Glucose colorimetric detection platform based on super-hydrophobic interface and preparation and application thereof
By depositing catalase-like catalysts and glucose oxidases on a superhydrophobic substrate to form reaction sites, the problems of difficult catalyst recovery and large sample reagent consumption are solved, realizing low-cost, reusable colorimetric detection of glucose.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-06-08
- Publication Date
- 2026-05-15
AI Technical Summary
Existing colorimetric methods for glucose detection involve catalysts that are difficult to recover and reuse, and require large quantities of samples and reagents, making them unsuitable for detecting rare or small quantities of samples.
A catalase-like catalyst is deposited on a superhydrophobic substrate to form reaction sites. After adding an aqueous solution of glucose oxidase, the catalyst is immobilized. The superhydrophobicity enables the catalyst to self-assemble and be immobilized, achieving detection with small sample sizes and reusability.
It enables rapid glucose detection with low sample volume and low reagent volume. The catalyst can be reused, reducing detection costs and making it suitable for detecting small numbers of samples.
Smart Images
Figure CN115165856B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of analytical chemistry technology, and more specifically, relates to a glucose colorimetric detection platform based on a superhydrophobic interface and its preparation and application, particularly to a glucose colorimetric detection method based on superhydrophobic microdroplets. Background Technology
[0002] Colorimetric analysis is a method that uses the color of the solution itself, or the color produced after adding reagents, to determine the concentration of the analyte in the solution by visually observing and comparing the color depth, or by measuring it with instruments. Colorimetric analysis is a visual detection method that requires no complex instruments and is convenient and rapid. Currently, most of the rapid glucose detection methods developed by researchers are based on colorimetric analysis. The principle of glucose colorimetric analysis is that glucose oxidase produces hydrogen peroxide, which then reacts with a colorimetric reagent under the action of an enzyme or enzyme-like catalyst. The quantitative analysis of glucose is achieved by observing the color change. For example, patent CN111239125 A first uses glucose oxidase to oxidize glucose to produce gluconic acid and hydrogen peroxide, and then uses platinum disulfide as a catalyst to activate the generated hydrogen peroxide, which reacts with a colorimetric reagent to detect the glucose concentration. Currently, researchers in this field generally focus on the development of catalysts, specifically peroxidase-like catalysts, as exemplified by patents CN108226074 A, CN104048957 A, and CN110609032 A. In this conventional colorimetric detection method, catalyst recovery and reuse are often difficult, increasing detection costs. Furthermore, the sample and reagent volumes used are typically above 100 microliters, making this method unsuitable for rare samples or samples with small single-sample volumes, such as sweat and saliva. Therefore, developing a detection platform that requires smaller sample volumes and is reusable is one of the goals currently being pursued by researchers in this field. Summary of the Invention
[0003] To address the shortcomings of existing technologies and the need for improvement, this invention provides a colorimetric glucose detection platform based on a superhydrophobic interface. A catalase-like catalyst is deposited on a superhydrophobic substrate to form reaction sites. An aqueous solution of glucose oxidase is then added to these reaction sites; after the water evaporates, the glucose oxidase remains immobilized at the reaction sites. This invention enables non-invasive and rapid detection of glucose content in human body fluids using this platform, requiring minimal sample volume and allowing for reusability. It solves the technical problems of existing colorimetric methods for glucose concentration detection, which require catalyst recovery and reuse, as well as large sample and reagent volumes.
[0004] According to a first aspect of the present invention, a method for preparing a glucose colorimetric detection platform based on a superhydrophobic interface is provided, comprising the following steps:
[0005] (1) A catalase-like catalyst solution is dropped onto a superhydrophobic substrate. Due to the hydrophobic effect of the substrate, the added solution stands on the substrate in the form of droplets. After the solvent in the droplets evaporates, the catalase-like catalyst gathers and is fixed on the substrate to form reaction sites.
[0006] (2) Add glucose oxidase aqueous solution to the reaction site described in step (1). After the water evaporates, the glucose oxidase is fixed on the reaction site, thus obtaining the glucose colorimetric detection platform.
[0007] Preferably, the catalase catalyst is Fe3O4 nanoparticles or a composite material of graphene and heme.
[0008] Preferably, the superhydrophobic substrate is prepared by the following method: dissolving oxide nanoparticles in water to form a first reagent; dissolving a low surface energy modifier in an organic solvent, wherein the low surface energy modifier is a silane compound, to form a second reagent; mixing the first and second reagents to obtain a third reagent; immersing the substrate in the third reagent, removing it and drying it to obtain the superhydrophobic substrate.
[0009] Preferably, the oxide nanoparticles are TiO2 nanoparticles, SiO2 nanoparticles, or ZnO nanoparticles.
[0010] Preferably, the silane compound is perfluorooctyltrichlorosilane, perfluorooctyltrimethoxysilane, hexadecyltrichlorosilane, or octadecyltrimethoxysilane.
[0011] According to another aspect of the present invention, a glucose colorimetric detection platform based on a superhydrophobic interface prepared by any of the methods described herein is provided.
[0012] According to another aspect of the present invention, the glucose colorimetric detection platform is provided for the application of detecting glucose concentration.
[0013] Preferably, the sample to be tested is added to the reaction site of the glucose colorimetric detection platform, followed by the addition of a colorimetric reagent; the glucose in the sample to be tested reacts with glucose oxidase to generate hydrogen peroxide, the hydrogen peroxide reacts with a catalase-like catalyst to generate hydroxyl radicals, and the colorimetric reagent is oxidized by the hydroxyl radicals to achieve color development; the concentration of glucose in the sample to be tested is calculated based on the displayed color.
[0014] Compared with existing glucose detection methods, the glucose colorimetric detection platform based on superhydrophobic microdroplets provided by this invention has the following advantages:
[0015] (1) The construction method of this detection platform is simple, without complicated steps and harsh reaction conditions, and can achieve efficient large-scale preparation.
[0016] (2) Using this method to construct a superhydrophobic substrate, droplets can stand upright on the substrate and form a perfect sphere. This spherical structure gives the microdroplets a high specific surface area and mass transfer performance, enabling efficient mass and energy transfer, reducing the mixing and reaction time of the sample inside the droplet, and enabling rapid detection.
[0017] (3) Due to the superhydrophobic effect of the substrate, the contact area between the catalyst-containing droplet and the substrate is very small. The catalyst particles complete self-assembly within the droplet through self-evaporation, and finally converge to a point and be fixed on the superhydrophobic substrate to form an active site. The active site formed by the catalyst is hydrophilic, which can firmly anchor the droplet of chromogenic agent and test sample solution to the reactive active site.
[0018] (4) The entire colorimetric reaction takes place in tiny droplets. When performing colorimetric analysis, the total consumption of the sample and colorimetric reagent is only a few microliters to tens of microliters, which reduces costs and allows the saved sample to be used for the analysis of other components.
[0019] (5) The catalyst particles are fixed on a superhydrophobic substrate. After the reaction droplets complete the color reaction on the catalyst surface, the reacted droplets can be removed by rolling or suction. Then, the reaction can continue on the original active sites to achieve the purpose of reuse. This solves the problem of difficult catalyst recovery or repeated use in the existing technical solutions, and has good economic benefits and application prospects.
[0020] (6) The intensity of the color after the droplet color reaction directly reflects the concentration of glucose in the sample. Therefore, the glucose concentration in the sample can be predicted by the naked eye without the aid of any instruments. In the later stage, a visual colorimetric card can be made based on the color change after different concentrations of glucose are developed, so as to facilitate rapid quantitative detection on site. Attached Figure Description
[0021] Figure 1 A schematic diagram of a glucose colorimetric detection platform based on a superhydrophobic interface.
[0022] Figure 2 Scanning electron microscope image of Fe3O4 nanoparticles deposited on a superhydrophobic substrate.
[0023] Figure 3 A standard curve was established between the glucose concentration in sweat and the absorbance of the droplets after color development.
[0024] Figure 4 The glucose visualization detection platform based on superhydrophobic microdroplets yields repeated detection results. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.
[0026] The technical solution adopted by the glucose colorimetric detection platform based on superhydrophobic microdroplets in this invention is as follows:
[0027] Step 1: Preparation of superhydrophobic substrate:
[0028] 100 mg of oxide nanoparticles were dispersed in 10 mL of water under ultrasonic assistance to form reagent ①; 20 μL of low surface energy modifier was dissolved in 10 mL of anhydrous ethanol to form reagent ②; reagent ① and reagent ② were mixed evenly under ultrasonic assistance to form reagent ③; clean substrate material was immersed in reagent ③ and immersed for 5 min under ultrasonic action. The substrate material was then removed and dried in an oven at 60 °C; the above steps were repeated three times to obtain a superhydrophobic substrate.
[0029] Step 2: Deposit catalase-like catalysts on a superhydrophobic substrate via self-assembly:
[0030] A 10 μL solution of an enzyme-like catalyst was dropped onto a superhydrophobic substrate. Due to the hydrophobic effect of the substrate, the catalyst droplet stood upright in a spherical shape. Under room temperature conditions or irradiation with an infrared heating lamp, the catalyst droplet continuously shrank until it completely evaporated, and finally all the catalyst converged to a single point, forming a reaction site on the superhydrophobic substrate.
[0031] Step 3: Detection of glucose in human body fluid samples:
[0032] A 10 μL droplet of glucose oxidase aqueous solution (1 g / L) was added to the reaction site on a superhydrophobic substrate. After the droplet evaporated naturally, the glucose oxidase was immobilized on the reaction site. A few μL of body fluid sample was added to the reaction site, followed by an equal volume of 3,3′,5,5′-tetramethylbenzidine (TMB) solution to initiate a colorimetric reaction. After 10 minutes, the color change of the droplet was observed, and a photograph of the droplet was taken using a smartphone. The RGB values of the colored droplet were then obtained using Photoshop software, allowing the glucose concentration in the sample to be read from the established standard curve. Alternatively, 2 μL of the colored droplet solution could be used to measure its absorbance at 652 nm using a Nanodrop micro spectrophotometer, and the glucose concentration in the sample could then be obtained from the established standard curve.
[0033] Preferably, the low surface energy modifier mentioned in step one is selected from one of perfluorooctyltrichlorosilane, perfluorooctyltrimethoxysilane, hexadecyltrichlorosilane, and octadecyltrimethoxysilane.
[0034] Preferably, the substrate material mentioned in step one can be white woven fabric or white nonwoven fabric, or it can be filter paper.
[0035] Preferably, the catalase-like catalyst mentioned in step two refers to a composite material of Fe3O4 nanoparticles, graphene, and heme.
[0036] Preferably, the concentration of the catalase-like catalyst in step two is 1–20 g / L.
[0037] Preferably, the size of the catalase-like catalyst particles described in step two is 20–500 nm.
[0038] Preferably, the concentration of the TMB solution in step three is 0.2–0.8 g / L.
[0039] Preferably, the human fluid sample mentioned in step three refers to sweat, urine, tears, or saliva.
[0040] Example 1
[0041] 100 mg of TiO2 nanoparticles were dispersed in 10 mL of water under ultrasonic assistance to form reagent ①; 20 μL of perfluorooctyltrichlorosilane was dissolved in 10 mL of anhydrous ethanol to form reagent ②; reagent ① and reagent ② were mixed evenly under ultrasonic assistance to form reagent ③; a clean white fabric was soaked in reagent ③ and ultrasonically treated for 5 min, then the fabric was removed and dried in an oven at 60 °C; the above steps were repeated three times to obtain the superhydrophobic fabric.
[0042] like Figure 1 As shown in Figure a, a 10 μL solution of 15 g / L Fe3O4 nanoparticles was dropped onto a superhydrophobic substrate. Due to the hydrophobicity of the substrate, the Fe3O4 droplets stood upright in a spherical shape. Under infrared heating lamp irradiation, the Fe3O4 droplets continuously shrank, while the Fe3O4 nanoparticles self-assembled and deposited on the substrate. Figure 1 (b) until completely evaporated, and finally all the Fe3O4 nanoparticles in the droplet converge at one point ( Figure 1 c) forms reactive sites on the superhydrophobic substrate. Figure 2 ).
[0043] A drop of 10 μL glucose oxidase aqueous solution (1 g / L) was added to the reaction site on the superhydrophobic substrate. Figure 1 In step d), after the droplets evaporate naturally, glucose oxidase is also immobilized at the reaction site. Figure 1 (e); Add 10 μL of human sweat sample to the reaction site, then add 10 μL of 0.4 g / L TMB solution to initiate a colorimetric reaction (e). Figure 1 (f in the text). After 10 minutes, observe the color change of the droplet. Take 2 μL of the color-developed droplet solution and measure its absorbance at 652 nm using a Nanodrop micro spectrophotometer. Figure 1 (g) or by taking a photo of the droplet with a smartphone, and then using Photoshop to obtain the RGB values of the droplet after color development. Figure 1 The concentration of glucose in the sample was obtained by referring to the established standard curve (h in the original text). In this embodiment, we tested a total of 3 human sweat samples, performed color development using the detection platform of this invention, and measured the absorbance at 652 nm using a Nanodrop micro-spectrophotometer. The concentration of glucose in the sample was then determined according to the established standard curve (h in the original text). Figure 3 The glucose concentrations in three samples were obtained and compared with the values measured by commercially available glucose detection standard kits to verify the accuracy of the results. The results are shown in Table 1. Experimental results show that the detection platform of this invention can achieve an accuracy of over 95% when detecting glucose in human sweat samples. Since this method uses colorimetry, the glucose concentration in the sample can also be predicted by observing the color intensity of the droplets. Figure 3 (Inner illustration).
[0044] To verify the stability and reusability of this superhydrophobic microdroplet glucose colorimetric detection platform, the H2O2 colorimetric reaction was repeatedly performed on the same reactive site formed by the Fe3O4 droplet. The experimental results show that after 40 repetitions, the absorbance values of the droplet did not change significantly in the first 30 repetitions (e.g., ...). Figure 4 This indicates that the microdroplet colorimetric detection platform can be used repeatedly.
[0045] Table 1. Detection of glucose content in sweat samples
[0046]
[0047] Example 2
[0048] 100 mg of SiO2 nanoparticles were dispersed in 10 mL of water under ultrasonic assistance to form reagent ①; 20 μL of octadecyltrimethoxysilane was dissolved in 10 mL of anhydrous ethanol to form reagent ②; reagent ① and reagent ② were mixed evenly under ultrasonic assistance to form reagent ③; laboratory cotton fiber filter paper was soaked in reagent ③ and ultrasonically treated for 5 min, then the filter paper was removed and dried in an oven at 60 °C; the above steps were repeated three times to obtain superhydrophobic filter paper.
[0049] In this embodiment, a 1 g / L graphene and heme complex was selected as the catalyst. A 10 μL catalyst solution was dropped onto superhydrophobic filter paper. Under the irradiation of an infrared heating lamp, the droplet continuously shrank until it was completely evaporated. Finally, all the nanoparticles in the droplet converged to one point, forming a reactive site on the superhydrophobic substrate.
[0050] A 10 μL droplet of glucose oxidase aqueous solution (1 g / L) was added to the reaction site on a superhydrophobic substrate. After the droplet evaporated naturally, the glucose oxidase was immobilized on the reaction site. A 10 μL human saliva sample was then added to the reaction site, followed by a 10 μL droplet of 0.8 g / L TMB solution to initiate a colorimetric reaction. The color change of the droplet was observed after 10 minutes, and a photograph of the droplet was taken using a smartphone. The RGB values of the colored droplet were then obtained using Photoshop software. The glucose concentration in the sample was then read from the established standard curve, yielding a glucose concentration of 205.2 μM in the sample.
[0051] Example 3
[0052] Superhydrophobic fabric was prepared as a substrate according to the method in Example 1. A 5 g / L graphene and heme complex was selected as a catalyst. A 10 μL catalyst solution was dropped onto the superhydrophobic filter paper. Under the irradiation of an infrared heating lamp, the droplet continuously shrank until it was completely evaporated. Finally, all the nanoparticles in the droplet converged to one point, forming a reactive site on the superhydrophobic substrate.
[0053] A 10 μL droplet of glucose oxidase aqueous solution (1 g / L) was added to the reaction site on a superhydrophobic substrate. After the droplet evaporated naturally, the glucose oxidase was immobilized on the reaction site. A 10 μL human urine sample was added to the reaction site, followed by a 10 μL droplet of 0.8 g / L TMB solution to initiate a colorimetric reaction. The color change of the droplet was observed after 10 minutes, and a photograph of the droplet was taken using a smartphone. The RGB values of the colored droplet were then obtained using Photoshop software. The glucose concentration in the sample was read from the established standard curve, and the glucose concentration in the sample was found to be 411.4 μM.
[0054] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A glucose colorimetric detection platform for repeated detection of glucose concentration, characterized in that, The glucose colorimetric detection platform is prepared through the following steps: (1) A catalase-like catalyst solution is dropped onto a superhydrophobic substrate. Due to the hydrophobicity of the substrate, the added solution stands upright on the substrate in the form of droplets. The droplets stand upright on the substrate to form spheres. This spherical structure gives the microdroplets a high specific surface area and mass transfer performance, which can carry out efficient mass and energy transfer and reduce the mixing and reaction time of the sample inside the droplet. After the solvent in the droplet evaporates, the catalase-like catalyst gathers and is fixed on the substrate to form reaction sites. The active sites formed by the catalyst are hydrophilic to anchor the chromogenic agent and the droplet of the sample solution to the active reaction sites. (2) Add glucose oxidase aqueous solution to the reaction site described in step (1). After the water evaporates, glucose oxidase is fixed on the reaction site, thus obtaining a glucose colorimetric detection platform. This glucose colorimetric detection platform can be used repeatedly. The specific application is as follows: the sample to be tested is dropped onto the reaction site of the glucose colorimetric detection platform, and then a colorimetric reagent is dropped on; the glucose in the sample to be tested reacts with glucose oxidase to generate hydrogen peroxide, the hydrogen peroxide reacts with a catalase-like catalyst to generate hydroxyl radicals, and the colorimetric reagent is oxidized by the hydroxyl radicals to achieve color development; the concentration of glucose in the sample to be tested is calculated based on the displayed color.
2. The application as described in claim 1, characterized in that, The catalase catalyst is Fe3O4 nanoparticles or a composite material of graphene and heme.
3. The application as described in claim 1 or 2, characterized in that, The superhydrophobic substrate is prepared by the following method: dissolving oxide nanoparticles in water to form a first reagent; dissolving a low surface energy modifier in an organic solvent, wherein the low surface energy modifier is a silane compound, to form a second reagent; mixing the first and second reagents to obtain a third reagent; immersing the substrate in the third reagent, removing it and drying it to obtain the superhydrophobic substrate.
4. The application as described in claim 3, characterized in that, The oxide nanoparticles are TiO2 nanoparticles, SiO2 nanoparticles, or ZnO nanoparticles.
5. The application as described in claim 3, characterized in that, The silane compound is perfluorooctyltrichlorosilane, perfluorooctyltrimethoxysilane, hexadecyltrichlorosilane, or octadecyltrimethoxysilane.