examples, the materials and reagents used are commercially available unless otherwise specified.

[0045] Example 1

[0046] A CuCDs capable of catalyzing the oxidation of TMB and NADH, the preparation method of the CuCDs is:

[0047] First, 1.051 g of citric acid monohydrate and 0.9 g of urea are dissolved in 20 mL of water, 2 mM of CuCl2·2H2O is added, and after ultrasonic dissolution, it is transferred to a high-pressure reaction kettle, and reacted at 200℃ for 10 h. Then centrifuge at 10000 rpm for 10 min, discard the precipitate, and filter with a 0.22 μm filter membrane. Finally, the filtrate is dialyzed for 48 h (the molecular weight of dialysis is 500), the product is collected, freeze-dried to obtain a solid powder, which is CuCDs.

[0048] Example 2

[0049] A CuCDs capable of catalyzing the oxidation of TMB and NADH, the preparation method of the CuCDs is:

[0050] First, 0.5 g of citric acid monohydrate and 0.8 g of urea were dissolved in 20 mL of water, 1 mM of CuCl2·2H2O was added, and after ultrasonic dissolution, it was transferred to a high-pressure reaction kettle, and reacted at 180°C for 9 h. Then, centrifuged at 10000 rpm for 10 min, the precipitate was discarded, and filtered using a 0.22 μm filter membrane. Finally, the filtrate was dialyzed for 48 h (the molecular weight of dialysis is 500), the product was collected, and after freeze-drying, a solid powder was obtained, which was CuCDs.

[0051] Example 3

[0052] A CuCDs capable of catalyzing the oxidation of TMB and NADH, the preparation method of the CuCDs is:

[0053] First, 2 g of citric acid monohydrate and 2 g of urea were dissolved in 20 mL of water, 4 mM of CuCl2·2H2O was added, and after ultrasonic dissolution, it was transferred to a high-pressure reaction kettle, and reacted at 210°C for 12 h. Then, centrifuged at 10000 rpm for 10 min, the precipitate was discarded, and filtered using a 0.22 μm filter membrane. Finally, the filtrate was dialyzed for 48 h (the molecular weight of dialysis is 500), the product was collected, and after freeze-drying, a solid powder was obtained, which was CuCDs.

[0054] Example 4

[0055] Enzymatic activity exploration of CuCDs obtained in Example 1

[0056] 4.1 NADH peroxidase activity

[0057] 50 μg / mL of CDs or CuCDs prepared in Example 1 were respectively reacted with 0.2 mM NADH and 2.0 mM H2O2 in 1 mL of 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 according to the decrease of absorbance at 340 nm (ΔA340 nm = A340 nm before reaction - A340 nm after reaction).

[0058] 50 μg / mL of CuCDs prepared in Example 1 were respectively reacted with 0.2 mM NADH and 2.0 mM H2O2 in 1 mL of 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, 70°C for 15 min, and the absorbance at 340 nm was measured. The influence of different pH and temperature on the peroxidation of NADH by CuCDs was analyzed according to the decrease of absorbance at 340 nm (ΔA340 nm).

[0059] CuCDs were prepared according to the method described in Example 1. 0.04 mM, 0.08 mM, 0.12 mM, 0.16 mM, 0.2 mM NADH were reacted with 50 μg / mL CuCDs 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 according to ΔA340 nm, and the double-reciprocal plot of 1 / c(NADH) vs 1 / v was drawn. The Michaelis-Menten constant KM was calculated according to the following equation: K m (NADH).

[0060] 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM, 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 according to ΔA340 nm, and the double-reciprocal plot of 1 / c(H2O2) vs 1 / v was drawn. The Michaelis-Menten constant KM was calculated according to the following equation: K m (H2O2).

[0061] Results analysis: CuCDs oxidized NADH in the presence of H2O2, i.e. CuCDs had NADH-like peroxidase activity. This was related to the presence of Cu in CuCDs, as CDs had no obvious peroxidation effect on NADH.

[0062] The NADH-like peroxidase activity of CuCDs was evaluated at different pH and temperature, and the results are shown as A and B in Figure 1 It was found that the activity of CuCDs decreased with increasing pH in the range of pH 3-8, and increased with increasing temperature in the range of 25-70 °C.

[0063] From C and D in Figure 1 , it can be seen that the reaction rate v increased with increasing [NADH] and [H2O2]. Due to the saturation of the active site of CuCDs, the reaction rate increased slowly. The double-reciprocal plots of reaction rate v and substrate concentration [NADH] or [H2O2] were drawn respectively (inserts in C and D in Figure 1 ). According to Michaelis-Menten calculation, the K m values of CuCDs for NADH and H2O2 were 0.12 mM and 0.16 mM respectively.

[0064] 4.2 TMB peroxidase activity

[0065] CuCDs were added into 1 mL of HAc-NaAc containing 1 mM TMB and 1 mM H2O2, respectively, 10 μg / mL of CDs or CuCDs prepared in Example 1 were added, the total reaction system was 1 mL, mixed thoroughly, 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 according to ΔA652 nm (ΔA652 nm = A652 nm after reaction - A652 nm before reaction).

[0066] CuCDs prepared in Example 1 were added into 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, respectively, the reaction time was 5 min, and the color change was recorded. The absorbance at 652 nm was measured, and the influence of different pH and temperature on CuCDs peroxidation of TMB was analyzed according to ΔA652 nm.

[0067] CuCDs prepared in Example 1 were added into 1 mL of HAc-NaAc containing 1.0 mM H2O2 at pH 5, 10 μg / mL of CuCDs prepared in Example 1 were dissolved with 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, 1.0 mM of TMB, 50 °C for 5 min, the reaction rate v was calculated according to ΔA652 nm, the relationship between 1 / c(TMB) and 1 / v was plotted by double reciprocal plot, and K m (TMB).

[0068] CuCDs prepared in Example 1 were added into 1 mL of HAc-NaAc containing 1.0 mM H2O2 at pH 5, 10 μg / mL of CuCDs prepared in Example 1 were dissolved with 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, 1.0 mM of TMB, 50 °C for 5 min, the reaction rate v was calculated according to ΔA652 nm, the relationship between 1 / c(TMB) and 1 / v was plotted by double reciprocal plot, and K m (H2O2).

[0069] Results analysis: CuCDs have peroxidase-like activity on TMB. The influence of pH and temperature on the activity of CuCDs was further studied. From Figure 2As shown in A and B, acidic environments and increased temperatures can promote the activity of CuCDs, reaching its peak at pH 5 and 60℃. Figure 2 C and D in the figure show the reaction rate v of CuCDs catalyzing TMB color development with different concentrations of TMB and H2O2. The reaction rate v increases with increasing [TMB] and [H2O2], and eventually the increase in reaction rate slows down because the activity of CuCDs tends to saturate. The reaction rate v and substrate concentration [TMB] or [H2O2] are plotted separately using double reciprocal plots (see Figure 1). Figure 2 (Illustrations in C and D). According to the Michaelis-Menten equation, CuCDs for TMB and H2O2 can be calculated. K m The values ​​were 0.40 mM and 0.14 mM, respectively.

[0070] Example 5

[0071] Experimental group (CuCDs+TMB+H2O2+NADH): 10 μg / mL of CuCDs obtained in Example 1 was added to 1 mL of pH5 HAc-NaAc containing 0.2 mM TMB, 0.1 mM H2O2 and 0.1 mM NADH.

[0072] Control group (CuCDs+TMB+H2O2): 10 μg / mL of CuCDs obtained in Example 1 was added to 1 mL of pH5 HAc-NaAc containing 0.2 mM TMB and 0.1 mM H2O2.

[0073] The experimental and control groups were reacted at room temperature (20℃~22℃) for 1 h, and the color changes of the reaction system were recorded. The absorbance at 652 nm was measured, and the effect of NADH on the oxidation of TMB by CuCDs was analyzed based on ΔA652 nm. The results are as follows: Figure 3 As shown, compared with the reaction system without the addition of 0.1 mM NADH, the blue color of oxTMB was weakened in the reaction system where NADH and TMB coexisted, indicating that the peroxidation process of TMB by CuCDs was inhibited, and this inhibition originated from NADH.

[0074] Example 6

[0075] Further investigation was conducted on the inhibitory effect of NADH on the oxidation of TMB by CuCDs.

[0076] 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 (21°C–23°C) for 75 min. The reaction without the addition of 0.1 mM NADH served as a control group. A652 nm was monitored every 30 s. The effect of NADH on CuCDs catalyzing TMB was analyzed based on A652 nm. The results are as follows: Figure 4 As shown in A, ΔA652 nm reaches its maximum value at 15 min, indicating that the inhibitory effect is most significant at 15 min.

[0077] 0.2 mM TMB, 0.1 mM H2O2, and 10 μg / mL CuCDs obtained in Example 1 were added to HAc-NaAc at pH 5. 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 to the total reaction system of 1 mL. The mixture was thoroughly mixed and reacted at room temperature (21℃~23℃) for 15 min. The absorbance at 652 nm was measured. The results are as follows: Figure 4 As shown in B, the inhibitory effect becomes stronger with increasing NADH concentration, and the color also shows a regular change.

[0078] Calculate the absorbance change at A652 nm, ΔA652 nm (ΔA652 nm is the difference between A652 nm without NADH and A652 nm with different concentrations of NADH), and plot the curve of [NADH] versus ΔA652 nm. The results are as follows: Figure 4 As shown in C, the fitting results indicate 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. According to... The detection limit can be calculated to be 3.6 μM.

[0079] Example 7

[0080] A colorimetric method for detecting NADH includes the following steps: the sample to be tested is mixed with a reaction solution and reacted at room temperature (21℃~23℃) for 15 min. The absorbance at 652 nm is measured to obtain ΔA652 nm. The concentration of NADH in the sample is calculated by substituting the absorbance into the formula ΔA652 nm=0.0081[NADH]+0.0326.

[0081] The ΔA652 nm value is the A652 nm value without the sample to be tested minus the A652 nm value after the sample to be tested is added;

[0082] The reaction solution is composed of HAc-NaAc with pH 5, 0.2 mM TMB, 0.1 mM H2O2 and 10 μg / mL CuCDs obtained in Example 1.

[0083] Example 8

[0084] A method for colorimetric detection of NADH, the specific steps are as follows: mixing the sample to be tested with the reaction solution, reacting at room temperature (the room temperature is 20℃-25℃) for 15 min, measuring the absorbance at 652 nm, obtaining ΔA652 nm, and substituting into the formula ΔA652 nm=0.0081[NADH]+0.0326 to calculate the concentration of NADH in the sample to be tested.

[0085] The ΔA652 nm value is the A652 nm value without the sample to be tested minus the A652 nm value after the sample to be tested is added;

[0086] The reaction solution is composed of HAc-NaAc with pH 6, 0.1 mM TMB, 0.2 mM H2O2 and 15 μg / mL CuCDs obtained in Example 2.

[0087] Example 9

[0088] An electrode CuCDs / CS / GCE, the preparation method is as follows:

[0089] Mixing 7.5 mg / mL CuCDs solution (the CuCDs are obtained by the preparation in Example 1, and the solvent of the CuCDs solution is water) with chitosan (CS) to obtain a mixed solution with a final mass / volume fraction of CS of 0.1%; polishing and cleaning a glassy carbon electrode (GCE), and then drying it under N2; dropping 5 μL of the mixed solution on the surface of the GCE electrode, and naturally drying it at room temperature to obtain the electrode CuCDs / CS / GCE.

[0090] Example 10

[0091] Mixing CuCDs solutions (the CuCDs are obtained by the preparation in Example 1) with different concentrations (2.5 mg / mL, 5.0 mg / mL, 7.5 mg / mL and 10 mg / mL) with CS to obtain mixed solutions with a final mass / volume fraction of CS of 0.1%; dropping 5 μL of the mixed solution on the surface of a GCE electrode which has been polished and cleaned, and naturally drying it at room temperature to obtain different electrodes CuCDs / CS / GCE. Taking the electrode without adding CuCDs as a control group, and recording it as CS / GCE.

[0092] The obtained different electrodes were placed in 0.1 M PBS containing 1 mM NADH, and CV (sweep i-t) was scanned, and the parameters were set as: 0-1 V, and the scanning rate was 10 mV / s. The results are shown in Figure 5 As shown in the table, except that the oxidation peak current of NADH on the CS / GCE was lower than that of the 2.5 mg / mL CuCDs, the peak current increased with the increase of the concentration, and the peak current of 7.5 mg / mL CuCDs reached the maximum. However, the peak current of NADH on the CuCDs decreased when the concentration continued to increase to 10.0 mg / mL. The results showed that too high or too low concentration would reduce the oxidation current performance of NADH on the GCE. In summary, 7.5 mg / mL CuCDs made the mass transfer and electron transfer in the process of electrocatalytic oxidation of NADH reach the best balance, and showed a relatively strong promoting effect on the electrocatalytic oxidation of NADH.

[0093] Example 11

[0094] The CuCDs / CS / GCE prepared in Example 9 was placed in 0.1 M PBS, and i-t was scanned, and the potential was set as: 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, and 0.8 V, respectively. After the current was stable, 1 mM NADH was added, and the influence of different oxidation potentials on the electrocatalytic NADH of CuCDs / CS / GCE was judged according to the increase amplitude of the oxidation peak current. The results are shown in Figure 6 As shown in the table, after the current was stable, NADH was added, and the current increased. However, the current response of NADH on the CuCDs / CS / GCE was different under different potentials. The current change amplitude was small under the fixed potentials of 0.3 V and 0.4 V, the current change amplitude obviously increased at 0.5 V, the current increase amplitude reached the maximum when the potential increased to 0.6 V, the current increase amplitude did not continue to increase at 0.7 V, and the current increase amplitude even decreased at 0.8 V. The results showed that the current response of NADH on the CuCDs / CS / GCE reached the highest at the fixed potential of 0.6 V, that is, the detection of NADH by the CuCDs / CS / GCE could reach the highest sensitivity when i-t was used at the fixed potential of 0.6 V.

[0095] Example 12

[0096] The CuCDs / CS / GCE prepared in Example 9 was scanned in 0.1 M PBS. 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 sequentially, with the next concentration added after each 10-second stabilization period. A linear curve was plotted based on the current increase Δi and the NADH concentration, and the relationship between Δi and c(NADH) was obtained by fitting the curve.

[0097] The results are as follows Figure 7 As shown, in 0.1 M PBS, NADH was added sequentially every 10 seconds; the current increased with increasing NADH concentration. From... Figure 7 As can be observed in Figure A, the response current is stable at low NADH concentrations, but the current fluctuation increases with increasing concentration. This is because excessively high NADH concentrations significantly disrupt the charge balance on the CuCDs / CS / GCE surface, leading to larger current fluctuations. Data fitting of NADH concentration [NADH] with current increment ΔI (ΔI is I after adding different amounts of NADH - initial I) reveals a linear relationship in the range of 0.25-120 μM (see Figure A). Figure 7 In the equation B), ΔI = 0.01757[NADH] + 0.00174, the linear correlation coefficient R0 2 =0.9965, sensitivity is 17.57 μA / mM, and the detection limit LOD can be calculated as 0.51 μM according to the formula LOD=3σ / k.

[0098] Example 13

[0099] An electrochemical method for detecting NADH is described below:

[0100] The CuCDs / CS / GCE electrode prepared in Example 9 was placed in 0.1 M PBS and scanned at it. The potential was set to 0.6 V. After the current stabilized, the sample to be tested was added, and the current increase ΔI was obtained. Substituting 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 is I after the sample to be tested is added - the initial I before the sample to be tested is added.

[0101] Example 14

[0102] Detecting bacteria E. coli NADH, will E. coli ( E. coliActivate the culture medium at an inoculum rate of 1‰ in LB medium. After 12 hours of culture, it can be transferred to the next generation. After three generations of activation, E. coli The cells were in stable growth and re-inoculated into a fresh culture medium. After culturing for 4 h, 8 h, 12 h, 24 h, and 48 h, the cells were centrifuged and weighed. The cells were then resuspended in 5 mL PBS, sonicated, and the supernatant was collected by centrifugation. The results were analyzed using the colorimetric method of Example 7 and the electrochemical method of Example 13, respectively. E. coli NADH was used. A liquid containing no E. coli served as the control group. Three parallel experiments were conducted.

[0103] The results of detecting NADH in different concentrations and weights of *E. coli* using the colorimetric method of Example 7 and the electrochemical method of Example 13 are as follows: Figure 8 As shown in Figures A through C, with increasing bacterial count, the NADH content increases, TMB color development is inhibited and the color becomes lighter, while the current signal increases. The NADH concentrations of 25 mg, 50 mg, and 100 mg of *E. coli* were 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).

[0104] NADH levels in bacteria of the same weight cultured for different durations were measured, and the results are as follows: Figure 8 As shown in Figures D to F. From 4 h to 12 h, the color development was increasingly inhibited while the current increase was also increasing. This is because the bacteria are in a proliferative state, their metabolism is faster, and the NADH content tends to increase. At 12 h, the bacterial growth reaches a plateau, the overall bacterial metabolism is at its fastest, the NADH content is at its highest, the degree of color development inhibition is greatest, and the magnitude of the current increase is also greatest. At 24 h and 48 h, due to nutrient deficiency, metabolism slows down, the NADH in the bacteria gradually decreases, the degree of color development inhibition decreases, and the magnitude of the current increase decreases. The NADH content in the same mass of bacteria at different times was further calculated based on the changes in color and current, as shown in Figures D to F. Figure 8 As shown in F, the NADH content 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).

[0105] Example 15

[0106] Mouse fibroblast L929 was resuscitated and cultured in DMEM containing 10% fetal bovine serum, and after the cell growth state was stable, it was subcultured for 24 h, 48 h and 72 h respectively, and then the cells were trypsinized, centrifuged, resuspended in PBS, counted, ultrasonically broken, centrifuged to obtain the supernatant, and 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 a control group. Three parallel tests were performed at the same time.

[0107] Result analysis: different amounts of cells cultured for 24 h were taken, and the NADH in L929 was detected by the colorimetric method of Example 7 and the electrochemical method of Example 13 respectively, and the results are shown as A~C in Figure 9 As the amount of cells increased, the degree of inhibition of the color development reaction was stronger, and the current signal increased, indicating that the NADH increased with the increase of the number of cells. It was further determined that 1×10 6 , 2×10 6 , and 3×10 6 cells corresponded to NADH contents of 4.07±0.06 nmol, 6.89±0.20 nmol, and 9.65±0.36 nmol (colorimetric method), and 4.08±0.09 nmol, 7.06±0.20 nmol, and 9.63±0.36 nmol (electrochemical method) respectively (see C in Figure 9 ).

[0108] The NADH contents in cells cultured for 24 h, 48 h and 72 h were detected by colorimetric and electrochemical methods respectively, and the results are shown as D~F in Figure 9 . The cells cultured for 24 h grew well and metabolized quickly, and their color was most strongly inhibited and the current signal increased most greatly. At 48 h and 72 h, due to insufficient nutrients, the cells metabolized slowly and became senescent, and the NADH content decreased, the color development was less inhibited, and the current signal increased less. It was further determined that 1×10 6 cells cultured for 24 h, 48 h and 72 h corresponded to NADH contents of 4.41±0.59 nmol, 3.93±0.59 nmol, and 3.67±0.52 nmol (colorimetric method), and 4.99±0.48 nmol, 4.30±0.60 nmol, and 3.80±0.86 nmol (electrochemical method) respectively (see F in Figure 9 ).

[0109] Comparative Example 1

[0110] An electrode CDs / CS / GCE, which differs from Example 9 in that the CuCDs of Example 9 are replaced by CDs, and the rest is the same as Example 9.

[0111] The CuCDs / CS / GCE obtained in Example 9 was compared with the CDs / CS / GCE obtained in this comparative example to explore the electrocatalytic effect of different electrodes on NADH.

[0112] The two different electrodes were placed in 0.1 M PBS containing 1 mM NADH, and i-t was scanned, with the parameters set as: 0-1 V, and the scanning rate was 10 mV / s.

[0113] As shown in the results Figure 10 , the oxidation potentials of NADH on CDs / CS / GCE and CuCDs / CS / GCE were 0.579 V and 0.533 V, respectively. From the oxidation peak potential, it can be seen that the oxidation of NADH on CuCDs / CS / GCE is easier, 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, so that more NADH can be oxidized on the electrode surface, resulting in a larger current.

[0114] Comparative Example 2

[0115] An electrode CS / GCE, which is different from Example 9 in that no CuCDs is added, and the rest is the same as Example 9.

[0116] 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, and i-t was scanned, with the potential set as: 0.6 V. After the current was stable, different concentrations of NADH were added, and the electrocatalytic effect of CS / GCE, CDs / CS / GCE, and CuCDs / CS / GCE on NADH was judged according to the increase in oxidation peak current.

[0117] As shown in the results Figure 11 , the response currents of NADH on CS / GCE, CDs / CS / GCE, and CuCDs / CS / GCE all increased with increasing concentration, but compared with CS / GCE, the response amplitude of NADH on CDs / CS / GCE was smaller, indicating that CDs had a certain inhibitory effect on the oxidation of NADH on the electrode. The response of NADH on CuCDs / CS / GCE was significantly improved compared with CS / GCE and CDs / CS / GCE, which was due to the electrocatalytic oxidation activity of CuCDs on NADH, making the response current more sensitive, and this also confirmed the previous CV results, further confirming that CuCDs had a promoting effect on the electrocatalytic oxidation of NADH.

[0118] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for colorimetric detection of NADH, characterized in that, The method comprises the following steps: mixing the sample to be tested with a reaction solution, reacting at room temperature for 15 min, measuring the absorbance A at 652 nm to obtain ΔA652 nm, and substituting the ΔA652 nm into the formula ΔA652 nm=0.0081[NADH]+0.0326 to calculate the concentration of NADH in the sample to be tested; the ΔA652 nm value is the A652 nm value without the sample to be tested 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 CuCDs; The CuCDs are prepared by the following method: dissolving citric acid monohydrate, urea and CuCl2·2H2O in water, reacting at 180-220 ℃ for 8-12 h, centrifuging to discard the precipitate, filtering to take the filtrate, dialyzing, collecting the product and freeze-drying to obtain the CuCDs; 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 CuCl2·2H2O is 1-4 mM.

2. A method for electrochemically detecting NADH, characterized by, The method comprises the following steps: placing the electrode CuCDs / CS / GCE in PBS, scanning i-t, setting the potential to 0.5-0.8 V, adding the sample to be tested after the current is stable, obtaining the current increment ΔI, substituting the ΔI into the formula ΔI=0.01757[NADH]+0.00174 to calculate the concentration of NADH in the sample to be tested; and the current increment ΔI is the initial I without the sample to be tested minus the I after the sample to be tested is added. The electrode CuCDs / CS / GCE is prepared by the following method: mixing the solution of CuCDs in claim 1 with CS to obtain a mixed solution; and dropping and coating the mixed solution on the surface of GCE to obtain the electrode CuCDs / CS / GCE. The concentration of the solution containing the CuCDs in claim 1 is 3-10 mg / mL, and the final mass-volume fraction of CS in the mixed solution is 0.05%-0.5%.

3. The method of claim 1 or claim 2, wherein, The sample to be tested comprises bacteria and / or cells.

Images