Micro-fluidic colorimetric chip for detecting creatinine in sweat as well as preparation method and application of micro-fluidic colorimetric chip

Abstract

The invention discloses a microfluidic colorimetric chip for sweat creatinine detection and a preparation method and application thereof, and belongs to the technical field of microfluidic detection chips, the microfluidic detection chip comprises a microfluidic channel layer, a detection layer and a packaging layer; the detection layer is filled on the micro-fluidic channel layer, and the encapsulation layer is positioned on the micro-fluidic channel layer; the microfluidic channel layer is provided with a sample inlet area, a shunting area, an explosion valve groove, a detection area and a blank control area; the detection layer comprises paper chips modified by two detection reagents, one is a paper chip modified by Cu < 2 + >, and the other is a paper chip jointly modified by H2MoO5 nano enzyme and 3, 3 ', 5, 5'-tetramethyl benzidine; a sample injection hole is formed in the packaging layer and corresponds to the sample injection port of the micro-fluidic channel layer. The microfluidic colorimetric detection chip for creatinine detection has the beneficial effects that the H2MoO5 nano enzyme-Cu < 2 + > catalytic system which is stable and low in preparation cost is used for replacing a biological enzyme catalytic system which is high in cost, difficult to store and easy to inactivate, and the stability and the storability are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic detection chips, and relates to a bio-enzyme-free creatinine detection microfluidic detection chip, and in particular to a wearable microfluidic colorimetric detection chip for creatinine detection in sweat, and a preparation method and application thereof. Background Art

[0002] Sweat is secreted by sweat glands and is rich in electrolytes (Na + , K + , Ca 2+ Sweat can be collected in situ on almost the entire skin surface in a non-invasive and continuous manner with high reproducibility, making it an ideal human fluid testing sample. Therefore, sweat testing technology has developed rapidly in recent years and has become a hot field that integrates multiple disciplines such as analysis, sensing, and medical health. However, traditional sweat testing is carried out in the laboratory, which often requires complex equipment and cumbersome steps, which greatly affects the portability and real-time performance of the test.

[0003] In recent years, with the development of micro-processing technology and flexible materials, wearable microfluidic chips have begun to be used for human sweat detection. Compared with traditional laboratory detection technology, wearable microfluidic detection chips integrate the sample transportation, separation, reaction, analysis and other functions carried out in traditional laboratories into a tiny chip of a few square centimeters, with the characteristics of small size, low cost, rapid response, continuous collection and easy operation.

[0004] Creatinine is a compound produced during the energy production process of muscles. The level of creatinine in the human body reflects the filtering function of the kidneys and is an important indicator for detecting kidney function. There are many methods for detecting creatinine, including chromatography, mass spectrometry, electrochemistry, and capillary zone electrophoresis. These methods require professionals to operate professional detection instruments and can only be performed in laboratories. The colorimetric method has the advantages of simplicity, low cost, and rapidity, and has important application prospects in the field of portable detection. The Jaffe method is a traditional colorimetric method for creatinine detection, but the Jaffe method has certain limitations in practical applications due to its poor specificity and susceptibility to interference from multiple substances.

[0005] The microfluidic colorimetric detection chips currently developed for creatinine detection mostly require the use of creatinine oxidase to catalyze the gradual hydrolysis of creatinine into hydrogen peroxide. The generated hydrogen peroxide further reacts with the colorimetric reagent to generate a colored substance for quantitative detection. Although the detection method based on biological enzymes has high selectivity, it has the disadvantages of high cost, difficult storage, easy inactivation, poor stability, and difficulty in recycling and reuse caused by biological enzymes. Therefore, the development of a new wearable microfluidic colorimetric detection chip that does not require biological enzymes for the detection of creatinine in sweat is expected to further improve the stability of wearable detection equipment, which is of great significance for the early diagnosis of kidney disease and health prevention and management. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to provide a bio-enzyme-free microfluidic colorimetric detection chip and its application in creatinine detection, so as to achieve in-situ, rapid and low-cost detection of creatinine in sweat. To achieve the above-mentioned goal, the present invention mainly includes the following technical solutions.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The first aspect of the present invention provides a microfluidic detection chip for sweat creatinine detection, the microfluidic detection chip comprising a microfluidic channel layer, a detection layer and a packaging layer; the detection layer is loaded on the microfluidic channel layer, and the packaging layer is located on the microfluidic channel layer;

[0009] The microfluidic channel layer is provided with an injection port area, a diversion area, a burst valve groove, a detection area and a blank control area;

[0010] The detection layer includes two paper chips modified with detection reagents, one of which is Cu 2+ modified paper chip, and the other is a paper chip co-modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB);

[0011] The packaging layer is provided with a sampling hole, and the sampling hole corresponds to the sampling port of the microfluidic channel layer.

[0012] Preferably, the microfluidic channel layer is made of PDMS.

[0013] Preferably, the material of the paper chip is any one or more of Whatman No. 1 filter paper, cellulose acetate paper, nitrocellulose membrane, and qualitative filter paper.

[0014] Preferably, the Cu 2+ The modified paper chip is Cu 2+The solution is dripped onto a filter paper; the paper chip co-modified with the H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) is obtained by dripping an acetic acid-sodium acetate buffer solution onto the filter paper, adding the H2MoO5 nanozyme, and then adding the TMB solution.

[0015] Preferably, the H2MoO5 nanozyme is prepared by the following process: ultrasonically dispersing Mo powder in water, adding H2O2 dropwise under ice-water bath conditions, adding glacial acetic acid, and fully reacting at room temperature until an orange-yellow solution appears, then filtering to obtain a yellow filtrate, drying, calcining, and grinding to obtain the H2MoO5 nanozyme.

[0016] Preferably, the H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) modified paper chip match the detection area and blank control area on the microfluidic channel layer; 2+ The modified paper chip matches the burst valve groove on the microfluidic channel layer.

[0017] The second aspect of the present invention provides a method for preparing the above-mentioned microfluidic detection chip for sweat creatinine detection, comprising the following steps: loading a paper chip modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) of the detection layer into the detection area and the blank control area on the microfluidic channel layer, 2+ The modified paper chip is loaded into the burst valve groove on the microfluidic channel layer, the injection hole of the encapsulation layer is aligned with the injection port of the microfluidic channel layer, and then pasted on the microfluidic flow channel layer filled with the paper chip to complete the encapsulation of the microfluidic channel.

[0018] The third aspect of the present invention proposes the use of the above-mentioned microfluidic detection chip for sweat creatinine detection in the detection of creatinine levels in sweat.

[0019] The fourth aspect of the present invention proposes a method for preparing H2MoO5 nanozyme, comprising the following steps: ultrasonically dispersing Mo powder in water, adding H2O2 dropwise under ice-water bath conditions, adding glacial acetic acid, and reacting fully at room temperature until an orange-yellow solution appears, then filtering to obtain a yellow filtrate, drying, calcining, and grinding to obtain H2MoO5 nanozyme.

[0020] The fifth aspect of the present invention provides the H2MoO5 nanozyme prepared by the above preparation method.

[0021] The sixth aspect of the present invention proposes the use of the above-mentioned H2MoO5 nanozyme in the preparation of a microfluidic detection chip for sweat creatinine detection.

[0022] The application of the wearable microfluidic colorimetric detection chip of the present invention is to use the microfluidic colorimetric detection chip for sweat creatinine detection, comprising the following steps:

[0023] Step 1: According to the above preparation method, a wearable microfluidic colorimetric detection chip is prepared.

[0024] Step 2: Add simulated sweat standard solutions containing different concentrations of creatinine to the injection hole. The simulated sweat enters the microfluidic colorimetric detection chip from the injection hole, flows through the blasting area after diversion, and then enters the detection area, where it reacts with the paper chip. After 10 minutes, take a photo with a smartphone to obtain a color picture of the microfluidic colorimetric detection chip.

[0025] Step 3: Use image editing software (such as ImageJ software) to obtain the RGB values ​​of the test area and the blank control area images, use the difference (△R) between the average R value of the two test areas and the R value of the blank control area as the detection signal, and obtain the linear curve for creatinine quantitative detection.

[0026] Step 4: Stick the wearable microfluidic colorimetric detection chip with the sample inlet facing downward on the sweat-prone skin surface (such as arms and back) of the athlete. After the sweat flows into the detection area and a color reaction occurs, take a photo to obtain the △R value. According to the above creatinine quantitative linear curve, calculate the actual creatinine concentration in sweat.

[0027] The beneficial effects of the present invention are:

[0028] 1. The microfluidic colorimetric detection chip for creatinine detection of the present invention uses a stable and low-cost H2MoO5 nanozyme-Cu 2+ The catalytic system replaces the biological enzyme catalytic system which is high in cost, difficult to store and easily inactivated, and has significantly improved stability and easy storage.

[0029] 2. The microfluidic colorimetric detection chip of the present invention has the advantages of simple operation, portability, softness, and small size. It can be directly worn on the skin surface to achieve real-time, non-invasive, and highly selective detection of creatinine in microliters of sweat, avoiding cumbersome sweat collection, pretreatment, and the use of large-scale detection instruments, and providing a new technology for sweat creatinine detection.

[0030] 3. The microfluidic colorimetric detection chip of the present invention can be extended to realize simultaneous, real-time and rapid detection of multiple markers in sweat, which is of great significance for personal health monitoring and early detection and monitoring of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a scanning electron microscope characterization image of the H2MoO5 nanozyme in Example 1.

[0032] Figure 2It is the UV-visible absorption spectrum diagram and the colorimetric solution picture of the colorimetric catalytic performance study of the H2MoO5 nanozyme in Example 1.

[0033] Figure 3 This is an assembly diagram for preparing a fan-shaped microfluidic colorimetric detection chip in Example 2.

[0034] Figure 4 This is the image data of the detection area when the microfluidic colorimetric detection chip in Example 3 is used for detecting simulated sweat containing different concentrations of creatinine.

[0035] Figure 5 It is a linear fitting curve diagram between the detection signal (△R) of the microfluidic colorimetric detection chip in Example 3 and the creatinine concentration.

[0036] Figure 6 This is a comparison chart of detection signals (△R) of the microfluidic colorimetric detection chip in Example 4 for detecting creatinine and multiple interfering test substances in sweat.

[0037] Figure 7 This is an assembly diagram of the circular microfluidic colorimetric detection chip used for detecting multiple markers in Example 6. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0040] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0041] Embodiment 1:

[0042] The H2MoO5 nanozyme was prepared and its morphology and catalytic performance were studied. The specific steps are as follows:

[0043] 1. Preparation of H2MoO5 nanozyme. Ultrasonic dispersion of 10.4mM Mo powder in 40mL ultrapure water, then add 30mL of 30% H2O2 dropwise to the above solution under ice-water bath conditions, and finally add 6mL of glacial acetic acid. Fully react at room temperature for about 18 hours until an orange-yellow solution appears. Subsequently, the yellow filtrate was filtered and dried in a 60°C vacuum drying oven for 8 hours. The dried product was calcined at 450°C for 1 hour at a heating rate of 5°C / min. The obtained solid was fully ground to successfully prepare yellow H2MoO5 powder.

[0044] 2. The morphology of the prepared H2MoO5 nanozyme was characterized using a scanning electron microscope. Figure 1 As shown, H2MoO5 nanozyme was successfully prepared. The material was foam-like entangled nanoparticles with uniform particle size.

[0045] 3. Test the catalytic performance of H2MoO5 nanozyme. TMB was selected as the color substrate. During the experiment, 1360 μL of acetic acid-sodium acetate buffer solution with a pH of 6.5 was added to centrifuge tubes 1, 2, 3, 4, 5, 6, and 7, and 120 μL of 200 μM Cu was added to centrifuge tubes 1, 2, 4, and 6. 2+ Then, add 200 μL of 100 μM creatinine (CR) solution to centrifuge tubes 1, 3, 4, and 7 respectively. 2+ Complexation, then continue to add 120μL of 0.1mg / mL H2MoO5 nanozyme to centrifuge tubes 1, 2, 3, and 5, and then add 200μL of 10Mm TMB solution to 7 centrifuge tubes, and add other volumes of ultrapure water to the above centrifuge tubes to keep the total volume of each tube at 2mL. Finally, the above solutions were fully mixed, reacted at room temperature for 20 minutes, and pictures of each reaction system were taken, and the UV-visible absorption spectrum in the range of 320nm to 800nm ​​was scanned.

[0046] according to Figure 2 The UV-visible absorption spectra of different reaction systems show that H2MoO5+Cu 2+ +TMB reaction system is sky blue, and a strong absorption peak belonging to TMB oxidation product also appears at 652nm. 2+ The system has good oxidase-like catalytic performance and can effectively catalyze TMB oxidation without adding other oxidants (hydrogen peroxide). 2+ The +CR+TMB reaction system is a deeper sky blue, and a stronger absorption peak appears at 652nm. It can be seen that after adding the target creatinine CR, a synergistic catalytic effect is produced, which significantly enhances the H2MoO5+Cu 2+The catalytic performance of the catalytic system effectively catalyzes the oxidation of TMB to produce a darker color. H2MoO5+CR+TMB, H2MoO5+TMB, Cu 2+ +CR+TMB、Cu 2+ +TMB and CR+TMB reaction systems are colorless and have no absorption peak at 652nm, which further proves that H2MoO5+Cu 2+ In the catalytic system, H2MoO5 nanozymes and Cu 2+ Neither is indispensable.

[0047] Embodiment 2:

[0048] Based on H2MoO5+Cu in Example 1 2+ A colorimetric catalytic system is designed and prepared to detect creatinine using a microfluidic colorimetric detection chip. The microfluidic detection chip includes a microfluidic channel layer, a detection layer and a packaging layer. The detection layer is loaded on the microfluidic channel layer, and the packaging layer is located on the microfluidic channel layer. The microfluidic channel layer is provided with an injection port area, a diversion area, a burst valve groove, a detection area and a blank control area. The detection layer includes two paper chips modified with detection reagents, one of which is Cu 2+ The first embodiment is a paper chip modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB). The encapsulation layer is provided with an injection hole, which corresponds to the injection port of the microfluidic channel layer.

[0049] The specific production and assembly process is as follows:

[0050] 1. Design the mold structure using AutoCAD software and prepare the mold using polytetrafluoroethylene (PTFE).

[0051] The mold base is composed of a sector with a central angle of 75 degrees, a radius of 3.5 cm, and a thickness of 1 cm. A protruding channel pattern is set on the base. The height of the pattern is 1 mm, and a fence with a height of 2 cm is added to the outside of the mold. The protruding pattern includes 1 circle with a protruding height of 1 mm and a diameter of 3 mm and 3 circles with a protruding height of 1 mm and a diameter of 5 mm. The protruding pattern also includes two protruding channels, starting from the center and extending in the radial direction until they are connected to the atmosphere. The channel width is 1 mm and the height is 1 mm. In addition, an isosceles triangle protruding pattern with a side length of 3 mm, a bottom angle of 30 degrees, and a depth of 1 mm is introduced into the channel.

[0052] 2. Prepare the PDMS microfluidic flow channel layer.

[0053] The main agent and curing agent for preparing PDMS were mixed in a ratio of 10:1, stirred in a beaker for 30 minutes at a speed of 150 rpm to form a PDMS prepolymer, and the PDMS prepolymer was placed in a vacuum chamber to remove excess bubbles, and finally poured evenly into the above-processed mold, placed in a drying oven at 70°C for 2 hours, and finally demolded to obtain a PDMS microfluidic flow channel layer.

[0054] The PDMS microfluidic flow channel layer is fan-shaped, with a central angle of 75 degrees, a radius of 2.5cm and a thickness of 1.2mm. The microfluidic channel layer is provided with an injection port area, a shunt area, a burst valve, a detection area and a blank control area. The injection port area includes a circular groove with a diameter of 3mm and a height of 1mm. The shunt area is a groove-type flow channel extending outward with an angle of 60 degrees from the injection port area, and the solution sample in the injection area is shunted into multiple, preferably 2. The shunt flow channel is 1mm wide, 1mm deep and 2cm long. An isosceles triangle-shaped burst valve with a side length of 3mm, a bottom angle of 30 degrees and a depth of 1mm is introduced at 5mm of each of the two groove-type flow channels. Two circular detection area grooves with a diameter of 5mm and a depth of 1mm are set at 1cm long of the two shunt flow channels. The two shunt flow channels continue to extend until they are connected to the atmosphere. A blank control zone groove is arranged between the two detection zone grooves. The size of the blank control zone groove is consistent with that of the detection zone groove, but is not connected with the diversion flow channel.

[0055] 3. Prepare H2MoO5 nanozyme and TMB modified colorimetric system paper chip.

[0056] In the first step, H2MoO5 nanozyme powder was prepared according to Example 1.

[0057] The second step is to prepare a paper chip co-modified with H2MoO5 nanozyme and TMB (the material of the paper chip is Whatman No. 1 filter paper). Use a plotter to batch prepare circular filter paper with a diameter of 5 mm. Add 5 μL of acetic acid-sodium acetate buffer solution with a pH value of 6.0 on the circular filter paper, wait for 5 minutes, then add 5 μL of 0.1 mg / mL H2MoO5 nanozyme, wait for 10 minutes, and then add 5 μL of 10mM TMB solution to prepare a paper chip co-modified with H2MoO5 nanozyme and TMB.

[0058] 4. Preparation of Cu 2+ Modified paper chips.

[0059] Use a plotter to batch prepare isosceles triangular paper pieces with a side length of 3 mm and a base angle of 30 degrees. 3 μL of 200 μM copper sulfate solution is dropped onto the above triangular paper pieces to prepare Cu 2+ Modified paper chips.

[0060] 5. Use a carving machine to carve the double-sided nano-gelatin into a fan-shaped shape that is the same as the above-mentioned PDMS microfluidic flow channel layer. Use a hole puncher to punch a hole with a diameter of 3 mm at the injection area, and align the position with the injection port of the microfluidic channel layer.

[0061] 6. Assembly scheme of microfluidic colorimetric detection chip Figure 3 As shown, the specific assembly steps are as follows:

[0062] Three H2MoO5 nanozymes and TMB-modified colorimetric paper chips were loaded into the two circular detection area grooves and one blank control area groove corresponding to the prepared microfluidic channel layer, and then two Cu 2+ The modified triangular paper chip is loaded into the groove of the burst valve. The central injection hole of the above encapsulation layer is aligned with the central area of ​​the injection port of the microfluidic channel layer, and then pasted on the microfluidic flow channel layer filled with the paper chip to complete the encapsulation of the microfluidic channel and prepare a wearable microfluidic colorimetric detection chip.

[0063] Embodiment 3:

[0064] The microfluidic colorimetric detection chip of Example 2 is used to simulate the detection of different concentrations of creatinine in sweat. The specific method is as follows:

[0065] 1. A microfluidic colorimetric detection chip prepared according to Example 2.

[0066] 2. Prepare simulated sweat standard solutions containing different concentrations of creatinine. Prepare simulated sweat: The simulated sweat contains 20 mg / mL sodium chloride, 17.5 mg / mL ammonium chloride, 5 mg / mL urea, 2.5 mg / mL acetic acid, and 15 mg / mL lactic acid. Then use the simulated sweat to prepare simulated sweat standard solutions containing 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM creatinine.

[0067] 3. Use a pipette to drop simulated sweat standard solutions containing different concentrations of creatinine into the injection port of the microfluidic colorimetric detection chip. After injection, the simulated sweat flows from the injection port area along the channel into the detection area, soaks the paper chip and reacts at room temperature for 10 minutes.

[0068] 4. Use a smartphone to take pictures and obtain color images of the microfluidic chip, such as Figure 4 As shown. Figure 4It can be seen that the color of the blank control area remains unchanged, while the detection area is blue, and as the creatinine concentration in the simulated sweat increases, the color of the paper chip in the detection area gradually deepens. Image J software is further used to obtain the RGB values ​​of the detection area and the blank control area images in the microfluidic colorimetric detection chip. The present invention preferably uses the R value as the quantitative standard, and uses the difference (△R) between the mean of the R values ​​of the two detection areas and the R value of the blank control area as the detection signal to draw a linear fitting curve between the simulated sweat creatinine concentration and the △R value of the microfluidic colorimetric detection chip. Figure 5 As shown, the linear equation is y = 1.02x + 6.95, and the correlation coefficient R 2 =0.9917.

[0069] Embodiment 4:

[0070] The microfluidic colorimetric detection chip of Example 2 is used to simulate the detection of other interfering substances in sweat. The specific method is as follows:

[0071] 1. Prepare a microfluidic colorimetric detection chip according to Example 2.

[0072] 2. Select common electrolyte ions and biological small molecules (Mg 2+ 、Na + , Ca 2+ , K + , glucose, lactic acid, uric acid) as interfering substances, and prepare relevant interference test solutions according to the concentration of each substance in sweat, including 10mM Mg 2+ , 10 mM Na + , 10 mM Ca 2+ , 10mM K + , 5mM glucose, 5mM lactic acid, 5mM uric acid. Each interfering solution was added dropwise to the sample inlet of the prepared microfluidic colorimetric detection chip. After the paper chip in the detection area was colored, a smartphone was used to take a picture. The R values ​​of the two detection areas and the blank control area were read using Image J software. △R was used as the detection signal to draw a comparative bar graph, as shown in Figure 6 As shown. Figure 6 It can be seen that the △R values ​​of various interfering substances are much smaller than creatinine, indicating that the H2MoO5 nanozyme-Cu 2+ The microfluidic colorimetric detection chip constructed by the catalytic TMB colorimetric system has high selectivity for sweat creatinine detection.

[0073] Embodiment 5:

[0074] To demonstrate the beneficial effects of the present invention, the microfluidic colorimetric detection chip of Example 2 was used to detect creatinine in actual sweat, and the specific method was as follows:

[0075] 1. Prepare a microfluidic colorimetric detection chip according to Example 2.

[0076] 2. Stick the prepared microfluidic colorimetric detection chip to the arms and other sweat-prone parts of the three athletes until the sweat soaks the paper chip in the detection area through capillary action. After 10 minutes of reaction, take a picture to obtain the image information of the microfluidic colorimetric detection chip. Use ImageJ software to read the R value of the detection area and the R value of the blank control area, and calculate △R as the detection signal.

[0077] 3. Further, according to the linear fitting curve of Example 3, the actual sweat creatinine concentration was calculated. The test results showed that the creatinine concentrations in the sweat of the above three athletes were 64.79 μmol / L, 72.36 μmol / L and 56.55 μmol / L, and the test results were consistent with the creatinine content in normal human sweat. Therefore, the microfluidic ratio detection chip of the present invention can be used for the quantitative detection of creatinine in actual sweat.

[0078] Embodiment 6:

[0079] In order to improve the practicality of the present invention, the sector-shaped microfluidic colorimetric detection chip of Example 2 is extended and extended to prepare a circular microfluidic colorimetric detection chip with multiple detection areas for simultaneous detection of multiple markers in sweat. The specific method is as follows:

[0080] 1. Redesign the mold structure using AutoCAD software and prepare the mold with polytetrafluoroethylene (PTFE).

[0081] The design parameters of the mold are as follows: the diameter of the circular mold base is 7cm, the thickness is 1cm, a protruding channel pattern is set on the base, the pattern protrusion height is 1mm, and a circle of 2cm high fence is added to the outside of the mold. The protruding pattern includes 3 circles with a protrusion height of 1mm and a diameter of 3mm and 9 circles with a protrusion height of 1mm and a diameter of 5mm. The protruding pattern also includes 6 protruding channels, starting from the center, extending in the radial direction until it is connected to the atmosphere, the channel width is 1mm, the height is 1mm, and the protruding side length is 3mm, the bottom angle is 30 degrees, and the protrusion height is 1mm. Isosceles triangle shape. Each protruding channel is connected to a circular protrusion with a diameter of 5mm. Simply put, it is similar to copying the circulation channel pattern in Example 2 as a whole 3 times and evenly distributing it in the circular mold.

[0082] 2. Prepare a circular PDMS microfluidic flow channel layer.

[0083] The main agent and curing agent for preparing PDMS are mixed in a ratio of 10:1, stirred in a beaker for 30min at a speed of 150rpm to form a PDMS prepolymer, and the PDMS prepolymer is placed in a vacuum chamber to remove excess bubbles, and finally poured evenly into the processed circular mold, placed in a drying oven at 70°C for 2 hours, and finally demolded to obtain a circular PDMS microfluidic circulation channel layer. The PDMS circulation channel layer is composed of a circle with a radius of 2.5cm and a thickness of 1.2mm. The circular PDMS circulation channel layer can be regarded as being prepared by assembling 3 fan-shaped microfluidic circulation channel layers identical to Example 2 into a circle. The circular PDMS circulation channel layer includes 3 injection ports, 3 groups of detection areas (2 detection holes in each group) and 3 blank control areas.

[0084] 3. Use a hole puncher to punch three injection holes with a diameter of 3 mm on a circular double-sided medical tape with a diameter of 5 cm. Align the three injection holes with the three injection ports of the circular PDMS microfluidic channel layer.

[0085] 4. Prepare paper chips modified with detection reagents suitable for corresponding target detection objects.

[0086] Paper chips modified with different detection reagents were prepared according to different targets. 2+ For example.

[0087] Referring to Example 2, two detection reagent modified paper chips for creatinine detection were prepared.

[0088] 0.1450 g of p-dimethylaminobenzaldehyde was dissolved in 4.0 mol / L sulfuric acid solution to prepare a urea detection reagent. 3 μL of urea detection reagent was added dropwise to a circular filter paper and dried for 3 min to prepare a urea detection paper chip.

[0089] Dissolve 2 mg / mL o-cresolphthalein complex and 2.5 mg / mL 8-hydroxyquinoline in 0.2 mol / L hydrochloric acid to prepare Ca 2+ Detection reagent. On the circular filter paper, first add 5 μL of ethanolamine buffer solution with a pH of 11.5, then add 5 μL of Ca 2+ Detection reagent, and dry for 3 minutes to prepare Ca 2+ Detection paper chip.

[0090] 5. Assembly and preparation scheme of circular microfluidic colorimetric detection chip Figure 7 As shown, the specific assembly scheme is as follows:

[0091] The circular PDMS microfluidic flow channel layer is divided into creatinine detection area, urea detection area and Ca 2+Detection area: In the creatinine detection area, two detection reagents for creatinine detection are modified into the paper chip, referring to Example 2, and are loaded into the burst valve groove, the detection area groove, and the blank control area groove, respectively.

[0092] Creatinine detection area: load the urea detection paper chip into the two detection area grooves and one blank control area groove of the urea detection area.

[0093] Ca 2+ Detection area, Ca 2+ Detection paper chip loaded with Ca 2+ The detection area has 2 detection zone grooves and 1 blank control zone groove.

[0094] The central injection hole of the circular double-sided nanogel was aligned with the injection port of the PDMS microfluidic flow channel layer, and then pasted on the microfluidic flow channel layer filled with paper chips to complete the encapsulation of the microfluidic channel. 2+ Circular multi-channel microfluidic colorimetric detection chip for simultaneous detection.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic detection chip for sweat creatinine detection, characterized in that: The microfluidic detection chip comprises a microfluidic channel layer, a detection layer and a packaging layer; the detection layer is loaded on the microfluidic channel layer, and the packaging layer is located on the microfluidic channel layer; The microfluidic channel layer is provided with an injection port area, a diversion area, a burst valve groove, a detection area and a blank control area; The detection layer includes two paper chips modified with detection reagents, one of which is Cu 2+ modified paper chip, and the other is a paper chip co-modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB); The packaging layer is provided with a sampling hole, and the sampling hole corresponds to the sampling port of the microfluidic channel layer.

2. The microfluidic detection chip for sweat creatinine detection according to claim 1, characterized in that: The microfluidic channel layer is made of PDMS; the material of the paper chip is any one or more of Whatman No. 1 filter paper, cellulose acetate paper, nitrocellulose membrane, and qualitative filter paper.

3. The microfluidic detection chip for sweat creatinine detection according to claim 1, characterized in that: The Cu 2+ The modified paper chip is Cu 2+ The solution is dripped onto a filter paper; the paper chip co-modified with the H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) is obtained by dripping an acetic acid-sodium acetate buffer solution onto the filter paper, adding the H2MoO5 nanozyme, and then adding the TMB solution.

4. The microfluidic detection chip for sweat creatinine detection according to claim 1, characterized in that: The H2MoO5 nanozyme is prepared by the following process: ultrasonically dispersing Mo powder in water, adding H2O2 dropwise under ice-water bath conditions, adding glacial acetic acid, and fully reacting at room temperature until an orange-yellow solution appears, then filtering to obtain a yellow filtrate, drying, calcining, and grinding to obtain the H2MoO5 nanozyme.

5. The microfluidic detection chip for sweat creatinine detection according to claim 1, characterized in that: The H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) modified paper chip are matched with the detection area and blank control area on the microfluidic channel layer; 2+ The modified paper chip matches the burst valve groove on the microfluidic channel layer.

6. The method for preparing the microfluidic detection chip for sweat creatinine detection according to any one of claims 1 to 5, characterized in that: The paper chip modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) of the detection layer was loaded into the detection area and blank control area on the microfluidic channel layer. 2+ The modified paper chip is loaded into the burst valve groove on the microfluidic channel layer, the injection hole of the encapsulation layer is aligned with the injection port of the microfluidic channel layer, and then pasted on the microfluidic flow channel layer filled with the paper chip to complete the encapsulation of the microfluidic channel.

7. Use of the microfluidic detection chip for detecting sweat creatinine according to any one of claims 1 to 5 and the microfluidic detection chip prepared by the preparation method of claim 6 in detecting creatinine levels in sweat.

8. A method for preparing H2MoO5 nanozyme, characterized in that: The method comprises the following steps: ultrasonically dispersing Mo powder in water, adding H2O2 dropwise in an ice-water bath, adding glacial acetic acid, and reacting fully at room temperature until an orange-yellow solution appears, then filtering to obtain a yellow filtrate, drying, calcining, and grinding to obtain H2MoO5 nanozyme.

9. The H2MoO5 nanozyme prepared by the preparation method according to claim 8.

10. Use of the H2MoO5 nanozyme according to claim 9 in preparing a microfluidic detection chip for sweat creatinine detection.

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