A microfluidic colorimetric chip for sweat choline detection and a preparation method and application thereof

Abstract

This invention discloses a microfluidic colorimetric chip for detecting sweat creatinine, its preparation method, and its application, belonging to the field of microfluidic detection chip technology. The microfluidic detection chip includes a microfluidic channel layer, a detection layer, and an encapsulation layer; the detection layer is filled on the microfluidic channel layer, and the encapsulation layer is located on top of the microfluidic channel layer; the microfluidic channel layer has an inlet area, a splitting area, a burst valve groove, a detection area, and a blank control area; the detection layer includes paper chips modified with two detection reagents, one of which is Cu 2+ Another type of modified paper chip is a paper chip co-modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine; the encapsulation layer has a sample inlet, which corresponds to the sample inlet of the microfluidic channel layer. Beneficial effects: This invention provides a microfluidic colorimetric detection chip for creatinine detection, using stable and low-cost H2MoO5 nanozyme-Cu... 2+ The catalytic system replaces the costly, difficult-to-store, and easily deactivated biological enzyme catalytic system, and has significantly improved stability and ease of storage.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic detection chip technology, and relates to a non-biological enzyme-based microfluidic detection chip for detecting creatinine, particularly a wearable microfluidic colorimetric detection chip for detecting creatinine in sweat, its preparation method, and its application. Background Technology

[0002] Sweat is secreted by sweat glands and is rich in electrolytes (Na+). + K + Ca 2+ Sweat contains molecules (such as glucose, lactic acid, and creatinine) and metabolic molecules (such as glucose, lactic acid, and creatinine), which can be used to indicate the body's health status at the molecular level, providing important information for understanding an individual's health condition and playing a significant role in the early detection, monitoring, and treatment of various diseases. Sweat can be collected non-invasively and continuously from almost the entire body's skin surface, achieving high reproducibility and in situ collection, making it an ideal sample for human body fluid testing. Therefore, sweat testing technology has developed rapidly in recent years, forming a hot interdisciplinary field integrating analysis, sensing, and medical health. However, traditional sweat testing is conducted in a laboratory setting, often requiring complex equipment and cumbersome procedures, which greatly affects the portability and real-time nature of the testing.

[0003] In recent years, with the development of microfabrication technology and flexible materials, wearable microfluidic chips have begun to be used for human sweat detection. Compared with traditional laboratory testing techniques, wearable microfluidic detection chips integrate the functions of sample transportation, separation, reaction, and analysis performed in traditional laboratories onto a tiny chip of just a few square centimeters. They are characterized by small size, low cost, rapid response, continuous data acquisition, and ease of operation.

[0004] Creatinine is a compound produced during muscle energy production. Creatinine levels in the human body reflect kidney filtration function and are an important indicator of kidney function. There are various methods for detecting creatinine, including chromatography, mass spectrometry, electrochemical methods, and capillary zone electrophoresis. These methods require specialized personnel to operate specialized instruments and can only be performed in a laboratory. Colorimetric methods have advantages such as simplicity, low cost, and speed, and have significant application prospects in portable detection. The Jaffe method is a traditional colorimetric method for creatinine detection; however, its poor specificity and susceptibility to interference from various substances limit its practical application.

[0005] Currently developed microfluidic colorimetric detection chips for creatinine detection mostly require the use of creatinine oxidase to catalyze the gradual hydrolysis of creatinine into hydrogen peroxide. The generated hydrogen peroxide then reacts with a colorimetric reagent to produce a colored substance for quantitative detection. While enzyme-based detection methods offer high selectivity, they suffer from drawbacks such as high cost, difficult storage, easy inactivation, poor stability, and difficulty in recycling and reuse. Therefore, developing novel enzyme-free wearable microfluidic colorimetric detection chips for the detection of creatinine in sweat is expected to further improve the stability of wearable detection devices and is of great significance for the early diagnosis and health prevention management of kidney diseases. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to provide a microfluidic colorimetric detection chip without biological enzymes 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 goal, the main technical solutions are as follows.

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

[0008] A first aspect of the present invention provides a microfluidic detection chip for detecting sweat creatinine, the microfluidic detection chip comprising a microfluidic channel layer, a detection layer, and an encapsulation layer; the detection layer is filled on the microfluidic channel layer, and the encapsulation layer is located on the microfluidic channel layer;

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

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

[0011] The encapsulation layer has an injection port, which corresponds to the injection port of the microfluidic channel layer.

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

[0013] Preferably, the paper chip is made of 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 made of Cu 2+The solution is prepared by dropping the solution onto filter paper. The paper chip modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) is obtained by dropping an acetate-sodium acetate buffer solution onto filter paper, adding H2MoO5 nanozyme, and then adding TMB solution.

[0015] Preferably, the H2MoO5 nanozyme is prepared by the following process: Mo powder is ultrasonically dispersed in water, H2O2 is added dropwise under ice-water bath conditions, glacial acetic acid is added, and the reaction is carried out at room temperature until an orange-yellow solution appears. Then, the solution is filtered to obtain a yellow filtrate, dried, calcined, and ground to obtain the H2MoO5 nanozyme.

[0016] Preferably, the paper chip modified with the H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) matches the detection area and blank control area on the microfluidic channel layer; the Cu 2+ The modified paper chip is matched with 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: filling a paper chip modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) in the detection layer into the detection area and blank control area on the microfluidic channel layer; and then filling the detection area and blank control area with Cu... 2+ The modified paper chip is filled into the rupture valve groove on the microfluidic channel layer. The injection port of the encapsulation layer is aligned with the injection port of the microfluidic channel layer and then pasted onto the microfluidic flow channel layer filled with the paper chip to complete the encapsulation of the microfluidic channel.

[0018] A third aspect of the present invention proposes the application 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 provides a method for preparing H2MoO5 nanozymes, comprising the following steps: ultrasonically dispersing Mo powder in water, adding H2O2 dropwise under ice-water bath conditions, adding glacial acetic acid, 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 nanozymes.

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

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

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

[0023] Step 1: Prepare a wearable microfluidic colorimetric detection chip according to the above preparation method.

[0024] Step 2: Add simulated sweat standard solutions containing different concentrations of creatinine to the injection well. The simulated sweat enters the microfluidic colorimetric detection chip through the injection well, flows through the bursting zone after splitting, and then enters the detection zone, where it reacts with the paper chip to produce a colorimetric reaction. After 10 minutes, take a picture with a smartphone to obtain a color image of the microfluidic colorimetric detection chip.

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

[0026] Step 4: Place the wearable microfluidic colorimetric detection chip with the inlet facing down on the surface of the athlete's sweaty skin (such as the arm or back). After the sweat flows into the detection area and a color reaction occurs, take a picture to obtain the ΔR value. Calculate the actual creatinine concentration in the sweat based on the above creatinine quantitative linear curve.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. The microfluidic colorimetric detection chip for creatinine detection in this invention uses stable and low-cost H2MoO5 nanozyme-Cu. 2+ The catalytic system replaces the costly, difficult-to-store, and easily deactivated biological enzyme catalytic system, and has significantly improved stability and ease of 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 realize real-time, non-invasive and highly selective detection of creatinine in micro-level sweat. It avoids the cumbersome sweat collection, pretreatment and use of large detection instruments, and provides a new technology for sweat creatinine detection.

[0030] 3. The microfluidic colorimetric detection chip of the present invention, through extension and expansion, can realize the 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. Attached Figure Description

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

[0032] Figure 2These are the UV-Vis absorption spectra and images of the colorimetric solution used in Example 1 to study the colorimetric catalytic performance of the H2MoO5 nanozyme.

[0033] Figure 3 This is an assembly diagram of the fan-shaped microfluidic colorimetric detection chip prepared 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 to detect simulated sweat containing different concentrations of creatinine.

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

[0036] Figure 6 This is a comparison diagram of the detection signals (ΔR) of the microfluidic colorimetric detection chip used in Example 4 for the detection of creatinine and various 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 Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0040] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0041] Example 1:

[0042] The preparation of H2MoO5 nanozymes and the study of their morphology and catalytic performance are as follows:

[0043] 1. Preparation of H₂MoO₅ nanozyme. 10.4 mM Mo powder was ultrasonically dispersed in 40 mL of ultrapure water. Then, 30 mL of 30% H₂O₂ was added dropwise to the solution under ice-water bath conditions, followed by 6 mL of glacial acetic acid. The reaction was allowed to proceed at room temperature for approximately 18 hours until an orange-yellow solution appeared. The resulting yellow filtrate was then filtered and dried in a vacuum drying oven at 60 °C for 8 hours. The dried product was then calcined at 450 °C for 1 hour at a heating rate of 5 °C / min. The resulting solid was thoroughly ground to successfully prepare yellow H₂MoO₅ powder.

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

[0045] 3. The catalytic performance of the H2MoO5 nanozyme was tested. TMB was selected as the chromogenic substrate. During the experiment, 1360 μL of pH 6.5 acetate-sodium acetate buffer solution was added to centrifuge tubes 1, 2, 3, 4, 5, 6, and 7, respectively. 120 μL of 200 μM Cu was added to centrifuge tubes 1, 2, 4, and 6, respectively. 2+ Solution, then, 200 μL of 100 μM creatinine (CR) solution was added to centrifuge tubes 1, 3, 4, and 7 respectively, and mixed with Cu 2+ After complexation, 120 μL of 0.1 mg / mL H₂MoO₅ nanozyme was added to centrifuge tubes 1, 2, 3, and 5. Then, 200 μL of 10 Mm TMB solution was added to each of the seven centrifuge tubes. The remaining volumes of ultrapure water were added to each tube to maintain a total volume of 2 mL. Finally, the solutions were thoroughly mixed, and the reaction was allowed to proceed for 20 min at room temperature. Images of each reaction system were then captured, and the UV-Vis absorption spectra in the range of 320 nm to 800 nm were scanned.

[0046] according to Figure 2 The UV-Vis absorption spectra of different reaction systems show that H2MoO5 + Cu 2+ The +TMB reaction system is sky blue, and a strong absorption peak belonging to the TMB oxidation product appears at 652 nm. This indicates that the H₂MoO₅ + Cu... 2+ The system exhibits excellent oxidase-like catalytic performance, effectively catalyzing the oxidation of TMB without the addition of other oxidants (hydrogen peroxide). H₂MoO₅ + Cu 2+ The +CR+TMB reaction system exhibits a deeper sky blue color and a stronger absorption peak at 652 nm. This demonstrates that the addition of the target compound creatinine (CR) produces a synergistic catalytic effect, significantly enhancing the H2MoO5+Cu reaction. 2+The catalytic performance of the catalytic system effectively catalyzes the oxidation of TMB to produce a deeper color. H₂MoO₅ + CR + TMB, H₂MoO₅ + TMB, Cu 2+ +CR+TMB、Cu 2+ Both the +TMB and CR+TMB reaction systems are colorless and show no absorption peak at 652 nm, further proving that H2MoO5+Cu 2+ In the catalytic system, H2MoO5 nanozyme and Cu 2+ None of them can be missing.

[0047] Example 2:

[0048] Based on H2MoO5+Cu in Example 1 2+ A colorimetric catalytic system was designed and fabricated to create a microfluidic colorimetric detection chip for creatinine detection. The microfluidic detection chip includes a microfluidic channel layer, a detection layer, and an encapsulation layer. The detection layer is packed onto the microfluidic channel layer, and the encapsulation layer is located above the microfluidic channel layer. The microfluidic channel layer has an inlet area, a splitting area, a burst valve groove, a detection area, and a blank control area. The detection layer includes two types of paper chips modified with different detection reagents, one of which is Cu. 2+ Another type of modified paper chip is a paper chip co-modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB); the encapsulation layer has a sample inlet, which corresponds to the sample inlet of the microfluidic channel layer.

[0049] The specific manufacturing 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 consists of a fan-shaped structure with a central angle of 75 degrees, a radius of 3.5 cm, and a thickness of 1 cm. A raised channel pattern is formed on the base, with each pattern protruding 1 mm in height. A 2 cm high fence surrounds the outside of the mold. The raised pattern includes one circle with a protrusion height of 1 mm and a diameter of 3 mm, and three circles with a protrusion height of 1 mm and a diameter of 5 mm. The raised pattern also includes two raised channels, each starting from the center and extending radially until it connects to the atmosphere. Each channel is 1 mm wide and 1 mm high, and each channel introduces an isosceles triangular raised pattern with a side length of 3 mm, a base angle of 30 degrees, and a depth of 1 mm.

[0052] 2. Preparation of PDMS microfluidic flow channel layer.

[0053] The main agent and curing agent for PDMS preparation were mixed in a ratio of 10:1 and stirred in a beaker for 30 minutes at a speed of 150 rpm to form a PDMS prepolymer. The PDMS prepolymer was placed in a vacuum chamber to remove excess air bubbles and then poured evenly into the prepared mold. The mold was then placed in a drying oven at 70°C for 2 hours and finally demolded to obtain the 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 2.5 cm, and a thickness of 1.2 mm. The microfluidic channel layer includes an inlet area, a split area, a burst valve, a detection area, and a blank control area. The inlet area contains a circular groove with a diameter of 3 mm and a height of 1 mm. The split area consists of two outward-extending groove-type flow channels with an included angle of 60 degrees, originating from the inlet area, splitting the solution sample in the inlet area into multiple channels, preferably two. The split flow channels are 1 mm wide, 1 mm deep, and 2 cm long. A burst valve in the shape of an isosceles triangle with a side length of 3 mm, a base angle of 30 degrees, and a depth of 1 mm is introduced into each of the two groove-type flow channels at a distance of 5 mm. Two circular detection area grooves with a diameter of 5 mm and a depth of 1 mm are located at a distance of 1 cm from the length of the two split flow channels. The two split flow channels continue to extend until they are open to the atmosphere. Between the two detection zone grooves, a blank control zone groove is set. The blank control zone groove has the same size as the detection zone groove, but it is not connected to the diversion flow channel.

[0055] 3. Preparation of H2MoO5 nanozyme and TMB-modified colorimetric paper chip system.

[0056] The first step was to prepare H2MoO5 nanozyme powder according to Example 1.

[0057] The second step involves preparing paper chips co-modified with H2MoO5 nanozyme and TMB (the paper chips are made of Whatman No. 1 filter paper). Circular filter paper discs with a diameter of 5 mm are prepared in batches using a cutting machine. 5 μL of a pH 6.0 acetate-sodium acetate buffer solution is added to each circular filter paper disc, and after 5 min, 5 μL of 0.1 mg / mL H2MoO5 nanozyme is added. After 10 min, 5 μL of 10 mM TMB solution is added to complete the preparation of the paper chips co-modified with H2MoO5 nanozyme and TMB.

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

[0059] Isosceles triangular paper pieces with sides of 3 mm and base angles of 30 degrees were mass-produced using a lettering machine. 3 μL of a 200 μM copper sulfate solution was added dropwise to each of these triangular paper pieces to obtain Cu. 2+ Modified paper chips.

[0060] 5. Use an engraving machine to engrave the double-sided nano-adhesive into a fan shape identical to the PDMS microfluidic flow channel layer described above. Use a hole punch to drill a 3mm diameter hole at the sample inlet area, aligned with the sample inlet of the microfluidic channel layer.

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

[0062] Three H2MoO5 nanozyme and TMB co-modified colorimetric system paper chips were respectively filled into the two circular detection area grooves and one blank control area groove corresponding to the prepared microfluidic channel layer. Then, two Cu 2+ The modified triangular paper chip is inserted into the groove of the rupture valve. The central injection port of the above-mentioned encapsulation layer is aligned with the central region of the injection port of the microfluidic channel layer, and then pasted onto the microfluidic flow channel layer filled with paper chips to complete the encapsulation of the microfluidic channel and prepare a wearable microfluidic colorimetric detection chip.

[0063] Example 3:

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

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

[0066] 2. Preparation of simulated sweat standard solutions containing different concentrations of creatinine. Simulated sweat was prepared as follows: The simulated sweat contained 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, 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 were prepared using the simulated sweat.

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

[0068] 4. Use a smartphone to take a picture to obtain a colorimetric image of the microfluidic chip, such as... Figure 4 As shown. From Figure 4As can be seen, the color of the blank control area remains constant, while the detection area is blue. Furthermore, the color of the paper chip in the detection area gradually deepens as the concentration of creatinine in the simulated sweat increases. ImageJ software was used to obtain the RGB values ​​of the detection area and blank control area images in the microfluidic colorimetric detection chip. This invention preferably uses the R value as the quantitative standard, and uses the difference (ΔR) between the mean R value of the two detection areas and the R value of the blank control area as the detection signal to plot a linear fitting curve between the creatinine concentration of simulated sweat and the ΔR value of the microfluidic colorimetric detection chip. Figure 5 As shown, the linear equation is y = 1.02x + 6.95, and the correlation coefficient R0 is... 2 =0.9917.

[0069] Example 4:

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

[0071] 1. A microfluidic colorimetric detection chip was prepared according to Example 2.

[0072] 2. Select common electrolyte ions and small biological molecules (Mg) found in sweat. 2+ Na + Ca 2+ K + (In addition to glucose, lactic acid, and uric acid) were used as interfering substances. Relevant interference test solutions were prepared according to the concentrations of each substance in sweat, including 10 mM Mg... 2+ 10mM Na + 10mM Ca 2+ 10mM K + 5 mM glucose, 5 mM lactic acid, and 5 mM uric acid were added dropwise to the inlet of the prepared microfluidic colorimetric detection chip. After the detection area of ​​the paper chip developed color, images were captured using a smartphone. ImageJ software was used to read the R values ​​of the two detection areas and the blank control area, respectively. Using ΔR as the detection signal, a comparison histogram was plotted. Figure 6 As shown. From Figure 6 As can be seen, the ΔR values ​​of various interfering substances are much smaller than those of creatinine, indicating that the H2MoO5 nanozyme-Cu 2+ The microfluidic colorimetric detection chip constructed using the catalytic TMB colorimetric system exhibits high selectivity for the detection of creatinine in sweat.

[0073] Example 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. The specific method is as follows:

[0075] 1. A microfluidic colorimetric detection chip was prepared according to Example 2.

[0076] 2. The prepared microfluidic colorimetric detection chip was attached to the arms or other areas prone to sweating of three athletes until the paper chip in the detection area was wetted by the sweat through capillary action. After reacting for 10 minutes, the image information of the microfluidic colorimetric detection chip was obtained by taking a picture. Using ImageJ software, the R value of the detection area and the R value of the blank control area were read, and ΔR was calculated as the detection signal.

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

[0078] Example 6:

[0079] To improve the practicality of this invention, a circular microfluidic colorimetric detection chip with multiple detection zones is prepared based on the fan-shaped microfluidic colorimetric detection chip of Example 2 for simultaneous detection of multiple markers in sweat. The specific method is as follows:

[0080] 1. The mold structure was redesigned using AutoCAD software, and the mold was made of polytetrafluoroethylene (PTFE).

[0081] The design parameters of the mold are as follows: the circular mold base has a diameter of 7cm and a thickness of 1cm. A raised channel pattern is set on the base, with a raised height of 1mm. A 2cm high fence is added around the outside of the mold. The raised pattern includes three circles with a raised height of 1mm and a diameter of 3mm, and nine circles with a raised height of 1mm and a diameter of 5mm. The raised pattern also includes six raised channels, each starting from the center and extending radially until it connects to the atmosphere. The channel width is 1mm, the height is 1mm, and an isosceles triangle shape with a side length of 3mm, a base angle of 30 degrees, and a raised height of 1mm is introduced into the channel. Each raised channel connects to a circular raised piece with a diameter of 5mm. In short, it is similar to replicating the flow channel pattern from Example 2 three times and distributing it evenly within the circular mold.

[0082] 2. Preparation of circular PDMS microfluidic flow channel layer.

[0083] The PDMS preparation agent and curing agent were mixed in a 10:1 ratio and stirred in a beaker for 30 minutes at 150 rpm to form a PDMS prepolymer. The PDMS prepolymer was placed in a vacuum chamber to remove excess air bubbles, and then uniformly poured into the prepared circular mold. The mold was then dried in a 70°C oven for 2 hours, and finally demolded to obtain a circular PDMS microfluidic flow channel layer. The PDMS flow channel layer consists of circles with a radius of 2.5 cm and a thickness of 1.2 mm. This circular PDMS flow channel layer can be considered as being assembled from three fan-shaped microfluidic flow channel layers identical to those in Example 2. The circular PDMS flow channel layer includes three injection ports, three sets of detection zones (two detection wells per set), and three blank control zones.

[0084] 3. Use a punch to make three 3mm diameter injection holes in a 5cm diameter circular double-sided medical adhesive tape. 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 the corresponding target analytes.

[0086] Paper chips modified with different detection reagents were prepared based on the different target analytes. These chips were used to detect creatinine, urea, and calcium. 2+ For example.

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

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

[0089] 2 mg / mL o-cresolphthalein complex and 2.5 mg / mL 8-hydroxyquinoline were dissolved in 0.2 mol / L hydrochloric acid to prepare Ca 2+ Test reagents. On a circular filter paper disc, first add 5 μL of ethanolamine buffer solution at pH 11.5, then add 5 μL of Ca... 2+ The reagent was tested and dried for 3 minutes to prepare Ca. 2+ Detect paper chips.

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

[0091] The circular PDMS microfluidic flow channel layer was divided into a creatinine detection area, a urea detection area, and a Ca detection area. 2+Detection area. In the creatinine detection area, two types of test reagents for creatinine detection are used to modify paper chips, which are then filled into the burst valve groove, the detection area groove, and the blank control area groove, respectively, as in Example 2.

[0092] In the creatinine detection area, the urea detection paper chip is filled into the two detection area grooves and one blank control area groove of the urea detection area.

[0093] Ca 2+ In the detection area, Ca 2+ The detection paper chip is filled into Ca 2+ The detection area has two detection zone grooves and one blank control zone groove.

[0094] Align the central injection port of the aforementioned circular double-sided nano-adhesive with the injection port of the PDMS microfluidic flow channel layer, and attach it to the microfluidic flow channel layer filled with paper chips to complete the encapsulation of the microfluidic channel. This allows for the preparation of materials that can be used to process creatinine, urea, and calcium. 2+ Simultaneous detection of a circular multi-channel microfluidic colorimetric chip.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic detection chip for detecting creatinine in sweat, characterized in that, The microfluidic detection chip includes a microfluidic channel layer, a detection layer, and an encapsulation layer; the detection layer is filled on the microfluidic channel layer, and the encapsulation layer is located on top of the microfluidic channel layer; The microfluidic channel layer is provided with an inlet area, a diversion area, a burst valve groove, a detection area, and a blank control area; The detection layer comprises a paper chip modified with two detection reagents, one of which is Cu. 2+ Another type of modified paper chip is a paper chip co-modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB); The encapsulation layer has an injection port, which corresponds to the injection port of the microfluidic channel layer. The paper chip co-modified with the H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) matches the detection area and blank control area on the microfluidic channel layer; the Cu 2+ The modified paper chip is matched with the burst valve groove on 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 material.

3. The microfluidic detection chip for sweat creatinine detection according to claim 1, characterized in that, The paper chip is made of any one or more of Whatman No. 1 filter paper, cellulose acetate paper, and nitrocellulose membrane.

4. The microfluidic detection chip for sweat creatinine detection according to claim 3, characterized in that, The paper chip is made of Whatman No. 1 filter paper.

5. The microfluidic detection chip for sweat creatinine detection according to claim 1, characterized in that, The Cu 2+ The modified paper chip is made of Cu 2+ The solution is prepared by adding it dropwise onto a filter paper.

6. The microfluidic detection chip for sweat creatinine detection according to claim 1, characterized in that, The paper chip co-modified with the H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) was prepared by adding an acetate-sodium acetate buffer solution to filter paper, then adding the H2MoO5 nanozyme, and finally adding the TMB solution.

7. The microfluidic detection chip for sweat creatinine detection according to claim 1, characterized in that, The H2MoO5 nanozyme was prepared by the following process: Mo powder was ultrasonically dispersed in water, H2O2 was added dropwise under ice-water bath conditions, glacial acetic acid was added, and the reaction was carried out at room temperature until an orange-yellow solution appeared. Subsequently, the solution was filtered to obtain a yellow filtrate, dried, calcined, and ground to obtain the H2MoO5 nanozyme.

8. The method for preparing the microfluidic detection chip for sweat creatinine detection according to any one of claims 1-7, characterized in that, A paper chip co-modified with H2MoO5 nanozyme and 3,3',5,5'-tetramethylbenzidine (TMB) in the detection layer was loaded into the detection area and blank control area on the microfluidic channel layer. Cu 2+ The modified paper chip is filled into the rupture valve groove on the microfluidic channel layer. The sample inlet of the encapsulation layer is aligned with the sample inlet of the microfluidic channel layer and pasted onto the microfluidic flow channel layer filled with the paper chip to complete the encapsulation of the microfluidic channel, thus obtaining the microfluidic detection chip.

9. The application of the microfluidic detection chip for sweat creatinine detection according to any one of claims 1-7 in the detection of creatinine levels in sweat.

10. The application of H2MoO5 nanozyme in the preparation of the microfluidic detection chip for sweat creatinine detection according to any one of claims 1-7, characterized in that, The preparation method of the H2MoO5 nanozyme includes the following steps: ultrasonically dispersing Mo powder in water, adding H2O2 dropwise under ice-water bath conditions, adding glacial acetic acid, reacting fully 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.

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