Glucose oxidase-like combined peroxidase-like cascade system and application thereof

Abstract

The invention provides a glucose oxidase-like combined peroxidase-like cascade system and application thereof. The glucose oxidase-like combined peroxidase-like cascade system comprises glucose oxidase and peroxidase-like. The glucose oxidase-like combined peroxidase-like cascade system provided by the invention can effectively detect glucose in tobacco, is good in specificity, high in recovery rate and low in detectability, and has the characteristics of simplicity in operation, high sensitivity, rapidness and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical analysis and detection, and particularly relates to a glucose oxidase-like enzyme combined peroxidase-like enzyme cascade system and application thereof. BACKGROUND

[0002] Water-soluble sugar is an important component in tobacco and tobacco liquid, and glucose, as one of the important water-soluble sugars in tobacco, can react with basic combustion substances in the process of cigarette combustion, thereby coordinating the acid-base balance of smoke and reducing the irritancy of odor. It has been reported that glucose is a good flavor ingredient carrier, and esterification and glycosylation of glucose can enrich the aroma of cigarettes, reduce the odor, and improve the roundness of smoke. Therefore, it is of great significance to accurately determine the water-soluble reducing sugar in tobacco and its products, especially glucose and fructose. The detection methods of glucose in tobacco include high performance liquid chromatography (HPLC), ion chromatography (IC), near infrared spectroscopy (NIR), etc., which have the advantages of high sensitivity, high accuracy, etc., but require expensive instruments and well-trained personnel.

[0003] Nanocatalysts with intrinsic enzyme-like activity have attracted extensive research interest due to their low cost, high stability, excellent catalytic performance, and easy preparation. Currently, the cascade reaction for glucose determination mainly focuses on the cascade system composed of natural glucose oxidase and nanocatalysts. Nanocatalyst-nanocatalyst cascade is a unique multifunctional enzyme, which uses nanocatalysts to replace natural glucose oxidase, and has advantages in terms of stability and cost. However, how to improve the glucose oxidase-like activity of nanocatalysts has become a research difficulty.

[0004] Therefore, how to provide a nanocatalyst cascade system with high activity, high stability, and low cost for the detection of glucose in tobacco has become a problem to be solved. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a glucose oxidase-like enzyme combined peroxidase-like enzyme cascade system and application thereof. The cascade system provided by the present application can effectively detect glucose in tobacco, has good specificity, high recovery rate, and low detection, and has the characteristics of simple operation, high sensitivity, and rapidness.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a glucose oxidase-like enzyme combined peroxidase-like enzyme cascade system, which comprises a glucose oxidase-like enzyme and a peroxidase-like enzyme.

[0008] The glucose oxidase-like enzyme is prepared by a method comprising the following steps:

[0009] (1) mixing 2-amino terephthalic acid, ZrCl4, acetic acid under ultrasonic, and then reacting to obtain UiO-66-NH2;

[0010] (2) mixing UiO-66-NH2 obtained in step (1) with chloroauric acid and sodium borohydride under ultrasonic, and then stirring with AgNO3, PVP (polyvinylpyrrolidone) and sodium borohydride to obtain the glucose oxidase-like enzyme UiO-66-NH2@Au@Ag;

[0011] The peroxidase-like enzyme is prepared by a method comprising the following steps:

[0012] Mixing L-arginine, copper salt and cerium salt with water under ultrasonic, and then microwave digestion to obtain the peroxidase-like enzyme Ce,Cu / Arg-CDs.

[0013] The above-mentioned glucose oxidase-like enzyme combined with the peroxidase-like enzyme cascade system uses UiO-66-NH2 as a typical metal organic framework (MOF) material, which has the advantages of mild synthesis conditions, high specific surface area and good biocompatibility. Gold and silver nanoparticles (Au, AgNPs) with adjustable exposed crystal surfaces are controllably anchored on the UiO-66-NH2 hexahedron (UiO-66-NH2@Au@Ag), which exhibits glucose oxidase-like enzyme (GOx) activity. At the same time, a Ce,Cu / Arg-CDs nanoscale enzyme with double-metal synergistic enhancement is prepared by a simple and environmentally friendly one-pot aqueous phase method. Cu 2+ and Ce 3+ are combined with L-arginine through strong coordination. Due to electron transfer, Ce 3+ / Ce 4+ and Cu 2+ / Cu + oxidation-reduction pairs promote the conversion of H2O2 into more active free radical species, increase the peroxidase-like enzyme activity, and only have peroxidase-like enzyme activity without oxidase-like enzyme activity, which provides conditions for the cascade reaction. Finally, it can be effectively used for detecting glucose in tobacco, has good specificity, high recovery rate and low detection, and has the characteristics of simple operation, high sensitivity and rapidness.

[0014] Preferably, the mass ratio of 2-amino terephthalic acid to ZrCl4 in step (1) is (7-8):(9-12), wherein the fraction of 2-amino terephthalic acid can be 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, and the fraction of ZrCl4 can be 9, 9.5, 10, 10.5, 11, 11.5 or 12, but is not limited to the above-mentioned values. Other values not listed in the above-mentioned value range are also applicable.

[0015] Preferably, the feed ratio of 2-amino terephthalic acid to acetic acid in step (1) is (7-8): (120-160) g / mL, wherein the fraction of 2-amino terephthalic acid can be 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, etc., and the fraction of acetic acid can be 120, 130, 140, 150 or 160, etc., but is not limited to the above-mentioned values. Other values not listed within the above-mentioned value range are also applicable.

[0016] Preferably, in the mixing and stirring with chloroauric acid and sodium borohydride in step (2), chloroauric acid and sodium borohydride are mixed in the form of a solution.

[0017] Preferably, in the mixing and stirring with chloroauric acid and sodium borohydride in step (2), the feed ratio of UiO-66-NH2 to chloroauric acid solution to sodium borohydride solution is (20-30): (200-300): (100-150) mg / μL / μL, wherein the fraction of UiO-66-NH2 can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc., the fraction of chloroauric acid solution can be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300, etc., and the fraction of sodium borohydride solution can be 100, 110, 120, 130, 140 or 150, etc., but is not limited to the above-mentioned values. Other values not listed within the above-mentioned value range are also applicable.

[0018] Preferably, in the mixing and stirring with chloroauric acid and sodium borohydride in step (2), the concentration of the chloroauric acid solution is 40-60 mmol / L, and the concentration of the sodium borohydride solution is 8-12 mmol / L, wherein the concentration of the chloroauric acid solution can be 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L or 60 mmol / L, etc., and the concentration of the sodium borohydride solution can be 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L or 12 mmol / L, etc., but is not limited to the above-mentioned values. Other values not listed within the above-mentioned value range are also applicable.

[0019] Preferably, in the mixing and stirring with AgNO3, PVP and sodium borohydride in step (2), AgNO3, PVP and sodium borohydride are mixed in the form of a solution.

[0020] Preferably, in the mixing and stirring with AgNO3, PVP, sodium borohydride of step (2), the liquid ratio of UiO-66-NH2 to AgNO3 solution, PVP solution, sodium borohydride solution is (20-30):(400-600):(100-150):(100-150) g / mL / mL / mL, wherein the fraction of UiO-66-NH2 can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, etc., the fraction of AgNO3 solution can be 400, 420, 440, 460, 480, 500, 520, 540, 560, 580 or 600, etc., the fraction of PVP solution can be 100, 110, 120, 130, 140 or 150, etc., and the fraction of sodium borohydride solution can be 100, 110, 120, 130, 140 or 150, etc., but not limited to the above listed values, other values not listed within the above value range are also applicable.

[0021] Preferably, in the mixing and stirring with AgNO3, PVP, sodium borohydride of step (2), the concentration of AgNO3 solution is 8-12 mmol / L, the weight / volume ratio of PVP solution is 0.8-1.2%, and the concentration of sodium borohydride solution is 8-12 mmol / L, wherein the concentration of AgNO3 solution can be 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L or 12 mmol / L, etc., the weight / volume ratio of PVP solution can be 0.8%, 0.9%, 1%, 1.1% or 1.2%, etc., and the concentration of sodium borohydride solution can be 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L or 12 mmol / L, etc., but not limited to the above listed values, other values not listed within the above value range are also applicable.

[0022] Preferably, the mixing and stirring time with chloroauric acid and sodium borohydride of step (2) is 30-40 min, for example 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min or 40 min, etc., but not limited to the above listed values, other values not listed within the above value range are also applicable.

[0023] Preferably, the mixing and stirring time with AgNO3, PVP, sodium borohydride of step (2) is 2-3 h, for example 2 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, 2.6 h, 2.7 h, 2.8 h, 2.9 h or 3 h, etc., but not limited to the above listed values, other values not listed within the above value range are also applicable.

[0024] Preferably, the mass ratio of the L-arginine, copper salt, and cerium salt is (7-8):(2.5-2.8):(11-15), wherein the fraction of L-arginine can be 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8, etc., the fraction of copper salt can be 2.5, 2.6, 2.7, or 2.8, etc., and the fraction of cerium salt can be 11, 12, 13, 14, or 15, etc., but is not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0025] Preferably, the copper salt comprises CuCl2·2H2O.

[0026] Preferably, the cerium salt comprises Ce(NH4)2(NO3).

[0027] Preferably, the time of microwave digestion is 100-150 min, for example, 100 min, 110 min, 120 min, 130 min, 140 min, or 150 min, etc., but is not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0028] In a second aspect, the application provides the use of the glucose oxidase-like enzyme combined peroxidase-like enzyme cascade system as described above in the detection of glucose.

[0029] In a third aspect, the application further provides a glucose detection method, which comprises the following steps:

[0030] The sample to be tested is mixed with a first buffer solution and a UiO-66-NH2@Au@Ag solution, and then mixed with a second buffer solution, a Ce, Cu / Arg-CDs solution, and a TMB (3,3',5,5'-tetramethylbenzidine) solution, the absorbance of the solution is determined, and the glucose concentration in the sample to be tested is calculated according to the linear relationship between the glucose concentration and the absorbance.

[0031] The sample to be tested is a tobacco sample or a tobacco product.

[0032] The above-mentioned method uses glucose as a GOx-like substrate, which can be oxidized by UiO-66-NH2@Au@Ag into gluconic acid and H2O2, and then the peroxidase activity of Ce, Cu / Arg-CDs is used to produce blue oxTMB by taking TMB as a substrate, thus a new type of nano-enzyme-nano-enzyme cascade catalytic reaction is established for the detection of glucose, the detection limit is low, and the detection sensitivity is high. Other tobacco coexisting substances and water-soluble sugars do not interfere with the determination, the method has good selectivity, and can meet the detection requirements of glucose in tobacco.

[0033] Preferably, the volume ratio of the sample to be tested, the first buffer solution, and the UiO-66-NH2@Au@Ag solution is (80-120):(50-100):(200-400), wherein the sample to be tested can be 80, 90, 100, 110, or 120, etc., the UiO-66-NH2@Au@Ag solution can be 200, 250, 300, 350, or 400, etc., and the first buffer solution can be 50, 60, 70, 80, 90, or 100, etc., but is not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0034] Preferably, the first buffer solution is a HEPES buffer solution.

[0035] Preferably, the concentration of the UiO-66-NH2@Au@Ag solution is 0.8-1.2 mg / mL, for example, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, or 1.2 mg / mL, etc., but is not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0036] Preferably, the temperature for mixing the sample to be tested, the first buffer solution, and the UiO-66-NH2@Au@Ag solution is 36-38℃, and the time is 30-40 min, wherein the temperature can be 36℃, 36.5℃, 37℃, 37.5℃, or 38℃, etc., and the time can be 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, or 40 min, etc., but is not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0037] Preferably, the volume ratio of the sample to be tested, the second buffer solution, the Ce,Cu / Arg-CDs solution, and the TMB solution is (80-120):(200-400):(50-100):(50-100), wherein the sample to be tested can be 80, 90, 100, 110, or 120, etc., the second buffer solution can be 200, 250, 300, 350, or 400, etc., the Ce,Cu / Arg-CDs solution can be 50, 60, 70, 80, 90, or 100, etc., and the TMB solution can be 50, 60, 70, 80, 90, or 100, etc., but is not limited to the above-mentioned values, and other values not mentioned in the above-mentioned value range are also applicable.

[0038] Preferably, the second buffer solution is a HAc-NaAc buffer solution.

[0039] Preferably, the concentration of the Ce,Cu / Arg-CDs solution is 0.8-1.2 mg / mL, for example 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL or 1.2 mg / mL, etc., but not limited to the above listed values, other values not listed in the above value range are also applicable.

[0040] Preferably, the concentration of the TMB solution is 18-22 mmol / L, for example 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L or 22 mmol / L, etc., but not limited to the above listed values, other values not listed in the above value range are also applicable.

[0041] Preferably, the temperature for mixing reaction with the second buffer solution, the Ce,Cu / Arg-CDs solution and the TMB solution is 36-38℃, and the time is 10-15 min, wherein the temperature can be 36℃, 36.5℃, 37℃, 37.5℃ or 38℃, etc., and the time can be 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc., but not limited to the above listed values, other values not listed in the above value range are also applicable.

[0042] Preferably, the linear relationship between glucose concentration and absorbance is obtained by a method comprising the following steps:

[0043] Mixing a series of glucose standard solutions with the first buffer solution and the UiO-66-NH2@Au@Ag solution, and then mixing with the second buffer solution, the Ce,Cu / Arg-CDs solution and the TMB solution, measuring the absorbance of the solution, and determining the linear relationship between glucose concentration and absorbance according to the results.

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

[0045] 1、The present application uses UiO-66-NH2 as a typical metal organic framework (MOF) material, which has the advantages of mild synthesis conditions, high specific surface area and good biocompatibility, etc., and uses it as a carrier to anchor gold and silver nanoparticles (Au, AgNPs) with adjustable exposed crystal surfaces on the UiO-66-NH2 hexahedron (UiO-66-NH2@Au@Ag), which exhibits glucose oxidase (GOx) activity; at the same time, a Ce,Cu / Arg-CDs nanoscale enzyme with double metal synergistic enhancement is prepared by a simple and environmentally friendly one-pot aqueous phase method, wherein Cu 2+ and Ce 3+ binds with L-arginine through strong coordination, and Ce 3+ / Ce 4+ and Cu2 + / Cu + Redox couples promote the conversion of H2O2 into more active radical species, increasing its peroxidase-like activity, and only peroxidase-like activity, no oxidase activity, providing conditions for cascade reactions.

[0046] 2, glucose as a GOx-like substrate, which can be oxidized by UiO-66-NH2@Au@Ag to glucose acid and H2O2, and then using Ce, Cu / Arg-CDs peroxidase activity, TMB as a substrate, TMB binds H2O2 to produce blue oxTMB, therefore, a new type of nanoscale enzyme-nanoscale enzyme cascade catalytic reaction for the detection of glucose is established, the detection limit is low, which can meet the detection requirements of glucose in tobacco.

[0047] 3, the glucose detection method established by the application has high detection sensitivity, and other tobacco coexisting substances and water-soluble sugars do not interfere with the determination, and the method has good selectivity. DETAILED DESCRIPTION

[0048] Figure 1 TEM diagram of UiO-66-NH2@Au@Ag prepared in example 1 of the application;

[0049] Figure 2 TEM diagram of Ce, Cu / Arg-CDs prepared in example 1 of the application;

[0050] Figure 3 XPS diagram of Ce3d of Ce, Cu / Arg-CDs prepared in example 1 of the application;

[0051] Figure 4 XPS diagram of Cu2p of Ce, Cu / Arg-CDs prepared in example 1 of the application;

[0052] Figure 5 UV absorption spectrum diagram of H2O2 detection in the glucose oxidation system in example 1 of the application;

[0053] Figure 6 UV absorption spectrum diagram of glucose acid detection in the glucose oxidation system in example 1 of the application;

[0054] Figure 7 UV absorption spectrum diagram of Ce, Cu / Arg-CDs catalyzing TMB+H2O2 and TMB;

[0055] Figure 8 UV spectrum diagram of different concentrations of Glu based on UiO-66-NH2@Au@Ag and Ce, Cu / Arg-CDs cascade reaction in example 1 of the application;

[0056] Figure 9 Linear relationship curve of different concentrations of Glu based on UiO-66-NH2@Au@Ag and Ce, Cu / Arg-CDs cascade reaction in embodiment 1 of the present application;

[0057] Figure 10 Figure of influence result of coexisting substances of the method of the present application on Glu detection. DETAILED DESCRIPTION

[0058] The technical solutions of the present application are further illustrated by specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0059] In the following examples, deionized water is used as the solvent of the solution unless otherwise specified.

[0060] Determination of glucose in tobacco sample in embodiment 1

[0061] 1. 0.350 g of 2-amino terephthalic acid and 0.450 g of ZrCl4 were weighed, 120 mL of N,N-dimethylformamide (DMF) and 6 mL of acetic acid were added, and ultrasonic treatment was performed for 20 min. The mixture was transferred to a high-pressure reaction kettle, and reaction was performed in a muffle furnace at 120℃ for 24 h. After cooling to room temperature, the mixture was washed with DMF and acetone alternately for 3 times, and vacuum drying was performed, thereby obtaining UiO-66-NH2.

[0062] 2. 20 mg of UIO-66-NH2 was weighed, 20 mL of anhydrous ethanol was added, and ultrasonic treatment was performed for 20 min. 200 μL of a 50 mmol / L chloroauric acid solution and 100 μL of a 10 mmol / L sodium borohydride solution were added. After stirring at 20℃ for 30 min, 400 μL of a 10 mmol / L AgNO3 solution and 100 μL of a 1% PVP solution (weight / volume) were added. Finally, 100 μL of a 10 mmol / L sodium borohydride solution was added, and stirring was continued for 2 h. Centrifugation, deionized water washing, and vacuum drying were performed, thereby obtaining UiO-66-NH2@Au@Ag. The morphology of UiO-66-NH2@Au@Ag was characterized by TEM, and the results are shown in Figure 1 Ce-UiO-66 / Zn still maintained the overall hexahedral shape, but its surface became rougher, indicating that in-situ reduction of HAuCl4 and AgNO3 occurred on the surface of UiO-66-NH2. At the same time, Au, AgNPs were observed to be distributed on the surface of UiO-66-NH2, confirming the successful synthesis of UiO-66-NH2@Au@Ag.

[0063] 3. Weigh 0.700g L-arginine, 0.250g CuCl2·2H2O, and 1.100g Ce(NH4)2(NO3), add them to 15mL deionized water, and sonicate for 15min. Transfer the mixed solution to a polytetrafluoroethylene high-pressure reactor and microwave digest it at 1000W, reaching 120℃ within 1min and maintaining the temperature for 120min. After cooling the solution to 20℃, centrifuge and then filter through a 0.22μm filter membrane to remove solid particles, obtaining Ce,Cu / Arg-CDs as the filtrate. The morphology of Ce,Cu / Arg-CDs was characterized by TEM, and the results are as follows: Figure 2 Ce,Cu / Arg-CDs exhibit a nanoneedle-like structure; high-resolution XPS spectra of Ce 3d ( Figure 3 The results show that Ce, on the Ce and Cu / Arg-CDs surfaces, respectively, is represented by Ce. 4+ and Ce 3+ The coexistence of these values, 882.38 eV, 885.98 eV, and 900.48 eV, corresponds to Ce. 3+ 904.08 eV corresponds to Ce 4+ This confirms that Ce,Cu / Arg-CDs contain Ce. 4+ and Ce 3+ Coexistence; Cu2p ( Figure 4 The high-resolution XPS spectrum shows that Cu exists in multiple valence states (Cu... + Cu 2+ It exists, located at 933.08 eV in Cu 2p 1 / 2 Corresponding to Cu + Cu 2p at 934.78 eV 1 / 2 Characteristic peaks and Cu 2p at 954.68 eV 3 / 2 Corresponding to Cu 2+ .

[0064] 4. Evaluation of glucose oxidase activity of UiO-66-NH2@Au@Ag nanozymes: To detect the glucose oxidase activity of UiO-66-NH2@Au@Ag nanozymes, Fe... 3+The gluconic acid and H2O2 produced by glucose oxidation were detected by the hydroxylamine method and the horseradish peroxidase (HPR) / TMB colorimetric method. 200 μL of 1 mg / mL UiO-66-NH2@Au@Ag solution was added to a mixture containing 100 μL of 0.1 mol / L HEPES buffer solution (pH 7.4) and 200 μL of deionized water. Then, 400 μL of 500 μmol / L glucose solution was added to the mixture. After reacting at 37°C for 30 min, 50 μL of 1 mg / mL HPR solution, 400 μL of 0.1 mol / L HAc-NaAc pH 4.0 buffer solution, and 100 μL of 5 mmol / L TMB solution were added. The volume was then adjusted to 3 mL with deionized water, and the reaction was carried out at 37°C for 10 min. Figure 5 As shown, ox-TMB is generated, exhibiting significant absorbance at 654 nm, indicating the formation of H2O2. Separately, 100 μL of 1 mg / mL UiO-66-NH2@Au@Ag solution was added to a mixture containing 100 μL of 0.1 mol / L HEPES buffer solution (pH 7.4) and 200 μL of deionized water. Then, 50 μL of 500 μmol / L glucose solution was added to the mixture. After reacting at 37°C for 30 min, 1 mL of solution A (5 mmol / L EDTA and 0.15 mmol / L trimethylamine aqueous solution) and 100 μL of solution B (3 mol / L hydroxylamine solution) were added, and the mixture was incubated for 25 min. Then, 500 μL of solution C (1 mol / L HCl, 0.10 mol / L FeCl3 and 0.25 mol / L CCl3COOH) was injected into the mixture. After 5 min, the absorbance was measured. Figure 6 As shown, gluconic acid can react with hydroxylamine and Fe... 3+ The reaction produces a red complex, which significantly enhances the absorbance, confirming the formation of gluconic acid.

[0065] 5. Evaluation of Ce,Cu / Arg-CDs nanozyme peroxidase activity: Take 100 μL of 5 mmol / L TMB solution, add 100 μL of 50 mmol / L H2O2 solution and 50 μL of 1 mg / mL Ce,Cu / Arg-CDs nanozyme solution respectively, add 0.1 mol / L HAc-NaAc pH 4.0 buffer solution to make up to 3 mL, shake well, let stand for 10 min, and measure the absorbance at 654 nm. Figure 7The results show that the Ce, Cu / Arg-CDs nanoscale enzyme has good peroxidase activity simulation; under the same conditions, without adding H2O2, the Ce, Cu / Arg-CDs nanoscale enzyme oxidase activity is evaluated, and the results show that the Ce, Cu / Arg-CDs nanoscale enzyme has almost no oxidase activity, which provides conditions for glucose cascade determination.

[0066] 6. Glucose (Glu, Glucose) working curve preparation: 200 μL of 1 mg / mL UiO-66-NH2@Au@Ag solution was added to a mixture containing 100 μL of 0.1 mol / L pH 7.4 HEPES buffer solution and 200 μL of deionized water, and then 10-700 μmol / L of glucose standard solution was added to the above mixture, and after 30 min of reaction at 37°C, 50 μL of 1 mg / mL Ce, Cu / Arg-CDs nanoscale enzyme solution, 400 μL of 0.1 mol / L HAc-NaAc pH 4.0 buffer solution, and 100 μL of 5 mmol / L TMB solution were added, and the volume was made up to 3 mL with deionized water, and the reaction was carried out at 37°C for 10 min. The ultraviolet absorption spectrum was determined at 400-600 nm, as shown in Figure 8 , and the absorbance was determined at 654 nm. The standard curve was drawn with glucose concentration as the abscissa and absorbance at 654 nm as the ordinate, and the regression equation was obtained, as shown in Figure 9 ; the regression equation, correlation coefficient, relative standard deviation, and linear range are shown in Table 1.

[0067] Table 1 Linear equation, correlation coefficient, relative standard deviation, and linear range

[0068]

[0069] 7. Method specificity investigation: The selectivity of glucose determination was investigated. Glucose and other possible coexisting substances were mixed, and the influence of coexisting substances on the detection system was detected. The concentration of glucose was 100 μmol / L, and the concentration of the rest of the interfering substances was 500 μmol / L, Figure 10 is the influence of coexisting substances (maltose, sucrose, lactose, galactose, fructose, citric acid, xylose, maltose, arabinose, and glutamic acid) on glucose. From the figure, it can be seen that the nanoscale enzyme catalytic system only has obvious catalytic effect on glucose, and other substances have almost no effect, and the method has good selectivity.

[0070] 8. Determination of glucose in cigarette samples

[0071] (1) Sample pretreatment: refer to YC / T31-1996 "Preparation of tobacco and tobacco products and determination of moisture by oven method", place 3 different brand cigarette tobacco samples in an oven at (40±1) °C for 24 h. After taking out, crush, pass through a 60 mesh sieve, and place the intermediate trapped particles in a sealed bag. After equilibrating at 20 °C for 48 h, weigh (0.1±0.0001) g of the above sample into a 50 mL centrifuge tube, add 20 mL of ultrapure water, and then oscillate for extraction at 200 r / min on a constant temperature water bath oscillator at (30±0.5) °C for 30 min. Centrifuge and the supernatant is the determination solution.

[0072] (2) Determination of glucose in cigarette samples: add 200 μL of 1 mg / mL UiO-66-NH2@Au@Ag solution to a mixture containing 100 μL of 0.1 mol / L HEPES buffer solution at pH 7.4 and 200 μL of deionized water, then add 100 μL of sample determination solution prepared in step (1) to the above mixture, react at 37 °C for 30 min, then add 50 μL of 1 mg / mL Ce, Cu / Arg-CDs nanoscale enzyme solution, 400 μL of 0.1 mol / L HAc-NaAc buffer solution at pH 4.0, and 100 μL of 20 mmol / L TMB solution, and then dilute to 3 mL with deionized water, react at 37 °C for 10 min, and then measure the absorbance at 654 nm. Enter the regression equation and the content of glucose is 510 μmol / L.

[0073] Example 2 Determination of glucose in cigarette samples

[0074] 1. Weigh 0.370 g of 2-amino terephthalic acid and 0.500 g of ZrCl4, add 140 mL of N,N-dimethylformamide (DMF) and 7 mL of acetic acid, ultrasonic treat for 25 min, transfer to a high-pressure reaction kettle, react in a muffle furnace at 130 °C for 24 h, cool to room temperature, and then wash with DMF and acetone alternately for 3 times, and then vacuum dry at 60 °C for 24 h to obtain UiO-66-NH2. 2. Weigh 25 mg of UIO-66-NH2, add 25 mL of anhydrous ethanol and ultrasonic treat for 25 min, add 250 μL of 50 mmol / L chloroauric acid solution and 120 μL of 10 mmol / L sodium borohydride solution, stir at 20 °C for 35 min, then add 500 μL of 10 mmol / L AgNO3 solution, 120 μL of 1% PVP solution (weight / volume), and finally add 120 μL of 10 mmol / L sodium borohydride solution, continue to stir for 2.5 h, centrifuge at 8000 r / min for 10 min, wash with deionized water, and then vacuum dry at 60 °C for 24 h to obtain UiO-66-NH2@Au@Ag.

[0075] 3. 0.750 g of L-arginine, 0.265 g of CuCl2·2H2O, 1.250 g of Ce(NH4)2(NO3) were weighed and added to 20 mL of deionized water, and ultrasonic treatment was performed for 20 min. The mixed solution was transferred to a polytetrafluoroethylene high-pressure reaction kettle, and microwave digestion was performed at 1000 W. The temperature reached 120°C within 1 min and was maintained for 100 min. After the solution was cooled to 20°C, centrifugal treatment was performed at 8000 r / min for 10 min. Then, the solid particles were removed by filtration through a 0.22 μm filter membrane to obtain a filtrate Ce, Cu / Arg-CDs.

[0076] 4. Glucose (Glu, Glucose) working curve preparation: same as Example 1.

[0077] 5. Determination of glucose in a cigarette sample

[0078] (1) Sample pretreatment: same as Example 1.

[0079] (2) Determination of glucose in a cut tobacco sample: the determination result was 601 μmol / L.

[0080] Example 3 Determination of glucose in a cigarette sample

[0081] 1. 0.400 g of 2-amino terephthalic acid and 0.600 g of ZrCl4 were weighed and added to 150 mL of N,N-dimethylformamide (DMF) and 8 mL of acetic acid, and ultrasonic treatment was performed for 30 min. The mixture was transferred to a high-pressure reaction kettle, and a muffle furnace reaction was performed at 140°C for 24 h. After cooling to room temperature, the mixture was washed with DMF and acetone alternately for 3 times, and vacuum drying was performed at 60°C for 24 h to obtain UiO-66-NH2.

[0082] 2. 30 mg of UIO-66-NH2 was weighed and added to 30 mL of anhydrous ethanol and ultrasonic treatment was performed for 30 min. 300 μL of a 50 mmol / L chloroauric acid solution and 150 μL of a 10 mmol / L sodium borohydride solution were added, and stirring was performed at 20°C for 40 min. Then, 600 μL of a 10 mmol / L AgNO3 solution and 150 μL of a 1% PVP solution (weight / volume) were added, and finally 150 μL of a 10 mmol / L sodium borohydride solution was added. Stirring was continued for 3 h, and centrifugal treatment was performed at 8000 r / min for 10 min. Deionized water was used for washing, and vacuum drying was performed at 60°C for 24 h to obtain UiO-66-NH2@Au@Ag.

[0083] 3. Take 0.800 g L-arginine, 0.280 g CuCl2·2H2O, 1.500 g Ce(NH4)2(NO3) and add to 20 mL deionized water, ultrasonic treatment for 20 min, transfer the mixed solution to a polytetrafluoroethylene high-pressure reaction kettle, microwave digestion at 1000 W, reach 120℃ in 1 min and keep for 150 min, after the solution is cooled to 20℃, centrifugal treatment at 10000 r / min for 5 min, then remove solid particles by 0.22 μm filter membrane to obtain the filtrate Ce,Cu / Arg-CDs.

[0084] 4. Glucose (Glu, Glucose) working curve preparation: same as example 1.

[0085] 5. Determination of glucose in cigarette samples

[0086] (1) Sample pretreatment: same as example 1.

[0087] (2) Determination of glucose in cut tobacco sample: the determination result is 535 μmol / L.

[0088] Recovery rate and precision experiment: add 3 different concentrations of glucose standard solution in the cigarette samples of example 1-3 respectively; determine 3 times for each concentration, calculate the recovery rate of standard addition, and calculate the relative standard deviation RSD, the results are shown in table 2; the measured recovery rate of standard addition of glucose is 96.7-102.1%, and the RSD is 2.09-4.01%, the method has good accuracy and precision; at the same time, the sample is determined by high performance liquid chromatography, and the results have no significant difference.

[0089] Table 2 sample recovery rate of standard addition and RSD (n=3)

[0090]

[0091]

[0092] From the above data, it can be found that the glucose determination method established by the application has the advantages of less processing steps, short processing time, low processing cost, simple operation, method specificity, and no need for large-scale instrument equipment, and has strong advantages in actual detection.

[0093] The applicant declares that the above examples are used to illustrate the glucose oxidase-like enzyme combined peroxidase-like enzyme cascade system and its application of the application, but the application is not limited to the above examples, that is, it does not mean that the application must rely on the above examples to be implemented. Those skilled in the art should understand that any improvement of the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the application.

[0094] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0095] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combinations.

Claims

1. A glucose oxidase-like and peroxidase-like cascade system, characterized in that, The glucose oxidase-peroxidase cascade system includes glucose oxidase and peroxidase; The glucose oxidase-like enzyme was prepared by a method comprising the following steps: (1) Mix 2-aminoterephthalic acid, ZrCl4 and acetic acid and sonicate, then react to obtain UiO-66-NH2; (2) After sonicating the UiO-66-NH2 obtained in step (1), mix it with chloroauric acid and sodium borohydride, and then mix it with AgNO3, PVP and sodium borohydride to obtain the glucose oxidase UiO-66-NH2@Au@Ag. The peroxidase-like enzyme was prepared by a method comprising the following steps: L-arginine, copper salt, cerium salt and water were mixed and sonicated, followed by microwave digestion to obtain the peroxidase-like Ce,Cu / Arg-CDs.

2. The glucose oxidase-like and peroxidase-like cascade system according to claim 1, characterized in that, The mass ratio of 2-aminoterephthalic acid to ZrCl4 in step (1) is (7-8):(9-12); Preferably, the ratio of 2-aminoterephthalic acid to acetic acid in step (1) is (7-8):(120-160) g / mL.

3. The glucose oxidase-like and peroxidase-like cascade system according to claim 1 or 2, characterized in that, In step (2), during the mixing and stirring of chloroauric acid and sodium borohydride, chloroauric acid and sodium borohydride are mixed in solution form; Preferably, in step (2), during the mixing and stirring of UiO-66-NH2 with chloroauric acid solution and sodium borohydride solution, the material-to-liquid ratio is (20-30):(200-300):(100-150) mg / μL / μL. Preferably, in step (2), during the mixing and stirring of chloroauric acid and sodium borohydride, the concentration of the chloroauric acid solution is 40-60 mmol / L, and the concentration of the sodium borohydride solution is 8-12 mmol / L. Preferably, the mixing and stirring time with chloroauric acid and sodium borohydride in step (2) is 30-40 minutes; Preferably, the mixing and stirring time with AgNO3, PVP and sodium borohydride in step (2) is 2-3 hours.

4. The glucose oxidase-like and peroxidase-like cascade system according to any one of claims 1-3, characterized in that, In step (2), during the mixing and stirring of AgNO3, PVP, and sodium borohydride, AgNO3, PVP, and sodium borohydride are mixed in solution form. Preferably, in step (2), during the mixing and stirring of AgNO3, PVP, and sodium borohydride, the material-to-liquid ratio of UiO-66-NH2 to AgNO3 solution, PVP solution, and sodium borohydride solution is (20-30):(400-600):(100-150):(100-150)g / mL / mL / mL; Preferably, in step (2), during the mixing and stirring of AgNO3, PVP, and sodium borohydride, the concentration of the AgNO3 solution is 8-12 mmol / L, the weight-volume ratio of the PVP solution is 0.8-1.2%, and the concentration of the sodium borohydride solution is 8-12 mmol / L.

5. The glucose oxidase-like and peroxidase-like cascade system according to any one of claims 1-4, characterized in that, The mass ratio of L-arginine, copper salt, and cerium salt is (7-8):(2.5-2.8):(11-15); Preferably, the copper salt comprises CuCl2·2H2O; Preferably, the cerium salt comprises Ce(NH4)2(NO3); Preferably, the microwave digestion time is 100-150 min.

6. The application of a glucose oxidase-like and peroxidase-like cascade system according to any one of claims 1-5 in glucose detection.

7. A method for detecting glucose, characterized in that, The glucose detection method includes the following steps: The sample to be tested was mixed with the first buffer solution and UiO-66-NH2@Au@Ag solution and reacted. Then it was mixed with the second buffer solution, Ce,Cu / Arg-CDs solution and TMB solution and reacted. The absorbance of the solution was measured. The glucose concentration in the sample to be tested was calculated based on the linear relationship between glucose concentration and absorbance.

8. The detection method according to claim 7, characterized in that, The volume ratio of the sample to be tested to the first buffer solution and the UiO-66-NH2@Au@Ag solution is (80-120):(50-100):(200-400); Preferably, the first buffer solution is a HEPES buffer solution; Preferably, the concentration of the UiO-66-NH2@Au@Ag solution is 0.8-1.2 mg / mL; Preferably, the reaction temperature for mixing with the first buffer solution and the UiO-66-NH2@Au@Ag solution is 36-38°C, and the reaction time is 30-40 min.

9. The detection method according to claim 7 or 8, characterized in that, The volume ratio of the sample to be tested to the second buffer solution, Ce,Cu / Arg-CDs solution, and TMB solution is (80-120):(200-400):(50-100):(50-100); Preferably, the second buffer solution is an HAc-NaAc buffer solution; Preferably, the concentration of the Ce,Cu / Arg-CDs solution is 0.8-1.2 mg / mL; Preferably, the concentration of the TMB solution is 18-22 mmol / L; Preferably, the reaction temperature for mixing with the second buffer solution, Ce,Cu / Arg-CDs solution, and TMB solution is 36-38°C, and the reaction time is 10-15 min.

10. The detection method according to any one of claims 7-9, characterized in that, The linear relationship between glucose concentration and absorbance is obtained by a method including the following steps: A series of glucose standard solutions of different concentrations were mixed and reacted with a first buffer solution and a UiO-66-NH2@Au@Ag solution, and then mixed and reacted with a second buffer solution, a Ce,Cu / Arg-CDs solution, and a TMB solution. The absorbance of the solutions was measured, and the linear relationship between glucose concentration and absorbance was determined based on the results.

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