A non-invasive, portable, multimodal wearable sweat sensor device

Abstract

The present invention provides a non-invasive, portable, multimodal wearable sweat sensor device, belonging to the technical field of wearable biosensor devices. ZrCl4, a monobasic acid modifier, and terephthalic acid are dissolved in an organic solvent, followed by a hydrothermal reaction. After the hydrothermal reaction, the white precipitate is collected and treated in a mixed solution of hydrochloric acid and DMF, centrifuged, washed, and dried to obtain a metal organic framework (MOF) carrier. The MOF carrier, GOx, and HRP are added to a buffer solution, stirred, centrifuged, and washed to obtain an enzyme-encapsulated metal organic framework (MOF) material. The enzyme-encapsulated MOF material is fabricated into a detection point, which, together with a skin adhesion layer, a paper-based microfluidic layer, and an encapsulation layer, forms a non-invasive, portable, multimodal wearable sweat sensor device. The sensor device can be directly integrated with the skin surface to continuously and in real time quantify sweat loss and provide quantitative data on glucose in sweat.

Description

Technical Field

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

[0002] As a non-invasive alternative to blood testing, sweat has the potential to effectively detect multiple biomarkers in real time and is widely used in fields such as exercise status assessment, diabetes screening, and disease monitoring. However, the accurate and rapid detection of biomarkers in sweat remains a major 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 marker and lack sufficient sensitivity and multi-index analysis capabilities.

[0003] Chinese invention patent publication number CN115639260A discloses a wearable sweat sensor that uses sweat flowing to an electrode layer to generate electrical signals, enabling non-invasive, continuous sweat analysis. However, the device suffers from a complex preparation method and lacks quantitative detection of sweat rate. Chinese invention patent publication number CN118348089A discloses a wearable sweat sensor device, its preparation method, and its application. This sweat sensor device utilizes a microfluidic chip and a three-electrode system to detect biomarkers using nucleic acid aptamers, providing a biosensing interface. However, the device lacks nucleic acid protection, resulting in poor stability and similarly failing to achieve quantitative detection of sweat rate. Both wearable sweat sensor devices disclosed in CN115639260A and CN118348089A suffer from single detection methods, cumbersome preparation, inconvenient use, and stability issues. Therefore, there is an urgent need for a sweat sensor device that has a simple design, can accurately and quickly detect the concentration of biomarkers and sweat volume in sweat, and has excellent stability. Summary of the Invention

[0004] In view of this, the present invention aims to provide a non-invasive, portable, multimodal wearable sweat sensor. This device can be directly integrated with the skin surface, effectively absorb sweat, and continuously and in real time quantify sweat loss. Using a smartphone to take a photo and read the RGB values ​​of the detection points, it provides quantitative data on glucose in sweat. The multimodal wearable sweat sensor comprises a skin adhesion layer, a paper-based microfluidic layer, detection points, and a PDMS encapsulation layer, all of which are made of flexible materials and a flexible design. This greatly improves the overall flexibility of the patch and overall comfort for athletes. Furthermore, the present invention utilizes natural enzymes, a highly efficient catalyst, to perform cascade catalysis on the substrate glucose, thereby improving the accuracy of molecular detection. However, natural enzymes have poor stability. By manipulating the pore size of the UiO-66 MOF material, a hierarchical porous structure HP-UiO-66-XA with a size closely matching that of natural glucose oxidase (GOx) and horseradish peroxidase (HRP) was synthesized. This allows for effective encapsulation of the natural enzymes GOx and HRP, significantly enhancing enzyme activity and the stability of the multimodal wearable sweat sensor. At the same time, the amount of sweat can be assessed 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:

[0006] One of the technical solutions of the present invention is a method for preparing a metal-organic framework material encapsulating an enzyme, comprising the following steps:

[0007] ZrCl4 (zirconium tetrachloride), a monobasic acid regulator and terephthalic acid are dissolved in an organic solvent and then subjected to a hydrothermal reaction;

[0008] After the hydrothermal reaction is completed, the white precipitate is collected and treated in a mixed solution of hydrochloric acid and DMF (N,N-dimethylformamide), then centrifuged, washed, and dried to obtain a MOF support;

[0009] The MOF carrier, glucose oxidase and horseradish peroxidase are added to a buffer solution, stirred, centrifuged and washed to obtain the enzyme-encapsulated metal organic framework material.

[0010] The second technical solution of the present invention is a metal-organic framework material encapsulating an enzyme prepared by the above preparation method.

[0011] The third technical solution of the present invention is the use of the above-mentioned enzyme-encapsulated metal-organic framework material in detecting glucose in sweat.

[0012] A fourth technical solution of the present invention is a non-invasive, portable, multi-modal wearable sweat sensor device comprising 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 comprises detection points and a paper-based microfluidic layer, the detection points being connected to the paper-based microfluidic layer; the number of the detection points being ≥2;

[0013] The skin adhesive layer is provided with a sweat inlet, and the sweat inlet is located at an end point of the paper-based microfluidic layer;

[0014] The detection point is composed of the metal organic framework material encapsulating the enzyme, a chromogenic substrate and a carrier; the metal organic framework material encapsulating the enzyme and the chromogenic substrate are loaded on the surface of the carrier.

[0015] The present invention discloses the following technical effects:

[0016] (1) The present invention precisely controls the design of the hierarchical porous structure to achieve a high degree of match between the size of the mesopores within the material and the size of the enzyme molecules. When the size of the enzyme is close to the nanopore diameter, the appropriate size can achieve a good balance between diffusion efficiency and stability. This match allows the enzyme molecules to smoothly diffuse into the pores and remain there, while effectively preventing enzyme leakage, significantly improving the enzyme's stability and effectively maintaining its activity.

[0017] (2) The sweat sensor designed in this invention successfully integrates multiple detection points, achieving 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 multiple 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.

[0018] (3) The sweat sensor device of the present invention enables real-time and continuous monitoring of sweat volume and blood sugar levels. Users can take photos and read RGB values ​​through the mobile client to view blood sugar levels in real time. The measurement results are accurate, and the device provides a comfortable wearing experience and convenient data reading function.

[0019] (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 easy use. These features significantly lower the threshold for use and are particularly suitable for daily health monitoring and instant diagnosis.

[0020] (5) Paper-based materials have excellent hygroscopic properties and can effectively absorb sweat and trigger chemical reactions, enabling highly sensitive component detection. At the same time, paper-based materials do not contain harmful chemicals and are suitable for use in wearable devices that directly contact the skin, reducing potential risks to human health.

[0021] (6) The preparation of wearable sweat sensor devices has the advantages of low cost, environmental protection, comfort, sensitivity and multifunctionality, which gives them significant economic advantages in large-scale production and can become a strong competitor in the field of health monitoring and biosensor technology in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Schematic diagram of pore size regulation and enzyme cascade catalysis in the present invention, wherein a is a schematic diagram of the introduction of a monoacid regulator in the synthesis of UiO-66 to achieve continuous regulation from micropores to mesopores, b is a schematic diagram of enzyme encapsulation by microporous structure and hierarchical porous structure, and c is a diagram of the enzyme cascade catalysis mechanism after enzyme encapsulation;

[0024] Figure 2 Schematic diagram of the wearable sweat sensor device and the detection process of the present invention;

[0025] Figure 3 a is the cut snake-shaped cellulose paper, and b is the PDMS encapsulation layer;

[0026] Figure 4 This is the X-ray diffraction (XRD) pattern of the defective HP-UiO-66-6 prepared in Example 1;

[0027] Figure 5 This is a nitrogen adsorption-desorption (BET) curve of the defective HP-UiO-66-6 prepared in Example 1;

[0028] Figure 6 Transmission image (a), statistical data of internal pore size (b), and three-dimensional reconstruction images (c, d) of the defective HP-UiO-66-6 prepared in Example 1;

[0029] Figure 7 (a) shows the maximum distance that different volumes of water can move on the cellulose paper of the device, and (b) shows the linear relationship between water volume and movement distance.

[0030] Figure 8 It is the RGB calibration curve of glucose solution with different concentrations;

[0031] Figure 9 This is a diagram of the color reaction that occurs when a wearable sweat sensor device is worn on an athlete;

[0032] Figure 10This is a correlation diagram between sweat sugar and blood sugar obtained through a wearable sweat sensor device. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] In order to improve the detection accuracy and sensitivity of wearable devices, the introduction of 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 needs of high-precision, multi-index analysis. However, the disadvantage is that natural enzymes have poor stability and are prone to clustering in solution. In response to this, the present invention uses a metal-organic framework material (MOF) with high adjustability to encapsulate the enzyme. The metal-organic framework (MOF) material encapsulating natural enzymes 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, which can significantly improve the catalytic performance of the enzyme; (2) Adjustable pore size structure: The pore size of the MOF material can be flexibly controlled, thereby optimizing the catalytic reaction environment of the enzyme and improving the substrate selectivity and catalytic efficiency of the enzyme. At the same time, the pore structure can help the enzyme achieve better spatial adaptation between the substrate molecule and the enzyme active center; (3) Enhance the stability of the enzyme: The solid structure of MOF provides physical protection for the natural enzyme, preventing the enzyme molecules from being inactivated in extreme environments; (4) Prevent excessive aggregation and denaturation of the enzyme: The pore structure of MOF can prevent nonspecific aggregation between enzyme molecules, avoid the enzyme from losing activity or denaturing during the reaction, and maintain its catalytic efficiency; (5) Multifunctionality: In addition to being able to encapsulate enzymes, MOF materials can also give MOF materials new functions, thereby realizing multiple detection functions.

[0039] As macromolecular structures, enzymes are often large in size. The smaller pore sizes in the intrinsic MOF microporous structure make it impossible to encapsulate the enzyme, and the enzyme is usually attached to the surface of the material. By adjusting the MOF pores to highly match the pore size with the enzyme size, the enzyme's loading capacity, stability, and catalytic efficiency can be significantly improved, thereby improving the application effect of the enzyme in sensing detection or catalytic reactions. To this end, the present invention uses monoprotic acids to adjust the pore size of the MOF to achieve a perfect match between the pore size and the enzyme molecular size, promote the effective diffusion of the enzyme within the nanopores, and reduce leakage, thereby achieving efficient enzyme catalysis.

[0040] To address the limitations of most current biosensor devices in detecting a single indicator and establishing a relationship between blood and sweat glucose levels, the present invention utilizes precisely regulated MOF materials to encapsulate enzymes and integrate them with wearable devices, developing a more advanced quantitative sweat analysis technology that meets the demand for high-precision analysis in this field. The non-invasive, portable, multimodal wearable sweat sensor designed in this invention boasts a simple structure, a straightforward fabrication process, and ease of use. By integrating color change information with RGB analysis using a smartphone, the sensor demonstrates excellent stability and high accuracy in the non-invasive detection of glucose in sweat.

[0041] The first aspect of the present invention provides a method for preparing a metal-organic framework material encapsulating an enzyme, comprising the following steps:

[0042] Dissolving ZrCl4, a monobasic acid regulator and terephthalic acid in an organic solvent, and then performing a hydrothermal reaction;

[0043] After the hydrothermal reaction is completed, the white precipitate is collected and treated in a mixed solution of hydrochloric acid and DMF, and then centrifuged, washed, and dried to obtain a MOF support;

[0044] The MOF carrier, glucose oxidase (GOx) and horseradish peroxidase (HRP) are added to a buffer solution, stirred, centrifuged, and washed to obtain the enzyme-encapsulated metal organic framework material.

[0045] In a preferred embodiment of the present invention, the monoacid regulator is formic acid, acetic acid, n-butyric acid, hexanoic acid, n-octanoic acid, dodecanoic acid, hexadecanoic acid, nitric acid or hydrochloric acid; and the molar ratio of ZrCl4, monoacid regulator and terephthalic acid is 1:15~60:0.5~3.

[0046] The present invention imposes no particular restrictions on the choice of organic solvent; any organic solvent known to those skilled in the art that can dissolve ZrCl₄, the monoprotic acid modifier, and terephthalic acid, such as DMF, can be used. The present invention also imposes no particular restrictions on the amount of organic solvent used; the amount used is sufficient to dissolve ZrCl₄, the monoprotic acid modifier, and terephthalic acid and to allow the hydrothermal reaction to proceed.

[0047] The present invention uses monobasic acid to adjust the pore size of MOF, but this method is not applicable to all metal-organic framework materials. The principle of monobasic acid for pore size adjustment is: by adding monobasic acid during the synthesis process, it competes with metal ions for coordination, introduces ligand defects, and then adjusts 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 monobasic acid; (2) Differences in synthesis conditions: The synthesis conditions of different MOFs react differently to monobasic acid. Some MOFs cannot achieve the expected pore size adjustment effect under specific conditions. Therefore, the specific applicability of this method requires a comprehensive evaluation based on the structure, chemical properties and synthesis conditions of the MOF. The present invention successfully prepared a MOF material with adjustable pore size by selecting a specific metal ligand (ZrCl4), monobasic acid and organic ligand terephthalic acid (BDC).

[0048] In a preferred embodiment of the present invention, the temperature of the hydrothermal reaction is 80-150° C., and the time is 48 hours.

[0049] 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 undiluted commercially available concentrated hydrochloric acid; the treatment temperature is room temperature and the treatment time is 10 hours. Many organic modifiers are easily dissolved or degraded in an acidic environment. Hydrochloric acid provides hydrogen ions (H + ) help these regulators to be removed from the MOF structure during the next drying step.

[0050] In a preferred embodiment of the present invention, the drying temperature is 200° C. and the drying time is 12 hours.

[0051] The functions of drying in the present invention are: (1) drying; (2) removing the monobasic acid regulator in the MOF structure, promoting the generation of defects and forming hierarchical pores.

[0052] 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; the concentration of the PBS buffer is 0.1M, and the pH is 7.0.

[0053] The second aspect of the present invention provides an enzyme-encapsulated metal-organic framework material prepared by the above preparation method.

[0054] A third aspect of the present invention provides a use of the above-mentioned enzyme-encapsulated metal organic framework material in detecting glucose in sweat.

[0055] A fourth aspect of the present invention provides a non-invasive, portable, multi-mode wearable sweat sensor device comprising 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 comprises detection points and a paper-based microfluidic layer, the detection points being connected to the paper-based microfluidic layer; the number of the detection points being ≥2;

[0056] The skin adhesive layer is provided with a sweat inlet, and the sweat inlet is located at an end point of the paper-based microfluidic layer;

[0057] The detection point is composed of the metal organic framework material encapsulating the enzyme, a chromogenic substrate and a carrier; the metal organic framework material encapsulating the enzyme and the chromogenic substrate are loaded on the surface of the carrier.

[0058] In a preferred embodiment of the present invention, the paper-based microfluidic layer is cellulose paper; the encapsulation layer is a PDMS film; the color developing substrate is 2,2'-azino-bis-(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS); and the carrier is cellulose paper.

[0059] In a preferred embodiment of the present invention, the preparation method of the detection point is:

[0060] (1) immersing cellulose paper in a dispersion of a metal organic framework material uniformly dispersed with the enzyme, followed by washing and drying to obtain a cellulose paper loaded with the metal organic framework material;

[0061] (2) Applying a color-developing substrate solution to the surface of the cellulose paper loaded with the metal-organic framework material and drying the solution to obtain a detection point.

[0062] 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; and the concentration of the chromogenic substrate solution is 0.3-2M.

[0063] The present invention does not impose any specific restrictions on the shape of the detection point or the paper-based microfluidic layer. As an example, the detection point can be circular, and the paper-based microfluidic layer can be serpentine with a width of 1-5 mm. By connecting the detection point and the paper-based microfluidic layer with the same material as the paper-based microfluidic layer, sweat can enter the detection point smoothly for detection.

[0064] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0065] The present invention does not impose any particular limitation on the source of the PDMS film, and the PDMS film may be a commercially available product or prepared using a preparation method well known to those skilled in the art.

[0066] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0067] In the examples, the PDMS film (PDMS encapsulation layer) was prepared by mixing parts A and B of a Sylgard 184 silicone elastomer kit (commercially available, PDMS elastomer (Sylgard 184) purchased from Dow Corning) in a weight ratio of 10:1, degassing under vacuum for 15 minutes, and then using a 120 nm thick mold to doctor blade coat a 120 nm thick PDMS encapsulation layer.

[0068] Example 1

[0069] (1) Preparation of HP-UiO-66-6

[0070] ZrCl4 (0.343 mmol) and hexanoic acid (12 mmol) were dissolved in DMF (20 mL) and sonicated for 25 min. BDC (0.343 mmol) was then added and sonicated to form a homogeneous solution. The mixed solution was then transferred to a polytetrafluoroethylene-lined container and reacted at 120°C for 48 h. The mixture was then centrifuged and washed three times with DMF to obtain a white precipitate. The white precipitate was treated in a mixture of concentrated hydrochloric acid (0.4 mL) and DMF (40 mL) for 10 h, followed by centrifugation and washing. The resulting white powder was activated in a vacuum oven at 200°C for 12 h to obtain HP-UiO-66-6.

[0071] (2) Preparation of GOx&HRP@HP-UiO-66-6

[0072] HP-UiO-66 (5 mg) and GOx (1.5 mg) prepared above were added to 1 mL of PBS buffer (0.01 M, pH 7.0). The mixture was stirred at room temperature for 1.5 hours, and then HRP (1.5 mg) was added to the solution and stirred for another 1.5 hours. GOx & HRP@HP-UiO-66-6 was then obtained by centrifugation and washing.

[0073] (3) Preparation of detection points

[0074] Chromatographic grade cellulose paper was cut into circles with a diameter of 3 mm, and then the 3 mm diameter circles of chromatography grade cellulose paper were soaked in GOx&HRP@HP-UiO-66-6 solution (5 mg / mL) for 1 h to allow GOx&HRP@HP-UiO-66-6 to be uniformly adsorbed on the paper surface. Subsequently, the GOx&HRP@HP-UiO-66-6 paper was washed with deionized water to remove the loosely bound GOx&HRP@HP-UiO-66-6, and after natural drying at room temperature, 20 μL of ABTS (1 M) solution was dropwise coated on it and allowed to dry naturally.

[0075] (4) Preparation of snake-shaped cellulose paper

[0076] The pre-designed AutoCAD pattern was printed on chromatography-grade cellulose paper and cut into 2 mm wide snake shapes using a paper cutting tool.

[0077] Finally, the snake-shaped cellulose paper and the detection points were adhered to medical-grade double-sided tape (skin adhesion layer) and then encapsulated with PDMS film to obtain a wearable sweat sensor device.

[0078] The schematic diagram of the wearable sweat sensor device is as follows Figure 2The sensor consists of four components: medical tape, a paper-based serpentine channel (serpentine cellulose paper), four detection points, and a PDMS encapsulation layer. A small hole is provided in the bottom double-sided tape layer to guide the inflow of sweat from the local skin. Sweat enters the serpentine channel through this inlet and flows sequentially through the four detection points. At each detection point, encapsulated glucose oxidase first catalyzes glucose in sweat to produce gluconic acid and hydrogen peroxide. The resulting hydrogen peroxide, catalyzed by horseradish peroxidase, reacts with ABTS to produce green ABTS•+. By capturing the image with a mobile phone and reading the RGB values, the glucose content in sweat can be accurately assessed.

[0079] Figure 3 Figure a shows a cut paper-based serpentine channel. The figure shows that the serpentine design can greatly increase the flexibility of the device. Figure 3 In b, it can be seen that the prepared PDMS encapsulation layer has good flatness.

[0080] Example 2

[0081] The only difference from Example 1 is that hexanoic acid is replaced with formic acid to obtain HP-UiO-66-1. Other steps and parameters are the same as those in Example 1.

[0082] Example 3

[0083] The only difference from Example 1 is that hexanoic acid is replaced by acetic acid to obtain HP-UiO-66-2. Other steps and parameters are the same as those in Example 1.

[0084] Example 4

[0085] The only difference from Example 1 is that hexanoic acid is replaced with n-butyric acid to obtain HP-UiO-66-4. Other steps and parameters are the same as those in Example 1.

[0086] Example 5

[0087] The only difference from Example 1 is that hexanoic acid is replaced with n-octanoic acid to obtain HP-UiO-66-8. Other steps and parameters are the same as those in Example 1.

[0088] Example 6

[0089] The only difference from Example 1 is that hexanoic acid is replaced with dodecanoic acid to obtain HP-UiO-66-12. Other steps and parameters are the same as those in Example 1.

[0090] Comparative Example 1

[0091] The only difference from Example 1 is that the addition of hexanoic acid is omitted to obtain UiO-66. Other steps and parameters are the same as those in Example 1.

[0092] Comparative Example 2

[0093] The only difference from Example 1 is that 12 mmol of hexanoic acid is replaced by 1 mmol of hexanoic acid to obtain HP-UiO-66-6-1 mmol. The other steps and parameters are the same as those in Example 1.

[0094] Comparative Example 3

[0095] The only difference from Example 1 is that 12 mmol hexanoic acid is replaced with 25 mmol hexanoic acid to obtain 25 mmol HP-UiO-66-6-. Other steps and parameters are the same as in Example 1.

[0096] Characterization and effect verification:

[0097] 1. Figure 1 Figure a describes the continuous adjustment of UiO-66 from micropores to mesopores by introducing a monobasic acid regulator during synthesis. Glucose oxidase and horseradish peroxidase were loaded on the unregulated UiO-66 sample and the regulated HP-UiO-66 sample. Figure 1 From the model b, we can see that the enzyme can be protected only when the mesopores exist. Figure 1 Figure c shows the enzyme cascade catalytic mechanism after enzyme encapsulation.

[0098] 2. Figure 4 The XRD pattern of the HP-UiO-66-6 sample prepared in Example 1 is shown. As can be seen from the figure, the sample conditioned with the conditioning agent still maintains good crystallinity, with its diffraction peak positions essentially consistent with those of the unconditioned sample, and with clear peak shapes and high intensities, indicating that the sample did not experience significant loss of crystallinity or structural damage during the conditioning process.

[0099] 3. In order to adjust UiO-66 to a size suitable for enzyme loading, monocarboxylic acids with different chain lengths were selected as regulators (formic acid, acetic acid, butyric acid, hexanoic acid, octanoic acid, and lauric acid). By adding appropriate amounts, a series of defect-rich HP-UiO-66-XA derivatives were prepared. The N2 adsorption-desorption isotherms and the corresponding pore size distribution diagrams were used to analyze the effect of the carbon chain length of the monocarboxylic acid on the pore size of HP-UiO-66-XA. Figure 5 As shown in Table 1, the HP-UiO-66-XA sample has a hysteresis loop in the pressure range of 0.8-1.0, which is attributed to the presence of macropores, indicating that the micropores (<2nm) of the original UiO-66 have been successfully transformed into mesopores (2-20nm) after adjustment. Figure 5From the average mesopore diameters of a series of HP-UiO-66-XAs shown in the middle illustration and 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 8.1 nm), which is very suitable as a nanomaterial for exploring enzyme cascade reactions.

[0100] Table 1 shows the results of the pore sizes of different UiO-66 prepared in Examples 1-6 and Comparative Examples 1-3 and the corresponding enzyme loading amounts.

[0101] Table 1

[0102]

[0103] As shown in Table 1, with the increase of the regulator chain length, the size of the mesopores of the prepared defective HP-UiO-66 first increases and then decreases, while the micropores are not much different. 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 comparison, the HP-UiO-66-6 prepared in Example 1 has the size that best matches the two enzymes. When the size of the enzyme is close to the size of the pore, it is usually most beneficial for the enzyme to diffuse into the pore and also helps to reduce the leakage of the enzyme. This process involves multiple factors, mainly: (1) Matching of the pore size and the enzyme molecule size: When the size of the enzyme molecule is close to the pore size, the enzyme can smoothly enter the pore. This is because a good match is formed between the pore size and the enzyme molecule, and the enzyme molecule can diffuse into the pore smoothly. However, because the sizes are similar, the friction between the enzyme molecule and the pore wall or pore size is 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 size of the pore size, the enzyme molecule will be subject to the physical restriction and force of 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. This restriction effect helps to enhance the stability and activity of the enzyme; (3) Diffusion efficiency and restriction: When the size of the enzyme matches the pore size, the enzyme molecule can not only enter through the pore, but also diffuse effectively in the pore. This is because when the pore size and the enzyme molecule are well matched, the enzyme molecule can have better mobility in the pore, which helps to improve the efficiency of the enzyme reaction; (4) Van der Waals force and physical interaction: When the pore size and the enzyme molecule size are close, van der Waals force (including attraction and repulsion) will also play a role. These interactions help the enzyme molecule better bind to the pore wall, thereby reducing leakage. Therefore, a perfect match between pore size and enzyme molecular size facilitates efficient enzyme diffusion within the nanopores and minimizes leakage, which is key to improving enzyme stability and maintaining enzyme activity. Based on this principle, the present invention successfully fabricated a hierarchical porous structure, HP-UiO-66-6, that closely matches enzyme size through rational selection and careful design. This structure is optimal for enzyme encapsulation.

[0104] As shown in Example 1 and Comparative Examples 2-3 in Table 1, the amount of the regulator must be within an appropriate range. Too little regulator cannot regulate sufficient mesopores, while too much regulator will form clusters and produce larger pore sizes, both of which will deviate from the target size suitable for loading the enzyme.

[0105] 4. If Figure 6 As shown, we successfully visualized the three-dimensional pores in HP-UiO-66-6 by combining atomic-resolution structural characterization and transmission electron microscopy (TEM) three-dimensional electron tomography. Figure 6 Figure a shows the transmission image of HP-UiO-66-6, while Figure 6 The CD precisely segments the tomographic images and labels the internal structure in detail in three dimensions. Based on these images, the porosity inside the structure can be clearly and intuitively revealed. Figure 6 The bar graph in (b) shows the statistics of the internal pore size of HP-UiO-66-6. It can be seen from the figure that its mesopore diameter is mainly concentrated in the range of 6-10 nm, which is consistent with the BET test results, verifying the high match between the pore size and the enzyme size, and proving that the HP-UiO-66-6 we designed and synthesized is extremely suitable for the loading of GOx and HRP.

[0106] 5. By Figure 7 As shown in Figure 2, 5, 10, 15, and 20 μL of water were introduced into the sweat inlet, and the maximum distance that different volumes of water could move on the cellulose paper of the device increased with the increase of liquid volume ( Figure 7 The calculated volume and travel distance are summarized in Figure 7 In figure b, the two show a linear relationship: y = 4.478x + 1.157 (R² = 0.952). Based on this linear equation, the amount of sweat can be quantified by visualizing the distance and position of the fluid.

[0107] 6. If Figure 8 As shown, first, known glucose solutions of different concentrations are introduced into the wearable sweat sensor device prepared in Example 1. When the solution reaches each detection point, the device is photographed with a mobile phone, and the color picker software on the mobile phone is used to read the RGB of the detection point after the color change occurs, and a standard curve is drawn based on the captured RGB values. Figure 8 From the curve, we can see that the standard curve for R value is y = -15.07x + 93.83, the standard curve for G value is y = -6.630x + 93.42, and the standard curve for B value is y = -11.31x + 94.87. In subsequent actual tests, the concentration of glucose in sweat can be determined by comparing the actual RGB values ​​with the standard curve.

[0108] 7. Figure 9The wearable sweat sensor device prepared in Example 1 was attached to the forearm of an athlete. As the sweat secreted during exercise flowed on the cellulose paper inside the sensor device, significant color reactions occurred at each detection point. In addition, we selected more people for experiments and analyzed the RGB values ​​of a large number of forearms to further reveal the Figure 10 There is a close correlation between changes in sweat and blood glucose levels, 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 the significant correlation between glucose in sweat and blood glucose, and has broad application prospects in predicting blood glucose status by glucose levels in sweat.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a metal organic framework material encapsulating an enzyme, characterized in that: The following steps are involved: Dissolving ZrCl4, a monobasic acid regulator and terephthalic acid in an organic solvent, and then performing a hydrothermal reaction; After the hydrothermal reaction is completed, the white precipitate is collected and treated in a mixed solution of hydrochloric acid and DMF, and then centrifuged, washed, and dried to obtain a MOF support; The MOF carrier, glucose oxidase and horseradish peroxidase are added to a buffer solution, stirred, centrifuged and washed to obtain the enzyme-encapsulated metal organic framework material; The monoacid regulator is hexanoic acid; the molar ratio of ZrCl4, the monoacid regulator and terephthalic acid is 1:15~60:0.5~3; The temperature of the hydrothermal reaction is 80-150° C., and the time is 48 hours.

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

0.

3. A metal-organic framework material encapsulating an enzyme prepared by the preparation method according to claim 1.

4. Use of the enzyme-encapsulated metal-organic framework material according to claim 3 in detecting glucose in sweat.

5. A non-invasive, portable, multi-mode wearable sweat sensor device, characterized in that: The invention comprises 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 comprises detection points and a paper-based microfluidic layer, the detection points are connected to the paper-based microfluidic layer; the number of the detection points is ≥2; The skin adhesive layer is provided with a sweat inlet, and the sweat inlet is located at an end point of the paper-based microfluidic layer; The detection point is composed of the metal organic framework material encapsulating the enzyme according to claim 3, a chromogenic substrate and a carrier; the metal organic framework material encapsulating the enzyme and the chromogenic substrate are loaded on the surface of the carrier.

6. The non-invasive, portable, multi-mode wearable sweat sensor device according to claim 5, characterized in that: The paper-based microfluidic layer is cellulose paper; the encapsulation layer is a PDMS film; the color development substrate is 2,2'-azino-bis-(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt; and the carrier is cellulose paper.

7. The non-invasive, portable, multi-mode wearable sweat sensor device according to claim 5, characterized in that: The preparation method of the detection point is: (1) immersing cellulose paper in a dispersion of a metal organic framework material uniformly dispersed with the enzyme, followed by washing and drying to obtain a cellulose paper loaded with the metal organic framework material; (2) Coating a color-developing substrate on the surface of the cellulose paper loaded with the metal-organic framework material and drying the paper to obtain a detection point.

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