A method for quantitative detection of uric acid based on non-enzymatic colorimetric sensing

Abstract

The present invention provides a method for quantitative detection of uric acid based on enzyme-free colorimetric sensing, belonging to the technical field of analysis and detection. The present invention obtains a blue product TMBox by mixing a buffer solution, a porous Co3O4 catalyst and 3,3',5,5'-tetramethylbenzidine and then filtering; then uric acid liquid is added to reduce TMBox, and after the reaction is completed, the uric acid concentration is judged according to the absorbance change of TMBox. The Co3O4 prepared by the present invention can catalyze the oxidation of TMB without hydrogen peroxide, and the method for quantitative detection of uric acid by enzyme-free colorimetric sensing established based on this does not require natural enzymes and artificial enzymes, and the uric acid detection range is wide (0-600 μmol / L) and the detection limit is low (0.33 μmol / L). The method for quantitative detection of uric acid by enzyme-free colorimetric sensing constructed by the present invention is applied to quantitative detection of uric acid, and the detection method is simple, convenient for promotion and application, and the detection time period is only 2-4min, which can be widely used in the determination of uric acid content in body fluids.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and specifically relates to a method for quantitative detection of uric acid based on non-enzyme colorimetric sensing. Background Art

[0002] Uric acid (UA, 2,6,8-trihydroxypurine) is one of the final products of purine metabolism in the body. Humans lack the uricase gene, which leads to high and unstable UA concentrations in body fluids. The normal level of UA in human serum is 0.12-0.46M, and the normal level of UA in urine is 1.4-4.4mM (Ma et al., Biosens. Bioelectron., 2023, 237: 115495). Abnormal UA levels can lead to diseases such as gout, arthritis, kidney disease, coronary heart disease and Lesch-Nyhan syndrome.

[0003] Therefore, it is necessary to establish a fast, accurate and convenient method to monitor the UA content in the human body. Common methods for UA detection include: colorimetry (Chen et al., Anal. Chim. Acta, 2023, 1273: 341543.), electrochemical method (Yu et al., Colloids Surf A Physicochem Eng Asp, 2016, 494: 241-247), high performance liquid chromatography (Chen et al., Biomed. Chromatogr., 2021, 35: e5156), fluorescence spectroscopy (Cao et al., Spectrochim. Acta AMol. Biomol. Spectrosc., 2021, 263: 120182), liquid chromatography-mass spectrometry (Sakurai et al., Legal Medicine, 2022, 55: 102011), chemiluminescence (Ji et al., Anal. Chem., 2023, 95: 3267-3273), etc. All of the above methods can achieve a wide detection range, high sensitivity, and good accuracy, but some methods have problems such as complex sample pretreatment, cumbersome operation, long detection time, and high cost. Among them, the colorimetric method has the advantages of simple operation, fast detection speed, low experimental equipment requirements, wide application range, and can even be measured visually compared with other UA detection methods.

[0004] The traditional colorimetric detection method of UA is to decompose UA by uricase to produce hydrogen peroxide, and then use horseradish catalase to catalyze hydrogen peroxide to produce OH, which oxidizes colorless 3,3',5,5'-tetramethylbenzidine (TMB) to generate a blue complex product (TMBox) (He et al., Analytica Chimica Acta, 2018, 1021: 113-120), and the color of the solution changes from colorless to blue, and its UV-visible spectrum has a maximum absorption at 652nm. This method has strong selectivity, simple operation, no need for expensive instruments, and high accuracy, but the natural enzyme is expensive and has strict requirements on reaction conditions, which limits the application of traditional colorimetric detection of uric acid. In recent years, the study of artificial catalase replacing horseradish catalase has received widespread attention (Shi et al., New J. Chem., 2022, 46: 18718). Compared with natural enzymes, artificial enzymes have the advantages of low cost, convenient preparation, and good stability. With the rapid development of nanotechnology, various nano-type catalases have been developed and successfully applied to the colorimetric detection of UA in body fluids, with satisfactory detection results (Yuan et al., Spectrochim. Acta A Mol. Biomol. Spectrosc., 2021, 254: 119678). At present, the enzyme-free colorimetric method of UA is mainly based on gold nanorods, silver nanoparticles, and MnO2 nanosheet etching (Qin et al., Sens. Actuators B Chem., 2021, 333: 129638; Amjadi et al., Microchem. J., 2020, 154: 104642; Huang et al., Microchemical Journal, 2023, 190: 108719), and other enzyme-free colorimetric methods are rarely reported. However, the above-mentioned materials are costly and the preparation process is cumbersome. It is necessary to propose a new detection method to reduce the detection cost and have the characteristics of high efficiency, high accuracy, wide detection limit and simple operation. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for quantitative detection of uric acid based on non-enzyme colorimetric sensing, which uses Co3O4 as a catalyst, TMB as a color developer, and uric acid as a reducing agent, and can quantitatively detect uric acid quickly, at low cost, and with high accuracy.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for quantitatively detecting uric acid based on non-enzyme colorimetric sensing, comprising the following steps:

[0008] The buffer solution, porous Co3O4 catalyst and 3,3',5,5'-tetramethylbenzidine were mixed and reacted, and then filtered to obtain a blue product TMBox; then uric acid liquid was added to reduce TMBox, and after the reaction was completed, the uric acid concentration was determined based on the absorbance change of TMBox.

[0009] Preferably, the preparation method of the porous Co3O4 catalyst is as follows: glucose, Co(NO3)2·6H2O, glycine, and NH4NO3 are placed in a container respectively, dissolved in deionized water, heated and stirred until burned, and the porous material Co3O4 is obtained after combustion. Cobalt tetroxide (Co3O4) is an important transition metal oxide, which appears as a black powder with a spinel structure and high stability (Ni et al., Microchimica Acta, 2017, 184(10):3689-3695). Studies have shown that the enzyme-mimicking activity of Co3O4 is closely related to its morphology. Porous Co3O4 has a large specific surface area, many catalytic reaction sites, good biocompatibility, and a wide pH range. It can be used as an excellent material for colorimetric sensing. By adopting the above technical scheme, it is shown that the cobalt tetroxide prepared by the present invention has excellent catalytic performance in UA detection (see Figure 4 ).

[0010] More preferably, the Co3O4 is porous, with a pore size distribution between 3 and 8 nm, an average pore size of 3.383 nm, and a BET surface area of ​​94.34 m 2 / g, and the total pore volume is 0.219 cm 3 / g.

[0011] Preferably, the weight ratio of glucose, Co(NO3)2·6H2O, glycine and NH4NO3 is 1.3-1.6:6-8:0.3-0.5:0.8-1.2.

[0012] Preferably, the volume-to-weight ratio of the buffer solution, the porous Co3O4 catalyst and 3,3',5,5'-tetramethylbenzidine is 2-3 mL: 1-5 mg: 10-50 μL.

[0013] Preferably, the buffer solution is NaAc-HAc, with a concentration of 0.2 mol / L and a pH of 3-7; and the concentration of 3,3',5,5'-tetramethylbenzidine is 8 mmol / L.

[0014] Preferably, the mixing reaction method is vortex reaction at 20-50° C. for 2-10 min.

[0015] Preferably, the pore size of the filtering membrane is 0.22-0.45 μm.

[0016] Preferably, the reduction time is 2 to 4 minutes.

[0017] Preferably, the measuring wavelength range of the absorbance is 400-800 nm; more preferably, the measuring wavelength range is 600-700 nm; most preferably, it is 652 nm.

[0018] Preferably, the absorbance change is determined based on a standard curve.

[0019] Further preferably, the absorbance change judgment is specifically to calculate the absorbance change rate ΔA at 652nm after adding the uric acid liquid to be tested, wherein ΔA=(A0-A n ) / A0, A0 and A n Substitute ΔA into the ΔA-uric acid concentration standard working curve to calculate the uric acid concentration in the unknown sample.

[0020] Preferably, the concentration range of uric acid detection is 0 to 600 μmol / L.

[0021] Contains at least the following beneficial technical effects:

[0022] (1) The Co3O4 prepared by the present invention can catalyze the oxidation of TMB in the absence of hydrogen peroxide. The method for quantitative detection of uric acid by enzyme-free colorimetric sensing established based on the method does not require natural enzymes and artificial enzymes, has a wide uric acid detection range (0-600 μmol / L), and a low detection limit (0.33 μmol / L). The method for quantitative detection of uric acid by enzyme-free colorimetric sensing constructed by the present invention is applied to the quantitative detection of uric acid. The detection method is simple, convenient for promotion and application, and the detection time is as short as 2-4 minutes, which can be widely used in the determination of uric acid content in body fluids.

[0023] (2) The method for preparing Co3O4 of the present invention is simple and easy to operate, is synthesized by a one-step combustion method, has mild reaction conditions, does not require complex equipment, has extremely low equipment investment cost, is environmentally friendly, and has good stability and reproducibility.

[0024] (3) The Co3O4 prepared by the present invention has porosity, with a main pore size distribution between 3 and 8 nm, an average pore size of 3.383 nm, and a calculated BET surface area of ​​94.34 m 2 / g, and the total pore volume is 0.219 cm 3 / g. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative work.

[0026] Figure 1 This is a schematic diagram of the principle of uric acid detection;

[0027] Figure 2 This is the SEM image of Co3O4 described in Example 1;

[0028] Figure 3 is the BET diagram of Co3O4 described in Example 1;

[0029] Figure 4 This is a feasibility diagram of the uric acid colorimetric sensor described in Example 1;

[0030] Figure 5 This is the standard curve spectrum of the uric acid colorimetric sensor described in Example 1;

[0031] Figure 6 This is a comparison chart of the results of measuring uric acid in blood samples of 26 volunteers using the fully automatic biochemical analyzer and the non-enzyme colorimetric sensing method described in Example 8. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below by specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples, unless otherwise specified, are conventional methods. Unless otherwise specified, the reagents and materials used can all be obtained through commercial channels.

[0033] Example 1

[0034] Verifying the Porosity of Co3O4

[0035] Preparation of porous Co3O4 catalyst: Take 1.3g glucose, 6g Co(NO3)2·6H2O, 0.3g glycine, and 0.8g NH4NO3 in an evaporating dish respectively, dissolve them with 1 drop of deionized water, heat and stir until combustion, and porous material Co3O4 will be obtained after combustion.

[0036] The SEM image of Co3O4 is shown in the attached Figure 2 As shown in the figure, Co3O4 presents a loose porous structure with stacked layers and pores of different sizes. The main pore size distribution is between 3 and 8 nm, the average pore size is 3.383 nm, and the calculated BET surface area is 94.34 m 2 / g, and the total pore volume is 0.219 cm 3 / g, the nitrogen adsorption isotherm of Co3O4 is shown in the attached Figure 3 shown.

[0037] Example 2

[0038] Feasibility of colorimetric sensing of uric acid

[0039] Take 5 centrifuge tubes and add 2.5mL 0.2M NaAc-HAc buffer solution (pH=4.0) respectively, add 200μL H2O2 to centrifuge tube a; add 200μL H2O2 and 100μL TMB to centrifuge tube b; add 100μL TMB solution and 10mgCo3O4 to centrifuge tube c; add 200μL H2O2, 100μL TMB and 10mg Co3O4 to centrifuge tube d; add 100μL TMB, 10mgCo3O4 and 200μL uric acid to centrifuge tube e; add 100μL TMB solution and 10mg commercially available Co3O4 to centrifuge tube f; add 100μL TMB, 10mg commercially available Co3O4 and 200μL uric acid to centrifuge tube g; all of the above were placed in a 30℃ water bath for constant temperature reaction for 10min, and filtered with a 0.22μm filter membrane. Add 100 μL TMB and 10 mg Co3O4 to centrifuge tube e, mix well and react in a 30°C water bath for 10 min. After filtering, add 200 μL 200 mg·L -1 The uric acid solution was added and the reaction was continued for 5 min. The UV-visible spectrum of the above solution was measured in the range of 400-800 nm. Figure 4 As shown. H2O2 alone has no absorption at 652nm (with Figure 4 a); add H2O2 and TMB at the same time, the solution color is close to colorless, A 652 nm Very small (with Figure 4 b), indicating that H2O2 has a weak ability to oxidize TMB and cause it to change color. Figure 4 d) or without adding H2O2 (with Figure 4 c) Both can change the solution from colorless to dark blue, and the A 652 nm The color of the solution changes from dark blue to light blue when uric acid is added to the Co3O4-TMB colorimetric solution. 652 nm Also significantly reduced (with Figure 4 e), indicating that uric acid can reduce the blue TMBox to colorless TMB. 652 nm The coexistence of commercially available Co3O4 (purchased from Nanjing Chemical Reagent Co., Ltd.) and TMB solution can also change the solution from colorless to blue (see attached Figure 4f), uric acid was added to the commercially available Co3O4-TMB colorimetric solution, and the solution color changed from blue to light blue (see attached Figure 4 g), but the catalytic performance of commercially available Co3O4 is much lower than that of the Co3O4 prepared by the present invention (see Appendix Figure 4 c).

[0040] Example 3

[0041] Standard working curve and precision of uric acid quantitative detection

[0042] Take several centrifuge tubes and add 2.5mL 0.2M NaAc-HAc buffer solution (pH=4.0), 30μL TMB and 5mg Co3O4, mix well and react in a 30℃ water bath for 2min. After filtering, add 200μL 0-600μmol·L -1 Uric acid was vortexed at room temperature for 4 minutes, and the UV-Vis spectrum in the range of 400-800 nm was measured. The results are shown in the attached Figure 5 As shown. Uric acid has a good linear relationship in the range of 1-10 μmol / L and 10-600 μmol / L, and the linear equations are ΔA=0.00437C UA +0.00683(R 2 =0.994) and ΔA = 0.00111C UA +0.04984(R 2 =0.996), the detection limits (LOD) were 0.33μmol / L and 3.33μmol / L, respectively, which showed that the sensitivity of UA determination was very high. Four concentrations of UA solution (2.5μmol / L, 5μmol / L, 50μmol / L and 200μmol / L) were selected in the range of 1-10μmol / L and 10-600μmol / L, respectively, and the parallel determination was repeated 5 times, and the RSDs were 2.5%, 2.1%, 1.1% and 0.9%, respectively, indicating that the method has good precision.

[0043] Example 4

[0044] 1) Preparation of porous Co3O4 catalyst: 1.3 g of glucose, 6 g of Co(NO3)2·6H2O, 0.3 g of glycine, and 0.8 g of NH4NO3 were respectively placed in an evaporating dish, dissolved with 1 drop of deionized water, heated and stirred until combustion, and porous material Co3O4 was obtained after combustion.

[0045] 2) Add 2.5 mL of 0.2 M NaAc-HAc buffer solution (pH 4) and 30 μL of 8 mmol / L TMB solution to a centrifuge tube, mix well, then add 5 mg of Co3O4, vortex at 30°C for 2 min, and filter the test solution in the centrifuge tube with a 0.22 μm filter membrane;

[0046] 3) Add 200 μL of different concentrations of uric acid (0-600 μmol / L) to the filtrate, vortex for 4 min, and measure the UV-Vis spectrum in the range of 400-800 nm. Calculate the absorbance at 652 nm (A 652nm ) rate of change ΔA(ΔA=(A0-A n ) / A0), where A0 and A n Represent the absorbance without and with uric acid, respectively.

[0047] 4) Substitute the unknown sample for the uric acid solution in (3), determine ΔA, substitute it into the ΔA-uric acid concentration standard working curve, and calculate the uric acid concentration in the unknown sample.

[0048] Example 5

[0049] This embodiment provides a method for quantitative detection of uric acid based on non-enzymatic colorimetric sensing, the steps are:

[0050] 1) Preparation of porous Co3O4 catalyst: 1.5 g of glucose, 7 g of Co(NO3)2·6H2O, 0.4 g of glycine, and 1 g of NH4NO3 were respectively placed in an evaporating dish, dissolved with 2 drops of deionized water, heated and stirred until combustion, and porous material Co3O4 was obtained after combustion.

[0051] 2) Add 2 mL of 0.2 M NaAc-HAc buffer solution (pH 5) and 10 μL of 8 mmol / L TMB solution to a centrifuge tube, mix well, then add 3 mg of Co3O4, vortex for 2 min at 30°C, and filter the test solution in the centrifuge tube with a 0.45 μm filter membrane;

[0052] 3) Add 200 μL of different concentrations of uric acid (0-600 μmol / L) to the filtrate, vortex for 4 min, and measure the UV-Vis spectrum in the range of 400-800 nm. Calculate the absorbance at 652 nm (A 652nm ) change rate ΔA(ΔA=(A0-An) / A0, where A0 and An represent the absorbance without and with uric acid, respectively.

[0053] 4) Substitute the unknown sample for the uric acid solution in (3), determine ΔA, substitute it into the ΔA-uric acid concentration standard working curve, and calculate the uric acid concentration in the unknown sample.

[0054] Example 6

[0055] This embodiment provides a method for quantitative detection of uric acid based on non-enzymatic colorimetric sensing, the steps are:

[0056] 1) Preparation of porous Co3O4 catalyst: 1.6 g of glucose, 8 g of Co(NO3)2·6H2O, 0.2 g of glycine, and 1.2 g of NH4NO3 were respectively placed in an evaporating dish, dissolved with 2 drops of deionized water, heated and stirred until combustion, and porous material Co3O4 was obtained after combustion.

[0057] 2) Add 3 mL of 0.2 M NaAc-HAc buffer solution (pH 7) and 50 μL of 8 mmol / L TMB solution to a centrifuge tube, mix well, then add 1 mg of Co3O4, vortex for 2 min at 30°C, and filter the test solution in the centrifuge tube with a 0.22 μm filter membrane;

[0058] 3) Add 200 μL of different concentrations of uric acid (0-600 μmol / L) to the filtrate, vortex for 4 min, and measure the UV-Vis spectrum in the range of 400-800 nm. Calculate the absorbance at 652 nm (A 652nm ) rate of change ΔA(ΔA=(A0-A n ) / A0, where A0 and A n Represent the absorbance without and with uric acid, respectively.

[0059] 4) Substitute the unknown sample for the uric acid solution in (3), determine ΔA, substitute it into the ΔA-uric acid concentration standard working curve, and calculate the uric acid concentration in the unknown sample.

[0060] Experimental Example 7

[0061] Urine uric acid test

[0062] The method of Example 4 of the present invention was used to detect the UA content in the urine of three healthy adult volunteers (two men and one woman), and the determination was repeated three times in parallel, and a spike recovery experiment was performed. The results are shown in Table 1. The UA content in the urine of the three volunteers was 2.43, 2.04, and 1.77 mmol / L, respectively, all within the normal reference value of 1.4 to 4.4 mmol / L. The spike recovery rates of the three samples were 104%, 96.8%, and 102%, respectively, and the RSDs were 0.9%, 1.7%, and 0.8%, respectively, indicating that this method is accurate and reliable in determining the content of uric acid in urine, and the specific results are shown in Table 1.

[0063] Table 1 Actual urine sample determination and spike recovery test results

[0064]

[0065]

[0066] Experimental Example 8

[0067] Uric acid test in blood

[0068] The method of Example 4 of the present invention was used to test blood samples of 26 volunteers (3 females and 23 males) aged 20 to 59 years old. The results are as follows: Figure 6 The black dots in the figure are the uric acid results measured by the hospital's fully automatic biochemical analyzer, and the red dots are the uric acid results measured by this method. By comparison, it was found that the uric acid levels in the blood of the 26 volunteers were between 190 and 810 μmol·L -1 The results of the two methods are close and there is no significant difference, which shows that this method is highly accurate and practical for determining the uric acid content in the blood.

[0069] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for quantitative detection of uric acid based on non-enzymatic colorimetric sensing, characterized in that: The following steps are involved: The buffer solution, porous Co3O4 catalyst and 3,3',5,5'-tetramethylbenzidine are mixed and reacted, and then filtered to obtain a blue product TMBox; then uric acid liquid is added to reduce TMBox, and after the reaction is completed, the uric acid concentration is determined according to the absorbance change of TMBox; The preparation method of the porous Co3O4 catalyst is as follows: glucose, Co(NO3)2·6H2O, glycine, and NH4NO3 are placed in a container respectively, dissolved with deionized water, heated and stirred until burned, and porous material Co3O4 is obtained after burning; The weight ratio of glucose, Co(NO3)2·6H2O, glycine and NH4NO3 is 1.3-1.6:6-8:0.3-0.5:0.8-1.2; The volume-to-weight ratio of the buffer solution, the porous Co3O4 catalyst and 3,3',5,5'-tetramethylbenzidine is 2-3 mL: 1-5 mg: 10-50 μL; The buffer solution is NaAc-HAc, with a concentration of 0.2 mol / L and a pH of 3-7; the concentration of 3,3',5,5'-tetramethylbenzidine is 8 mmol / L.

2. The method for quantitative detection of uric acid according to claim 1, characterized in that: The mixing reaction method is to vortex react at 20-50° C. for 2-10 minutes.

3. The method for quantitative detection of uric acid according to claim 1, characterized in that: The pore size of the filtering membrane is 0.22-0.45 μm.

4. The method for quantitative detection of uric acid according to claim 1, characterized in that: The reduction time is 2 to 4 minutes.

5. The method for quantitative detection of uric acid according to claim 1, characterized in that: The absorbance measurement wavelength range is 400-800nm.

6. The method for quantitative detection of uric acid according to claim 1, characterized in that: The concentration range of the uric acid detection is 0 to 600 μmol / L.

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