Method for detecting uric acid in serum based on biochar magnetic nano-enzyme colorimetry

Abstract

The invention belongs to the field of analysis and detection, and provides a non-diagnostic method for detecting uric acid in serum based on biochar magnetic nano-enzyme colorimetry. According to the method, the biochar magnetic nano-enzyme is used for activating the potassium hydrogen peroxymonosulfate, then the 3, 3 ', 5, 5'-tetramethylbenzidine is oxidized to generate a blue characteristic product ox-TMB, and the uric acid can inhibit the reaction process, that is, within a certain quantity range, the concentration of the uric acid can be judged according to the change of the absorbance of a reaction system. The nano enzyme is easy to synthesize, low in cost and capable of being stored for a long time, and the uric acid detection method constructed by the invention is low in detection limit (3.85 mu mol / L), wide in linear range (50-1000 mu mol / L), relatively simple, rapid and cheap, and expected to be widely applied to serum uric acid detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical detection, and particularly relates to a method for detecting uric acid in serum by colorimetry based on biochar magnetic nanozyme for non-diagnostic purposes. Background Art

[0002] Uric acid (UA, 2,4,6-trihydroxypurine) is the end product of purine decomposition in the human body and is often widely distributed in the human blood circulation system in a free state. Under normal physiological conditions, the body's daily ability to metabolize uric acid and the corresponding amount excreted are limited. Excessive intake of foods with high purine content, long-term use of drugs that inhibit uric acid metabolism, or due to defects in physiological functions such as genetic metabolism, can cause abnormal serum uric acid levels in the body (reference value range: 210 - 420 μmol / L for adult males, 150 - 360 μmol / L for adult females). A large amount of uric acid accumulates throughout the body through the systemic circulation and precipitates in the form of urate, depositing in corresponding parts such as joint cavities or internal organs, further affecting various physiological and biochemical processes of the body by affecting the functions of various tissues and organs, and ultimately leading to damage to the body, presenting symptoms such as gout, secondary hypertension caused by kidney damage, type II diabetes, etc.

[0003] In the existing methods for detecting serum uric acid, high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) have high specificity, but require relatively complex pretreatment, high costs, and certain conditions for instrument storage and use, and the sample detection cycle is relatively long; the electrochemical method is restricted by the inherent characteristics of the materials modified on the electrode and has poor anti-interference ability and weak stability in the detection of actual samples; for the enzyme method, currently, natural peroxidase or synthetic mimetic peroxidase is commonly used to catalyze hydrogen peroxide (H 2 O 2 ) for detection. However, due to the poor stability of the enzyme itself and its easy inactivation, and H 2 O 2 is relatively unstable and is under control due to certain dangers, these factors will ultimately affect the convenience of detection, as well as the accuracy and stability of the results. Therefore, there is an urgent need to develop a method for detecting uric acid that is fast, convenient, low-cost, has relatively accurate inspection results, and is safe and green. Summary of the Invention

[0004] For the above reasons, the present invention aims to provide a method for detecting uric acid in serum by colorimetry based on biochar magnetic nanozyme for non-diagnostic purposes, using cobalt ferrite on biochar (BC / CoFe 2 O 4)Taking permonosulfate (PMS) as an activator, uric acid (UA) as a reducing agent, and 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic agent. This nanozyme is simple to synthesize, inexpensive, and can be stored for a long time, and can be prepared as needed. This method can quickly, relatively inexpensively and accurately detect the content of uric acid in serum.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The reaction system adopted by the method for detecting uric acid in serum based on the colorimetry of biochar magnetic nanozyme provided by the present invention includes biochar magnetic nanozyme - BMNE, permonosulfate - PMS and 3,3',5,5'-tetramethylbenzidine - TMB; wherein, biochar magnetic nanozyme - BMNE is an activator, permonosulfate - PMS is an oxidant, and it is mixed with 3,3',5,5'-tetramethylbenzidine - TMB for a chromogenic reaction to obtain blue ox - TMB. Uric acid - UA will inhibit the generation of ox - TMB in the reaction system, reduce its absorbance, and the uric acid concentration is correlated with the absorbance value to determine the uric acid concentration.

[0007] Preferably, after the buffer solution, BC / CoFe 2 O 4 activator, PMS, tert - butanol, and UA solution are fully mixed, TMB is added for color development, and after dilution and filtration, the absorbance is measured with a UV - visible spectrophotometer, and the concentration of uric acid in the serum sample to be measured is obtained by substituting it into the standard curve of uric acid UV absorbance.

[0008] Preferably, the volume ratio of the buffer solution, BC / CoFe 2 O 4 activator, PMS, tert - butanol, UA solution and TMB is 100 μL: 20 - 140 μL: 40 μL: 15 μL: 85 μL: 60 μL.

[0009] Preferably, the buffer solution is NaAc - HAc with a concentration of 0.2 mol / L and a pH of 6 - 8; Na 2 HPO 4 -NaH 2 PO 4 buffer solution with a concentration of 0.1 mol / L; MES buffer solution with a concentration of 0.5 mol / L;

[0010] Preferably, the activator BC / CoFe 2 O 4The preparation method is as follows: Take clean biological straws (such as reed straws, corn straws, etc.), cut them into small pieces, wash, dry, crush, and sieve them. Take the above powdery material, calcine it in a tubular furnace, and then sieve it to obtain biochar (BC). Mix BC, Co(NO 3 ) 2 ·6H 2 O, Fe(NO 3 ) 3 ·9H 2 O by ultrasonic waves until evenly mixed, adjust the pH value to an appropriate level with NaOH solution, and use the hydrothermal method or ultrasonic precipitation method to prepare BC / CoFe 2 O 4 . Add cetyltrimethylammonium bromide (CTAB) to prepare an aqueous solution, and then BC / CoFe 2 O 4 activator can be prepared. Its surface structure is good, CoFe 2 O 4 is evenly distributed on BC, which can effectively reduce the aggregation caused by the magnetism of CoFe 2 O 4 , increase the reaction contact area, and it has rich activation sites and small diffusion resistance of nanoenzymes, which is beneficial to the activation of PMS.

[0011] Preferably, the concentration of the NaOH is 1.0 mol / L.

[0012] Preferably, for the hydrothermal method, use a hydrothermal reaction kettle to react the mixture at 160 °C for ten hours to obtain the target product; for the ultrasonic method, ultrasonically react the mixture for 1 hour to synthesize the target product.

[0013] Preferably, the mass-volume ratio of BC / CoFe 2 O 4 , CTAB and water is 33 mg: 0.1 - 0.6 g: 50 mL.

[0014] Preferably, the concentration of PMS is 0.5 - 4.5 g / L.

[0015] Preferably, the concentration of tert-butanol is 0.4 g / mL.

[0016] Preferably, the concentration of TMB is 7.5 - 27.5 mmol / L.

[0017] Preferably, the reaction conditions for the mixing are vortexing at 25 °C for 0.5 min.

[0018] Preferably, the color development time is 1 - 6 min.

[0019] Preferably, the final volume after dilution is 4.0 mL

[0020] Preferably, the pore size of the filter membrane of the filter head is 0.22 to 0.45 μm.

[0021] Preferably, the ultraviolet absorbance wavelength measurement range is 400 to 800 nm, and the optimal wavelength is 652 nm.

[0022] Preferably, the standard curve of uric acid ultraviolet absorbance is calculated as follows: Measure the absorbance ΔA at 652 nm after color development of standard samples with uric acid concentrations of 50, 125, 250, 375, and 500 μmol / L by this method, and perform linear fitting on it, that is, obtain the standard curve of uric acid ultraviolet absorbance, where ΔA = A 1 -A 0 , A 1 and A 0 respectively represent the actual absorbance of the uric acid standard or sample under this condition and the absorbance of the reagent blank under this condition; measure ΔA of the actual sample by this method and substitute it into the standard curve, that is, obtain the uric acid concentration in the actual sample.

[0023] Preferably, the concentration range for uric acid detection is 50 to 1000 μmol / L. Preferably, the present invention also provides a kit for detecting uric acid in serum, which includes biochar magnetic nanozyme - BMNE, potassium monopersulfate - PMS, and 3,3’,5,5’ - tetramethylbenzidine - TMB. The mixed reagent also includes a buffer solution, PMS, and tert - butanol.

[0024] The present invention has at least the following advantages compared with the existing related detection methods:

[0025] (1) The present invention provides a method for detecting uric acid in serum based on the colorimetry of biochar magnetic nanozyme. It uses safe and harmless PMS to replace hydrogen peroxide (H 2 O 2 ) in the traditional color development system as an oxidant. The biochar magnetic nanozyme (BMNE) activates PMS to generate various reactive oxygen species (ROS), which then oxidize 3,3’,5,5’ - tetramethylbenzidine (TMB) to form a characteristic blue oxidation product ox - TMB. Uric acid inhibits this color development process due to its reducibility, reducing the absorbance of the reaction system. That is, within a certain range, the uric acid concentration in the sample is inversely proportional to the absorbance of the reaction system in a linear relationship. This technical solution constructs an innovative BMNE - PMS - TMB color development system and successfully applies it to the determination of uric acid in serum, avoiding the use of highly corrosive and unstable H 2 O 2Technical defects caused by being a chromogenic oxidant. The detection system uses an environmentally friendly reagent system, reducing the potential harm during detection while reducing reagent costs. The method can be completed using a conventional ultraviolet-visible spectrophotometer without complex instrument equipment. The operation process is simple and fast, with a detection time of 4 - 6 minutes. This method has a wide linear detection range (50 - 1000 μmol / L) and a low detection limit (3.85 μmol / L), and can be widely used in the determination of uric acid in serum. It shows good anti-interference performance against various interfering substances, such as: K + 、Na + 、Cl - 、NH 4 + 、glucose, ascorbic acid, glycine, etc.

[0026] (2) The magnetic nanozyme BC / CoFe 2 O 4 prepared in the present invention has a simple synthesis method, low cost, strong stability, can be stored for a long time, has good activation performance, low dosage, and is environmentally friendly.

[0027] (3) The BC / CoFe 2 O 4 prepared in the present invention has certain mesoporous characteristics and good structure. Through experimental verification, various biological tissues such as reed straw, corn straw, and tea residue can be prepared into biochars with good performance, and most of the modified BMNE synthesized by replacing different BCs or substituting metal elements have good activation performance and a wide application range. Description of the Drawings

[0028] Figure 1 is the actual image of the sample in Example 1, where a is BC (reed straw source); b is one of the biochar magnetic nanozymes: BC (reed straw source) / CoFe 2 O 4 ; c is the distribution of BC (reed straw source) / CoFe 2 O 4 in aqueous solution without a magnetic field; d is the magnetic aggregation phenomenon of BC (reed straw source) / CoFe 2 O 4 when attracted by a magnet.

[0029] Figure 2 is the picture of the feasibility of colorimetric determination of uric acid (UA) in Example 2.

[0030] Figure 3 is the picture of the ultraviolet absorbance of different uric acid (UA) concentration reaction systems at wavelengths of 400 - 800 nm in Example 3.

[0031] Figure 4 It is a picture of the anti-interference degree test for the quantitative detection of uric acid (UA) in Example 6.

[0032] Figure 5 It is a picture of the activation performance of different types of BMNE in Example 8, where a is the BC (reed stalk source) / CoFe 2 O 4 detection system; b is the BC (corncob source) / CoFe 2 O 4 detection system; c is the BC (corncob source) / CuFe 2 O 4 detection system; d is the BC (reed stalk source) / CoFe 2 O 4 detection system, with 1M UA added for color development; e is the BC (corncob source) / CoFe 2 O 4 detection system, with 1M UA added for color development; f is the BC (corncob source) / CuFe 2 O 4 detection system, with 1M UA added for color development. Detailed implementation manners

[0033] The technical solutions of the present invention will be described in detail below to more clearly illustrate the present invention and its technical features, etc. The specific implementation scope is not limited thereto.

[0034] Example 1

[0035] Specific conditions of biochar magnetic nanozyme (BMNE)

[0036] Biochar can be prepared by drying and calcining various plant tissues, such as: reed straw biochar (attached Figure 1 a), corn straw biochar, tea residue biochar, etc., all of which have good loading properties. The synthesized BC (reed straw source) / CoFe 2 O 4 (attached Figure 1 b) often appears as black and uniform powder. Due to the presence of the Fe 2 O 4 group, BC (reed straw source) / CoFe 2 O 4 has strong magnetism, and this property can be used to recover it, which is convenient for saving resources and reuse. When preparing BMNE, different BC and metal elements can be changed to modify it. Experiments show that various different types of BMNE all have good activation performance.

[0037] Example 2

[0038] Feasibility of Colorimetric Determination of Uric Acid (UA)

[0039] The reagent specifications for this example are as follows: NaAc-HAc buffer solution, 0.2 M, pH = 6.5; BC / CoFe 2 O 4 activator (C CTAB = 6 g / L), 0.66 g / L; PMS, 3.0 g / L; tert-butanol, 0.4 g / L; TMB, 22.5 mmol / L. Take a certain number of centrifuge tubes and divide them into 6 groups evenly, and add 100 μL of NaAc-HAc buffer solution and 15 μL of tert-butanol to each group respectively.

[0040] Add 100 μL of BC / CoFe 2 O 4 to the a centrifuge tube;

[0041] Add 40 μL of PMS to the b centrifuge tube;

[0042] Add 40 μL of PMS and 60 μL of TMB to the c centrifuge tube;

[0043] Add 100 μL of BC / CoFe 2 O 4 , 40 μL of PMS and 60 μL of TMB to the d centrifuge tube;

[0044] Add 100 μL of BC / CoFe 2 O 4 , 40 μL of PMS, 60 μL of TMB and 85 μL of 500 μmol / L UA to the e centrifuge tube;

[0045] Add 100 μL of BC / CoFe 2 O 4 , 40 μL of PMS, 60 μL of TMB and 85 μL of 250 μmol / L UA to the f centrifuge tube;

[0046] After filtering the above reaction systems with a 0.22 μm filter membrane, measure their absorbance at a wavelength of 400 - 800 nm.

[0047] The individual BC / CoFe 2 O 4 and PMS have no absorbance (Appendix Figure 2 a, 2b); when PMS and TMB are added simultaneously, the absorbance is weak and the solution does not show the characteristic blue color of ox-TMB (Appendix Figure 2 c), indicating that PMS alone has little oxidation effect on TMB; while when BC / CoFe 2 O 4 , PMS and TMB are added simultaneously, the solution turns blue (AppendixFigure 2 d), indicating BC / CoFe 2 O 4 has a strong ability to activate PMS, which can cause the reaction system to develop color; on this basis, when 500 μmol / L or 250 μmol / L of UA is added, the absorbance of the reaction system decreases compared to the absorbance of the blank reaction system, and the higher the uric acid concentration, the lower the absorbance of the reaction system (attached Figure 2 e, 2f), proving that UA can inhibit the oxidation of TMB to ox-TMB, and the higher the UA concentration, the stronger the inhibitory effect; the above reaction can prove that the relationship between the UA concentration and the absorbance in this reaction system is inversely proportional, and the method for detecting the uric acid concentration in an unknown sample is feasible.

[0048] Example 3

[0049] Selection of the optimal ultraviolet detection wavelength

[0050] The reagent specifications for this example are as follows: NaAc-HAc buffer solution, 0.2 M, pH = 6.85; BC / CoFe 2 O 4 activator (C CTAB = 6 g / L), 0.66 g / L; PMS, 1.0 g / L; tert-butanol, 0.4 g / L; TMB, 10.0 mmol / L. Take a certain number of centrifuge tubes and divide them into 5 groups evenly. Add 100 μL of NaAc-HAc buffer solution, 100 μL of BC / CoFe 2 O 4 activator, 100 μL of PMS (3.0 g / L) and 15 μL of tert-butanol (0.4 g / L) into each test tube, mix well, then add 85 μL of five different concentrations of UA solutions (50, 100, 150, 200, 250 μmol / L) according to the groups and mix well. Finally, add 60 μL of TMB to each test tube, vortex and mix well for a color reaction for 1.5 min, filter, and then use a UV-visible spectrophotometer to perform a full-band scan detection in the wavelength range of 400 - 800 nm. The results are as Figure 3 shown. As Figure 3 shown, the test results indicate that each reaction system shows a characteristic absorption peak at a wavelength of 652 nm, and the absorbance response value at this wavelength has a significant correlation with the concentration of the analyte (R 2 = 0.9998). Therefore, 652 nm is selected as the characteristic detection wavelength for quantitative analysis.

[0051] Example 4

[0052] Absorbance standard curve and precision for uric acid quantitative detection

[0053] A certain number of centrifuge tubes were evenly divided into 5 groups. 100 μL of 0.2 M NaAc-HAc buffer solution, 100 μL of BC / CoFe 2 O 4 activator (0.66 g / L, C CTAB = 6.0 g / L), 100 μL of PMS (3.0 g / L), and 15 μL of tert-butanol (0.4 g / L) were mixed evenly. Then, 85 μL of five different concentrations of UA solutions (50, 250, 500, 750, 1000 μmol / L) were added according to the groups and mixed evenly. Finally, 60 μL of TMB (22.5 mmol / L) was added to each test tube, and it was vortexed and mixed evenly for a color reaction for 1.5 min. After filtration, the absorbance of the solution was measured at 652 nm, and the average value was taken. Its linear equation was ΔA = -0.0013c UA + 1.8888 (R 2 = 0.9996). The detection limit (LOD) and the quantification limit (LOQ) were 3.85 μmol / L and 11.54 μmol / L respectively, indicating that this method had a relatively high sensitivity for the determination of UA. Three serum samples were selected to detect their UA content using this method, and each sample was measured in parallel 5 times. The average values of the detections were 193.38, 256.46, and 275.62 μmol / L, and their relative standard deviations were 1.43, 1.04, and 1.98% respectively. The spiked recovery rates were 92.00 - 109.54%. The above results showed that this method had good precision.

[0054] Table 1 Results of the determination and recovery of standard uric acid samples

[0055]

[0056] Example 5

[0057] In this example, the serum uric acid quantitative detection method based on the activation colorimetry of the nanozyme was elaborated in detail. Only the preparation of the activator using biochar obtained by calcining reed straw and BC / CoFe 2 O 4 synthesized by ultrasonic method was taken as an example, and the specific steps were as follows:

[0058] 1) Preparation of BC / CoFe 2 O 4 activator: Take the dried and clean reed stalks, crush and sieve them, place them in a tube furnace, use nitrogen to isolate the air, calcine at 600 °C for 1.5 h, then cool and sieve to obtain BC; take BC, Co(NO 3 ) 2 ·6H 2 O, Fe(NO 3 ) 3 ·9H 2O and distilled water were placed in a container, and their mass-volume ratio was: 93.8 mg: 116.4 mg: 323.2 mg: 35 mL. They were mixed evenly by ultrasonic for 30 min, and the pH value of the solution was adjusted to 10.00 with NaOH solution (1 mol / L), then reacted ultrasonically for 1 h, washed with deionized water and dried, thus preparing BC / CoFe 2 O 4 ; Take BC / CoFe 2 O 4 、CTAB and water were mixed evenly to prepare BC / CoFe 2 O 4 activator, and its mass-volume ratio was 33 mg: 300 mg: 50 mL.

[0059] 2) Add 100 μL of 0.2 M NaAc-HAc (pH = 6.5) and 100 μL of BC / CoFe 2 O 4 activator into a centrifuge tube and mix evenly, then add 40 μL of 3.0 g / L PMS and mix well, then add 15 μL of 0.4 g / L tert-butanol and mix well, then add 85 μL of the actual sample and vortex to mix evenly, finally add 60 μL of 22.5 mmol / L TMB and vortex for color reaction for 0.5 min, filter, measure its absorbance at 652 nm, and substitute it into the absorbance standard curve for uric acid quantitative detection, then the concentration of uric acid in the sample can be obtained.

[0060] Example 6

[0061] Determination of the anti-interference degree of uric acid quantitative detection

[0062] Replace the actual sample in Example 4 with an equal volume of distilled water (blank), UA standard solution or a mixture of uric acid standard solution and other interfering substances, and measure the interference situation of its determination of uric acid according to Example 5. Among them, in the UA standard solution or mixed solution, the UA concentration is 500 μmol / L; the concentrations of other interfering substances are as follows: K + 、Na + 、Cl — and glucose concentration are 5 mmol / L, urea and NH 4 + are 250 μmol / L, glycine and ascorbic acid (AA) are 100 μmol / L, and phenylalanine is 50 μmol / L. Calculate the difference in absorbance between the blank sample and the uric acid sample and between the mixed solution and the uric acid sample. The experimental results are as Figure 4 shown, and these results indicate that this reaction system has good anti-interference characteristics.

[0063] Example 7

[0064] BC / CoFe2 O 4 Performance Test of Magnetic Nanozyme

[0065] 1) Add 100 μL of 0.2 M NaAc-HAc (pH = 6.5) and 100 μL of BC / CoFe 2 O 4 activator (660 mg / L, C CTAB = 6 g / L) into a centrifuge tube, mix well, then add 40 μL of PMS with different concentrations and mix evenly. Next, add 15 μL of 0.4 g / L tert-butanol and 85 μL of distilled water, vortex to mix well. Finally, add 60 μL of 10 mmol / L TMB, vortex for color development reaction for 2.5 min, filter, and measure the absorbance at 652 nm. The PMS concentrations include 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5 g / L.

[0066] 2) Add 100 μL of 0.2 M NaAc-HAc (pH = 6.5) and 100 μL of BC / CoFe 2 O 4 activator (660 mg / L, C CTAB = 6 g / L) into a centrifuge tube, mix well, then add 40 μL of 3.0 g / L PMS and mix evenly. Next, add 15 μL of 0.4 g / L tert-butanol and 85 μL of distilled water, vortex to mix well. Finally, add 60 μL of TMB with different concentrations, vortex for color development reaction for 2.5 min, filter, and measure the absorbance at 652 nm. The TMB concentrations include 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, 25.0, and 27.5 mmol / L.

[0067] 3) Substitute the above data into the equation and perform double-reciprocal curve fitting. Here, V max (mol / min) is the reaction rate unit when the enzyme is completely saturated by the substrate, and the higher its value, the better the enzyme activity; K m (mol / L) is the Michaelis constant, which is the substrate (S) concentration when the enzyme-catalyzed reaction reaches half of the maximum rate (V max ), and the lower its value, the better the enzyme activity; [S] (mol / L) is the substrate concentration; is the current reaction rate, where A 样品 (L / (g·cm)) is the absorbance of the sample at 652 nm, A 空白(L / (g·cm)) is the absorbance of the reagent at 652 nm, ε is the molar absorptivity of TMB at 652 nm which is 39000 L / (mol·cm), l is the optical path length of the cuvette which is 1 cm, and t (min) is the color development reaction time.

[0068] 4) The calculation results show that when using PMS as the substrate, the K 2 O 4 of nanozyme BC / CoFe m = 0.611 mmol / L, and V max = 22.667*10 -7 mol / (L·S); when using TMB as the substrate, the K 2 O 4 of nanozyme BC / CoFe m = 0.062 mmol / L, and V max = 4.254*10 -7 mol / (L·S). The results show that the magnetic nanozyme BC / CoFe 2 O 4 has good performance.

[0069] Example 8

[0070] Activation performance of different types of BMNE;

[0070] According to the test method in Example 5, using distilled water instead of the actual sample as the blank group and 1M UA instead of the actual sample as the control group, the absorbance of the reaction system was measured by replacing different types of BMNE, including: BC (reed stalk source) / CoFe 2 O 4 , BC (corncob source) / CoFe 2 O 4 and BC (corncob source) / CuFe 2 O 4 , with other conditions remaining unchanged.

[0070] Among them, the blank group BC (reed stalk source) / CoFe 2 O 4 (Appendix Figure 5 a), BC (corncob source) / CoFe 2 O 4 (Appendix Figure 5 b) and BC (corncob source) / CuFe 2 O 4 (Appendix Figure 5 c) all have good color development; while the control group BC (reed stalk source) / CoFe 2 O 4 (Appendix Figure 5d), BC (corncob source) / CoFe 2 O 4 (attached Figure 5 e) and BC (corncob source) / CuFe 2 O 4 (attached Figure 5 f) After adding UA to the reaction system, the absorbance decreased significantly. The absorbance of its reaction system is shown in Table 2. The above results indicate that various different types of BMNE all have relatively good activation performance. Among them, the color development effect of the BC (corncob source) / CuFe 2 O 4 reaction system is weaker than that of the other two reaction systems. The color development effect of the BC (reed stalk source) / CoFe 2 O 4 reaction system is slightly stronger than that of the BC (corncob source) / CoFe 2 O 4 reaction system. Due to the material characteristics, the BC (reed stalk source) / CoFe 2 O 4 reaction system has stronger stability, a larger linear range, higher sensitivity, and the reed stalks are inexpensive. In summary, BC (reed stalk source) / CoFe 2 O 4 has good activation performance and low cost, and is more suitable as the activator of this detection system.

[0071] Table 2 Absorbance of reaction systems of different types of BMNE;

[0072]

[0073] The above is only the preferred embodiment of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry, characterized in that: The reaction system used included biochar magnetic nanozyme-BMNE, potassium permonosulfate-PMS, and 3,3',5,5'-tetramethylbenzidine-TMB; Among them, biochar magnetic nanozyme-BMNE is an activator and potassium permonosulfate-PMS is an oxidant, which is mixed with 3,3',5,5'-tetramethylbenzidine-TMB for color development reaction to obtain blue ox-TMB. Uric acid-UA will inhibit the formation of ox-TMB in the reaction system, reduce its absorbance, and the uric acid concentration is correlated with the absorbance value to determine the uric acid concentration.

2. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 1, characterized in that: The buffer solution, biochar magnetic nanozyme-BMNE activator, potassium permonosulfate-PMS, tert-butanol, uric acid sample and 3,3',5,5'-tetramethylbenzidine-TMB are vortex mixed for color development reaction to obtain blue ox-TMB. Uric acid-UA will inhibit the formation of ox-TMB in the reaction system, reducing its absorbance, and the higher the uric acid concentration, the stronger the inhibitory effect. According to this principle, the solution after the reaction is filtered, its absorbance value is measured, and the corresponding uric acid ultraviolet absorption standard curve is drawn and inserted into it to obtain the uric acid concentration.

3. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 2, characterized in that: The volume ratio of the buffer solution, BC / CoFe2O4 activator, PMS, tert-butyl alcohol, uric acid sample and TMB is 100 μL: 20-140 μL: 40 μL: 15 μL: 85 μL: 60 μL; The buffer solution is a NaAc-HAc buffer solution with a concentration of 0.2 mol / L and a pH of 6-8; or a Na2HPO4-NaH2PO4 buffer solution with a concentration of 0.1 mol / L; or a MES buffer solution with a concentration of 0.5 mol / L.

4. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 2, characterized in that: The concentration of potassium permonosulfate-PMS is 0.5-4.5 g / L; The concentration of tert-butyl alcohol is 0.4 g / mL; The concentration of TMB is 7.5-27.5 mmol / L.

5. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 1, characterized in that: The color development time is 1 to 6 minutes; The absorbance measurement wavelength range is 400-800nm; The concentration range of uric acid detection is 50-1000μmol / L.

6. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 2, characterized in that: The pore size of the filter membrane used for filtration is 0.22 to 0.45 μm.

7. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 1, characterized in that: The biochar magnetic nanozyme-BMNE has good activation performance, and the biochar magnetic nanozyme-BMNE is biochar-based cobalt ferrite-BC / CoFe2O4.

8. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 7, characterized in that: The preparation method of the BC / CoFe2O4 activator is as follows: biochar-BC, Co(NO3)2·6H2O, and Fe(NO3)3·9H2O are ultrasonically mixed, adjusted to a suitable pH value with a NaOH solution, synthesized BC / CoFe2O4 by a hydrothermal method or an ultrasonic method, and cetyltrimethylammonium bromide-CTAB is added to prepare an aqueous solution, thereby preparing the biochar BC / CoFe2O4 activator; The weight ratio of the BC to CoFe2O4 is 1:4 to 4:1; the concentration of the NaOH solution is 1.0 mol / L; the suitable pH value of the solution is 10; the solid-liquid ratio of the biochar BC / CoFe2O4, hexadecyltrimethylammonium bromide-CTAB and water is 33 mg:100 to 600 mg:50 mL.

9. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 8, characterized in that: The biochar is prepared by high-temperature calcination of various plant straws, and the plant straws are corn straws, reed straws or tea residue straws.

10. The method for detecting uric acid in serum based on biochar magnetic nanoenzyme colorimetry according to claim 8, characterized in that: BC / CoFe2O4 was synthesized by using a hydrothermal reactor at 160°C for 10 hours or ultrasonic treatment for 1 hour.

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