Non-invasive portable multi-mode wearable sweat sensing device

Abstract

The invention provides a non-invasive portable multi-mode wearable sweat sensing device, and belongs to the technical field of wearable biosensing devices. The preparation method comprises the following steps: dissolving ZrCl4, a monoacid regulator and terephthalic acid in an organic solvent, and then carrying out hydrothermal reaction; after the hydrothermal reaction is finished, collecting white precipitates, treating the white precipitates in a mixed solution of hydrochloric acid and DMF (Dimethyl Formamide), and then centrifuging, washing and drying to obtain an MOF carrier; and adding the MOF carrier, GOx and HRP into a buffer solution, stirring, centrifuging, and washing to obtain the enzyme-coated metal organic framework material. A detection point is prepared from an enzyme-coated metal organic framework material, and the detection point, a skin adhesion layer, a paper-based micro-fluidic layer and a packaging layer jointly form a non-invasive portable multi-mode wearable sweat sensing device. The sensing device can be directly integrated with the skin surface, continuously quantifies sweat loss in real time, and provides quantitative data of glucose in sweat.

Description

Technical Field

[0001] The present invention relates to the technical field of wearable biosensor devices, and particularly to a non-invasive, portable, multi-mode wearable sweat sensor device. Background Art

[0002] Sweat, as a non-invasive alternative to blood testing, has the potential to detect multiple biomarkers in real time and effectively, and is widely used in fields such as sports state assessment, diabetes screening, and disease monitoring. However, accurately and rapidly detecting biomarkers in sweat remains the main challenge in its clinical application. Current detection methods, such as absorbent pads or laboratory analysis, have problems such as high cost, slow speed, and dependence on large equipment, which limit their real-time application. In contrast, wearable sensor devices can solve these problems, but most wearable devices can only detect a single biomarker and lack sufficient sensitivity and multi-index analysis capabilities.

[0003] The Chinese patent invention document with the publication number CN115639260A discloses a wearable sweat sensor. This device realizes non-invasive and continuous sweat analysis by the flow of sweat to the electrode layer according to the generated electrical signals, but it has a complex preparation method and lacks quantitative detection of the sweating rate. The Chinese patent invention document with the publication number CN118348089A discloses a wearable sweat sensing device, its preparation method and application. This sweat sensing device uses a microfluidic chip and a three-electrode system to detect biomarkers through aptamers and provide a biosensing interface, but its device lacks protection for nucleic acids, resulting in poor stability and also fails to achieve quantitative detection of the sweating rate. The two wearable sweat sensing devices disclosed in CN115639260A and CN118348089A both have problems of single detection, cumbersome preparation, inconvenient use and stability. Therefore, there is an urgent need for a sweat sensor device with a simple design that can accurately and rapidly detect the concentration of biomarkers and the amount of sweating in sweat, and has excellent stability. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a non-invasive, portable, multi-mode wearable sweat sensor device. This device can be directly integrated with the skin surface, effectively absorb sweat, continuously and real-time quantify sweat loss, and read the RGB values of the detection points by taking pictures with a smart phone to provide quantitative data of glucose in sweat. The multi-mode wearable sweat sensor device includes a skin adhesion layer, a paper-based microfluidic layer, detection points, and a PDMS encapsulation layer, all of which adopt flexible materials and flexible designs, which greatly improves the overall flexibility of the patch and overall improves the comfort of the wearer. In addition, the present invention uses natural enzymes, which are highly efficient catalysts, to perform cascade catalysis on the substrate glucose to improve the accuracy of molecular detection. However, natural enzymes have poor stability. By regulating the pore size of the UiO-66 MOF material, a hierarchically porous structure HP-UiO-66-XA with extremely consistent sizes with natural glucose oxidase (GOx) and horseradish peroxidase (HRP) is synthesized to effectively encapsulate natural enzymes GOx and HRP, greatly improving the enzyme activity and the stability of the multi-mode wearable sweat sensor device. At the same time, the amount of sweating can be evaluated by observing the flow distance of sweat on the serpentine channel.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: One of the technical solutions of the present invention is a preparation method of a metal-organic framework material encapsulating enzymes, including the following steps: Dissolve ZrCl4 (zirconium tetrachloride), a monobasic acid regulator, and terephthalic acid in an organic solvent, and then perform a hydrothermal reaction; After the hydrothermal reaction is completed, collect the white precipitate, and treat the white precipitate in a mixed solution of hydrochloric acid and DMF (N,N-dimethylformamide), and then centrifuge, wash, and dry to obtain a MOF support; Add the MOF support, glucose oxidase, and horseradish peroxidase to a buffer solution, stir, and then centrifuge and wash to obtain the metal-organic framework material encapsulating enzymes.

[0006] Another technical solution of the present invention is a metal-organic framework material encapsulating enzymes prepared by the above preparation method.

[0007] Another technical solution of the present invention is the application of the above metal-organic framework material encapsulating enzymes in detecting glucose in sweat.

[0008] Another technical solution of the present invention is a non-invasive, portable, multi-mode wearable sweat sensor device, including a skin adhesion layer, a detection layer, and an encapsulation layer; the detection layer is located between the skin adhesion layer and the encapsulation layer; the detection layer includes detection points and a paper-based microfluidic layer, and the detection points are connected to the paper-based microfluidic layer; the number of detection points ≥ 2; A sweat inlet is provided in the skin adhesion layer, and the sweat inlet is located at the end point of the paper-based microfluidic layer; The detection point is composed of the above-mentioned metal-organic framework material encapsulating the enzyme, the chromogenic substrate and the carrier; the metal-organic framework material encapsulating the enzyme and the chromogenic substrate are loaded on the surface of the carrier.

[0009] The present invention discloses the following technical effects: (1) By precisely regulating the design of the hierarchical porous structure, the mesopore size inside the material in the present invention is highly matched with the enzyme molecule size. When the size of the enzyme is close to the nano-pore size, the appropriate size can achieve a good balance between diffusion efficiency and stability. This match enables the enzyme molecules to smoothly diffuse into the pores and stay therein, while effectively preventing the leakage of the enzyme, not only significantly improving the stability of the enzyme, but also effectively maintaining its activity.

[0010] (2) The sweat sensor device designed in the present invention successfully integrates multiple detection points, realizing the integration and miniaturization of flexible multi-channel wearable sensor devices. This sensor device can not only accurately measure the glucose concentration in sweat, but also integrate the detection of various physiological parameters such as temperature, pH value, and ion concentration by optimizing the design of the paper-based sensor device, providing multi-dimensional data support for comprehensive health monitoring.

[0011] (3) The sweat sensor device of the present invention realizes real-time continuous monitoring of the sweating volume and blood glucose level. Users can take pictures through the mobile phone client to read the RGB value and view the blood glucose level in real time. The measurement results are accurate, and it has a comfortable wearing experience and convenient data reading function.

[0012] (4) The sweat sensor device of the present invention has excellent stability and high accuracy, and has a simple structure, reasonable design, simple preparation process, convenient storage and convenient use. These characteristics significantly reduce the use threshold and are especially suitable for daily health monitoring and point-of-care diagnosis.

[0013] (5) The paper-based material has excellent moisture absorption performance, can effectively absorb sweat and trigger chemical reactions, and realizes highly sensitive component detection. At the same time, the paper-based material has no harmful chemical substances and is suitable for use as a wearable device in direct contact with the skin, reducing the potential risk to human health.

[0014] (6) The preparation of the wearable sweat sensor device has the advantages of low cost, environmental protection, comfort, sensitivity and multi-function, making it have significant economic advantages in large-scale production and becoming a strong competitor in the future health monitoring and biosensing technology fields. Brief Description of the Drawings

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0016] Figure 1 It is a schematic diagram of pore size regulation and enzyme cascade catalysis in the present invention. Among them, a is a schematic diagram of realizing continuous regulation from micropores to mesopores by introducing a monobasic acid regulator in the synthesis of UiO-66, b is a schematic diagram of enzyme coating by microporous structure and hierarchical porous structure, and c is a schematic diagram of the enzyme cascade catalysis mechanism after encapsulating the enzyme; Figure 2 It is an anatomical diagram and a schematic diagram of the detection process of the wearable sweat sensor device in the present invention; Figure 3 In it, a is the cut snake-shaped cellulose paper, and b is the PDMS encapsulation layer; Figure 4 It is the X-ray diffraction (XRD) pattern of the defective HP-UiO-66-6 prepared in Example 1; Figure 5 It is the nitrogen adsorption and desorption (BET) curve of the defective HP-UiO-66-6 prepared in Example 1; Figure 6 It is the transmission diagram a of the defective HP-UiO-66-6 prepared in Example 1, the statistical data diagram b of the internal pore size, and the three-dimensional reconstruction diagrams c and d; Figure 7 In it, a is the maximum distance that different volumes of water can move on the device cellulose paper, and b is the linear relationship diagram between the water volume and the moving distance; Figure 8 It is the RGB calibration curve of different concentration glucose solutions; Figure 9 It is the color reaction diagram that occurs when the wearable sweat sensor device is worn on the athlete; Figure 10 It is the correlation diagram of sweat sugar and blood sugar obtained through the wearable sweat sensor device. Detailed implementation manners

[0017] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0018] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0020] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and embodiments of the present invention are merely exemplary.

[0021] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0022] To improve the detection accuracy and sensitivity of wearable devices, introducing natural enzymes with high specificity and high catalytic efficiency can significantly enhance the performance of sensors, promote the further development of sweat analysis technology, and meet the requirements for high-precision and multi-index analysis. However, the disadvantage is that natural enzymes have poor stability and are prone to aggregation in solution. In response to this, the present invention uses metal-organic framework materials (MOFs) with high tunability to encapsulate enzymes. The encapsulation of natural enzymes by metal-organic framework (MOF) materials has the following advantages: (1) High specific surface area: The extremely high specific surface area of MOF provides more contact and reaction areas for natural enzymes, significantly improving the catalytic performance of enzymes; (2) Tunable pore structure: The pore size of MOF materials can be flexibly regulated, thereby optimizing the catalytic reaction environment of enzymes, enhancing the substrate selectivity and catalytic efficiency of enzymes. At the same time, the pore structure can help achieve better spatial adaptation between substrate molecules and enzyme active centers; (3) Enhanced enzyme stability: The robust structure of MOF provides physical protection for natural enzymes, preventing enzyme molecules from inactivating in extreme environments; (4) Preventing excessive aggregation and denaturation of enzymes: The pore structure of MOF can prevent non-specific aggregation between enzyme molecules, avoid the loss of activity or denaturation of enzymes during the reaction process, and maintain their catalytic efficiency; (5) Versatility: In addition to being able to encapsulate enzymes, MOF materials can endow MOF materials with new functions, thereby achieving multiple detection functions.

[0023] As a macromolecular structure, enzymes are often large in size and cannot be encapsulated in the smaller pore sizes of the intrinsic MOF microporous structure. Enzymes are usually attached to the material surface. By adjusting the MOF pore channels to highly match the pore size with the enzyme size, the enzyme loading capacity, stability, and catalytic efficiency can be significantly improved, thereby enhancing the application effect of enzymes in sensing detection or catalytic reactions. For this purpose, the present invention uses a monobasic acid to adjust the pore size of MOF to achieve a perfect match between the pore size and the enzyme molecule size, promote the effective diffusion of enzymes in the nanopores and reduce leakage, thereby achieving efficient catalysis of enzymes.

[0024] Aiming at the problems that most current biosensor devices have a single detection index and there are still certain limitations in establishing the relationship between glucose levels in blood and sweat, the present invention encapsulates the above-mentioned precisely regulated MOF materials with enzymes and assembles them with wearable devices to develop a more advanced quantitative sweat analysis technology to meet the requirements for high-precision analysis in this field. The non-invasive, portable, and multi-mode wearable sweat sensor device designed in the present invention has the advantages of simple structure, simple preparation process, and convenient use. By combining with smartphone and RGB analysis, the color change information is digitized, showing excellent stability and high accuracy in the non-invasive detection of glucose in sweat.

[0025] The first aspect of the present invention provides a preparation method of a metal-organic framework material encapsulating an enzyme, comprising the following steps: Dissolve ZrCl4, a monobasic acid regulator, and terephthalic acid in an organic solvent, and then carry out a hydrothermal reaction; After the hydrothermal reaction is completed, collect the white precipitate, and treat the white precipitate in a mixed solution of hydrochloric acid and DMF, then centrifuge, wash, and dry to obtain the MOF support; Add the MOF support, glucose oxidase (GOx), and horseradish peroxidase (HRP) to a buffer solution, stir, and then centrifuge and wash to obtain the enzyme-encapsulated metal-organic framework material.

[0026] In a preferred embodiment of the present invention, the monobasic acid regulator is formic acid, acetic acid, n-butyric acid, hexanoic acid, n-octanoic acid, dodecanoic acid, hexadecanoic acid, nitric acid or hydrochloric acid; the molar ratio of ZrCl4, the monobasic acid regulator and terephthalic acid is 1∶15~60∶0.5~3.

[0027] The present invention does not make special limitations on the selection of the organic solvent. Any organic solvent well-known to those skilled in the art that can dissolve ZrCl4, the monobasic acid regulator and terephthalic acid can be used, for example: DMF. The present invention does not make special limitations on the amount of the organic solvent. The amount thereof can sufficiently dissolve ZrCl4, the monobasic acid regulator and terephthalic acid, and can meet the amount required for the hydrothermal reaction to proceed.

[0028] The present invention uses a monobasic acid to adjust the pore size of the MOF, but this method is not applicable to all metal-organic framework materials. The principle of pore size adjustment by the monobasic acid is as follows: by adding a monobasic acid during the synthesis process, competing for coordination with metal ions and introducing ligand defects, thereby adjusting the pore size. However, the applicability of this method is limited by the following factors: (1) Framework stability: Different MOF materials have different combinations of metal nodes and ligands. MOFs with poor stability will cause the framework to collapse due to the introduction of the monobasic acid; (2) Synthesis condition differences: The synthesis conditions of different MOFs have different reactions to the monobasic acid. Some MOFs cannot achieve the expected pore size adjustment effect under specific conditions. Therefore, whether this method is specifically applicable needs to be comprehensively evaluated according to the structure, chemical properties and synthesis conditions of the MOF. The present invention has successfully prepared a MOF material with adjustable pore size by selecting specific metal ligands (ZrCl4), a monobasic acid and the organic ligand terephthalic acid (BDC).

[0029] In a preferred embodiment of the present invention, the temperature of the hydrothermal reaction is 80-150°C and the time is 48 h.

[0030] In a preferred embodiment of the present invention, the volume ratio of hydrochloric acid to DMF is 1:100. The hydrochloric acid in the present invention is concentrated hydrochloric acid obtained by purchase without dilution; the temperature of the treatment is room temperature and the time is 10 h. Many organic regulators are prone to dissolution or degradation in an acidic environment. Hydrochloric acid helps these regulators to be removed from the MOF structure in the next drying step by providing hydrogen ions (H + ).

[0031] In a preferred embodiment of the present invention, the temperature of the drying is 200 °C and the time is 12 h.

[0032] The functions of drying in the present invention are: (1) drying function; (2) removing the monobasic acid regulator in the MOF structure, promoting the generation of defects and forming hierarchical pores.

[0033] In a preferred embodiment of the present invention, the mass ratio of the MOF carrier, glucose oxidase and horseradish peroxidase is 1:0.1 - 1:0.1 - 2; the buffer solution is PBS buffer solution; the concentration of the PBS buffer solution is 0.1 M and pH = 7.0.

[0034] The second aspect of the present invention provides an enzyme-encapsulated metal-organic framework material prepared by the above preparation method.

[0035] The third aspect of the present invention provides an application of the above enzyme-encapsulated metal-organic framework material in detecting glucose in sweat.

[0036] The fourth aspect of the present invention provides a non-invasive, portable and multi-mode wearable sweat sensor, which includes a skin adhesion layer, a detection layer and a packaging layer; the detection layer is located between the skin adhesion layer and the packaging layer; the detection layer includes detection points and a paper-based microfluidic layer, and the detection points are connected to the paper-based microfluidic layer; the number of the detection points ≥ 2; There is a sweat inlet provided in the skin adhesion layer, and the sweat inlet is located at the end point of the paper-based microfluidic layer; The detection point is composed of the above enzyme-encapsulated metal-organic framework material, a chromogenic substrate and a carrier; the enzyme-encapsulated metal-organic framework material and the chromogenic substrate are loaded on the surface of the carrier.

[0037] In a preferred embodiment of the present invention, the paper-based microfluidic layer is cellulose paper; the packaging layer is a PDMS film; the chromogenic substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); the carrier is cellulose paper.

[0038] In a preferred embodiment of the present invention, the preparation method of the detection point is as follows: (1) Immerse the cellulose paper in a dispersion of metal-organic framework material uniformly dispersing and encapsulating the enzyme, then wash and dry to obtain the cellulose paper loaded with the metal-organic framework material; (2) Coat the surface of the cellulose paper loaded with the metal-organic framework material with the chromogenic substrate solution and dry to obtain the detection point.

[0039] In a preferred embodiment of the present invention, the concentration of the metal-organic framework material encapsulating the enzyme in the dispersion is 2 - 10 mg / mL; the concentration of the chromogenic substrate solution is 0.3 - 2 M.

[0040] The present invention does not make special limitations on the shape of the detection point and the shape of the paper-based microfluidic layer. As an example, the shape of the detection point can be selected as circular, and the shape of the paper-based microfluidic layer can be selected as serpentine with a width of 1 - 5 mm. Connect the detection point and the paper-based microfluidic layer with a material of the same material as the paper-based microfluidic layer to ensure that sweat can smoothly enter the detection point for detection.

[0041] The technical solutions of the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or are publicly available.

[0042] The present invention does not make special limitations on the source of the PDMS film. Commercially available products can be selected, or it can be prepared by a preparation method well-known to those skilled in the art.

[0043] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments, but the content of the present invention is not limited to the following embodiments only.

[0044] In the embodiment, the preparation method of the PDMS film (PDMS encapsulation layer) is: Mix part A and part B of the Sylgard 184 silicone elastomer kit (commercially available, PDMS elastomer (Sylgard 184) is purchased from Dow Corning) in a weight ratio of 10:1, degas under vacuum for 15 min, and then scrape and coat with a 120 nm thick mold to prepare a PDMS encapsulation layer with a thickness of 120 nm.

[0045] Example 1 (1) Preparation of HP-UiO-66-6 ZrCl4 (0.343 mmol) and hexanoic acid (12 mmol) were dissolved in DMF (20 mL), and ultrasonicated for 25 min. Subsequently, BDC (0.343 mmol) was added. After ultrasonicating to form a homogeneous solution, the mixed solution was transferred to a polytetrafluoroethylene liner and reacted at 120 °C for 48 h. Then, it was centrifuged and washed three times with DMF to obtain a white precipitate. The above white precipitate was treated in a mixed solution of concentrated hydrochloric acid (0.4 mL) and DMF (40 mL) for 10 h, followed by centrifugation and washing. The obtained white powder was activated in a vacuum oven at 200 °C for 12 h to obtain HP-UiO-66-6.

[0046] (2) Preparation of GOx&HRP@HP-UiO-66-6 The prepared HP-UiO-66 (5 mg) and GOx (1.5 mg) were added to 1 mL of PBS buffer (0.01 M, pH = 7.0). The mixture was stirred at room temperature for 1.5 h, and then HRP (1.5 mg) was added to the above solution and stirred for another 1.5 h. Then, GOx&HRP@HP-UiO-66-6 was obtained by centrifugation and washing.

[0047] (3) Preparation of detection points Chromatographic-grade cellulose paper was cut into circles with a diameter of 3 mm, and then the 3-mm-diameter circular chromatographic-grade cellulose paper was soaked in a GOx&HRP@HP-UiO-66-6 solution (5 mg / mL) for 1 h to uniformly adsorb GOx&HRP@HP-UiO-66-6 on the paper surface. Subsequently, the GOx&HRP@HP-UiO-66-6 paper was washed with deionized water to remove loosely bound GOx&HRP@HP-UiO-66-6, and after natural drying at room temperature, 20 µL of ABTS (1 M) solution was drop-coated on it and allowed to dry naturally.

[0048] (4) Preparation of serpentine cellulose paper By printing a pre-designed AutoCAD pattern on chromatographic-grade cellulose paper and using a paper-cutting tool to cut the chromatographic-grade cellulose paper into a serpentine shape with a width of 2 mm.

[0049] Finally, the serpentine cellulose paper and the detection points were adhered to a medical-grade double-sided tape (skin adhesion layer), and then encapsulated with a PDMS film to obtain a wearable sweat sensor device.

[0050] The schematic diagram of the wearable sweat sensor device is shown in Figure 2Shown as follows: The sensor consists of four parts: a medical tape, a paper-based serpentine channel (serpentine cellulose paper), 4 detection points, and a PDMS encapsulation layer. A small hole is opened on the double-sided tape at the bottom layer for guiding the sweat of local skin to flow in. The sweat enters the serpentine channel through this inlet and flows through the four detection points in sequence. At each detection point, the encapsulated glucose oxidase first catalyzes the glucose in the sweat to generate gluconic acid and hydrogen peroxide. The generated hydrogen peroxide reacts with ABTS under the catalytic action of horseradish peroxidase to generate green ABTS•+. By taking a photo with a mobile phone and reading the RGB values in the image, the glucose content in the sweat can be accurately evaluated.

[0051] Figure 3 As shown in a of, the cut paper-based serpentine channel is presented. It can be seen from the figure that the serpentine design can greatly increase the flexibility of the device; Figure 3 As shown in b of, it can be seen that the prepared PDMS encapsulation layer has good flatness.

[0052] Example 2 The difference from Example 1 is only that caproic acid is replaced by formic acid to obtain HP-UiO-66-1. Other steps and parameters are the same as those in Example 1.

[0053] Example 3 The difference from Example 1 is only that caproic acid is replaced by acetic acid to obtain HP-UiO-66-2. Other steps and parameters are the same as those in Example 1.

[0054] Example 4 The difference from Example 1 is only that caproic acid is replaced by n-butyric acid to obtain HP-UiO-66-4. Other steps and parameters are the same as those in Example 1.

[0055] Example 5 The difference from Example 1 is only that caproic acid is replaced by n-octanoic acid to obtain HP-UiO-66-8. Other steps and parameters are the same as those in Example 1.

[0056] Example 6 The difference from Example 1 is only that caproic acid is replaced by dodecanoic acid to obtain HP-UiO-66-12. Other steps and parameters are the same as those in Example 1.

[0057] Comparative Example 1 The difference from Example 1 is only that the addition of caproic acid is omitted to obtain UiO-66. Other steps and parameters are the same as those in Example 1.

[0058] Comparative Example 2 The difference from Example 1 is only that 12 mmol of hexanoic acid is replaced by 1 mmol of hexanoic acid to obtain HP-UiO-66-6-1mmol. Other steps and parameters are the same as those in Example 1.

[0059] Comparative Example 3 The difference from Example 1 is only that 12 mmol of hexanoic acid is replaced by 25 mmol of hexanoic acid to obtain HP-UiO-66-6-25mmol. Other steps and parameters are the same as those in Example 1.

[0060] Characterization and effect verification: 1. Figure 1 In a, it is described that under the condition of introducing a monocarboxylic acid regulator in the synthesis of UiO-66, continuous regulation from micropores to mesopores is achieved. The unregulated UiO-66 sample and the regulated HP-UiO-66 sample are loaded with glucose oxidase and horseradish peroxidase. From Figure 1 Model b, it can be seen that the protection of the enzyme can be achieved only when mesopores exist. In addition, Figure 1 In c shows the enzyme cascade catalysis mechanism diagram after encapsulating the enzyme.

[0061] 2. Figure 4 The XRD pattern of the HP-UiO-66-6 sample prepared in Example 1 is shown. It can be seen from the figure that the sample after being regulated by the regulator still maintains good crystallinity. The positions of its diffraction peaks are basically the same as those of the unregulated sample, and the peak shapes are clear and the intensities are high, indicating that there is no obvious decrease in crystallinity or structural damage during the regulation process of this sample.

[0062] 3. In order to adjust UiO-66 to a size suitable for enzyme loading, monocarboxylic acids with different chain lengths (formic acid, acetic acid, butyric acid, hexanoic acid, octanoic acid, and lauric acid) are selected as regulators. Through reasonable addition amounts, a series of HP-UiO-66-XA derivatives rich in defects are prepared. The N2 adsorption-desorption isotherm and the corresponding pore size distribution diagram are used to analyze the influence of the carbon chain length of the monocarboxylic acid on the pore size of HP-UiO-66-XA. As Figure 5 shown in and Table 1, the HP-UiO-66-XA samples have a hysteresis loop in the pressure range of 0.8-1.0, which is attributed to the existence of large pores, indicating that the micropores (<2nm) of the original UiO-66 have been successfully transformed into mesopores (2-20nm) after regulation. From Figure 5 the inset in and the average mesopore diameters of a series of HP-UiO-66-XA in Table 1, it can be seen that HP-UiO-66-6 regulated by hexanoic acid has the most matching pore size (average pore diameter of 8.1nm), which is very suitable as a nanomaterial for exploring enzyme cascade reactions.

[0063] Table 1 shows the results of the pore size and the corresponding enzyme loading of different UiO-66 prepared in Examples 1-6 and Comparative Examples 1-3.

[0064] Table 1

[0065] As can be seen from Table 1, with the increase of the chain length of the regulator, the mesopore size of the prepared defective HP-UiO-66 first increases and then decreases, while the micropores show little difference. The size of GOx is (6.0*5.2*7.7 nm), and the size of HRP is (4.0*4.4*6.8 nm). In contrast, HP-UiO-66-6 prepared in Example 1 has the most suitable size for the two enzymes. When the size of the enzyme is close to the pore size, it is usually most beneficial for the diffusion of the enzyme into the pores, and at the same time, it helps to reduce the leakage of the enzyme. This process involves multiple factors, mainly: (1) Matching of pore size and enzyme molecule size: When the size of the enzyme molecule is close to the pore size, the enzyme can smoothly enter the pores. This is because a good match is formed between the pore size and the enzyme molecule, and the enzyme molecule can diffuse smoothly into the pore size. However, due to the similar size, the friction between the enzyme molecule and the pore wall or pore size is relatively large, so it is easy to stay in the pore; (2) Slowing down leakage: When the size of the enzyme molecule is almost the same as the pore size, the enzyme molecule will be physically restricted and affected by the pore wall, and may stay in the pore for a longer time, reducing the chance of leakage from the pore. Especially in nanopores, the enzyme molecule may be "trapped" in the pore channel, and this confinement effect helps to enhance the stability and activity of the enzyme; (3) Diffusion efficiency and confinement: When the size of the enzyme matches the pore size, the enzyme molecule can not only enter through the pore, but also effectively diffuse in the pore. This is because when the cooperation between the pore size and the enzyme molecule is good, the mobility of the enzyme molecule in the pore is better, which helps to improve the efficiency of the enzyme reaction; (4) Van der Waals forces and physical interactions: When the pore size is close to the size of the enzyme molecule, Van der Waals forces (including attractive and repulsive forces) also play a role. These interactions help the enzyme molecule to better bind to the pore wall, thereby reducing leakage. Therefore, the perfect match between the pore size and the enzyme molecule size helps the enzyme to effectively diffuse in the nanopore and reduce leakage, which is the key to improving the enzyme stability and maintaining the enzyme activity. Based on the above principles, through reasonable selection and careful design, the present invention successfully prepared a hierarchically porous structure HP-UiO-66-6 with a high degree of match with the enzyme size, which is most suitable for enzyme encapsulation.

[0066] As can be seen from Example 1 and Comparative Examples 2-3 in Table 1, the amount of the regulator needs to be within a suitable range. Too little cannot play a role in regulating enough mesopores, and too much regulator will form clusters, resulting in a relatively large pore size, both of which will deviate from the target size suitable for loading enzymes.

[0067] 4. Such as Figure 6As shown, by combining atomic-resolution structural characterization and three-dimensional electron tomography of transmission electron microscopy (TEM), we successfully visualized the three-dimensional pore size in HP-UiO-66-6. Figure 6 Figure a in Figure 6 shows the transmission image of HP-UiO-66-6, while Figures c-d in Figure 6 perform detailed three-dimensional labeling of its internal structure by precisely segmenting the tomographic images. Based on these images, the internal porosity can be clearly and intuitively revealed. The histogram in Figure b in

[0068] shows the statistical data of the internal pore size of HP-UiO-66-6. As can be seen from the figure, the mesopore size is mainly concentrated in the range of 6-10 nm, which is consistent with the BET test results, verifying the high matching of the pore size and the enzyme size, and proving that the HP-UiO-66-6 designed and synthesized by us is extremely suitable for the loading of GOx and HRP. Figure 7 As shown, water with volumes of 5, 10, 15, and 20 μL was introduced into the sweating inlet respectively. The maximum distance that different volumes of water can move on the cellulose paper of the device increases with the increase of the liquid volume (Figure a in Figure 7 ). The calculated volume and the moving distance are summarized in Figure b in Figure 7 , and the two show a linear relationship, with the linear relationship being y = 4.478x + 1.157 (R2 = 0.952). According to this linear equation, the sweating volume can be quantified by visualizing the position of the moving distance of the fluid.

[0069] 6. As shown in Figure 8 , first, glucose solutions with known different concentrations were introduced onto the wearable sweat sensor device prepared in Example 1. When the solution reached each detection point, the device was photographed with a mobile phone, and the RGB values of the detection points after color change were read using the color picker software on the mobile phone, and a standard curve was plotted based on the captured RGB values. As shown in the curve in Figure 8 , the standard curve of the R value is y = -15.07x + 93.83, the standard curve of the G value is y = -6.630x + 93.42, and the standard curve of the B value is y = -11.31x + 94.87. In subsequent actual tests, according to the actual obtained RGB values and the standard curve, the concentration of glucose in sweat can be known.

[0070] 7. Figure 9 shows the wearable sweat sensor device prepared in Example 1 attached to the forearm of an athlete. As the sweat secreted during exercise flows on the cellulose paper inside the sensor device and significant color reactions occur at each detection point. In addition, we selected more people for experiments, and through data analysis by reading a large number of RGB values on the forearms, we further revealed Figure 10The close correlation between the changes in glucose content in sweat and blood, and the linear relationship between the two is: y = 0.1418x - 0.6068 (R 2 = 0.847). Therefore, the wearable sweat sensor device prepared by the present invention verifies that there is a significant correlation between glucose in sweat and blood glucose, and has broad application prospects in predicting blood glucose status through the glucose level in sweat.

[0071] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a metal-organic framework material encapsulating an enzyme, characterized in that, It includes the following steps: Dissolve ZrCl4, a monobasic acid regulator, and terephthalic acid in an organic solvent, and then carry out a hydrothermal reaction; After the hydrothermal reaction is completed, collect the white precipitate, and treat the white precipitate in a mixed solution of hydrochloric acid and DMF, then centrifuge, wash, and dry to obtain the MOF support; Add the MOF support, glucose oxidase, and horseradish peroxidase to a buffer solution, stir, and then centrifuge and wash to obtain the enzyme-encapsulated metal-organic framework material.

2. The preparation method according to claim 1, characterized in that, The monobasic acid regulator is formic acid, acetic acid, n-butyric acid, hexanoic acid, n-octanoic acid, dodecanoic acid, hexadecanoic acid, nitric acid, or hydrochloric acid; the molar ratio of ZrCl4, the monobasic acid regulator, and terephthalic acid is 1∶15 - 60∶0.5 - 3.

3. The preparation method according to claim 1, characterized in that, The temperature of the hydrothermal reaction is 80 - 150 °C, and the time is 48 h.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the MOF support, glucose oxidase, and horseradish peroxidase is 1∶0.1 - 1∶0.1 - 2; the buffer solution is a PBS buffer solution; the concentration of the PBS buffer solution is 0.1 M, and pH = 7.

0.

5. An enzyme-encapsulated metal-organic framework material prepared by the preparation method according to any one of claims 1 - 4.

6. An application of the enzyme-encapsulated metal-organic framework material according to claim 5 in detecting glucose in sweat.

7. A non-invasive, portable, multi-mode wearable sweat sensor device, characterized in that, It includes a skin adhesion layer, a detection layer, and a packaging layer; the detection layer is located between the skin adhesion layer and the packaging layer; the detection layer includes detection points and a paper-based microfluidic layer, and the detection points are connected to the paper-based microfluidic layer; the number of detection points ≥ 2; There is a sweat inlet provided in the skin adhesion layer, and the sweat inlet is located at the end point of the paper-based microfluidic layer; The detection point is composed of the enzyme-encapsulated metal-organic framework material according to claim 5, a chromogenic substrate, and a carrier; the enzyme-encapsulated metal-organic framework material and the chromogenic substrate are loaded on the surface of the carrier.

8. The non-invasive portable multi-mode wearable sweat sensor device according to claim 7, characterized in that, The paper-based microfluidic layer is cellulose paper; the packaging layer is a PDMS film; the chromogenic substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; the carrier is cellulose paper.

9. The non-invasive portable multi-mode wearable sweat sensor device according to claim 7, wherein The preparation method of the detection point is: (1) Immerse cellulose paper in a dispersion liquid uniformly dispersing the enzyme-encapsulated metal-organic framework material, and then wash and dry to obtain cellulose paper loaded with the metal-organic framework material; (2) Coat the chromogenic substrate on the surface of the cellulose paper loaded with the metal-organic framework material and dry to obtain the detection point.

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