Wearable sweat microfluidic analysis chip and preparation method and application thereof

Abstract

The application provides a wearable sweat micro-fluidic analysis chip and a preparation method and application thereof. The wearable sweat micro-fluidic analysis chip is sequentially provided with a sweat inlet layer 1, a branch channel layer 3 and a cover layer 5 from top to bottom. The sweat inlet layer is provided with a sweat inlet 6. The branch channel layer 3 is provided with a detection chamber 10, a channel 17 and a valve chamber 9. The sweat inlet 6, the valve chamber 9, the channel 17 and the detection chamber 10 are sequentially communicated. The cover layer 5 is provided with a sweat outlet 14. A water droplet type polymer valve is placed in the valve chamber 9. The opening time of the water droplet type polymer valve can be flexibly controlled by simply adjusting the polymer concentration. The sweat sample can be sequentially collected, and the contact time of the sweat sample and the detection reagent can be accurately controlled. A reliable premise is provided for subsequent accurate analysis. The application can be applied to the detection of cortisol and the detection of uric acid.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, particularly to a microfluidic analysis chip based on an irreversible reaction system for sweat time-series analysis, and especially relates to a wearable sweat microfluidic analysis chip, its preparation method and application. Background Technology

[0002] Sweat, as an easily accessible non-invasive bodily fluid, contains various metabolites, hormones, and inorganic ions, providing a wealth of physiological information. Wearable sweat sensors, due to their integration and portability, enable in-situ, dynamic tracking of target analytes, offering a new tool for personalized health management. Currently, wearable sweat sensors have made significant progress in detecting single or multiple analytes. However, the secretion rate and concentration of sweat components are not constant but dynamically change with exercise intensity, ambient temperature, and individual physiological differences. Traditional single-point sampling methods struggle to capture this temporal information, leading to the loss of crucial dynamic data and limiting their application in continuous health monitoring.

[0003] To achieve continuous dynamic analysis of sweat composition, the development of integrated wearable devices with continuous monitoring capabilities has become crucial. A mainstream strategy for continuous monitoring relies on reversible biomolecular recognition reactions; however, the design of these sensing interfaces is challenging, and many high-affinity reactions with high sensitivity and specificity (such as certain antibody-antigen bindings) are difficult to directly apply to the dynamic detection of sweat components due to their slow or irreversible binding processes. Time-series measurement technology solves the problem of using irreversible chemical processes for continuous detection. This technology integrates programmable valves with functional units such as microchannels and reservoirs to construct devices for continuous sweat monitoring. Passive valves, due to their ease of integration and lack of external power supply, are often used for fluid control and distribution. Currently, liquid bridge valves, capillary burst valves, and Tesla valves have been used for dynamic monitoring of sweat components, but they often rely on sophisticated fluid design, increasing the fabrication difficulty of microfluidic analysis chips. Some polymer valves that achieve timing functions based on the swelling, shrinkage, or dissolution properties of materials require a portion of the polymer to undergo neutralization, cross-linking, and gelation steps to form a gel. This gel is then evenly spread and dried at 70°C for 24 hours. The dried material needs to be manually ground and finely sieved to obtain polymer powder with uniform particle size, and finally manually loaded into the corresponding positions on the microfluidic analysis chip. This preparation process is cumbersome. Another type of polymer can be directly used for paper-based chip fabrication, which is relatively simpler. However, paper-based chips are susceptible to environmental humidity and have limited mechanical strength, thus restricting their practical application. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a wearable sweat microfluidic analysis chip, its preparation method and application.

[0005] To address the aforementioned technical problems, this invention provides a wearable sweat microfluidic analysis chip. The wearable sweat microfluidic analysis chip, from top to bottom, comprises a sweat inlet layer 1, a branch channel layer 3, and a cover layer 5. The sweat inlet layer 1 has a sweat inlet 6, and the branch channel layer 3 has a detection chamber 10, a channel 17, and a valve chamber 9. The sweat inlet 6, valve chamber 9, channel 17, and detection chamber 10 are sequentially connected, and the cover layer 5 has a sweat outlet 14. The valve chamber 9 contains a teardrop-shaped polymer valve. The manufacturing process of this teardrop-shaped polymer valve is simple; it only requires casting the polymer onto a teardrop-shaped filter paper with a diameter of 2 mm to 3 mm and air-drying it at room temperature. This process requires no complex operations and does not involve expensive equipment, facilitating large-scale production. The teardrop-shaped polymer valve controls the opening time by adjusting the polymer concentration, enabling not only sequential collection of sweat samples but also effective control of the contact time between the sample and the detection reagent. The polymer is one or more of polyvinyl alcohol, polyethylene glycol, polystyrene sulfonic acid, hyaluronic acid, and bovine serum albumin. The filter paper is one or more of cellulose filter paper, glass fiber filter paper, quartz fiber filter paper, mixed fiber filter paper, and synthetic polymer filter paper. The detection chamber 10 contains the detection reagent.

[0006] Furthermore, the aforementioned wearable sweat microfluidic analysis chip also includes a central channel and outlet layer 2 and a pore layer 4, wherein the sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover plate layer 5 are arranged sequentially from top to bottom. The central channel and the outlet layer 2 are provided with a central channel 7 around the sweat inlet 6 and communicate with the sweat inlet 6. A branch channel 8 is provided around the central channel 7 and communicates with the central channel 7, so that sweat flows through the central channel 7 first. Whether it can enter the branch channel 8 depends only on whether the water droplet polymer valve is open. Once the sweat enters the branch channel 8, it will not flow back, thereby avoiding cross-contamination between new and old sweat samples. The branch channel layer 3 is provided with multiple valve chambers 9 that communicate with different branch channels 8. Each valve chamber 9 is provided with an independent channel 17 and a detection chamber 10. The detection chamber 10, the channel 17 and the valve chamber 9 are connected. Each valve chamber 9 contains a droplet-shaped polymer valve with different concentrations. The pore layer 4 is provided with pores 13 for communicating with the air in the environment. The pores 13 are in communication with the detection chamber 10 to ensure that sweat flows from the sweat inlet 6 to the detection chamber 10.

[0007] Furthermore, in the aforementioned wearable sweat microfluidic analysis chip, the droplet-shaped polymer valve is positioned 2 mm away from the sweat inlet 6, so that the valve opening time has a linear relationship with the polymer concentration within the range of 0 to 90 minutes.

[0008] Furthermore, the wearable sweat microfluidic analysis chip described above can meet the detection requirements of a constant signal response and can collect sweat samples at different times as needed. The detection reagent is a reagent for detecting cortisol. The reagent for detecting cortisol is Ap-MFDN, which is prepared using the following method: H1-cDNA, H2 and Aptamer were annealed at 96 °C for 3 min, and then Trigger, aptamer, H1 and H2 were mixed and incubated. The gene sequence of the Aptamer is shown in SEQ ID NO.1; the gene sequence of the H1-cDNA is shown in SEQ ID NO.2; the gene sequence of the H2 is shown in SEQ ID NO.3; and the gene sequence of the Trigger is shown in SEQ ID NO.4. The wearable microfluidic chip operates as follows: An athlete uses a sweatband to attach the microfluidic analysis chip to their forehead, with the sweat collection inlet 6 facing the skin surface. During continuous exercise, valves open sequentially at different times (T1~T5), guiding sweat from different times to different detection chambers 10, causing the pre-stored Ap-MFDN dry powder to re-dissolve and generate a fluorescent signal related to the target. The principle behind the fluorescence signal generation is as follows: When a trigger is present, two hairpin probes (H1-cDNA-BHQ and H2) trigger an HCR reaction, alternately hybridizing to form the double-stranded DNA backbone of Ap-MFDN. Without a trigger, H1 and H2 remain in a hairpin state and cannot assemble into a double-stranded DNA backbone. Next, a nucleic acid aptamer labeled with a fluorescent group (FAM) (Aptamer-FAM) hybridizes complementaryly with cDNA-BHQ extending from H1, thereby assembling onto the double-stranded DNA backbone to form a multivalent DNA nanostructure (Ap-MFDN). At this point, the fluorescence of FAM is quenched by BHQ. After the target compound was added, competitive binding separated the aptamer and cDNA, and the fluorescence of FAM was restored; The detection chamber 10, F0 calibration chamber 11 and F B The calibration chamber 12 generates F, F0 and F respectively. B The signal is used for sample detection, reagent blank control, and background control, and is expressed as relative fluorescence intensity (FF).B ) / (F0-F B () as a signal value.

[0009] Based on a general technical concept, the present invention provides a method for fabricating the wearable sweat microfluidic analysis chip, the method comprising the following steps: SA-1, cut the PET film to obtain sweat inlet layer 1, central channel and outlet layer 2, branch channel layer 3, pore layer 4 and cover plate layer 5. SA-2. Adhere the sweat inlet layer 1, the central channel and the outlet layer 2 together. Add a hydrophilic reagent to treat the central channel 7 and the interface with the surrounding branch channels 8. Adhere the branch channel layer 3, the pore layer 4 and the cover plate layer 5 in order from top to bottom. Treat the valve chamber 9 with fluorinated oil to make it hydrophobic. After the fluorinated oil evaporates, use a cotton swab to apply a hydrophilic reagent to the detection chamber 10 for local hydrophilic treatment. SA-3, Place the teardrop-shaped polymer valve in the valve chamber 9 and encapsulate the chip; SA-4 and Ap-MFDN reagents are injected into the detection chamber 10 through the pore 13 of the microfluidic analysis chip, and the pore 13 is sealed with a PTFE membrane (breathable but not water-permeable). The sweat inlet 6 is sealed with transparent tape. The reagents in the detection chamber are freeze-dried at -60°C for at least 2 hours to form Ap-MFDN dry powder.

[0010] The fabrication method of the above-mentioned wearable sweat microfluidic analysis chip further includes the following: the sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover layer 5 are all fabricated by laser cutting; the cutting parameters of the laser cutting are: maximum light intensity 60%~65%, minimum light intensity 20%~25%, and cutting speed 10 mm / s~15 mm / s.

[0011] Based on a general technical concept, the present invention provides an application of the wearable sweat microfluidic analysis chip in the detection of cortisol.

[0012] To address the aforementioned technical problems, this invention provides a wearable sweat microfluidic analysis chip. The wearable sweat microfluidic analysis chip, from top to bottom, comprises a sweat inlet layer 1, a branch channel layer 3, and a cover layer 5. The sweat inlet layer has a sweat inlet 6, and the branch channel layer 3 has a detection chamber 10, a channel 17, and a valve chamber 9. The sweat inlet 6, valve chamber 9, channel 17, and detection chamber 10 are sequentially connected, and the cover layer 5 has a sweat outlet 14. The valve chamber 9 contains a teardrop-shaped polymer valve. The manufacturing process of this teardrop-shaped polymer valve is simple; it only requires casting the polymer onto a teardrop-shaped filter paper with a diameter of 2 mm to 3 mm and air-drying it at room temperature. This process requires no complex operations and does not involve expensive equipment, facilitating large-scale production. The teardrop-shaped polymer valve controls the opening time by adjusting the polymer concentration, enabling not only sequential collection of sweat samples but also effective control of the contact time between the sample and the detection reagent. The polymer is one or more of polyvinyl alcohol, polyethylene glycol, polystyrene sulfonic acid, hyaluronic acid, and bovine serum albumin. The filter paper is one or more of cellulose filter paper, glass fiber filter paper, quartz fiber filter paper, mixed fiber filter paper, and synthetic polymer filter paper. The detection chamber 10 contains the detection reagent.

[0013] Furthermore, the aforementioned wearable sweat microfluidic analysis chip also includes a central channel and outlet layer 2 and a pore layer 4, wherein the sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover plate layer 5 are arranged sequentially from top to bottom. The central channel and the outlet layer 2 are provided with a central channel 7 communicating with the sweat inlet 6 around the sweat inlet 6, and a branch channel 8 communicating with the central channel 7 around the central channel 7; so that sweat flows through the central channel 7 first, and whether it can enter the branch channel 8 depends only on whether the water droplet polymer valve is open, and once the sweat enters the branch channel 8, it will not flow back, thereby avoiding cross-contamination between new and old sweat samples; On the branch channel layer 3, each branch channel layer 8 is provided with two valve chambers 9, one detection chamber 10, and one sweat storage chamber 15. The two valve chambers are respectively the first valve chamber 9-1 and the second valve chamber 9-2. The first valve chamber 9-1 is connected to the sweat storage chamber 15, the second valve chamber 9-2, and the detection chamber 10 in sequence. Multiple first valve chambers 9-1 contain droplet-shaped polymer valves with progressively increasing concentrations; multiple second valve chambers 9-2 contain droplet-shaped polymer valves with progressively decreasing concentrations.

[0014] The pore layer 4 is provided with pores 13 for communicating with the air in the environment. The pores 13 are in communication with the detection chamber 10 to ensure that sweat flows from the sweat inlet 6 to the detection chamber 10.

[0015] Furthermore, the wearable sweat microfluidic analysis chip described above can meet the detection requirements of signal change-type reactions. In addition to being able to sample samples at different times as needed, it can also control the release of samples at a preset time to allow them to come into contact with and mix with the detection reagent, which is a uric acid detection reagent. The uric acid detection reagent is loaded onto filter paper and prepared using the following method: Cut the filter paper into circles with a diameter of 4-6 mm, add 5-10 µL of uric acid detection reagent onto the filter paper, and then dry the filter paper with nitrogen gas to prepare a reagent pad; The uric acid detection reagent comprises: 100 mM PBS buffer, 100 U / mL uricase, 100 U / mL horseradish peroxidase, 5 mM 4-aminoantipyrine, 10 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, 1 mg / mL bovine serum albumin, and 5 wt% trehalose. The wearable microfluidic chip operates as follows: An athlete uses a sweatband to secure the microfluidic analysis chip to their forehead. During continuous exercise, sweat enters the chip through sweat inlet 6, first contacting the time-sequential sampling polymer valves T1-T4, and then the sample release polymer valves T1'-T4'. These valves are made of polystyrene sulfonic acid (PSS) of varying concentrations. The opening times of T1-T4 increase sequentially, while the opening times of T1'-T4' decrease sequentially, thus eliminating the time difference between the sweat collected at different times and the detection reagent. During the use of the microfluidic analysis chip, sweat spontaneously enters the detection chamber 10 from the sweat inlet 6 and comes into contact with the reagent pad pre-stored with the detection reagent, resulting in a color reaction. No additional reagents are required during the process. The color reaction principle is as follows: uric acid generates hydrogen peroxide (H2O2) under the action of uricase. Subsequently, H2O2 reacts with 4-aminoantipyrine (4-APP) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) under the catalysis of horseradish peroxidase (HRP) to generate a red quinone compound.

[0016] Based on a general technical concept, the present invention provides a method for fabricating the wearable sweat microfluidic analysis chip, the method comprising the following steps: SB-1. Cut the PET film to obtain the sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover plate layer 5; SB-2. Adhere the sweat inlet layer 1, the central channel and the outlet layer 2 together. Add a hydrophilic reagent to treat the central channel 7 and the interface with the surrounding branch channels 8. Adhere the branch channel layer 3, the pore layer 4 and the cover plate layer 5 in order from top to bottom. Treat the valve chamber 9 with fluorinated oil to make it hydrophobic. After the fluorinated oil evaporates, use a cotton swab to apply a hydrophilic reagent to the detection chamber 10 for local hydrophilic treatment. SB-3. Place the droplet-shaped polymer valve and the filter paper loaded with uric acid detection reagent into the corresponding chamber and encapsulate the chip.

[0017] Furthermore, in the above-mentioned method for fabricating the wearable sweat microfluidic analysis chip, the sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover layer 5 are all fabricated by laser cutting; the cutting parameters of the laser cutting are: maximum light intensity 60% to 65%, minimum light intensity 20% to 25%, and cutting speed 10 mm / s to 15 mm / s.

[0018] Based on a general technical concept, the present invention provides an application of the wearable sweat microfluidic analysis chip in the detection of uric acid.

[0019] Compared with the prior art, the advantages of the present invention are as follows: (1) The present invention provides a wearable sweat microfluidic analysis chip. The droplet-shaped polymer valve used has a very simple preparation process. It only requires drop-coating polymers of different concentrations onto filter paper and air-drying them at room temperature, and then embedding them into the corresponding positions of the microfluidic analysis chip. It does not require complex equipment or stringent process conditions and has significant advantages in mass production and industrial application.

[0020] (2) The present invention provides a wearable sweat microfluidic analysis chip, which can flexibly control the opening time of the droplet-shaped polymer valve by simply adjusting the polymer concentration. This enables both the time-sequential collection of sweat samples and precise control of the contact time between the sweat sample and the detection reagent, providing a reliable basis for subsequent accurate analysis.

[0021] (3) The present invention provides a wearable sweat microfluidic analysis chip. The microfluidic analysis chip adopts a partitioned structure of central channel and branch channel. Sweat flows through the central channel first and enters the branch channel only when the corresponding valve is opened, and there is no backflow. The structure eliminates cross-contamination between new and old sweat samples. At the same time, with the layout design of 2 to 2.5 mm diameter teardrop-shaped valve and 2 to 3 mm away from the sweat inlet, the retention of old sweat can be reduced, and the valve opening time is linearly related to the polymer concentration within 0 to 90 minutes. The control accuracy is high and the stability is good.

[0022] (4) This invention provides a wearable sweat microfluidic analysis chip. Relying on the collaborative design of droplet-shaped polymer valves and main branch channels, it can automatically divide new and old sweat at preset time intervals and complete independent analysis. It can meet the needs of signal constant and signal change-type reaction time dynamic monitoring of high-sensitivity, high-specificity non-reversible reaction systems, and can also flexibly adapt to human dynamic health monitoring and personalized health management during exercise. It has strong application scenarios and high practical value. Attached Figure Description

[0023] 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 with reference to the accompanying drawings.

[0024] Figure 1 This is a schematic diagram illustrating the fabrication of the polymer valve in Example 1 of the present invention.

[0025] Figure 2 This is a schematic diagram of a single-channel chip equipped with a valve placement chamber in Embodiment 1 of the present invention.

[0026] Figure 3 This is a graph showing the ability of different polymer valves to isolate liquids in Example 1 of the present invention.

[0027] Figure 4 This is an observation diagram examining the optimization of different valve shapes in Embodiment 1 of the present invention.

[0028] Figure 5 This is an exploded view of the microfluidic analysis chip for cortisol detection according to Embodiment 2 of the present invention.

[0029] Figure 6 This is a schematic diagram illustrating the working principle of the microfluidic analysis chip used for cortisol detection in sweat in Embodiment 2 of the present invention.

[0030] Figure 7 This is a diagram showing the cross-contamination of liquids in each branch channel in Embodiment 2 of the present invention.

[0031] Figure 8 This is a diagram showing the timing sampling capability of the microfluidic analysis chip in Embodiment 2 of the present invention.

[0032] Figure 9 This is a kinetic response diagram of Ap-MFDN to cortisol in Example 2 of the present invention.

[0033] Figure 10 This is a calibration curve for cortisol detection in Example 2 of the present invention.

[0034] Figure 11 This is a graph showing the changes in cortisol content in human sweat at different times in Example 2 of the present invention.

[0035] Figure 12 This is an exploded view of the microfluidic analysis chip for uric acid detection according to Embodiment 3 of the present invention.

[0036] Figure 13 This is a schematic diagram illustrating the working principle of the microfluidic analysis chip used for uric acid detection in sweat in Embodiment 3 of the present invention.

[0037] Figure 14This is a diagram showing the cross-contamination of liquids in each branch channel in Embodiment 3 of the present invention.

[0038] Figure 15 This is a diagram showing the timing sampling capability of the microfluidic analysis chip in Embodiment 3 of the present invention.

[0039] Figure 16 This is a diagram showing the kinetic response and condition optimization for uric acid detection by uricase in Example 3 of the present invention.

[0040] Figure 17 This is a calibration curve for uric acid detection in Example 3 of the present invention.

[0041] Figure 18 This is a graph showing the change in uric acid content in human sweat at different times in Example 3 of the present invention.

[0042] Legend: 1. Sweat inlet layer; 2. Central channel and outlet layer; 3. Branch channel layer; 4. Pore layer; 5. Cover layer; 6. Sweat inlet; 7. Central channel; 8. Branch channel; 9. Valve chamber; 10. Detection chamber; 11. F0 calibration chamber; 12. FB calibration chamber; 13. Pores; 14. Sweat outlet; 15. Sweat storage chamber; 17. Channel. Detailed Implementation

[0043] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0044] The materials, reagents, and instruments used in the following examples were all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Units mM and μM are mmol / L and μmol / L, respectively.

[0045] Example 1 A polymer valve, the manufacturing process of which is described in [reference needed]. Figure 1 Specifically, this includes: dripping polystyrene sulfonic acid of different concentrations onto cellulose filter paper in the shape of a water droplet with a diameter of 2 mm, and air-drying it at room temperature to obtain a polymer valve.

[0046] The filter paper can also be selected from one or more of the following: cellulose filter paper, glass fiber filter paper, quartz fiber filter paper, mixed fiber filter paper, and synthetic polymer filter paper. The polymer can also be selected from one or more of the following: polyvinyl alcohol, polyethylene glycol, polystyrene sulfonic acid, hyaluronic acid, and bovine serum albumin. These polymers are all water-soluble / water-dispersible polymers (BSA is a natural protein polymer), possessing the function of blocking channels and isolating liquids. Positioning the polymer valve 2 mm from the sweat inlet reduces the retention of old sweat. This design ensures that the valve opening time is linearly related to the polymer concentration within 0–90 minutes; as time progresses, the polymer dissolves, and the valve opens.

[0047] Experiment 1: Investigating the ability of different polymer valves to isolate liquids.

[0048] Polymer valves act as a soluble barrier, sealing channels and isolating liquids. Over time, the polymer dissolves, opening the valve.

[0049] Experimental steps: (1) According to Figure 2 The design shown is a single-channel chip equipped with a valve placement chamber. The single-channel microfluidic chip has, from top to bottom, a sweat inlet layer 1, a branch channel layer 3, and a cover layer 5. The sweat inlet layer has a sweat inlet 6, and the branch channel layer 3 has a valve chamber 9, a channel 17, and a detection chamber 10. (2) Polyvinyl alcohol (PVA), polyethylene glycol (PEG), polystyrene sulfonic acid (PSS), hyaluronic acid (HA), and bovine serum albumin (BSA) were selected as valve materials to prepare polymer solutions with different concentration gradients. The polymer solutions of each concentration were dripped onto cellulose filter paper in the shape of water droplets, air-dried at room temperature, and then embedded into the valve placement chamber of the single-channel chip to form a soluble polymer barrier valve.

[0050] (3) Test liquid was introduced into the single-channel chip loaded with polymer valves of different types and concentrations. The sealing and isolation status of the valves to the liquid was observed and recorded in real time. The opening time of each group of valves and the time it took for the liquid to fill the valve chamber were accurately measured. For the BSA polymer valve group, a papain addition experimental group was set up. The above liquid isolation test procedure was repeated, and the changes in valve opening time and chamber filling time under enzyme addition and no enzyme addition conditions were compared and recorded.

[0051] See the experimental results. Figure 3In the figure, A represents the valve opening time and the time it takes for the liquid to fill the valve chamber for PVA solutions of different concentrations. It can be seen from the figure that PVA with a concentration of 15% to 20% only has short-term liquid isolation capability, with a valve opening time of <3 min; when the concentration is below 15%, there is no isolation effect, and the time control range is extremely narrow, which cannot meet the timing control requirements.

[0052] B in the figure represents the valve opening time and the time it takes for the liquid to fill the valve chamber for different concentrations of PEG solution. As can be seen from the figure, PEG has virtually no liquid isolation capability in the concentration range of 10% to 20% and cannot be used as a soluble barrier valve.

[0053] In the figure, C represents the valve opening time and the time it takes for the liquid to fill the valve chamber for PSS solutions of different concentrations. The figure shows that within the test concentration range, the PSS valve opening time increases from 10 min to 60 min with increasing concentration, exhibiting a wide control range and stable response. Its molecular repeating unit contains -SO3. - It is more likely to interact with the filter paper groups and be adsorbed. When the liquid is flushed, it stacks forward along the pores and is not easily dispersed. It has the best liquid isolation and time resolution effect.

[0054] In the figure, D represents the valve opening time and the time it takes for the liquid to fill the valve chamber for HA solutions of different concentrations. It can be seen from the figure that when the concentration is 0.1% to 1%, the isolation effect of the HA valve is similar and the opening time is <2 min; when the concentration is >1%, the valve still does not open after 1 hour, and the opening time is either too short or too long, resulting in extremely poor control flexibility.

[0055] In the figure, E represents the valve opening time and the time for the liquid to fill the valve chamber for BSA solutions of different concentrations. It can be seen from the figure that when the concentration is 0 to 0.8%, the valve opening time and the time for the chamber to fill increase with the increase of concentration.

[0056] In the figure, F represents the valve opening time and the time it takes for the liquid to fill the valve chamber after adding papain to BSA solutions of different concentrations. As can be seen from the figure, the valve opening time is not significantly shortened after adding papain, but the chamber filling time is shortened, and the shortening is more significant at higher concentrations. This proves that the protease can degrade BSA and accelerate dissolution, but its overall time resolution is still inferior to that of PSS.

[0057] Comparing the liquid isolation capabilities, opening time control range, and response characteristics of the five polymers, PSS exhibits the best overall performance. Therefore, PSS was selected as the material for preparing the polymer valve of this invention.

[0058] Experiment 2: Investigating the effect of valve shape.

[0059] Since soluble polymer valves open by immersing themselves in sweat, the contact area between the valve and sweat has a certain impact on their isolation effect. Therefore, the relationship between teardrop-shaped and circular valves and opening time was investigated.

[0060] (1) Two shapes of PSS valves were prepared: teardrop and circular, both using the same substrate material (filter paper, 3 mm in diameter). PSS solutions with concentrations of 10%, 12%, 15%, 18%, 20%, 24%, 25%, and 30% were prepared respectively. The PSS solutions of different concentrations were dripped onto the filter paper and air-dried at room temperature to produce valves of uniform thickness. The two shapes and different concentrations of PSS valves were embedded into single-channel microfluidic chips equipped with valve placement chambers. The distance between the valve and the sweat inlet was uniformly set to the same reference value to ensure that other experimental conditions (such as chip channel size, test liquid composition, and ambient temperature) were consistent. Simulated sweat was introduced into the chip sweat inlet, and the effect of each group of valves in delaying liquid flow and the valve opening time were observed and recorded in real time. The performance differences between the teardrop and circular valves at different PSS concentrations were compared. The corresponding experimental results are as follows: Figure 4 As shown.

[0061] As can be seen from Figure A, the droplet-type PSS valve can stably achieve liquid delayed flow control within the PSS concentration range of 0% to 24%, with a delay time covering 0 to 90 minutes, which can meet the basic requirements for time-series sweat sample collection.

[0062] As can be seen from B in the figure, even when the PSS concentration of the circular PSS valve is increased to 30% (higher than the highest test concentration of the teardrop valve), its effect of delaying liquid flow is still not better than that of the teardrop valve. It cannot achieve wide-range and stable delay control, indicating that the teardrop structure is more conducive to the PSS valve to perform liquid isolation and delayed opening functions.

[0063] (2) To address the issue of old sweat retention in the valves, the diameters of the teardrop-shaped valves were set to 3 mm and 2 mm respectively, with the distance between the valve and the sweat inlet maintained at 2 mm. A series of PSS solutions with concentrations ranging from 0% to 24% were prepared, and corresponding valves were fabricated and embedded in the chip. Simulated sweat was introduced, and the delay time for liquid flow in each group of valves was recorded to verify whether the preset delay time range could be achieved by adjusting the PSS concentration. At the same time, the retention of old sweat was observed.

[0064] In the figure, C represents the opening time of a 3 mm diameter valve and the time it takes for liquid to fill the valve chamber, while D represents the opening time of a 2 mm diameter valve and the time it takes for liquid to fill the valve chamber. The figures show that compared to valves located directly next to the sweat inlet, a valve 2 mm away from the sweat inlet exhibits a more linear relationship between valve opening time and PSS concentration. However, some "old sweat" may remain within the valve itself, affecting subsequent test results. Therefore, reducing the valve diameter to 2 mm and examining its effect on delaying liquid flow was investigated. Simply increasing the PSS concentration in parallel achieves the same delay effect (0–90 min), a time range sufficient for most sports activities. Therefore, a 2 mm diameter teardrop-shaped valve, designed to be 2 mm away from the inlet, was used to isolate the liquid.

[0065] Example 2 A wearable sweat microfluidic chip with an integrated droplet-shaped polymer valve that meets the requirements of constant signal response detection is used for the detection of cortisol in sweat.

[0066] See Figure 5 The wearable sweat microfluidic chip is arranged from top to bottom as follows: sweat inlet layer 1, central channel and outlet layer 2, branch channel layer 3, pore layer 4, and cover plate layer 5.

[0067] The sweat inlet layer 1 has three sweat inlets 6, one of which is a large circle with a radius of 1.5 mm; the other two are small circles with a radius of 0.5 mm, serving as artificial sweat inlets to generate F0 and F2. B Signal, F0 is the reagent blank control, F B For background contrast.

[0068] The central channel and outlet layer 2 have corresponding inlets at the same locations as the sweat inlet layer 1. Simultaneously, a central channel 7 (0.5 mm wide) communicating with the large sweat inlet 6 is provided around it, and branch channels 8 communicating with it are provided around the central channel 7. The dimensions of each branch channel 8 are 2 mm × 0.3 mm. Sweat flows in from the sweat inlet 6 and preferentially flows through the central channel 7.

[0069] The branch channel layer 3 is provided with a valve chamber 9 for placing valves and a detection chamber 10 for placing detection solutions, as well as two independent calibration chambers 11 and 12 for calibration. B Calibration chamber 12 and F0 calibration chamber 11 are used for the calibration of F0. B Calibration chamber 12 is used for F B The valve chamber 9 has a diameter of 2 mm, and the detection chamber 10, F0 calibration chamber 11, and F... BThe diameter of each calibration chamber 12 is 3 mm. Pre-stored lyophilized test reagents are stored in both the detection chamber 10 and the F0 calibration chamber 11. B There are no reagents in calibration chamber 12.

[0070] The pore layer 4 is provided with pores 13 for communicating with the air in the environment, so as to ensure that sweat flows from the sweat inlet 6 to the detection chamber 10. The pores are circles with a diameter of 0.5 mm.

[0071] The central channel, outlet layer 2, branch channel layer 3, pore layer 4, and cover layer 5 all have sweat outlets 14 at the same location, each with a diameter of 2 mm. Excess liquid flowing out of the central channel 7 can flow out through the sweat outlets 14. Each layer of the chip is a fan-shaped structure with a radius of 18 mm and an included angle of 154.16°.

[0072] In this embodiment, the chip is partitioned into a central channel 7 and branch channels 8, which allows for the collection of sweat samples at different times as needed. During the collection process, sweat flows first through the central channel 7. As it flows into the branch channels 8, the opening time of the droplet-shaped polymer valve is controlled by adjusting the polymer concentration, thus achieving the sequential collection of sweat samples. Once the sweat passes through the droplet-shaped polymer valve, it will not flow back, thereby avoiding cross-contamination between new and old sweat samples.

[0073] A method for fabricating a wearable sweat microfluidic chip according to this embodiment specifically includes the following steps: S1. Based on the structure of each layer of the microfluidic analysis chip, the layers are drawn on a 0.2 mm thick PET film with 3M adhesive backing to obtain the sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover layer 5. The sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover layer 5 are all prepared by laser cutting; the laser cutting parameters are: maximum light intensity 65%, minimum light intensity 20%, and cutting speed 15 mm / s.

[0074] S2. Adhere the sweat inlet layer 1, the central channel and the outlet layer 2 together. Add a hydrophilic reagent to treat the central channel 7 and the interface with the surrounding branch channels 8. Adhere the branch channel layer 3, the pore layer 4 and the cover plate layer 5 in order from top to bottom. Treat the valve chamber 9 with fluorinated oil to make it hydrophobic. After the fluorinated oil evaporates, use a cotton swab to apply a hydrophilic reagent to the detection chamber 10 for local hydrophilic treatment.

[0075] S3. Place the teardrop-shaped polymer valve from Example 1 into the valve chamber 9 and encapsulate the chip.

[0076] S4. The Ap-MFDN reagent is injected into the detection chamber 10 through the pore 13 of the microfluidic analysis chip, and the pore 13 is sealed with a PTFE membrane (breathable but not water-permeable). The sweat inlet 6 is sealed with transparent tape. The reagent in the detection chamber is freeze-dried at -60°C for at least 2 hours to form Ap-MFDN dry powder.

[0077] The specific preparation method for the Ap-MFDN (multivalent fluorescent DNA nanostructures functionalized with aptamers based on hybridization chain reaction) reagent in S4 is as follows: Prepare a 100µM stock solution of H1-cDNA, H2, trigger, and aptamer powder using PBS buffer and store at 4℃ for later use. Anneal H1-cDNA, H2, and aptamer at 96℃ for 3 min. Then, mix the trigger, aptamer, H1, and H2 to final concentrations of 0.25 µM, 5 µM, 25 µM, and 25 µM, respectively, and incubate at 25℃ in a metal constant-temperature shaking bath for 24 h to form AP-MFDN.

[0078] The PBS buffer solution is prepared as follows: Weigh 3.5816 g Na2HPO4·12H2O, 0.3520 g KH2PO4, 8.006 g NaCl and 0.2010 g KCl, dissolve them in 800 mL of ultrapure water, then add 1.0160 g MgCl2·6H2O, dissolve the solution, transfer it to a 1 L volumetric flask and bring the volume to a final volume. Adjust the pH to 7.5, filter the solution in a clean bench using a syringe and a 0.22 µm filter, dispense the solution, and store it at -20 ℃ for later use.

[0079] Based on the fact that the biomarker to be tested is cortisol, the nucleotide sequence of the cortisol nucleic acid aptamer Aptamer-FAM is shown in SEQ ID No. 1, the nucleotide sequence of H1-cDNA-BHQ is shown in SEQ ID No. 2, the H2 sequence is shown in SEQ ID No. 3, and the trigger sequence is shown in SEQ ID No. 4, as detailed in Table 1.

[0080] Table 1: DNA strand sequences used in Example 2 of the present invention

[0081] Figure 6This embodiment describes the process of using a wearable sweat microfluidic analysis chip with an integrated droplet-shaped polymer valve for detecting cortisol in sweat. As shown in the figure, the athlete uses an antiperspirant to fix the microfluidic analysis chip to their forehead, with the sweat collection inlet 6 facing the skin surface. During continuous exercise, the valve opens sequentially at different times (T1-T5), guiding the sweat at different times to different detection chambers 10. This causes the pre-stored Ap-MFDN dry powder to re-dissolve, generating a fluorescent signal related to the target analyte. The principle of fluorescence signal generation is as follows: when a trigger is present, two hairpin probes (H1-cDNA-BHQ and H2) trigger an HCR reaction, alternately hybridizing to form the double-stranded DNA backbone of Ap-MFDN. Without a trigger, H1 and H2 remain in a hairpin state and cannot assemble into a double-stranded DNA backbone. Next, the nucleic acid aptamer labeled with a fluorescent group (FAM) (Aptamer-FAM) hybridizes complementaryly with the cDNA-BHQ extended from H1, thereby assembling onto the double-stranded DNA backbone to form a multivalent DNA nanostructure (Ap-MFDN). At this point, the fluorescence of FAM is quenched by BHQ. After the target analyte is added, competitive binding separates the aptamer and cDNA, and the fluorescence of FAM is restored. Detection chamber 10, F0 calibration chamber 11, and F... B The calibration chamber 12 generates F, F0 and F respectively. B The signal is used for sample detection, reagent blank control, and background control, and is expressed as relative fluorescence intensity (FF). B ) / (F0-F B () as a signal value.

[0082] Experiment 3: Investigate the cross-contamination of liquids in each branch channel.

[0083] (1) Verification of cross-contamination prevention capability: Absorbent paper was placed at the outlet of the microfluidic analysis chip to simulate the function of a sports sweatband, assisting in the renewal of sweat in the central channel and ensuring that the experimental conditions fit the actual application scenario. Artificial sweat containing indicators, artificial sweat containing methylene blue dye, artificial sweat containing different colored dyes, and colorless artificial sweat were injected into the microfluidic analysis chip at a rate of 10 µL / min through a syringe pump until each chamber of the chip was filled with the artificial sweat. The injection pump was then used to continue to introduce artificial sweat containing methylene blue dye into the chip until the solution volume reached 200 µL. The diffusion of methylene blue dye was observed in real time, and the chip image was recorded. Using ImageJ image analysis software, five chambers on the chip were selected as the analysis objects, and the gray values ​​of each chamber at three time points (T1, T2, and T3) were measured to analyze the gray value change pattern.

[0084] Figure 7The results are as follows. Figure A shows that after continuously introducing 200µL of artificial sweat containing methylene blue dye into a chip that was already filled with artificial sweat containing the indicator, the blue dye did not diffuse into the chip chamber, indicating that the subsequently introduced sweat did not enter the already filled chamber.

[0085] Figure B shows that Image J's analysis of the grayscale values ​​at time points T1–T3 in the five chambers revealed no significant differences in grayscale values ​​across the chambers at different time points. This stability of the grayscale values ​​further confirms that the solution within the chambers did not mix with the subsequently introduced methylene blue dye solution.

[0086] (2) Verification of anti-backflow capability: Artificial sweat containing different colored dyes was sequentially introduced into different chambers of the microfluidic analysis chip, ensuring that each chamber was filled with artificial sweat of the corresponding color. Colorless artificial sweat was introduced into the central channel of the chip. After continuous introduction for a period of time, the colored solution in each chamber was observed in real time to see if it flowed back into the central channel, and the chip image was recorded. Using the same Image J analysis method as above, five chambers were selected, and the gray values ​​of each chamber were measured at three time points: T1, T2, and T3, and the differences in gray value changes were analyzed.

[0087] Figure C shows that after colorless artificial sweat was introduced into the central channel of a chamber filled with artificial sweat dyed with different colors, no backflow of the colored solution in the chamber into the central channel was observed, indicating that an effective liquid flow isolation was formed between the chamber and the central channel.

[0088] In the figure, D represents the grayscale analysis results: Image J's analysis of the grayscale values ​​of the five chambers at time points T1 to T3 shows that there is no significant difference in the grayscale values ​​of each chamber, indicating that the solution concentration in the chamber has not changed, further proving that no solution backflow occurred.

[0089] Combining the results of two verification experiments, the microfluidic analysis chip successfully achieved the dual effects of "preventing cross-contamination" and "preventing solution backflow" through the structural design of the outlet absorbent paper to assist sweat renewal and the chamber and central channel. This ensures the independence of sweat samples in each chamber, meets the needs of sweat time-series collection and accurate detection, and is suitable for application scenarios of dynamic health monitoring during exercise.

[0090] Experiment 4: Examine the chip's timing sampling capability.

[0091] First, the prepared PSS valves of different concentrations were placed in their corresponding positions on the microfluidic analysis chip. Then, an absorbent paper was placed at the chip outlet to simulate the function of a sports sweatband, assisting in the renewal of sweat within the central channel. Using a syringe pump, artificial sweat containing lemon yellow dye was pumped into the microfluidic analysis chip at a rate of 10 µL / min. During the process, the flow of artificial sweat and the opening status of the valves in each branch channel were closely observed, and optical images at each stage were recorded, especially the stages before the artificial sweat completely filled each chamber of the chip. In addition, the times when sweat entered the branch channels from the central channel were preset, T1 to T5, corresponding to 0, 10, 30, 60, and 90 minutes, respectively, to observe whether the sweat entered the branch channels at these preset times.

[0092] Figure 8 The results show the chip's timing capability evaluation. Figure A shows that sweat in the central channel enters the branch channels at preset times, T1 to T5, which are 0, 10, 30, 60, and 90 min, respectively. Artificial sweat containing lemon yellow dye is pumped into the microfluidic analysis chip at a rate of approximately 10 µL / min. Figure B shows optical images of each stage before the artificial sweat completely fills the chambers of the microfluidic analysis chip. The results show that the artificial sweat opens the valves and fills the chambers sequentially according to the preset order. This demonstrates that different concentrations of PSS valves can achieve timing control of the sweat, achieving our preset experimental objective.

[0093] Experiment 5: Investigate the kinetic response of Ap-MFDN to cortisol.

[0094] Prepare cortisol standard solutions of varying concentrations: 0 nM, 20 nM, 200 nM, 500 nM, 1000 nM, 2000 nM, 3000 nM, and 5000 nM. Add the cortisol solutions of different concentrations to the Ap-MFDN substrate system and immediately start fluorescence intensity monitoring. Continuously record the changes in fluorescence intensity from 0 to 3000 s.

[0095] like Figure 9 As shown, when detecting cortisol based on multivalent DNA nanostructures, the detection signal shows a trend of first increasing and then remaining constant over time.

[0096] Experiment 6: Detecting cortisol in sweat at different times.

[0097] Plotting a standard curve for cortisol detection: Cortisol was added to artificial sweat at final concentrations of 0 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, and 1000 nM. Artificial sweat containing cortisol was introduced into F0 and the detection chamber through the inlet, while artificial sweat without the target substance was introduced into F0. BAfter incubation in the chamber for 10 minutes, the chip was placed in a dark box, and the RGB signal was extracted by taking a picture with a smartphone. The relative fluorescence intensity (FF) was then fitted. B ) / (F0-F B The relationship curve between signal and cortisol concentration, Figure 10 The resulting calibration curve is: y = 0.0020x + 1.0972, where y is the relative fluorescence intensity value, x is the cortisol concentration value, and R0 is the relative fluorescence intensity value. 2 =0.9848.

[0098] The valve was set to open at five time points: 0 min, 15 min, 30 min, 40 min, and 50 min to collect sweat. A microfluidic analysis chip was worn on the volunteer's forehead using a sweatband, allowing the volunteer to move freely and maintain a sweating state. After exercise, artificial sweat was dripped onto F0 and F... B In the calibration chamber, the microfluidic analysis chip was placed in a dark box, photographed, and its RGB values ​​were read and converted into relative fluorescence intensity (FF). B ) / (F0-F B The cortisol concentration can be obtained by substituting the signal into the calibration curve y=0.0020x + 1.0972.

[0099] Figure 11 The graph shows the changes in cortisol levels in human sweat at different times. As can be seen from the graph, the cortisol levels of both volunteers showed an increasing trend over time. However, at time T5, the cortisol level of volunteer 1 decreased slightly, which may be due to the dilution of the marker concentration caused by the large amount of sweat.

[0100] Example 3 A wearable sweat microfluidic chip with an integrated droplet-shaped polymer valve that meets the requirements of signal change-type response detection is used for the detection of uric acid in sweat.

[0101] See Figure 12 The wearable sweat microfluidic chip is arranged from top to bottom as follows: sweat inlet layer 1, central channel and outlet layer 2, branch channel layer 3, pore layer 4, and cover plate layer 5.

[0102] The sweat inlet layer 1 has a sweat inlet 6, which is a circle with a radius of 1.5 mm.

[0103] The central channel and outlet layer 2 have corresponding inlets, similar to those in the sweat inlet layer 1. A central channel 7 (0.5 mm wide) is located to one side of each inlet, and branch channels 8 connect to the central channel. Each branch channel 8 measures 2 mm × 0.3 mm. Sweat preferentially flows through the central channel 7.

[0104] Each branch channel (8) is provided with two valve chambers (9), one detection chamber (10), and one sweat storage chamber (15). The two valve chambers (9) are respectively the first valve chamber (9-1) and the second valve chamber (9-2). The first valve chamber (9-1) is connected to the sweat storage chamber (15), the second valve chamber (9-2), and the detection chamber (10) in sequence. Multiple first valve chambers (9-1) contain droplet-shaped polymer valves with progressively increasing concentrations; multiple second valve chambers (9-2) contain droplet-shaped polymer valves with progressively decreasing concentrations. Valve chamber 9 has a diameter of 2 mm, while the sweat storage chamber 15 and detection chamber 10 both have a diameter of 4 mm. Detection chamber 10 is pre-stored with filter paper loaded with uric acid detection reagent.

[0105] The pore layer 4 is provided with pores 13 for communicating with the air in the environment, so as to ensure that sweat flows from the sweat inlet 6 to the detection chamber 10. The pores are circles with a diameter of 0.4 mm.

[0106] The central channel, outlet layer 2, branch channel layer 3, pore layer 4, and cover layer 5 all have sweat outlets 14 at the same location, each with a diameter of 2 mm. Excess liquid flowing out of the central channel 7 can flow out through the sweat outlets 14. Each layer of the chip is a fan-shaped structure with a radius of 22.33 mm and an included angle of 184.43°.

[0107] In this embodiment, the chip is partitioned into a central channel 7 and branch channels 8. This design allows for the collection of sweat samples at different times as needed, and also enables controlled sample release at preset times to allow for contact and mixing with the detection reagents. During collection, sweat preferentially flows through the central channel 7. As it flows into the branch channels 8, a droplet-shaped polymer valve controls the opening time by adjusting the polymer concentration, achieving sequential collection of sweat samples. Once sweat passes through the droplet-shaped polymer valve, it will not flow back, thus avoiding cross-contamination between new and old sweat samples.

[0108] A method for fabricating a wearable sweat microfluidic chip according to this embodiment specifically includes the following steps: S1. Based on the structure of each layer of the microfluidic analysis chip, the layers are drawn on a 0.2 mm thick PET film with 3M adhesive backing to obtain the sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover layer 5. The sweat inlet layer 1, the central channel and outlet layer 2, the branch channel layer 3, the pore layer 4, and the cover layer 5 are all prepared by laser cutting; the laser cutting parameters are: maximum light intensity 65%, minimum light intensity 20%, and cutting speed 15 mm / s.

[0109] S2. Adhere the sweat inlet layer 1, the central channel and the outlet layer 2 together. Add a hydrophilic reagent to treat the central channel 7 and the interface with the surrounding branch channels 8. Adhere the branch channel layer 3, the pore layer 4 and the cover plate layer 5 in order from top to bottom. Treat the valve chamber 9 with fluorinated oil to make it hydrophobic. After the fluorinated oil evaporates, use a cotton swab to apply a hydrophilic reagent to the detection chamber 10 for local hydrophilic treatment.

[0110] S3. Place the droplet-shaped polymer valve and the filter paper loaded with uric acid detection reagent into the corresponding chamber and encapsulate the chip to obtain a wearable sweat microfluidic analysis chip with integrated droplet-shaped polymer valve.

[0111] The filter paper loaded with uric acid test reagent is prepared as follows: cut the filter paper into a circle with a diameter of 4 mm, add 5 μL of uric acid test reagent to the filter paper, and then dry the filter paper with nitrogen gas to prepare a test pad. The uric acid test reagent contains 100 mM PBS buffer, 100 U / mL uricase (UO), 100 U / mL horseradish peroxidase (HRP), 5 mM 4-aminoantipyrine (4-APP), 10 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), 1 mg / mL bovine serum albumin (BSA), and 5% trehalose.

[0112] The PBS buffer solution is prepared as follows: Weigh 3.5816 g Na2HPO4·12H2O, 0.3520 g KH2PO4, 8.006 g NaCl and 0.2010 g KCl, dissolve them in 800 mL of ultrapure water, then add 1.0160 g MgCl2·6H2O, dissolve, transfer to a 1 L volumetric flask and bring to volume. Adjust the pH to 7.4, filter using a syringe and a 0.22 µm filter in a clean bench, dispense, and freeze at -20 ℃ for later use.

[0113] Figure 13This is a schematic diagram illustrating the working principle of the microfluidic analysis chip used for uric acid detection in sweat in Embodiment 3 of the present invention. An athlete uses a sweatband to fix the microfluidic analysis chip to their forehead. During continuous exercise, sweat enters the chip through the sweat inlet 6, first contacting the time-sequential sampling polymer valves T1-T4, and then contacting the sample release polymer valves T1'-T4'. These valves are made of polystyrene sulfonic acid (PSS) of different concentrations. The opening times of T1-T4 increase sequentially, while the opening times of T1'-T4' decrease sequentially, thereby eliminating the time difference in contact between the sweat collected at different times and the detection reagent. Sweat spontaneously enters the detection chamber 10 through the sweat inlet 6 and comes into contact with the reagent pad pre-stored with the test reagent, resulting in a color reaction. No additional reagents are needed during the process. The color reaction principle is as follows: uric acid generates hydrogen peroxide (H2O2) under the action of uricase. Subsequently, H2O2 reacts with 4-aminoantipyrine (4-APP) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) under the catalysis of horseradish peroxidase (HRP) to generate a red quinone compound.

[0114] Experiment 7: Investigate the cross-contamination of the liquid in each branch channel in Example 4.

[0115] (1) Verification of cross-contamination prevention capability: The experimental procedure is the same as in Experiment 3. For example... Figure 14 As shown in Figure A, even after introducing 200 µL of methylene blue dye solution, the blue color did not diffuse into the chamber. Figure B shows that ImageJ analysis of the grayscale value changes from T1 to T3 in the four chambers of the chip image revealed no significant difference in grayscale values. This indicates that once the chamber is filled with solution, subsequent sweat will not enter the chamber and cause cross-contamination.

[0116] (2) Verification of anti-backflow capability: The experimental procedure was the same as in Experiment 3. Figure C shows that artificial sweat containing different colored dyes was sequentially introduced into different chambers of the microfluidic analysis chip, and then colorless artificial sweat was introduced into the central channel. Figure D shows that the grayscale value changes of the four chambers of the chip image from T1 to T3 were analyzed using ImageJ, and there was no significant difference in grayscale values. This indicates that the solution in the chambers will not flow back into the central channel over time.

[0117] Experiment 8: To examine the timing sampling and timed incubation capabilities of the chip in Example 4.

[0118] The opening times for valves 1 through 4 were set to 0, 10, 30, and 60 min, respectively, and the opening times for valves 1' through 4' were set to 60, 30, 10, and 0 min, respectively. Artificial sweat containing lemon yellow dye was introduced into the microfluidic analysis chip at a rate of approximately 10 μL / min. Figure 15 As shown in the figure, A is the flowchart, and B represents the optical images of each stage before the artificial sweat completely fills the chambers of the microfluidic analysis chip. The results show that the artificial sweat opens the valves and fills the chambers in a preset order. Guided by the valves, the liquid first enters the sweat storage chamber, stays there, and then enters the sweat detection chamber simultaneously after making up the time difference.

[0119] Experiment 9: Kinetic response of uric acid by enzymatic method.

[0120] In enzyme reaction systems, the generated signals change dynamically over time due to the kinetic characteristics of enzymes, so precise control of reaction time is often required.

[0121] Figure 16 This figure shows the kinetic response and condition optimization of uric acid detection using uricase in Example 3 of the present invention. As shown in Figure A, when detecting uric acid based on uricase and horseradish peroxidase, the detection signal exhibits a trend of first increasing and then decreasing over time. Therefore, the reaction time and uricase concentration were subsequently investigated.

[0122] As shown in Figure B, the grayscale value gradually increases within 10 minutes as the reaction time increases, and then begins to decrease slightly after 10 minutes. This also indicates that selecting an appropriate incubation time is crucial to ensuring the stability and accuracy of the detection signal.

[0123] As shown in Figure C, the signal response increases with increasing enzyme concentration and reaches a plateau at 100 U / mL. Therefore, the optimal incubation time was determined to be 10 minutes, and a uricase concentration of 100 U / mL was selected for the preparation of the uric acid detection reagent.

[0124] Experiment 10: Detecting uric acid in sweat at different times.

[0125] To construct a standard curve for uric acid detection: Uric acid was added to artificial sweat at final concentrations of 0 µM, 20 µM, 40 µM, 80 µM, 100 µM, 150 µM, and 200 µM. The uric acid-containing artificial sweat was introduced into the sweat storage chamber through the inlet, and simultaneously the valve was opened to allow it to enter the detection chamber. After incubation at 25 °C for 10 min, the chip was placed in a dark box, and grayscale values ​​were extracted using a smartphone. The relationship between grayscale values ​​and uric acid concentration was then fitted to obtain the standard curve. Figure 17The calibration curve shown is y = 0.1624x + 115.3544, where y is the gray value, x is the uric acid concentration, and R0 is the gray value. 2 = 0.9933.

[0126] The microfluidic analysis chip was worn on the forehead of the volunteer using an antiperspirant. The volunteer moved freely and continued to sweat. The chip was set with valves to collect sweat at four time points: 0 min, 15 min, 30 min, and 60 min. The movement ended after the chamber was completely filled. After incubating at 25 ℃ for 10 min, the microfluidic analysis chip was placed in a dark box to take a picture and read the grayscale value. Then, it was substituted into the calibration curve y=0.1624x + 115.3544 to obtain the uric acid concentration.

[0127] The results are as follows Figure 18 As shown, the uric acid levels of both volunteers increased during the T1 to T3 time period, but decreased slightly at T4 time, possibly due to the dilution of the marker concentration caused by their large amount of sweating.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A wearable sweat microfluidic analysis chip, characterized in that, The wearable sweat microfluidic analysis chip is provided with a sweat inlet layer (1), a branch channel layer (3) and a cover layer (5) from top to bottom. The sweat inlet layer (1) is provided with a sweat inlet (6). The branch channel layer (3) is provided with a detection chamber (10), a channel (17) and a valve chamber (9). The sweat inlet (6), valve chamber (9), channel (17) and detection chamber (10) are connected in sequence. The cover layer (5) is provided with a sweat outlet (14). A water droplet-shaped polymer valve is placed in the valve chamber (9). The water droplet-shaped polymer valve is obtained by dripping polymer onto water droplet-shaped filter paper and air-drying it at room temperature. The polymer is one or more of polyvinyl alcohol, polyethylene glycol, polystyrene sulfonic acid, hyaluronic acid, and bovine serum albumin. The detection chamber (10) is filled with detection reagents.

2. The wearable sweat microfluidic analysis chip according to claim 1, characterized in that, The wearable sweat microfluidic analysis chip also includes a central channel and outlet layer (2) and a pore layer (4). The sweat inlet layer (1), the central channel and outlet layer (2), the branch channel layer (3), the pore layer (4), and the cover plate layer (5) are arranged sequentially from top to bottom. The central channel and outlet layer (2) are provided with a central channel (7) communicating with the sweat inlet (6) around the sweat inlet (6), and branch channels (8) communicating with the central channel (7) around the central channel (7). The branch channel layer (8) is provided with multiple valve chambers (9) communicating with different branch channels (8). Each valve chamber (9) is provided with an independent channel (17) and a detection chamber (10). The detection chamber (10), channel (17) and valve chamber (9) are connected. Each valve chamber (9) contains a droplet-shaped polymer valve with different polymer concentrations. Sweat flows preferentially through the central channel (7). In the branch channel (8), the droplet-shaped polymer valve controls the opening time by controlling the polymer concentration to realize the time-sequential collection of sweat samples. The pore layer (4) is provided with pores (13) for communicating with air in the environment, and the pores (13) are in communication with the detection chamber (10).

3. The wearable sweat microfluidic analysis chip according to claim 2, characterized in that, In the branch channel layer (3), each branch channel (8) is provided with two valve chambers (9), one detection chamber (10), and one sweat storage chamber (15). The two valve chambers (9) are respectively the first valve chamber (9-1) and the second valve chamber (9-2). The first valve chamber (9-1) is connected to the sweat storage chamber (15), the second valve chamber (9-2), and the detection chamber (10) in sequence. Multiple first valve chambers (9-1) contain droplet-shaped polymer valves with progressively increasing concentrations; multiple second valve chambers (9-2) contain droplet-shaped polymer valves with progressively decreasing concentrations.

4. The wearable sweat microfluidic analysis chip according to claim 1, characterized in that, The teardrop-shaped polymer valve is located 2 mm away from the sweat inlet (6), and the diameter of the teardrop-shaped polymer valve is 2 mm to 3 mm.

5. The wearable sweat microfluidic analysis chip according to any one of claims 1 to 4, characterized in that, The detection reagent is a reagent for detecting cortisol; The reagent for detecting cortisol is Ap-MFDN, which is prepared using the following method: H1-cDNA, H2 and Aptamer were annealed at 96°C for 3 min, and then Trigger, aptamer, H1 and H2 were mixed and incubated. The gene sequence of the Aptamer is shown in SEQ ID NO.1; the gene sequence of the H1-cDNA is shown in SEQ ID NO.2; the gene sequence of the H2 is shown in SEQ ID NO.3; and the gene sequence of the Trigger is shown in SEQ ID NO.

4.

6. The wearable sweat microfluidic analysis chip according to any one of claims 1 to 4, characterized in that, The test reagent is a test reagent for detecting uric acid; The reagent for detecting uric acid includes: 100 mM PBS buffer, 100 U / mL uricase, 100 U / mL horseradish peroxidase, 5 mM 4-aminoantipyrine, 10 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, 1 mg / mL bovine serum albumin, and 5 wt% trehalose.

7. A method for preparing the wearable sweat microfluidic analysis chip according to claim 5, characterized in that, The preparation method includes the following steps: SA-1. Cut the PET film to obtain the sweat inlet layer (1), the central channel and outlet layer (2), the branch channel layer (3), the pore layer (4), and the cover plate layer (5). SA-2, the sweat inlet layer (1), the central channel and the outlet layer (2) are attached together, and a hydrophilic reagent is added to the central channel (7) and the interface with the surrounding branch channels (8) for hydrophilic treatment. The branch channel layer (3), the pore layer (4) and the cover plate layer (5) are attached in order from top to bottom. The valve chamber (9) is hydrophobically treated with fluorinated oil. After the fluorinated oil evaporates, the detection chamber (10) is locally hydrophilically treated with a hydrophilic reagent. SA-3, Place the droplet-shaped polymer valve in the valve chamber (9) and encapsulate the chip; SA-4 and Ap-MFDN reagents are injected into the detection chamber (10) through the pores (13) of the microfluidic analysis chip, and the pores (13) are sealed with a PTFE membrane. The sweat inlet (6) is sealed with transparent tape. The reagents in the detection chamber are freeze-dried to form Ap-MFDN dry powder.

8. A method for preparing the wearable sweat microfluidic analysis chip according to claim 6, characterized in that, The preparation method includes the following steps: SB-1. Cut the PET film to obtain a sweat inlet layer (1), a central channel and outlet layer (2), a branch channel layer (3), a pore layer (4), and a cover layer (5); SB-2, attach the sweat inlet layer (1), the central channel and the outlet layer (2) together, add a hydrophilic reagent to treat the central channel (7) and the interface with the surrounding branch channels (8) to hydrophilicity, attach the branch channel layer (3), the pore layer (4) and the cover plate layer (5) together in order from top to bottom, treat the valve chamber (9) with fluorinated oil to make it hydrophobic, and after the fluorinated oil evaporates, dip it in the hydrophilic reagent to treat the detection chamber (10) locally to make it hydrophilic; SB-3. Place the droplet-shaped polymer valve and the filter paper loaded with uric acid detection reagent into the corresponding chamber and encapsulate the chip.

9. The application of the wearable sweat microfluidic analysis chip of claim 5 in the detection of cortisol.

10. The application of the wearable sweat microfluidic analysis chip of claim 6 in the detection of uric acid.

Images