Silver nanocomposite for rapid detection of uric acid

Abstract

The present application relates to the field of nanobiotechnology, and particularly relates to a silver nanocomposite for rapid detection of uric acid, and the preparation steps include: (1) adding diatomite and deionized water into a round bottom flask in sequence, stirring to make them completely dispersed, adjusting the pH of the suspension with sodium hydroxide solution, and obtaining diatomite suspension by water bath heating; (2) adding the diatomite suspension and anhydrous ethanol into a polytetrafluoroethylene liner, then adding sodium citrate aqueous solution and AgNO3 aqueous solution in sequence, and stirring to make them uniformly mixed; loading the liner into an autoclave and placing it into an oven, after hydrothermal reaction, cooling to room temperature, centrifugally separating the reaction product, washing and centrifugally separating the sediment, and drying in a vacuum drying box to obtain the silver nanocomposite. The silver nanocomposite prepared by the application is applied to the detection of uric acid, the detection method is simple, convenient for popularization and application, and can be widely applied to the determination of uric acid content in body fluids.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanobiotechnology, and particularly to a silver nanocomposite for rapid detection of uric acid. BACKGROUND

[0002] Uric acid (UA) is the main end product of purine metabolism in human urine and serum. 98% of uric acid is dissolved in blood in the form of urate, and the normal range of uric acid in blood is 180-420 μmol / L for men and 120-360 μmol / L for women. The daily excretion of uric acid by the kidney accounts for 70% of the total excretion of uric acid, and the normal level of uric acid in urine of healthy adults is 1.49-4.46 mmol / (L·24h). After filtration, reabsorption, secretion and reabsorption of uric acid by the kidney, it is very easy to precipitate and form crystals under acidic conditions. The crystallization of uric acid can cause a series of damage to the renal tubules, leading to kidney damage in hyperuricemia. Therefore, the content of UA in the human body can be indicative of hyperuricemia, gout (Liu T, Health World, 2022, (004), 13-15), renal failure (Wan D M et al, Laboratory Test, 2017, 36(10): 1132-1135) and other diseases. In order to prevent the above diseases and reduce the occurrence of related complications, it is very necessary to develop a rapid, simple and visual method for daily monitoring of UA levels in blood and urine.

[0003] At present, there are many methods for determining UA, such as colorimetric analysis (fluorescence, ultraviolet absorption) (Luo J et al, NANO, 2018, 13:1850085; He Y, Anal. Chim. Acta, 2018, 1021, 113), chemiluminescence method, electrochemical method, high performance liquid chromatography (HPLC) method (Sun L P et al, Journal of Electronic Materials, 2021, 50:3907-3915). Among them, colorimetric analysis is based on the catalytic decomposition of UA by uricase to generate allantoin and H2O2, and H2O2 oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) to form a blue product under the action of catalase. Colorimetric analysis is simple, rapid and sensitive, but the determination process uses two natural enzymes (uricase and catalase), which are expensive and not suitable for daily on-site rapid detection by the public. In order to solve this problem, various artificially synthesized nanoenzymes have been developed to replace natural enzymes.

[0004] Nanoprotease has similar catalytic efficiency and enzymatic reaction kinetics to natural enzymes, and has the advantages of high stability, low cost and easy storage compared with natural enzymes. In recent years, many nanomaterials have been found to have peroxide mimetic enzyme activity, such as many iron oxides Fe3O4, α-Fe2O3, MFe2O4, copper oxide (CuO), vanadium pentoxide (V2O5), manganese oxide nanomaterials, ruthenium oxide, cerium oxide and other metal oxides, noble metal nanoparticles such as gold, platinum and palladium (Cai S F, et al, Nanoscale, 2016, 8(6)), carbon-based nanomaterials (Zeng Y, et al, Appl. Sci., 2017, 7(9): 924-935), and metal organic framework MOFs nanomaterials. However, there are relatively few nanomaterials with oxidase mimetic enzyme catalytic activity. The reported main ones are: CeO2 oxidation mimetic enzyme promotes the oxidation of dopamine in citric acid solution at pH 4 within a few minutes, producing an orange solution (Atul A, et al, Angew. Chem. Int. Ed., 2009, 48: 2308-2312); In the H-Co-ZIF / TMB reaction system, the activity of H-Co-ZIF oxidation mimetic enzyme is inhibited in the presence of dopamine, making the solution color lighter or faded, establishing a colorimetric analysis for determining dopamine (Wang Wei, Master's thesis of Southwest University, 2018); Cluster platinum nanoprotease is prepared by using protamine as a modifier and stabilizer, and the nanoprotease has high uricase-like enzyme activity (Liu Yan, Yangzhou University, Patent Application No. 202010043846.9).

[0005] Diatomite is a natural non-metallic mineral with abundant reserves, which is a diatom fossil deposited by single-cell aquatic plants through a long geological process, and the main component is diatomite (Jiang Shanyu, China Non-Metallic Mineral Industry Journal, 2020(01): 1-4+16). Diatomite has a unique microporous structure, which is composed of a large number of small pores and channels, forming a high specific surface area, and the inside of the body has countless nanoscale micropores, and the surface is rich in silyl hydroxyl and hydrogen bonds. These characteristics make diatomite widely used in the field of adsorbents or filtration materials (Tao X M, et al, Water Air Soil Pollut, 2018, 229: 322).

[0006] At present, there is no related research report on diatomite / Ag nanomaterial as an oxidation mimetic enzyme or peroxide mimetic enzyme. SUMMARY

[0007] In view of the problems existing in the prior art, the purpose of the present application is to provide a metal nanocomposite with uric acid oxidase-like and hydrogen peroxidase-like activity, which can be directly applied to rapid detection of uric acid content in body fluids.

[0008] The object of the present application is achieved by adopting the following technical solutions:

[0009] In a first aspect, the present application provides a silver nanocomposite for rapid detection of uric acid, and the preparation steps thereof include:

[0010] (1) diatomite and deionized water are sequentially added in a round-bottom flask, and stirred to make them completely dispersed, and a sodium hydroxide solution is used to adjust the pH of the suspension, and a water bath is heated to obtain a diatomite suspension;

[0011] (2) the diatomite suspension and anhydrous ethanol are added in a polytetrafluoroethylene liner, and then a citric acid aqueous solution and an AgNO3 aqueous solution are sequentially added, and stirred to make them uniformly mixed; the liner is loaded into an autoclave and placed in an oven, and after hydrothermal reaction, it is cooled to room temperature, and the reaction product is centrifuged to separate, and the precipitate is washed and centrifuged to separate, and then placed in a vacuum drying box to dry to obtain a silver nanocomposite.

[0012] Preferably, in step (1), the solid-liquid ratio of the diatomite to the deionized water is (0.4-1.0) g: 50 mL.

[0013] Preferably, in step (1), the stirring is performed by a magnetic stirring method, and the stirring speed is 150-600 rpm.

[0014] Preferably, in step (1), the concentration of the sodium hydroxide solution is 0.2-0.5 mol / L, and the pH of the suspension is adjusted to 9.0.

[0015] Preferably, in step (1), the water bath temperature is 60-90℃, and the water bath time is 30 min.

[0016] Preferably, in step (2), the amount of the anhydrous ethanol used is 15-40 mL, the mass concentration of the citric acid aqueous solution is 3%-10%, the concentration of the AgNO3 aqueous solution is 0.1-0.3 mol / L, the total volume of the citric acid aqueous solution and the AgNO3 aqueous solution is 11-17 mL, and the volume ratio of the citric acid aqueous solution to the AgNO3 aqueous solution is 1:1-1:4.

[0017] Preferably, the temperature of the hydrothermal reaction is 100-140℃, and the time is 2-4 hours.

[0018] Preferably, the centrifugation is performed at a speed of 6000-10000 rpm for 3-10 min.

[0019] Preferably, the washing is performed by using deionized water for 3-5 times.

[0020] Preferably, the temperature of the vacuum drying box is 40-70℃, and the drying time is 12 h.

[0021] In a second aspect, the present application provides a silver nanocomposite for rapid detection of uric acid.

[0022] The present application has the following advantages:

[0023] (1) The silver nanocomposite prepared by the present application has uric acid oxidase-like activity and hydrogen peroxidase-like activity, which is comparable to natural hydrogen peroxidase and uric acid oxidase (detection limit of 0.07 μmol / L).

[0024] (2) The preparation method of the present application is simple and easy to operate, and is a one-step hydrothermal synthesis method, which has mild reaction conditions, only requires normal pressure operation, low equipment investment cost, is environmentally friendly, and has good stability and reproducibility.

[0025] (3) The silver nanocomposite prepared by the present application has porosity, and the calculated BET surface area is 157.14 m 2 / g, and the total pore volume is 0.044 cm 3 / g.

[0026] (4) The silver nanocomposite prepared by the present application is applied to uric acid detection, and the detection method is simple and convenient for popularization and application, and can be widely applied to determination of uric acid content in body fluids. BRIEF DESCRIPTION OF DRAWINGS

[0027] The present application is further described with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the following drawings.

[0028] Figure 1 is the SEM image of the silver nanocomposite prepared in Example 1 of the present application.

[0029] Figure 2 is the BET image of the silver nanocomposite prepared in Example 1 of the present application.

[0030] Figure 3 is the enzyme-like activity experimental result graph of the silver nanocomposite prepared in Example 1 of the present application. In the graph, a. H2O2; b. H2O2+TMB; c. TMB+Ag nanocomposite; d. H2O2+TMB+diatomite; e. H2O2+TMB+Ag nanocomposite; f. Uric acid+TMB+Ag nanocomposite.

[0031] Figure 4 is the standard working curve graph of the method for detecting urine by using the silver nanocomposite prepared in Example 1 of the present application.

[0032] Figure 5is a result chart of the silver nanocomposite prepared by the embodiment 1 of the present application applied to urine detection of thirteen volunteers. DETAILED DESCRIPTION

[0033] In order to more clearly illustrate the present application, to have a more clear understanding of the technical features, objects and beneficial effects of the present application, the technical solutions of the present application are described in detail as follows, but cannot be understood as limiting the scope of the present application.

[0034] The raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods, unless otherwise specified.

[0035] The present application is further described in conjunction with the following examples.

[0036] Example 1

[0037] A silver nanocomposite for rapid detection of uric acid, the preparation steps include:

[0038] (1) In a 150 mL round bottom flask, add 0.8 g of diatomite, 50 mL of deionized water, and stir at 600 rpm to make it completely dispersed. Adjust the pH of the suspension to 9.0 with 0.4 M sodium hydroxide solution, and heat in a water bath to 80℃, and stir at constant temperature for 30 min to obtain a diatomite suspension.

[0039] (2) In a polytetrafluoroethylene liner, add the above diatomite suspension and 25 mL of anhydrous ethanol, and then add 4 mL of 7% citric acid aqueous solution and 11.2 mL of 0.2M AgNO3 aqueous solution (total volume of the two is 15.2 mL, volume ratio = 1:2.8) in sequence, and stir to mix evenly. Put the liner into an autoclave and put it into an oven, and react at 100℃ for 2 hours, and cool to room temperature. Centrifuge the reaction at 8000 rpm for 5 min, wash the sediment with deionized water for 3-5 times and centrifuge, and then place it in a vacuum drying oven at 50℃ for 12 h to obtain a silver nanocomposite.

[0040] The microstructure and pore size distribution are shown in Figure 1 and Figure 2 . The calculated BET surface area is 157.14 m 2 / g, and the total pore volume is 0.044 cm 3 / g.

[0041] Example 2

[0042] A silver nanocomposite for rapid detection of uric acid, the preparation steps include:

[0043] (1) In a 150 mL round bottom flask, 0.4-1.0 g of diatomite, 50 mL of deionized water, and 0.2-0.5 M NaOH solution were added, and the diatomite was completely dispersed under magnetic stirring at 150-600 rpm. The pH of the suspension was adjusted to 9.0, and the suspension was heated to 60-90 °C in a water bath and stirred at constant temperature for 30 min to obtain a diatomite suspension.

[0044] (2) 3-10% citric acid aqueous solution and 0.1-0.3 M AgNO3 aqueous solution (V:V = 1:1-1:4) were added to the diatomite suspension, and then 15-40 mL of anhydrous ethanol was added. The reaction was carried out for 1-4 h, and the product was cooled and centrifuged at 6000-10000 rpm for 3-10 min. The sediment was washed with deionized water for 3-5 times and centrifuged at 6000-10000 rpm for 3-10 min, and then placed in a vacuum drying oven at 30-70 °C for 12 h to obtain the silver nanocomposite.

[0045] Experimental Example 1

[0046] Enzyme-like activity of the silver nanocomposite prepared in Example 1:

[0047] Six centrifuge tubes were taken, and 2.5 mL of 0.2 M NaAc-HAc buffer solution (pH = 4.0) was added to each of the centrifuge tubes. 200 μL of H2O2 was added to centrifuge tube 1, 200 μL of H2O2 and 100 μL of TMB were added to centrifuge tube 2, 100 μL of TMB solution and 10 mg of Ag nanocomposite were added to centrifuge tube 3, 200 μL of H2O2, 100 μL of TMB, and 10 mg of diatomite were added to centrifuge tube 4, 200 μL of H2O2, 100 μL of TMB, and 10 mg of Ag nanocomposite were added to centrifuge tube 5, and 200 μL of 200 mg·L-1 uric acid solution, 100 μL of TMB, and 10 mg of Ag nanocomposite (H2O2 concentration: 200 μM; TMB concentration: 8 mM) were added to centrifuge tube 6. After ultrasonic mixing for 60 s, the reaction was carried out at 30 °C for 10 min. The test solution in the centrifuge tube was filtered with a 0.22 μm filter membrane, and the absorbance of the filtrate was measured at a wavelength of 652 nm with UV-Vis. The results are shown in -1 Figure 3 It can be seen from Figure 3 that the embedded graph is the color change of oxTMB (TMB oxidation product) solution, and the color gradually deepens from left to right (a→f), and the absorbance value gradually increases. The H2O2 solution alone has no characteristic absorption peak (see Figure 3 a); Figure 3 b) shows that TMB and H2O2 solution coexist without characteristic absorption peak and no obvious color change; Figure 3 c shows that the phenomenon of Ag nanocomposite directly oxidizing TMB to change its color is not obvious;​Figure 3 d indicates that diatomite can catalyze the oxidation discoloration of TMB, but the absorbance value at 652 nm is not very high, and the activity is not strong; Figure 3 e indicates that when the Ag nanocomposite nanomaterial is added, the H2O2 oxidizes the TMB solution to discolor, and there is a stronger characteristic absorption peak at a wavelength of 652 nm, which indicates that the Ag nanocomposite nanomaterial can oxidize TMB by catalyzing H2O2 to generate hydroxyl radicals, the synthesized Ag nanocomposite nanomaterial has a horseradish peroxidase-like activity, and the catalytic ability is 2 times that of diatomite; from Figure 3 f can be seen that the Ag nanocomposite nanomaterial also has the activity of uric acid oxidase and can catalyze the decomposition of uric acid to produce H2O2, and H2O2 generates hydroxyl radicals under the action of the Ag nanocomposite nanomaterial to oxidize TMB to discolor.

[0048] Experimental Example 2

[0049] The silver nanocomposite material prepared in Example 1 is applied to the standard working curve for the detection of uric acid:

[0050] 0.0200 g of uric acid powder is dissolved in 0.4 M NaOH solution, and then deionized water is added to 100 mL to obtain a uric acid standard stock solution, which is placed in a refrigerator for cold storage for standby use. The stock solution is diluted to obtain a uric acid solution with a concentration ranging from 0.2 to 200 μmol·L -1 , 2.5 mL of 0.2 M NaAc-HAc buffer solution (pH = 4.0) and 100 μL of 8 mmol·L -1 TMB color developing solution are added to 300 μL of the uric acid solution with different concentrations, respectively, to form a mixed system, and then 15 mg of the Ag nanocomposite material is added, and the reaction is carried out in a water bath constant temperature at 35℃ for 15 min. The test solution in the centrifugal tube is filtered by a 0.22 μm filter membrane, and the filtrate is measured by UV-Vis for A652nm value. The standard working curve obtained is shown in Figure 4 It can be seen from Figure 4 that the linear equation is y = 0.02375 + 0.00208x (μM) (R = 0.9979), and the detection limit is 0.07 μmol·L -1 .

[0051] Experimental Example 3

[0052] The silver nanocomposite material prepared in Example 1 is applied to the rapid detection of uric acid in human urine samples

[0053] The urine samples of thirteen volunteers (eight women and five men) with an age range of 20-45 years old are selected, and the silver nanocomposite material is used as a uric acid simulation enzyme and a peroxidase simulation enzyme for determination, and a commercially available household medical grade uric acid detector (Kew Medical Technology Co., Ltd.) is used for control determination.

[0054] Urine sample processing method: take 10 mL urine sample in centrifuge tube, centrifuge at 8000 rpm for 5 min. Take supernatant urine, add excess silver nitrate solution, precipitate to remove Cl - , K + , Ag + , Na + in urine. Take 500 μL urine supernatant and dilute 10 times with 0.2 M phosphate buffer solution (pH = 7.8), ready for use. Take 300 μL diluted urine supernatant, add 2.5 mL 0.2 M NaAc-HAc buffer (pH = 4.0), 100 μL 8 mmol·L -1 TMB solution, 15 mg diatomite / Ag composite material, react in 35℃ water bath constant temperature for 15 min, filter the test solution in centrifuge tube with 0.22 μm filter membrane, then measure A 652nm value of sample, parallel determination for 3 times, calculate the content of uric acid in sample by standard curve. At the same time, use KF-UA01-C type uric acid detector (Kew Medical Technology Co., Ltd.) for control determination.

[0055] The determination results are shown in Figure 5 . Black dots in the figure are the uric acid concentrations measured by the patent method, and red dots are the uric acid concentrations measured by the uric acid detector. By comparison, it is found that the uric acid values of the thirteen volunteers are 190-450 μmol·L -1 , and the determination results of the two methods are close, without significant difference, indicating that the silver nano composite material described in the application can be used for detecting the content of uric acid in body fluid.

[0056] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not a limitation on the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A silver nanocomposite material for rapid detection of uric acid, characterized in that, The preparation steps of the silver nanocomposite include: (1) adding diatomite and deionized water in a round-bottom flask in sequence, stirring to make them completely dispersed, adjusting the pH of the suspension with sodium hydroxide solution, and heating in a water bath to obtain a diatomite suspension; (2) adding the diatomite suspension and anhydrous ethanol in a polytetrafluoroethylene liner, then adding sodium citrate aqueous solution and AgNO3 aqueous solution in sequence, stirring to make them uniformly mixed; loading the liner into an autoclave and placing it in an oven, after hydrothermal reaction, cooling to room temperature, centrifugally separating the reaction product, washing and centrifugally separating the sediment, and drying in a vacuum drying box to obtain the silver nanocomposite; The amount of the anhydrous ethanol used is 15-40 mL, the mass concentration of the sodium citrate aqueous solution is 3%-10%, the concentration of the AgNO3 aqueous solution is 0.1-0.3 mol / L, the total volume of the sodium citrate aqueous solution and the AgNO3 aqueous solution is 11-17 mL, and the volume ratio of the sodium citrate aqueous solution to the AgNO3 aqueous solution is 1:1-1:4; In step (1), the concentration of the sodium hydroxide solution is 0.2-0.5 mol / L, and the pH of the suspension is adjusted to 9.

0. The temperature of the hydrothermal reaction is 100-140℃, and the time is 2-4 hours. 2.The silver nanocomposite for rapid detection of uric acid according to claim 1, characterized in that, In step (1), the solid-liquid ratio of the diatomite to the deionized water is (0.4-1.0) g: 50 mL. 3.The silver nanocomposite for rapid detection of uric acid according to claim 1, characterized in that, In step (1), the stirring is performed by a magnetic stirring method, and the stirring speed is 150-600 rpm. 4.The silver nanocomposite for rapid detection of uric acid according to claim 1, characterized in that, In step (1), the water bath temperature is 60-90℃, and the water bath time is 30 min. 5.The silver nanocomposite for rapid detection of uric acid according to claim 1, characterized in that, The washing is performed by using deionized water, and the washing is performed for 3-5 times; the centrifugation is performed at a speed of 6000-10000 rpm for 3-10 min. 6.The silver nanocomposite for rapid detection of uric acid according to claim 1, characterized in that, The temperature of the vacuum drying box is 40-70℃, and the drying time is 12 h.

7. Application of the silver nanocomposite for rapid detection of uric acid according to claim 1 in the detection of uric acid.

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