Microfluidic colorimetric detection chip and application thereof in uric acid detection

Abstract

The application discloses a microfluidic colorimetric detection chip and application thereof in uric acid detection, and the microfluidic colorimetric detection chip comprises a polydimethylsiloxane (PDMS) flexible microfluidic flow channel layer and test reagent modified filter paper; wherein the test reagent modified filter paper comprises two kinds, the first kind of test reagent modified filter paper is a TMB color developing system paper chip obtained by loading MnO2@Co nanoscale enzyme and TMB on a circular filter paper; and the second kind of test reagent modified filter paper is an ABTS ·+ modified ABTS color developing system paper chip. The microfluidic colorimetric detection chip combines two kinds of uric acid colorimetric detection analysis methods without biological enzymes, so that accurate, rapid, low-cost and portable detection of uric acid in sweat is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microfluidic detection chip, and relates to a microfluidic detection chip special for uric acid detection, in particular to a microfluidic colorimetric detection chip for detecting uric acid in sweat and a preparation method and application thereof. BACKGROUND

[0002] Uric acid is the final circulating metabolite of purine nucleotides in the human body, and its imbalance is related to cardiovascular diseases, gout and kidney diseases. Excessive consumption of foods rich in purines will have an adverse effect on uric acid levels. Sweat can be collected in situ on almost the entire skin surface in a completely non-invasive manner, and is an ideal detection sample. Studies have shown that there is a certain correlation between the uric acid level in blood and the uric acid content in sweat. Therefore, developing a wearable detection device to monitor the uric acid content in sweat is of great significance for reducing the risk of gout and hyperuricemia in the clinical detection field.

[0003] Microfluidic chip technology integrates sample preparation, reaction, separation, detection and other operations involved in conventional chemical or biological laboratory on a chip with a size of only a few square centimeters or even smaller. Microfluidic chip can realize in-situ collection, sampling, transmission and detection of sweat, and has important application prospects in the field of human sweat detection. In-situ collection and detection of sweat can also improve the accuracy of sweat detection. Colorimetric analysis method can be directly observed by naked eye to realize qualitative and semi-quantitative analysis. It can also be photographed by a smart phone, and data analysis can be carried out by combining image processing software and smart phone detection software to realize quantitative detection. The combination of microfluidic chip technology and colorimetric analysis method has the advantages of simple device, easy to carry, convenient operation and low cost, and occupies an important position in the development field of wearable detection devices. At present, there are some microfluidic electrochemical detection chips for detecting uric acid in sweat. However, there is no report on wearable microfluidic colorimetric detection chip for detecting uric acid in sweat.

[0004] In addition, the microfluidic colorimetric detection chip developed at present for detecting uric acid in urine or blood samples mostly needs to use uric acid oxidase to catalyze the oxidation of uric acid to produce hydrogen peroxide, and further needs to use peroxidase to catalyze the oxidation between the color developing reagent and hydrogen peroxide to produce a distinguishable color signal. These natural biological enzymes have the disadvantages of easy deactivation, high cost, poor stability, inconvenience of storage and the like. Developing a new type of microfluidic colorimetric detection chip without biological enzymes for detecting uric acid in sweat is expected to further improve the stability of wearable detection equipment, and is of great significance for personal health monitoring. SUMMARY

[0005] The present application aims to provide a microfluidic colorimetric detection chip and its application in uric acid detection. The microfluidic colorimetric detection chip of the present application combines two uric acid colorimetric detection methods without biological enzymes to achieve accurate, rapid, low-cost and portable detection of uric acid in sweat.

[0006] The microfluidic colorimetric detection chip of the present application comprises a polydimethylsiloxane (PDMS) flexible microfluidic flow channel layer and a filter paper modified with a detection reagent.

[0007] The PDMS flexible microfluidic flow channel layer is provided with a central hole in the center of the layered structure, and four recessed flow channels are respectively arranged from the central hole and extend to the boundary of the layered structure in different directions and are in communication with the outside (for example, if the PDMS flexible microfluidic flow channel layer is square or rectangular, the four recessed flow channels are arranged from the central hole to the four diagonal directions and are in communication with the outside; if the PDMS flexible microfluidic flow channel layer is circular, the four recessed flow channels are arranged from the central hole to the radial directions and are in communication with the outside); recessed detection holes are respectively arranged on the four recessed flow channels, and the four recessed detection holes are not in communication with each other. The PDMS flexible microfluidic flow channel layer is also provided with four recessed blank control holes, which are not in communication with each other and with the four recessed flow channels.

[0008] The filter paper modified with a detection reagent comprises two kinds, the first kind of filter paper modified with a detection reagent is a TMB color developing system paper chip obtained by co-modifying a circular filter paper with MnO2@Co nanoscale enzyme and 3,3',5,5'-tetramethylbenzidine (TMB); and the second kind of filter paper modified with a detection reagent is an ABTS ·+ modified ABTS color developing system paper chip.

[0009] The first kind of filter paper modified with a detection reagent is prepared by the following method:

[0010] In the first step, MnO2@Co nanoscale enzyme is synthesized. A calculated amount of Mn 2+ salt and Co 2+ salt is dissolved in 100 mL of distilled water, and a required amount of hydroxide base is added dropwise under constant stirring to co-precipitate, so that the final pH of the solution is 9; the obtained suspension is stirred vigorously for a period of time, then 30% H2O2 solution is added, and the color of the whole mixture turns black. The mixture is stirred for more than 3 hours, the obtained solid is filtered out with a large amount of distilled water and dried overnight, the obtained solid is ground thoroughly and calcined at 400℃ for 10 hours with a temperature rising rate of 10℃ / min, and the obtained solid is treated by grinding to obtain MnO2@Co nanoscale enzyme.

[0011] The preferred solution is to dissolve 990 mg of manganese chloride tetrahydrate and 7.9 mg of cobalt acetate in 100 mL of distilled water.

[0012] The hydroxide base is preferably sodium hydroxide.

[0013] Second, prepare MnO2@Co nanoscale enzyme and TMB modified paper chip. Use a lettering machine to prepare a large number of circular filter paper pieces with a diameter of 5 mm. On the circular filter paper pieces, first add 5 μL of a phosphate buffer with a pH value of 7.0, wait for 3 min, then add 5 μL of 0.3 mg / mL MnO2@Co nanoscale enzyme, wait for 15 min, then add 5 μL of 3 mM TMB solution, react for 15 min, then add 5 μL of phosphate buffer again to make the paper piece color more uniform, then wait for 15 min to achieve the best color development state, and prepare a TMB color development system paper chip co-modified by MnO2@Co nanoscale enzyme and TMB.

[0014] The second detection reagent modified filter paper is prepared by the following method:

[0015] Take 22 mg of ABTS and dissolve it in 4 mL of deionized water, take 7.6 mg of potassium persulfate and dissolve it in 8 mL of deionized water, then mix the two at a volume ratio of 1:1, and react for 12 h to obtain 5 mM ABTS ·+ solution; add 6 μL of ABTS ·+ solution to a 5 mm circular filter paper piece, dry for 10 min, and prepare an ABTS ·+ modified ABTS color development system paper chip.

[0016] The circular filter paper has a diameter of 5 mm, corresponding to the pore diameters of the detection holes and blank holes of the PDMS microfluidic flow channel layer. The filter paper is preferably Whatman No. 1 filter paper.

[0017] The assembly method of the microfluidic colorimetric detection chip of the present application is as follows:

[0018] Transfer 4 pieces of TMB color development system paper chips and 4 pieces of ABTS color development system paper chips to the corresponding grooved circular holes of the prepared PDMS microfluidic flow channel layer. Specifically, place two pieces of TMB color development system paper chips and two pieces of ABTS color development system paper chips in the four grooved detection holes, respectively, and then place two pieces of TMB color development system paper chips and two pieces of ABTS color development system paper chips in the four grooved blank control holes, respectively.

[0019] For example, when the PDMS flexible microfluidic flow channel layer is square or rectangular, two of the four TMB color developing system paper chips are placed in the two circular detection hole recesses on the two flow channels in one diagonal direction, and the other two are placed in the two blank control hole recesses not connected with the flow channels. Two of the four ABTS color developing system paper chips are placed in the two circular detection hole recesses on the two flow channels in the other diagonal direction, and the other two are placed in the two blank control hole recesses not connected with the flow channels.

[0020] Further, in order to fix the detection reagent modified filter paper, a double-sided adhesive medical tape can be adhered to the PDMS microfluidic flow channel layer of the assembled paper chip, so as to fix the TMB color developing system paper chip and the ABTS color developing system paper chip inside the microfluidic detection chip. The double-sided adhesive medical tape is provided with a hollow sample injection hole. The sample injection hole is directly punched by a puncher. The diameter of the sample injection hole is preferably 3 mm. The center sample injection hole of the double-sided adhesive medical tape is aligned with the center hole of the PDMS. The double-sided adhesive medical tape can be replaced by other double-sided adhesive films, double-sided adhesive tapes and the like.

[0021] The preparation process of the PDMS flexible microfluidic flow channel layer is as follows.

[0022] Firstly, the mold structure is designed by SolidWorks software. The mold structure comprises a base, and the base is 50mm*50mm*5mm. The mold is additionally provided with a fence with an inner diameter of 30mm*30mm, an outer diameter of 40mm*40mm and a height of 10mm. The inside of the fence on the base of the mold is a convex platform with a size of 30mm*30mm*6mm, and a convex pattern is arranged on the platform. The convex pattern comprises eight circular convexes with a diameter of 5mm and a convex height of 1mm. The eight circular convexes with a diameter of 5mm are uniformly distributed in eight directions of the square convex platform. The convex pattern further comprises four convex channels, which respectively extend in four diagonal directions from the center, and the channel width is 1mm and the height is 1mm. Four circular convexes with a diameter of 5mm are located in the four diagonal direction channels. The mold is made of polytetrafluoroethylene

[0023] Secondly, the main agent and curing agent for preparing PDMS film (both commercially available) were mixed in a ratio of 10:1, stirred in a beaker for 30 min, then the obtained mixed reagent was ultrasonically treated to remove excess air bubbles, and finally poured into the polytetrafluoroethylene mold and placed in a dry box at 60°C for drying for 3 hours. Finally, the flexible PDMS microfluidic flow channel layer was demolded. The size of the PDMS microfluidic flow channel layer was 30mm*30mm, and the thickness was 2.5mm, wherein the thickness of 1.5mm part was the base film part without pores. The PDMS microfluidic flow channel layer base film contained 8 circular grooves with a diameter of 5mm and a depth of 1mm. The PDMS microfluidic flow channel layer base also contained four groove flow channels extending from the center to the four diagonal lines, with a width of 1mm and a depth of 1mm. The four flow channels communicated with the four circular grooves, and then communicated with the atmosphere.

[0024] The application of the microfluidic colorimetric detection chip is for uric acid detection, including the following steps:

[0025] Step 1: Simulated sweat solution containing different concentrations of uric acid is injected from the center hole, the sweat is divided from the center hole to the four flow channels, and the paper chip on the channel is soaked;

[0026] Step 2: After injection, react for 1min, take a picture to obtain the picture of the microfluidic colorimetric detection chip, and further use the picture editing software (such as Image J software) to obtain the gray value data of the four detection holes and four blank control holes of the microfluidic colorimetric detection chip;

[0027] Step 3: Drawing of standard curve

[0028] 3a, add the gray values of the two detection holes of the TMB color developing system on the microfluidic colorimetric detection chip and take the average to obtain the average gray value of the TMB color developing system detection hole; add the gray values of the two blank control holes of the TMB color developing system on the microfluidic colorimetric detection chip and take the average to obtain the average gray value of the TMB color developing system blank control hole; subtract the average gray value of the TMB color developing system blank control area from the average gray value of the TMB color developing system detection hole to obtain the gray value difference, which is used as the detection signal of the TMB color developing system of the microfluidic colorimetric detection chip, and the relationship between the TMB color developing system detection signal and the uric acid concentration is plotted to obtain the linear curve of the TMB color developing system uric acid detection;

[0029] 3b, add the gray scale values of the two detection holes of the ABTS color developing system on the microfluidic colorimetric detection chip and take the average to obtain the average gray scale value of the ABTS color developing system detection hole; add the gray scale values of the two blank control holes of the ABTS color developing system on the microfluidic colorimetric detection chip and take the average to obtain the average gray scale value of the ABTS color developing system blank control hole; subtract the average gray scale value of the ABTS color developing system blank control area from the average gray scale value of the ABTS color developing system detection hole to obtain the gray scale value difference, which is used as the detection signal of the ABTS color developing system of the microfluidic colorimetric detection chip, and a relationship diagram between the detection signal of the ABTS color developing system and the concentration of uric acid is drawn to obtain a linear curve for the detection of uric acid by the ABTS color developing system;

[0030] 3c, use the sum of the detection signal of the TMB color developing system and the detection signal of the ABTS color developing system as the joint quantitative detection signal, draw a relationship diagram between the joint quantitative detection signal and the concentration of uric acid, and obtain a joint quantitative linear curve for the quantitative detection of uric acid.

[0031] Step 4: In the actual test process, the sample to be tested is introduced through the center hole and enters the microfluidic colorimetric detection chip by capillary action and wets the paper chip, and after 1 min, the picture information of the microfluidic colorimetric detection chip is obtained by taking a picture; referring to step 3, the TMB color developing system detection signal and the ABTS color developing system detection signal of the microfluidic colorimetric detection chip are obtained respectively, and the concentration of uric acid is calculated according to the TMB color developing system linear curve, the ABTS color developing system linear curve and the joint quantitative linear curve respectively. According to the average value of the uric acid concentration calculated by the three linear curves, the final concentration of uric acid in the actual sweat is calculated.

[0032] Compared with the prior art, the beneficial effects of the present application are as follows:

[0033] 1. The microfluidic colorimetric detection chip of the present application can be directly attached to the surface of the skin for rapid detection of uric acid in sweat, without the need for complex pretreatment steps such as sampling and sample preparation, and has the advantages of small amount of reagent and sample, simple method, low cost, rapidness, portability and the like, and has important significance for rapid detection of sweat uric acid and monitoring and prevention of related diseases.

[0034] 2. The microfluidic colorimetric detection chip for uric acid detection of the present application does not require the use of any biological enzyme, overcoming the problems of poor stability, harsh storage and use conditions caused by the use of biological enzymes in traditional uric acid colorimetric analysis methods.

[0035] 3. The microfluidic colorimetric detection chip of the present application uses the TMB color developing system based on MnO2@Co nanocatalysis and the ABTS ·+The color developing systems are used for high-selectivity colorimetric detection of uric acid for the first time, and provide two feasible technical solutions for new uric acid colorimetric detection methods without biological enzymes.

[0036] 4、The microfluidic colorimetric detection chip of the application is equipped with two colorimetric detection systems without biological enzymes, and the two colorimetric detection systems are used in combination to improve the detection accuracy. In addition, for each color developing system on the microfluidic colorimetric detection chip, two diagonal detection holes and two opposite blank control holes are arranged, and the gray value mean of the two detection holes is subtracted from the gray value mean of the two blank holes as the detection signal, so that the experimental error can be effectively overcome, and the interference of the external environment on the detection can be reduced. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a mold structure pattern used for preparing the PDMS microfluidic flow channel layer of the microfluidic colorimetric detection chip in Example 1.

[0038] Figure 2 is a structure pattern of the PDMS microfluidic flow channel layer of the microfluidic colorimetric detection chip in Example 1.

[0039] Figure 3 is an assembly scheme for preparing the microfluidic colorimetric detection chip in Example 1.

[0040] Figure 4 is picture data of the microfluidic colorimetric detection chip for detecting uric acid with different concentrations in Example 2.

[0041] Figure 5 is a linear fitting curve graph of the TMB color developing system catalyzed by the MnO2@Co nanoscale enzyme for uric acid detection in the microfluidic colorimetric detection chip in Example 2.

[0042] Figure 6 is a linear fitting curve graph of the ABTS color developing system for uric acid detection in the microfluidic colorimetric detection chip in Example 2.

[0043] Figure 7 is a combined quantitative linear fitting curve graph of the TMB color developing system and the ABTS color developing system in the microfluidic colorimetric detection chip for uric acid quantification in Example 2.

[0044] Figure 8 is picture data and a detection signal comparison graph of the TMB color developing system in the microfluidic colorimetric detection chip for detecting uric acid and various interfering substances in sweat in Example 3.

[0045] Figure 9is the picture data and detection signal comparison chart of ABTS color system for the detection of uric acid and various interfering substances in sweat using microfluidic colorimetric detection chip in Example 3. DETAILED DESCRIPTION

[0046] The application will be further described in detail below in conjunction with the drawings and examples, but the scope of protection of the application is not limited to these examples. All other examples obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the application.

[0047] Example 1:

[0048] The specific manufacturing and assembly process of the microfluidic colorimetric detection chip is as follows:

[0049] 1) First, the mold structure diagram for preparing the PDMS microfluidic flow channel layer of the microfluidic colorimetric detection chip is designed by using the SolidWorks drawing software (as shown in the accompanying Figure 1 The design parameters of the mold for preparing the PDMS microfluidic flow channel layer are as follows:

[0050] The mold base is 50mm*50mm*5mm. The mold is additionally provided with a fence with an inner diameter of 30mm*30mm, an outer diameter of 40mm*40mm, and a height of 10mm. A 30mm*30mm*6mm raised platform is arranged inside the fence on the mold base, and a raised pattern is arranged on the platform. The raised pattern includes eight circular protrusions with a hole diameter of 5mm, and the protrusion height is 1mm. The eight circular protrusions with a hole diameter of 5mm are uniformly distributed in eight directions of the square protrusion platform. The raised pattern also includes four protruding channels, which respectively extend from the center to four diagonal directions until they are in communication with the atmosphere, with a channel width of 1mm and a height of 1mm. The four circular protrusions with a hole diameter of 5mm are located in the four diagonal direction channels. The mold is made of polytetrafluoroethylene.

[0051] 2) The flexible PDMS microfluidic flow channel layer is prepared by using the reverse mold method, and the structure of the PDMS microfluidic flow channel is as shown in the accompanying Figure 2 The steps for preparing the PDMS microfluidic channel are as follows:

[0052] The main agent and curing agent of the Dow Corning 184 transparent slow-drying potting adhesive for preparing PDMS were mixed at a ratio of 10:1, stirred in a beaker for 30 min at a speed of 150 rpm, then the obtained mixed reagent was ultrasonically treated to remove excess air bubbles, finally 3.3 mL of the mixed reagent was poured into the prepared polytetrafluoroethylene mold, the mold was placed in a dry box at 60°C for drying for 3 hours, and finally the cured PDMS film was demolded from the mold to prepare a flexible PDMS microfluidic flow channel layer with a groove type channel. The size of the PDMS microfluidic flow channel layer is 30mm*30mm, and the thickness is 2.5mm, wherein the 1.5mm part is a complete and non-porous base film part. The PDMS microfluidic flow channel layer base film contains 8 circular grooves with a diameter of 5mm and a depth of 1mm. The PDMS microfluidic flow channel layer base also contains four groove type flow channels extending from the center to the four diagonal lines, with a width of 1mm and a depth of 1mm. The four flow channels are communicated with the four circular grooves, and then communicated with the atmosphere.

[0053] 3) Use a puncher to punch a circular hole in the middle of the double-sided adhesive medical tape. The size of the double-sided adhesive medical tape is 3cm*3cm, consistent with the size of the PDMS film. The diameter of the circular sample inlet hole on the double-sided adhesive medical tape is 3mm.

[0054] 4) Preparation of MnO2@Co nanoscale enzyme and TMB modified detection paper chip. The preparation steps are as follows:

[0055] Step 1: Synthesis of MnO2@Co nanoscale enzyme. Dissolve 990mg of manganese chloride tetrahydrate and 7.9mg of cobalt acetate in 100mL of distilled water. Under constant stirring, add 0.1M sodium hydroxide solution dropwise until the solution pH is 9. Stir the obtained suspension vigorously for a period of time, then add 30% H2O2 until the solution turns brown-black, stir the mixture for 3 hours, filter the obtained solid with a large amount of distilled water and dry overnight. After drying, the obtained solid is ground with a mortar and pestle and calcined at 400°C for 10 hours at a heating rate of 10°C / min, and the obtained solid is treated by grinding to obtain MnO2@Co nanoscale enzyme.

[0056] Step 2: Preparation of the MnO2@Co nanozyme and TMB-modified detection paper chip. Using a cutting machine, Whatman No. 1 filter paper was prepared into circular filter paper discs with a diameter of 5 mm. On the circular filter paper disc, 5 μL of pH 7.0 phosphate buffer was added first, and the reaction was allowed to proceed for 3 min. Then, 5 μL of 0.3 mg / mL MnO2@Co nanozyme was added, and the reaction was allowed to proceed for 15 min. Finally, 5 μL of phosphate buffer was added again to ensure more uniform color development on the paper disc. The reaction was allowed to proceed for another 15 min to reach the optimal color development state, thus preparing the MnO2@Co nanozyme and TMB-modified TMB colorimetric detection paper chip.

[0057] 5) Preparation of ABTS ·+ The modified detection paper chip is prepared by the following steps:

[0058] First, dissolve 22 mg of ABTS in 4 mL of deionized water, and dissolve 7.6 mg of potassium persulfate in 8 mL of deionized water. Then, mix the two solutions at a volume ratio of 1:1 and react for 12 h to obtain 5 mM ABTS. ·+ Solution. Take 6 μL of ABTS. ·+ The solution was dropped onto a 5 mm circular filter paper and dried for 10 min to prepare ABTS. ·+ Modified detection paper chip.

[0059] 6) The assembly and fabrication scheme of the microfluidic colorimetric detection chip is attached. Figure 3 As shown. A detection paper chip modified with PDMS microfluidic flow channel layer, MnO2@Co nanozyme and TMB, and ABTS were used. ·+ A microfluidic colorimetric detection chip was prepared by assembling modified detection paper chips and double-sided adhesive tape. The specific assembly scheme is as follows:

[0060] 4 ABTS ·+ The modified detection paper chips were placed in the recessed circular wells numbered 1, 2, 5, and 6 of the PDMS microfluidic flow channel layer. Wells 2 and 6 correspond to the detection wells of the ABTS colorimetric system, while wells 1 and 5 correspond to the blank control wells of the ABTS colorimetric system. Four MnO2@Co nanozyme and TMB-modified detection paper chips were placed in the recessed circular wells numbered 3, 4, 7, and 8 of the PDMS microfluidic flow channel layer. Wells 4 and 8 correspond to the detection wells of the TMB colorimetric system, while wells 3 and 7 correspond to the blank control wells of the TMB colorimetric system.

[0061] After aligning the center sample injection hole of the double-sided adhesive medical tape with the center hole of the PDMS, the tape is adhered to the PDMS film with the assembled paper chip, so as to fix the detection paper chip inside the microfluidic detection chip, and a microfluidic colorimetric detection chip is prepared. When in use, if the sample is directly injected, the tape is upward, or the tape can not be used; if it is used by being attached to the skin, one side of the tape is adhered to the PDMS film, and the other side is adhered to the surface of the skin.

[0062] Example 2:

[0063] In order to prove the beneficial effects of the present application, the microfluidic colorimetric detection chip of Example 1 is used for the detection of simulated sweat uric acid, and the specific method is as follows:

[0064] 1) First, the microfluidic colorimetric detection chip is prepared according to Example 1.

[0065] 2) Prepare simulated sweat standard solutions containing different concentrations of uric acid. Prepare simulated sweat: the simulated sweat contains 20 mg / mL of sodium chloride, 17.5 mg / mL of ammonium chloride, 5 mg / mL of urea, 2.5 mg / mL of acetic acid and 15 mg / mL of lactic acid. Then use the simulated sweat to prepare 20 μM, 50 μM, 80 μM, 110 μM, 140 μM, 170 μM, 200 μM uric acid standard solutions.

[0066] 3) Inject the simulated sweat standard solutions containing different concentrations of uric acid into the center sample injection hole of the microfluidic colorimetric detection chip, respectively. After injection, the simulated sweat is divided into four diagonal flow channels from the center hole, and then the paper chip on each flow channel is wetted, and the reaction is carried out at room temperature for 1 min.

[0067] 4) Use a smart phone to take a picture of the microfluidic colorimetric detection chip (see Figure 4 It can be seen from the figure that the TMB color developing system is blue-green, and the ABTS color developing system is green. Uric acid has an inhibitory fading effect on the TMB and ABTS color developing systems. Therefore, with the increase of the concentration of uric acid in the simulated sweat, the color of the detection area of the microfluidic colorimetric detection chip TMB and ABTS color developing system gradually becomes light, and the color of the blank control area of the TMB and ABTS color developing system remains unchanged. The color change of the microfluidic colorimetric detection chip can be recognized by the naked eye, and the semi-quantitative detection of uric acid is realized. Further, the Image J software is used to obtain the color value information of the eight color developing regions of the microfluidic colorimetric detection chip, which is used for the accurate quantitative detection of uric acid.

[0068] 5) The average gray value of the TMB color developing detection area at positions 4 and 8 of the microfluidic colorimetric detection chip was subtracted from the average gray value of the TMB color developing system blank control area at positions 3 and 7, and the difference in gray value was taken as the gray value detection signal of the TMB color developing system. The experimental data were processed using origin software, and a linear fitting curve between the simulated sweat uric acid concentration and the colorimetric signal of the TMB color developing system in the microfluidic colorimetric detection chip was plotted (as shown in the accompanying Figure 5 TMB = 0.63 + 0.08x, and the linear correlation coefficient was R 2 = 0.975.

[0069] 6) The average gray value of the ABTS color developing detection area at positions 2 and 6 of the microfluidic colorimetric detection chip was subtracted from the average gray value of the ABTS color developing system blank control area at positions 1 and 5, and the difference in gray value was taken as the gray value detection signal of the ABTS color developing system. The experimental data were processed using origin software, and a linear fitting curve between the simulated sweat uric acid concentration and the colorimetric signal of the ABTS color developing system in the microfluidic colorimetric detection chip was plotted (as shown in the accompanying Figure 6 ABTS = 21.65 + 0.12x, and the linear correlation coefficient was R 2 = 0.980. Each color developing system of the microfluidic colorimetric detection chip was provided with two detection holes and two corresponding blank control holes to reduce test errors.

[0070] 7) The colorimetric signal of the TMB color developing system of the microfluidic colorimetric detection chip and the colorimetric signal of the ABTS color developing system of the microfluidic colorimetric detection chip were combined as a joint quantitative colorimetric signal, and a joint quantitative linear fitting curve of the TMB color developing system and the ABTS color developing system in the microfluidic colorimetric detection chip for uric acid quantification was plotted (as shown in the accompanying Figure 7 (TMB+ABTS) = 20.78 + 0.21x, and the linear correlation coefficient was R 2 = 0.985. The linear curve obtained by combining the two color developing systems has higher sensitivity.

[0071] 8) The linear range of the microfluidic colorimetric detection chip for uric acid detection in sweat was 20 μM to 200 μM, the lower limit of detection was 6.6 μM, and the standard deviation of detection was less than 4.18%. The prepared microfluidic colorimetric detection chip has a wide detection range and good reproducibility, and can meet the detection of uric acid in human sweat.

[0072] Example 3:

[0073] ​​​To demonstrate the beneficial effects of the present invention, the microfluidic colorimetric detection chip of Example 1 was used to simulate the detection of other interfering substances in sweat. The specific method is as follows:

[0074] 1) First, prepare a microfluidic colorimetric detection chip according to Example 1.

[0075] 2) Select substances coexisting in sweat, including glucose, lactic acid, creatinine, and Cl-. - Na + K + Ca 2+ Mg 2+ Eight substances were used as interfering agents, and corresponding interference test solutions were prepared according to the concentrations of each substance in sweat, including 2 mM glucose, 20 mM lactic acid, 133 μM creatinine, and 50 mM Cl. – 50mM Na + 50mM K + 50mM Ca 2+ 50mM Mg 2+ The microfluidic colorimetric detection chip prepared in step 1 was used to test various interfering solutions. ImageJ software was used to read and calculate the grayscale detection signals of the TMB colorimetric system and the ABTS colorimetric system, respectively, and a comparative bar chart was plotted. The results for the TMB colorimetric system are attached. Figure 8 As shown in the attached figure. Results of the ABTS colorimetric system are as follows. Figure 9 As shown, the colors of the detection areas of the microfluidic colorimetric detection chip with the addition of eight interfering substances (TMB and ABTS) are basically consistent with the blank control area, with no obvious fading. However, the microfluidic colorimetric detection chip with the addition of uric acid shows significant and large-area color fading in the detection areas of the TMB and ABTS systems. This further verifies that uric acid exhibits a significant inhibitory effect on fading in both the constructed MnO2@Co nanozyme-catalyzed TMB and ABTS colorimetric systems, and the resulting grayscale detection signals are significantly greater than those of the interfering substances and the blank. Therefore, when the prepared microfluidic colorimetric detection chip is used for uric acid detection in sweat, the interfering substances that may be present in sweat have almost no effect on uric acid detection, and the prepared microfluidic colorimetric detection chip has high selectivity for uric acid detection in sweat.

[0076] Example 4:

[0077] To demonstrate the beneficial effects of the present invention, the microfluidic colorimetric detection chip of Example 1 was used for the detection of uric acid in actual sweat. The specific method is as follows:

[0078] 1) First, prepare a microfluidic colorimetric detection chip according to Example 1.

[0079] 2) The prepared microfluidic colorimetric detection chip is attached to the arm of the exerciser until the sweat wets the 4 paper chips in the detection area of the microfluidic colorimetric detection chip by capillary action, and after 1 min, the picture information of the microfluidic colorimetric detection chip is obtained by taking a picture. The average value of the gray value of the TMB color developing detection area of the microfluidic colorimetric detection chip is subtracted from the average value of the gray value of the corresponding TMB color developing system blank control area, and the obtained gray value difference is used as the detection signal of the TMB color developing system. The average value of the gray value of the ABTS color developing detection area of the microfluidic colorimetric detection chip is subtracted from the average value of the gray value of the corresponding ABTS color developing system blank control area, and the obtained gray value difference is used as the detection signal of the ABTS color developing system. The detection signal of the TMB color developing system is added to the detection signal of the ABTS color developing system to obtain the final quantitative detection signal. According to the linear curve for the quantitative determination of uric acid, the concentration of uric acid in the actual sweat is calculated. The detection results show that the above-mentioned actual sweat contains 0.17 mmol / L of uric acid. The above-mentioned detection results are consistent with the uric acid content in the sweat of normal people. Therefore, the microfluidic colorimetric detection chip of the present application can realize the quantitative detection of uric acid in sweat.

[0080] 3) In addition, by developing a smart phone detection APP, after taking a picture by the smart phone detection APP, the gray values of each detection area and the blank control area can be automatically recognized, and according to the linear equation built in the APP, the concentration of uric acid can be automatically calculated, and the detection results can be displayed on the result interface, so that intelligent sweat uric acid detection is realized.

[0081] The detection accuracy of the microfluidic colorimetric detection chip of the present application is further investigated by using the standard addition recovery experiment. The simulated sweat standard solution of 0.1 mmol / L uric acid is quantitatively added to the above-mentioned actual sweat detection area, the uric acid content in the added sweat is detected, and the recovery rate and the standard deviation between the detection data are calculated. The results show that the recovery rate is 110%, and the standard deviation is less than 10%, and the data of the standard addition recovery experiment is ideal. The above-mentioned data show that the microfluidic colorimetric detection chip of the present application can realize the detection of uric acid in actual sweat, and the detection results are accurate.

Claims

1. A microfluidic colorimetric detection chip, characterized in that: the microfluidic colorimetric detection chip comprises a polydimethylsiloxane (PDMS) flexible microfluidic flow channel layer and a test reagent modified filter paper; the PDMS flexible microfluidic flow channel layer is provided with a central hole in the center of a layered structure, and four recessed flow channels are respectively arranged from the central hole and extend to the boundary of the layered structure in different directions and are communicated with the outside; recessed detection holes are respectively arranged on the four recessed flow channels, and the four recessed detection holes are not communicated with each other; the PDMS flexible microfluidic flow channel layer is further provided with four recessed blank control holes, the four recessed blank control holes are not communicated with each other and with the four recessed flow channels; two pieces of TMB color developing system paper chips and two pieces of ABTS color developing system paper chips are respectively placed in the four recessed detection holes, and two pieces of TMB color developing system paper chips and two pieces of ABTS color developing system paper chips are respectively placed in the four recessed blank control holes; the first test reagent modified filter paper is prepared by the following method: The detection reagent modified filter paper includes two kinds, the first detection reagent modified filter paper is a TMB color developing system paper chip obtained by co-modifying a circular filter paper with MnO2@Co nanoscale enzyme and TMB; the second detection reagent modified filter paper is an ABTS ·+ modified ABTS color developing system paper chip; in the second step, 5 μL of a phosphate buffer with a pH value of 7.0 is added to the circular filter paper piece, and after waiting for 3 min, 5 μL of 0.3 mg / mL MnO2@Co nanoscale enzyme is added, and after waiting for 15 min, 5 μL of 3 mM TMB solution is added, and after reacting for 15 min, 5 μL of phosphate buffer is added again to make the paper piece develop color more uniformly, and then after waiting for 15 min to reach the best color developing state, a TMB color developing system paper chip modified by MnO2@Co nanoscale enzyme and TMB is prepared; the second test reagent modified filter paper is prepared by the following method: First step, Mn 2+ salt and Co 2+ The salt is dissolved in distilled water, and the desired amount of hydroxide base is added dropwise under constant stirring to coprecipitate, so that the final pH of the solution is 9; the obtained suspension is stirred for a period of time, then a 30% H2O2 solution is added, and the color of the mixture turns black; the mixture is stirred for more than 3 hours, the obtained solid is filtered off with distilled water and dried, the obtained solid is ground thoroughly and calcined at 400°C for 10 hours, and the obtained solid is treated by grinding to obtain MnO2@Co nanoszyme; 2. The microfluidic colorimetric detection chip according to claim 1, characterized in that: in the first step, 990 mg of manganese chloride tetrahydrate and 7.9 mg of cobalt acetate are dissolved in 100 mL of distilled water. Take 22 mg of ABTS dissolved in 4 mL of deionized water, take 7.6 mg of potassium persulfate dissolved in 8 mL of deionized water, then mix the two at a volume ratio of 1:1, and react for 12 h to obtain 5 mM of ABTS ·+ solution; take 6 μL of ABTS ·+ solution and drop it on a circular filter paper sheet, dry for 10 min, and prepare ABTS ·+ Modified ABTS color developing system paper chip. comprising the following steps: Step 1: The simulated solution containing different concentrations of uric acid is injected from the central hole, and the simulated solution is divided into four flow channels from the central hole, wetting the paper chips on the channels; 3. Use of the microfluidic colorimetric detection chip according to claim 1 or 2 for the detection of uric acid, characterized in that Step 2: After injection, react for 1 min, take a picture to obtain the picture of the microfluidic colorimetric detection chip, and further use the picture editing software to obtain the gray value data of the four detection holes and the four blank control holes of the microfluidic colorimetric detection chip; Step 3: Drawing of the standard curve 3a, the gray values of the two detection holes of the TMB color developing system on the microfluidic colorimetric detection chip are added and averaged to obtain the average gray value of the TMB color developing system detection hole; the gray values of the two blank control holes of the TMB color developing system on the microfluidic colorimetric detection chip are added and averaged to obtain the average gray value of the TMB color developing system blank control hole; ​ ​ The gray value difference calculated by subtracting the average gray value of the blank control area of ​​the TMB colorimetric system from the average gray value of the detection wells of the TMB colorimetric system is used as the detection signal of the TMB colorimetric system of the microfluidic colorimetric detection chip. The relationship between the detection signal of the TMB colorimetric system and the uric acid concentration is plotted to obtain the linear curve of uric acid detection by the TMB colorimetric system. 3b. Sum the gray values ​​of the two detection wells of the ABTS colorimetric system on the microfluidic colorimetric detection chip and take the average to obtain the average gray value of the detection wells of the ABTS colorimetric system; sum the gray values ​​of the two blank control wells of the ABTS colorimetric system on the microfluidic colorimetric detection chip and take the average to obtain the average gray value of the blank control wells of the ABTS colorimetric system; use the difference between the average gray value of the detection wells of the ABTS colorimetric system and the average gray value of the blank control area of ​​the ABTS colorimetric system as the detection signal of the ABTS colorimetric system on the microfluidic colorimetric detection chip; plot the relationship between the detection signal of the ABTS colorimetric system and the uric acid concentration to obtain the linear curve of uric acid detection by the ABTS colorimetric system. 3c. Use the sum of the detection signals from the TMB colorimetric system and the ABTS colorimetric system as the detection signal for combined quantification, plot the relationship between the detection signal for combined quantification and uric acid concentration, and obtain the combined quantification linear curve for uric acid quantification. Step 4: During the actual test, the sample to be tested is injected through the central hole and enters the microfluidic colorimetric detection chip through capillary action, wetting the paper chip. After 1 minute, an image of the microfluidic colorimetric detection chip is captured. Referring to Step 3, the detection signals of the TMB colorimetric system and the ABTS colorimetric system of the microfluidic colorimetric detection chip are obtained respectively. The uric acid concentration is calculated according to the linear curves of the TMB colorimetric system, the ABTS colorimetric system, and the combined quantitative linear curve. The final concentration of uric acid in the actual sweat is calculated based on the average of the uric acid concentrations calculated from the three linear curves. The microfluidic colorimetric detection chip has a linear range of 20 μM to 200 μM for uric acid detection.

4. The application according to claim 3, characterized in that: The detection limit of the microfluidic colorimetric detection chip is 6.6 μM.

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