Nano-enzyme cascade catalysis sweat glucose colorimetric detection patch as well as preparation method and application thereof

Abstract

The invention discloses a nano-enzyme cascade catalysis sweat glucose colorimetric detection patch and a preparation method and application thereof, and belongs to the technical field of glucose colorimetric detection patches, the detection patch mainly comprises a water absorption layer, a paper chip detection layer, a hydrophobic array layer and an adhesive layer; the hydrophobic array layer is filled with the paper chip detection layer, the water absorption layer is located on the hydrophobic array layer, and the pasting layer is located below the hydrophobic array layer. The nano-enzyme cascade catalysis sweat glucose colorimetric detection patch has the beneficial effects that the nano-enzyme cascade catalysis sweat glucose colorimetric detection patch does not need to use biological enzyme, the nano-enzyme stability of the used Au-coated Cu composite nanosheet is high, and the detection sensitivity is high; the nano-enzyme prepared by the method can be stored at room temperature for a long time, cascade catalytic reaction and glucose detection can be realized only by using one nano-enzyme, and the problems of instability, harsh storage conditions, short storage time, strict use conditions and the like caused by the defects of the bio-enzyme in a glucose detection method based on the bio-enzyme are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of glucose colorimetric detection patches, and specifically relates to a glucose colorimetric detection patch based on nanozyme cascade catalysis and without the use of biological enzymes, and particularly relates to a patch for detecting glucose in sweat, and a preparation method and application thereof. Background Art

[0002] With the development of society, people's attention to personal health is increasing. Abnormal glucose content in human body fluids is related to a series of diseases such as hypoglycemia, diabetes, and heart disease. Therefore, the detection and monitoring of glucose content in human body fluids is of great significance for the prevention and control of related diseases. Although commercial blood glucose meters can achieve rapid detection of glucose content in the blood, repeated acupuncture sampling during blood glucose meter testing may cause discomfort and fear in patients. Sweat can be collected in situ with high reproducibility on almost the entire skin surface in a completely non-invasive way, making it an ideal human body fluid test sample. Studies have shown that the glucose content in sweat has a certain correlation with the glucose level in the blood, and abnormal glucose content in sweat can also indicate the risk of diseases such as hypoglycemia and diabetes.

[0003] Wearable sweat detection patches can realize in-situ real-time collection and detection of sweat. They are easy to operate, quick to respond, low cost, and easy to carry. They play an important role in the development of wearable detection devices. The colorimetric analysis method can be directly observed by the naked eye to achieve qualitative and semi-quantitative analysis, or it can be taken by a smartphone and combined with image processing software and smartphone detection software for data analysis to achieve quantitative detection. Therefore, the development of a wearable sweat glucose colorimetric detection patch to achieve simple, rapid, low-cost, non-invasive, and convenient detection of glucose in sweat is of great significance for clinical testing and personal health monitoring.

[0004] The sweat glucose colorimetric detection patch currently developed all needs to use glucose oxidase. After using glucose oxidase to oxidize glucose to produce hydrogen peroxide, it further uses horseradish peroxidase or a nanozyme with simulated peroxidase activity to construct a cascade catalytic reaction, generate a visual colorimetric signal, and realize the colorimetric detection of glucose. Biological enzymes such as glucose oxidase have the disadvantages of high cost, easy inactivation, poor stability, and inconvenient storage. Therefore, the existing sweat glucose detection patch based on biological enzymes (glucose oxidase and horseradish peroxidase) has the problems of high preparation cost, difficulty in long-term storage at room temperature, and harsh use conditions. Therefore, it is of great significance to develop a new sweat glucose colorimetric detection patch that does not require the use of biological enzymes, further improve the stability and easy storage of wearable sweat glucose colorimetric detection patches, and reduce their preparation costs at the same time. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide a nanozyme cascade catalyzed sweat glucose colorimetric detection patch without the use of biological enzymes, and a preparation method and application thereof.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The first aspect of the present invention provides a nanozyme cascade catalytic sweat glucose colorimetric detection patch, which mainly includes a water absorption layer, a paper chip detection layer, a hydrophobic array layer and an adhesive layer; the paper chip detection layer is loaded on the hydrophobic array layer, the water absorption layer is located above the hydrophobic array layer, and the adhesive layer is located below the hydrophobic array layer;

[0008] The paper chip detection layer includes two sites, one is a color detection site and the other is a blank control site; each site is composed of two layers of paper chips stacked together; the color detection site is composed of a paper chip modified with tetramethylbenzidine (TMB) and a paper chip modified with Au@Cu composite nanosheet nanozyme stacked together; the blank control site is composed of two layers of unmodified filter paper stacked together;

[0009] The hydrophobic array layer is a nano-scale polytetrafluoroethylene (EPTFE) microporous composite membrane.

[0010] Preferably, the hydrophobic array layer is provided with detection holes and control holes; the detection holes and control holes are matched with the color development detection sites and blank control sites of the paper chip detection layer, respectively.

[0011] Preferably, the modified paper chip is obtained by immobilizing and modifying the corresponding reagent on a filter paper.

[0012] Preferably, the tetramethylbenzidine (TMB) modified paper chip is obtained by dropping a TMB solution onto a filter paper; the Au@Cu composite nanosheet nanozyme modified paper chip is obtained by dropping an Au@Cu composite nanosheet nanozyme solution onto a filter paper, and then dropping an acetic acid solution.

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

[0014] Preferably, the Au@Cu composite nanosheet nanozyme is prepared by the following process:

[0015] First, copper nitrate, trifluoroacetic acid and polyvinyl pyrrolidone are dissolved in a mixed solution 1 of N,N-dimethylformamide and ethanol, and TCPP(Fe) is dissolved in a mixed solution 2 of N,N-dimethylformamide and ethanol. After the mixture is completely dissolved, the mixed solution 2 is added dropwise into the mixed solution 1;

[0016] Secondly, ultrasonic treatment is performed, and after a period of reaction, the mixture is centrifuged, washed, dried, ground, and redispersed in water to obtain a Cu-TCPP(Fe) mixed solution;

[0017] Finally, chloroauric acid was added to the Cu-TCPP(Fe) mixture, stirred evenly, and then sodium borohydride was added. The mixture was washed by centrifugation and redispersed in water to obtain the Au@Cu composite nanosheet nanozyme.

[0018] Preferably, the water-absorbing layer is any one or more of a non-woven absorbent cotton pad, gauze, cotton fabric, and absorbent paper.

[0019] Preferably, the adhesive layer is any one or more of single-sided adhesive nano glue, medical tape, etc.

[0020] The second aspect of the present invention proposes a method for preparing the above-mentioned nanozyme cascade catalyzed sweat glucose colorimetric detection patch, comprising the following steps: pasting a hydrophobic array layer on an adhesive layer, loading a paper chip detection layer on the hydrophobic array layer, completely covering the hydrophobic array layer with a water-absorbing layer, and pasting and fixing the four sides of the water-absorbing layer on the adhesive layer.

[0021] The third aspect of the present invention proposes the use of the above-mentioned nanozyme cascade catalyzed sweat glucose colorimetric detection patch in the detection of glucose content in sweat.

[0022] The fourth aspect of the present invention provides a method for preparing Au@Cu composite nanosheet nanozyme, comprising the following steps:

[0023] (1) dissolving copper nitrate, trifluoroacetic acid and polyvinyl pyrrolidone in a mixed solution 1 of N,N-dimethylformamide and ethanol, dissolving TCPP(Fe) in a mixed solution 2 of N,N-dimethylformamide and ethanol, and after the mixture is completely dissolved, adding the mixed solution 2 dropwise into the mixed solution 1;

[0024] (2) performing ultrasonic treatment, reacting for a period of time, centrifuging, washing, drying, grinding, and redispersing in water to obtain a Cu-TCPP (Fe) mixed solution;

[0025] (3) Add chloroauric acid to the Cu-TCPP(Fe) mixed solution, stir evenly, then add sodium borohydride, centrifuge and wash, and redisperse in water to obtain Au@Cu composite nanosheet nanozyme.

[0026] Preferably, in step (1), the molar ratio of copper nitrate, trifluoroacetic acid and polyvinyl pyrrolidone is 1:0.4:9.

[0027] The fifth aspect of the present invention provides the Au@Cu composite nanosheet nanozyme prepared by the above preparation method.

[0028] The sixth aspect of the present invention proposes the use of the above-mentioned Au@Cu composite nanosheet nanozyme in the preparation of a sweat glucose colorimetric detection patch.

[0029] The nanozyme cascade catalyzed sweat glucose colorimetric detection patch of the present invention is applied to sweat glucose detection, comprising the following steps:

[0030] 1) According to the above steps, a nanozyme cascade catalytic sweat glucose colorimetric detection patch was prepared. A simulated sweat standard solution containing different concentrations of glucose was added dropwise to the detection patch. After reacting for 10 minutes, a photo was taken with a smartphone to obtain image data of the detection patch.

[0031] 2) Read the colorimetric information of the colorimetric detection area and the blank control area of ​​the sweat glucose detection patch. Use the difference in the colorimetric value between the colorimetric detection area and the blank control area of ​​the detection patch as the detection signal to obtain a linear curve for quantitative glucose detection.

[0032] 3) In the actual sweat detection process, the prepared nanozyme cascade catalytic sweat glucose colorimetric detection patch is pasted on the surface of the athlete's skin until the sweat passes through the water-absorbing layer and completely soaks the paper chip inside the detection patch. After 10 minutes of reaction, take a picture to obtain the image data of the sweat detection patch. Read the color value difference between the color detection area of ​​the detection patch and the blank control area as the detection signal, and calculate the actual glucose concentration in the sweat according to the glucose quantitative linear curve.

[0033] The mechanism of the nanozyme cascade catalyzed sweat glucose colorimetric detection patch of the present invention for detecting glucose is as follows:

[0034] Nanozyme cascade catalysis of sweat glucose colorimetric detection patch The Au@Cu composite nanosheet nanozyme modified on the paper chip inside has the activity of simulating glucose oxidase, which catalyzes the oxidation of glucose to produce hydrogen peroxide in the presence of oxygen. At the same time, the Au@Cu composite nanosheet nanozyme also has the catalytic activity of simulating peroxidase, further catalyzing hydrogen peroxide to produce hydroxyl radicals, which oxidize the TMB modified on the paper chip inside the detection patch to generate blue TMB oxidation products, realizing the colorimetric detection of glucose in sweat.

[0035] The beneficial effects of the present invention are:

[0036] 1) The nanozyme cascade catalyzed sweat glucose colorimetric detection patch of the present invention combines a superhydrophobic-superhydrophilic microarray with a nanozyme catalyzed colorimetric detection method, has the advantages of simple preparation method and easy operation, can realize in situ collection and real-time non-invasive detection of sweat glucose, and provides a new technology for sweat glucose detection.

[0037] 2) The nanozyme cascade catalytic sweat glucose colorimetric detection patch of the present invention does not require the use of biological enzymes. The Au@Cu composite nanosheet nanozymes used have high stability and can be stored at room temperature for a long time. Only one nanozyme is used to achieve cascade catalytic reactions and glucose detection, overcoming the problems of glucose detection methods based on biological enzymes due to the shortcomings of biological enzymes, such as instability, harsh storage conditions, short storage time, and strict use conditions.

[0038] 3) The nanozyme cascade catalyzed sweat glucose colorimetric detection patch of the present invention has a single-piece production cost of about 0.286 yuan / piece. Therefore, the nanozyme cascade catalyzed sweat glucose colorimetric detection patch of the present invention has a low preparation cost and a high cost performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the transmission electron microscope characterization image of the Au@Cu composite nanosheet nanozyme in Example 1.

[0040] Figure 2 It is the UV-visible absorption spectrum and the color development solution picture of the Au@Cu composite nanosheets simulating the properties of glucose oxidase in Example 1.

[0041] Figure 3 This is a three-dimensional structural diagram of the nanozyme cascade catalyzed sweat glucose colorimetric detection patch prepared in Example 2.

[0042] Figure 4 This is a photo of the bending and curling of the nanozyme cascade catalyzed sweat glucose colorimetric detection patch prepared in Example 2.

[0043] Figure 5 is a linear fitting curve diagram between the detection signal of the nanozyme cascade catalyzed sweat glucose colorimetric detection patch and the glucose concentration in Example 3, wherein Figure 5 a is a picture of the color development detection area of ​​the test patch in the presence of simulated sweat containing different concentrations of glucose; Figure 5 b is the linear fitting curve.

[0044] Figure 6 This is a graph of the change in detection signal of the prepared detection patch at different temperatures, in which nanozymes and biological enzymes are used to prepare the glucose colorimetric detection patch in Comparative Example 1.

[0045] Figure 7 This is a graph showing changes in detection signals of a glucose colorimetric detection patch prepared using nanozymes and biological enzymes, respectively, at room temperature in Comparative Example 1, after the prepared detection patch is stored for different periods of time.

[0046] Figure 8It is a comparison chart of the detection effects of using or not using the nano-EPTFE microporous composite membrane as the hydrophobic layer in Example 2, and using a water-absorbing layer instead of the hydrophobic layer to prepare the nanoenzyme cascade catalytic glucose colorimetric detection patch. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0049] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0050] Embodiment 1:

[0051] A method for preparing Au@Cu composite nanosheet nanozyme for colorimetric detection of glucose was described, and its characterization and performance testing were performed. The specific steps are as follows:

[0052] (1) Dissolve 24 mg of copper nitrate trihydrate, 400 μL of 1.0 mol / L trifluoroacetic acid, and 100 mg of polyvinyl pyrrolidone in a mixture of 90 mL of N,N-dimethylformamide and 30 mL of ethanol. Then, dissolve 4.4 mg of TCPP(Fe) in a mixture of 30 mL of N,N-dimethylformamide and 10 mL of ethanol. After the mixture is completely dissolved, add the mixture dropwise to the mixture.

[0053] (2) the obtained mixed solution was subjected to ultrasonic treatment for 15 minutes, reacted at 80° C. for 4 hours, the obtained mixture was centrifuged and washed twice with ethanol, and after drying, the obtained solid was fully ground into powder, and redispersed in an aqueous solution to obtain a Cu-TCPP (Fe) solution;

[0054] (3) Add 1 mL of 10 mmol / L chloroauric acid to 100 mL of 0.1 mg / mL Cu-TCPP(Fe) solution, stir evenly, and then add 250 μL of 0.1 mol / L sodium borohydride. The resulting mixture is centrifuged and washed twice with ultrapure water, and redispersed in water to obtain Au@Cu composite nanosheet nanozyme.

[0055] The Au@Cu composite nanosheet nanozyme prepared in this example was characterized and its performance tested:

[0056] Transmission electron microscopy characterization of Au@Cu composite nanosheet nanozymes, such as Figure 1 As shown, it can be seen that the Au@Cu composite nanosheet nanoenzyme material was successfully prepared. The material has a sheet-like structure, and gold nanoparticles with a diameter of about 20 nm exist on the sheet-like structure.

[0057] The catalytic performance of Au@Cu composite nanosheet nanozyme was tested:

[0058] Take 4 centrifuge tubes and label them a, b, c and d. Add 1 mL of 200 μmoL / L glucose solution to tube a. Add 0.5 mL of 200 μmoL / L glucose solution and 0.5 mL of Au@Cu composite nanosheet solution to tube b. Add 0.5 mL of Au@Cu composite nanosheet solution to tube c. Add 0.5 mL of 200 μmoL / L glucose solution and 0.5 mL of Au@Cu composite nanosheet solution to tube d.

[0059] Place tubes a, b, c and d at 37°C for 30 minutes. Centrifuge tube b to remove the nanozyme precipitate, and take its supernatant back to tube b. Then add 1mL of pH=4 acetate buffer solution to tubes a, b, c and d respectively, and after 5 minutes of reaction, add 1mL of 5mmoL / L TMB solution to tubes a, b, c and d respectively. Finally, transfer the solutions of tubes a, b, c and d to a cuvette, let it stand for 5 minutes, and use a UV spectrophotometer to detect its UV-visible absorption spectrum.

[0060] The oxidation product of TMB can produce a characteristic absorption peak at 652nm.

[0061] The results are as follows Figure 2 As shown, the solution in tube a is colorless and has no absorption peak at 652 nm, indicating that glucose does not react with TMB.

[0062] Tube b: After the Au@Cu composite nanosheets react with glucose for a period of time, the Au@Cu composite nanosheets precipitate is removed, and the supernatant is taken to react with TMB. The solution turns blue and an absorption peak is generated at 652nm. This indicates that the Au@Cu composite nanosheets have the activity of simulating glucose oxidase, and can catalyze the oxidation of glucose to produce hydrogen peroxide in the first step. After removing the nanozyme in the centrifugal precipitate, the residual hydrogen peroxide in the supernatant can oxidize TMB to produce a characteristic absorption peak.

[0063] In tube c, the solution turns blue and an absorption peak appears at 652 nm, indicating that the Au@Cu composite nanosheets have simulated oxidase activity and can catalyze the oxidation of TMB, causing the solution to turn blue and produce a characteristic absorption peak.

[0064] Tube d, Au@Cu composite nanosheets acted with glucose for a period of time, and then TMB was added. Compared with tube c without glucose, the color of the solution was significantly deepened, and the absorption peak at 652nm was significantly enhanced. This shows that the Au@Cu composite nanosheets prepared by the present invention have both simulated glucose oxidase and simulated peroxidase activities. When glucose and TMB are present at the same time, a cascade catalytic reaction occurs, first catalyzing the oxidation of glucose to produce hydrogen peroxide, further catalyzing the decomposition of hydrogen peroxide, oxidizing TMB, and producing a large amount of blue TMB oxidation products, which can be used for colorimetric detection of glucose.

[0065] Embodiment 2:

[0066] Prepare a nanozyme cascade catalyzed sweat glucose colorimetric detection patch, the structure of which is as follows Figure 3 As shown, the preparation and assembly technical scheme is as follows:

[0067] 1) Use a mold to prepare a Band-Aid shaped template, and then use this template to cut a single-sided adhesive nano-glue into a Band-Aid shape as the third adhesive layer of the test patch.

[0068] 2) Use a plotter to batch prepare circular filter paper with a diameter of 3 mm. Add 3 μL of 5 mM TMB solution to the circular filter paper with a diameter of 3 mm to prepare a paper chip modified with TMB reagent. Prepare the Au@Cu composite nanosheet solution according to Example 1, add 3 μL of the Au@Cu composite nanosheet solution to the circular filter paper with a diameter of 3 mm, wait for 1 minute, and then add 3 μL of pH = 5 acetate buffer solution to prepare the Au@Cu composite nanosheet nanozyme modified paper chip.

[0069] 3) Use a plotter to carve two circular holes with a pore size of 4 mm on the nanoscale EPTFE microporous composite membrane, named as the detection hole and the control hole respectively. Cut the nanoscale EPTFE microporous composite membrane into a rectangle with a width of 1.5 cm and a length of 2 cm around the circular hole area. Paste the cut rectangular thin sheet of nanoscale EPTFE microporous composite membrane as the second hydrophobic layer to the center area of ​​the above adhesive layer. Paste the paper chip modified with Au@Cu composite nanosheet nanozyme to the bottom layer of the detection hole, and then stack the paper chip modified with TMB reagent on the paper chip modified with Au@Cu composite nanosheet nanozyme. Stack two unmodified circular filter paper sheets on the control hole. Nanoscale EPTFE microporous composite membrane and paper chip to construct a superhydrophilic-superhydrophobic array.

[0070] 4) Cut the non-woven absorbent cotton pad into a rectangle with a width of 2 cm and a length of 2.5 cm, and use it as the first water-absorbing layer, which is completely covered on the second layer of nano-scale EPTFE microporous composite membrane, and is pasted on the first adhesive layer on all sides to achieve the encapsulation of the detection patch, and obtain a nanoenzyme cascade catalytic sweat glucose colorimetric detection patch. The water-absorbing layer fits the skin, absorbs sweat, and can prevent the detection reagent on the paper chip from directly contacting the skin.

[0071] The prepared nanozyme cascade catalyzed sweat glucose colorimetric detection patch can be stretched, folded, twisted, etc., and it can still return to its original shape. Figure 4 As shown, it indicates that the prepared detection patch has good flexibility and morphological stability.

[0072] Embodiment 3:

[0073] The nanozyme cascade catalytic sweat glucose colorimetric detection patch prepared in Example 2 was used to simulate the quantitative detection of glucose in sweat. The specific method is as follows:

[0074] 1) Prepare a nanozyme cascade catalyzed sweat glucose colorimetric detection patch according to Example 2.

[0075] 2) Prepare simulated sweat standard solutions containing different concentrations of glucose. Prepare simulated sweat: The simulated sweat contains 20 mg / mL sodium chloride, 17.5 mg / mL ammonium chloride, 5 mg / mL urea, 2.5 mg / mL acetic acid, and 15 mg / mL lactic acid. Then use the simulated sweat to prepare simulated sweat standard solutions containing 0 μM, 20 μM, 40 μM, 80 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, and 200 μM glucose.

[0076] 3) Take 10 test patches and drop simulated sweat standard solutions containing different concentrations of glucose onto the test patches. After 10 minutes of reaction, take a photo with a smartphone to obtain a picture of the test area of ​​the sweat test patch.

[0077] 4) If Figure 5 As shown in a, the color of the paper chip in the detection area of ​​the detection patch gradually deepens as the glucose concentration in the simulated sweat increases. Image J software is further used to read the chromaticity value information of the detection hole and the control hole of the sweat detection patch. The present invention preferably uses the R value in the RGB value as the quantitative standard, and uses the difference (△R) between the R value of the detection hole and the R value of the blank control hole as the detection signal to draw a linear fitting curve between the simulated sweat glucose concentration and the △R value of the sweat detection patch detection signal. Figure 5 As shown in b, the linear equation is: y = 25.38 + 0.46x, and the linear correlation coefficient is: R 2 =0.9927.

[0078] Embodiment 4:

[0079] To demonstrate the beneficial effects of the present invention, the nanozyme cascade catalytic sweat glucose colorimetric detection patch prepared in Example 2 was used for quantitative detection of actual sweat glucose in the human body, and the specific method was as follows:

[0080] 1) Prepare a nanozyme cascade catalyzed sweat glucose colorimetric detection patch according to Example 2.

[0081] 2) The prepared nanozyme cascade catalytic sweat glucose colorimetric detection patch is attached to the arms and back of the neck of the athlete, which are prone to sweating. After the sweat completely soaks the detection patch through the non-woven absorbent cotton pad, it reacts for 10 minutes, takes a photo to obtain the image data of the sweat detection patch, reads the R value of the color detection area of ​​the detection patch and the R value of the blank control area, and calculates △R as the detection signal.

[0082] 3) The actual glucose concentration in sweat was calculated based on the glucose quantitative linear curve obtained in Example 3. According to the calculation results, the glucose concentrations in the sweat of the arms and back of the neck of the athlete were 84.14 μmol / L and 88.24 μmol / L, respectively. The test results are consistent with the glucose content in normal sweat.

[0083] Therefore, the nanozyme cascade catalyzed sweat glucose colorimetric detection patch of the present invention can be used for the quantitative detection of glucose in human sweat.

[0084] Embodiment 5:

[0085] The nanozyme cascade catalytic sweat glucose colorimetric detection patch of Example 2 is used for quantitative detection of glucose in sweat of healthy people under fasting and full stomach conditions, respectively. The specific method is as follows:

[0086] 1) Prepare a nanozyme cascade catalyzed sweat glucose colorimetric detection patch according to Example 2.

[0087] 2) Referring to Example 3, the prepared sweat detection patch was used to detect the glucose concentration in the sweat of 4 volunteers under fasting and full stomach conditions. The test results are shown in Table 1.

[0088] The table below shows that the sweat glucose concentration of the four volunteers was between 33.75-47.07 μmol / L when fasting, and between 66.85-82.08 μmol / L when full. The glucose concentration in the sweat of the four volunteers when full was significantly higher than that when fasting. This indicates that the prepared nanozyme cascade catalyzed sweat glucose colorimetric detection patch can be used to detect different glucose concentrations in actual sweat.

[0089] Table 1. Nanozyme cascade catalytic sweat glucose colorimetric detection patch for glucose detection in sweat of healthy subjects under fasting and full conditions

[0090]

[0091]

[0092] Comparative Example 1:

[0093] This comparative example refers to the technical solution of Example 2, only the enzyme used is changed, specifically the combined use of glucose oxidase and horseradish peroxidase, instead of the Au@Cu composite nanosheet nanozyme used in Example 2, to prepare a sweat glucose detection patch based on biological enzymes. Specifically, on a circular filter paper, first add 3μL of 1ku / mL glucose oxidase, wait for 1min, then add 3μL of 20ng / mL horseradish peroxidase, and finally add 3μL of pH=5 acetate buffer solution to prepare a paper chip modified with biological enzymes. With reference to the preparation method of Example 2, only the bottom paper chip in the detection area is changed, and the paper chip modified with biological enzymes is replaced with the paper chip modified with Au@Cu composite nanosheet nanozyme in Example 2 to prepare a sweat glucose detection patch based on biological enzymes. Compare the stability.

[0094] 1) Temperature stability comparison: The paper chip modified with bioenzyme and the paper chip modified with Au@Cu composite nanosheet nanozyme were stored at 5℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃ and 100℃ for 1 hour, and then the sweat glucose detection patch based on bioenzyme and the sweat glucose detection patch based on nanozyme were prepared according to the preparation and assembly steps of Example 2, and used for the detection of glucose in the same simulated sweat. The test results are as follows Figure 6 As shown, the detection signal of the sweat glucose detection patch based on biological enzymes drops rapidly as the temperature rises. When the temperature rises to above 30°C, the detection signal is basically consistent with the blank, indicating that the biological enzyme has been completely inactivated, and the detection patch prepared based on the paper chip modified by the biological enzyme has poor high temperature resistance and stability. The detection signal of the sweat glucose detection patch prepared using the paper chip modified by nanozyme decreases slightly as the temperature rises. However, the detection signal change rate of the detection patch prepared by the nanozyme-modified paper chip is within 10% within the common room temperature range of 5-40°C. After the nanozyme-based paper chip has been exposed to a high temperature environment of 100°C, the detection patch prepared still maintains a high detection signal. The comparison shows that the nanozyme-based paper chip has significantly improved high temperature resistance and good stability compared with the bioenzyme-based paper chip detection system.

[0095] 2) Storage stability comparison: The paper chip modified with bioenzyme and the paper chip modified with Au@Cu composite nanosheet nanozyme were stored at room temperature for 1 day, 3 days, 5 days, 10 days, 15 days, 20 days, 30 days, and 40 days, respectively. Then, referring to the preparation and assembly steps of Example 2, sweat glucose detection patches based on bioenzyme and sweat glucose detection patches based on nanozyme were prepared, respectively, and used for the detection of glucose in the same simulated sweat. The test results are shown in Figure 2. Figure 7 As shown, after 40 days of storage at room temperature, the detection signal of the paper chip modified with Au@Cu composite nanosheet nanozyme remained basically stable, with a change rate of less than 15%. After 1 day of storage at room temperature for the paper chip modified with biological enzyme, the detection signal was greatly reduced. After 3 days of storage at room temperature, the detection signal was basically the same as the blank, indicating that the biological enzyme had been inactivated. The comparison shows that compared with the paper chip detection system based on biological enzyme, the paper chip based on nanozyme has significantly improved storage ease, storage time and storage stability.

[0096] Comparative Example 2:

[0097] In order to demonstrate the usefulness and necessity of the second hydrophobic layer in the sweat glucose colorimetric detection patch structure prepared by the present invention, detection patches were prepared with or without using a nano-scale EPTFE microporous composite membrane as the hydrophobic layer, and detection patches were prepared using an absorbent non-woven absorbent cotton pad instead of the nano-scale EPTFE microporous composite membrane as the second layer, and a comparative study was carried out.

[0098] like Figure 8 As shown, Figure a shows the nanozyme cascade catalytic sweat glucose colorimetric detection patch prepared in reference to Example 2, which is attached to the skin surface of the athlete to detect the actual glucose concentration in the sweat. It can be seen that the color development detection area of ​​the detection patch a appears light blue under the action of glucose in the sweat. Compared with the control well, the color development effect is obvious, and the detection patch is intact.

[0099] Figure b is a reference to Example 2, but instead of using a nano-scale EPTFE microporous composite membrane as the hydrophobic layer, a non-woven absorbent cotton pad of the same size and perforated is used instead of the nano-scale EPTFE microporous composite membrane as the second layer to prepare a nanoenzyme cascade catalytic sweat glucose colorimetric detection patch, which is attached to the same skin surface of the same athlete to detect the actual glucose concentration in sweat. Since the second layer of the detection patch b is a non-woven absorbent cotton pad, it has hydrophilicity and water absorption like the paper chip, and it is impossible to construct a super-hydrophobic-super-hydrophilic array. The second layer of non-woven absorbent cotton pad absorbs part of the sweat, and the amount of sweat immersed in the paper chip is reduced, so the color development effect of the color development detection area is not obvious.

[0100] Figure c is a reference to Example 2, but without using a nano-scale EPTFE microporous composite membrane as a hydrophobic layer, in the absence of a second hydrophobic layer, a nanoenzyme cascade catalytic sweat glucose colorimetric detection patch is directly prepared with an adhesive layer, a paper chip and a water-absorbing layer, and the glucose concentration in the actual sweat is detected after being attached to the same skin surface of the same athlete. After the sweat is absorbed by the water-absorbing layer of the detection patch c, it is immersed in the paper chip, and the color development detection area has a certain color development effect, but due to the lack of a hydrophilic-hydrophobic array constructed by the second hydrophobic layer, sweat is not easy to gather on the paper chip, so the color of the color development detection area is lighter than that of the detection patch a. In addition, due to the lack of micropores for fixing the paper chip constructed by the second hydrophobic layer, the two layers of paper chips in the detection area are prone to movement and dislocation due to the action of sweat, and the paper chip is accompanied by watermarks around it, which affects the reading of the color development signal in the detection area.

[0101] The above comparative case shows that the use of nano-scale EPTFE microporous composite membrane as a hydrophobic layer and the construction of a super-hydrophobic-super-hydrophilic array with a hydrophilic paper chip to prepare a sweat detection patch is beneficial to the enrichment of sweat in the paper chip and improves the colorimetric detection effect.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanozyme cascade catalyzed sweat glucose colorimetric detection patch, characterized in that: The detection patch mainly includes a water absorption layer, a paper chip detection layer, a hydrophobic array layer and an adhesive layer; the paper chip detection layer is loaded on the hydrophobic array layer, the water absorption layer is located above the hydrophobic array layer, and the adhesive layer is located below the hydrophobic array layer; The paper chip detection layer includes two sites, one is a color detection site and the other is a blank control site; each site is composed of two layers of paper chips stacked together; the color detection site is composed of a paper chip modified with tetramethylbenzidine (TMB) and a paper chip modified with Au@Cu composite nanosheet nanozyme stacked together; the blank control site is composed of two layers of unmodified filter paper stacked together; The hydrophobic array layer is a nano-scale polytetrafluoroethylene (EPTFE) microporous composite membrane.

2. The nanozyme cascade catalyzed sweat glucose colorimetric detection patch according to claim 1, characterized in that: The hydrophobic array layer is provided with detection holes and control holes; the detection holes and control holes are matched with the color development detection sites and blank control sites of the paper chip detection layer respectively.

3. The nanozyme cascade catalyzed sweat glucose colorimetric detection patch according to claim 1, characterized in that: The modified paper chip is obtained by immobilizing and modifying the corresponding reagent on a filter paper. Specifically, the tetramethylbenzidine (TMB) modified paper chip is obtained by dripping a TMB solution onto a filter paper; the Au@Cu composite nanosheet nanozyme modified paper chip is obtained by dripping an Au@Cu composite nanosheet nanozyme solution onto a filter paper and then dripping an acetic acid solution.

4. The nanozyme cascade catalyzed sweat glucose colorimetric detection patch according to claim 1, characterized in that: The Au@Cu composite nanosheet nanozyme is prepared by the following process: First, copper nitrate, trifluoroacetic acid and polyvinyl pyrrolidone are dissolved in a mixed solution 1 of N,N-dimethylformamide and ethanol, and TCPP(Fe) is dissolved in a mixed solution 2 of N,N-dimethylformamide and ethanol. After the mixture is completely dissolved, the mixed solution 2 is added dropwise into the mixed solution 1; Secondly, ultrasonic treatment is performed, and after a period of reaction, the mixture is centrifuged, washed, dried, ground, and redispersed in water to obtain a Cu-TCPP(Fe) mixed solution; Finally, chloroauric acid was added to the Cu-TCPP(Fe) mixed solution, stirred evenly, and then sodium borohydride was added. The mixture was centrifuged and washed, and then redispersed in water to obtain the Au@Cu composite nanosheet nanozyme.

5. The nanozyme cascade catalyzed sweat glucose colorimetric detection patch according to claim 1, characterized in that: The water-absorbing layer is any one or more of non-woven absorbent cotton pad, gauze, cotton fabric, and absorbent paper; the adhesive layer is any one or more of single-sided adhesive nano glue and medical tape.

6. The method for preparing the nanozyme cascade catalyzed sweat glucose colorimetric detection patch according to any one of claims 1 to 5, characterized in that: The hydrophobic array layer is pasted on the adhesive layer, the paper chip detection layer is filled on the hydrophobic array layer, the water-absorbing layer is completely covered on the hydrophobic array layer, and the water-absorbing layer is pasted and fixed on the adhesive layer around the periphery.

7. Use of the nanozyme cascade catalyzed sweat glucose colorimetric detection patch described in any one of claims 1 to 5 or the detection patch prepared by the preparation method described in claim 6 in the detection of glucose content in sweat.

8. A method for preparing Au@Cu composite nanosheet nanozyme, characterized in that: The following steps are involved: (1) dissolving copper nitrate, trifluoroacetic acid and polyvinyl pyrrolidone in a mixed solution 1 of N,N-dimethylformamide and ethanol, dissolving TCPP(Fe) in a mixed solution 2 of N,N-dimethylformamide and ethanol, and after complete dissolution, adding the mixed solution 2 dropwise into the mixed solution 1; the molar ratio of the copper nitrate, trifluoroacetic acid and polyvinyl pyrrolidone is 1:0.4:9; (2) performing ultrasonic treatment, reacting for a period of time, centrifuging, washing, drying, grinding, and redispersing in water to obtain a Cu-TCPP (Fe) mixed solution; (3) Add chloroauric acid to the Cu-TCPP(Fe) mixed solution, stir evenly, then add sodium borohydride, centrifuge and wash, and redisperse in water to obtain Au@Cu composite nanosheet nanozyme.

9. The Au@Cu composite nanosheet nanozyme prepared by the preparation method according to claim 8.

10. Use of the Au@Cu composite nanosheet nanozyme according to claim 9 in the preparation of a sweat glucose colorimetric detection patch.

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