A paper-based enzyme biosensor for hypoxanthine detection and a preparation method thereof

Abstract

The application provides a paper-based enzyme biosensor for hypoxanthine detection and a preparation method thereof, and comprises paper-based chip manufacturing, enzyme solution, silicon precursor solution and mineralization system solution required by a synthesis xanthine oxidase silicon enzyme method, color developing system configuration, paper-based chip modification and packaging. Through the technical scheme of the application, xanthine oxidase (XOD) is combined with color developing agent nitro blue tetrazolium chloride (NBT), one-step color development is realized, the operation process is significantly simplified, and the stability of the color developing system is improved. The enzyme biosensor constructed based on the biomimetic mineralization immobilization system can maintain high enzyme activity after being stored at room temperature for 21 days, exhibits excellent room temperature storage stability, is more convenient for practical application and storage and transportation, and greatly reduces the use amount of enzymes, thereby saving costs while ensuring high sensitivity and reliable quantitative detection performance.

Description

Technical Field

[0001] This invention relates to the field of rapid detection technology for food / biological samples, and more specifically, to a paper-based enzyme biosensor for hypoxanthine detection and its preparation method. The hypoxanthine paper-based biosensor employs a biomimetic mineralization immobilization enzyme strategy to achieve rapid and visualized detection of hypoxanthine. Background Technology

[0002] Currently, there are various methods for detecting hypoxanthine, such as colorimetric methods, electrochemical methods, and chemiluminescence methods. The publicly disclosed patent technology, "Fabrication and Application of a Paper-Based Chip for Fish Freshness Detection Based on Microfluidic Aggregation," proposes a paper-based enzyme detection scheme. The core technology of this scheme involves layer-by-layer modification of a nitrocellulose membrane (NC membrane) with xanthine oxidase (XOD) and horseradish peroxidase (HRP) and polysaccharides of different molecular weights, achieving visual detection of hypoxanthine through enzymatic catalysis. However, this method has the following significant drawbacks: 1. Complex operation and high cost: The multi-layer modification process of the enzyme is cumbersome, and the large amount of enzyme used as the core signal recognition element significantly increases the preparation and detection costs, especially hindering large-scale detection of actual samples. 2. Poor storage stability: The enzyme activity in the paper-based sensor is easily degraded and must be stored strictly at low temperatures of 4°C or -20°C, resulting in a short shelf life. 3. Susceptible to environmental influences: This sensor is highly sensitive to external environmental factors such as air, humidity, and temperature, and its storage and usage conditions are demanding, which seriously affects its application potential in commercial test strips. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention provides a paper-based enzyme biosensor for hypoxanthine detection and its preparation method. This addresses the problems of excessive enzyme usage and insufficient sensor stability at room temperature in existing technologies.

[0004] This invention is achieved through the following technical solution: a method for preparing a paper-based enzyme biosensor for hypoxanthine detection, specifically including the following steps:

[0005] Step S1, Paper-based chip fabrication: The enzyme solution, silicon precursor solution, and mineralization system solution required for the synthesis of xanthine oxidase silicon enzyme specifically include the following steps:

[0006] Step S1-1: Preparation of xanthine oxidase (XOD) solution: Dissolve XOD in ultrapure water to prepare xanthine oxidase aqueous solution;

[0007] Step S1-2, Preparation of silicon precursor solution: Take tetraethyl orthosilicate (TEOS), propyltriethoxysilane (TEOPS), and (3-aminopropyl)triethoxysilane (APTES), dissolve them in ultrapure water, and vortex mix.

[0008] Step S1-3, Preparation of mineralization system solution: Dissolve xanthine oxidase aqueous solution in silicon precursor solution to form a mixture; place the mixture at 4℃ for 6 hours to obtain silicon enzyme XOD@Si;

[0009] Step S2, Colorimetric System Configuration

[0010] Step S2-1, Preparation of colorimetric solution: Weigh nitrotetrazolium chloride (NBT) powder, dissolve it in ultrapure water, and prepare an NBT solution for later use;

[0011] Step S2-2, Polysaccharide immobilization system solution: accurately dissolve pullulan or seaweed polysaccharide in 1 mL of ultrapure water to prepare pullulan or seaweed polysaccharide solution for later use;

[0012] Step S2-3, Modification solution: Mix the above pullulan polysaccharide or seaweed polysaccharide solution, XOD@Si solution and NBT solution evenly in a volume ratio of 2:3:5;

[0013] Step S2-4, Specific modification process: Cut the nitrocellulose NC membrane into 1 cm * 1 cm squares to serve as the paper base for the chip. Add 2 μL of modification solution to the NC membrane and dry at room temperature for 15 min to complete the modification. Using the same method, up to 3 detection sites can be modified on a single paper base.

[0014] Step S3: Paper-based chip modification and packaging

[0015] Pre-packaging of paper-based chips: Five finished paper-based chips are assembled into a 6 cm*10 cm aluminum foil vacuum packaging bag, and vacuum-sealed using a vacuum machine.

[0016] As a preferred embodiment, the concentration of the xanthine oxidase aqueous solution in step S1-1 is 6~10 mg / mL.

[0017] As a preferred embodiment, in steps S1-2, 10 μL of tetraethyl orthosilicate (TEOS), propyltriethoxysilane (TEOPS), and (3-aminopropyl)triethoxysilane (APTES) are dissolved in 10 mL of ultrapure water in a volume ratio of 4:2:4.

[0018] As a preferred embodiment, in steps S1-3, 9 μL of 10 mg / mL xanthine oxidase aqueous solution is dissolved in 250 μL of silicon precursor solution to form a mixture.

[0019] As a preferred embodiment, in step S2-1, nitrotetrazole blue (NBT) powder is dissolved in 3 mL of ultrapure water to prepare an NBT solution with a concentration of 4-8 mg / mL.

[0020] As a preferred embodiment, in step S2-2, pullulan or seaweed polysaccharide is dissolved in 1 mL of ultrapure water to prepare a pullulan or seaweed polysaccharide solution with a concentration of 6-12 wt%.

[0021] A paper-based enzyme biosensor for hypoxanthine detection is prepared by any of the methods described above.

[0022] By employing the above technical solutions, this invention has the following beneficial effects compared to existing technologies:

[0023] 1. Simplified colorimetric operation and improved stability: Existing technologies for detecting hypoxanthine rely on the synergistic catalysis of xanthine oxidase (XOD) and horseradish peroxidase (HRP) for colorimetric development, which is relatively complex and the stability of the colorimetric system is easily affected. This invention uses xanthine oxidase (XOD) in combination with the colorimetric reagent nitrotetrazole blue chloride (NBT) to achieve one-step colorimetric development, which significantly simplifies the operation process and improves the stability of the colorimetric system. 2. Significantly Enhanced Room Temperature Storage Stability: Existing enzyme biosensors require low-temperature storage (4℃ or -20℃) to maintain activity, limiting their portability and practicality. The enzyme biosensor of this invention, constructed based on a biomimetic mineralization immobilization system, maintains high enzyme activity even after up to 21 days of room temperature storage, demonstrating excellent room temperature storage stability and facilitating practical applications and transportation. 3. Significantly Reduced Enzyme Dosage and Excellent Detection Performance: Compared to existing technologies that typically require high enzyme concentrations for effective detection, this invention successfully detects hypoxanthine while significantly reducing the concentration of xanthine oxidase (XOD), and the detection signal exhibits a good linear relationship with the hypoxanthine concentration. This indicates that this invention significantly reduces enzyme usage, saving costs while maintaining high sensitivity and reliable quantitative detection performance.

[0024] Additional aspects and advantages of the invention will become apparent in the following description or may be learned by practice of the invention. Attached Figure Description

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0026] Figure 1 To plot the detection curves and standard curves of hypoxanthine at different concentrations based on the biomimetic mineralized immobilized enzyme paper-based chip, the linear range was 0.0195-0.625 mM, and the correlation coefficient was 0.9955.

[0027] Figure 2The graph shows the effect of enzyme amount on color development intensity between the biomimetic mineralized enzyme immobilized hypoxanthine paper-based chip and the ordinary hypoxanthine paper-based chip. The amount of enzyme used in the biomimetic mineralized enzyme immobilized hypoxanthine paper-based chip is significantly lower than that in the ordinary hypoxanthine paper-based chip.

[0028] Figure 3 The graph shows the color intensity changes of a biomimetic mineralized immobilized enzyme hypoxanthine paper-based chip and a conventional hypoxanthine paper-based chip stored at room temperature for different times. The stability of the biomimetic mineralized immobilized enzyme hypoxanthine paper-based chip is significantly better than that of the conventional hypoxanthine paper-based chip. Detailed Implementation

[0029] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0030] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0031] The following is combined with Figures 1 to 3 The paper-based enzyme biosensor for hypoxanthine detection and its preparation method according to embodiments of the present invention will be described in detail.

[0032] This invention proposes a method for preparing a paper-based enzyme biosensor for hypoxanthine detection, specifically including the following steps:

[0033] Step S1, Paper-based chip fabrication: The enzyme solution, silicon precursor solution, and mineralization system solution required for the synthesis of xanthine oxidase silicon enzyme specifically include the following steps:

[0034] Step S1-1: Preparation of xanthine oxidase (XOD) solution: Dissolve XOD in ultrapure water to prepare a 6-10 mg / mL xanthine oxidase aqueous solution;

[0035] Step S1-2, Preparation of silicon precursor solution: Take 10 μL of tetraethyl orthosilicate (TEOS), propyltriethoxysilane (TEOPS), and (3-aminopropyl)triethoxysilane (APTES) in a volume ratio of 4:2:4, dissolve them in 10 mL of ultrapure water, and vortex mix.

[0036] Steps S1-3: Preparation of mineralization system solution: Dissolve 9 μL of 10 mg / mL xanthine oxidase aqueous solution in 250 μL of silicon precursor solution to form a mixture; place the mixture at 4℃ for 6 hours to obtain silicon enzyme XOD@Si;

[0037] Step S2, Colorimetric System Configuration

[0038] Step S2-1, Preparation of colorimetric solution: Weigh nitrotetrazolium chloride (NBT) powder and dissolve it in 3 mL of ultrapure water to prepare an NBT solution with a concentration of 4-8 mg / mL for later use;

[0039] Step S2-2, Polysaccharide immobilization system solution: accurately dissolve pullulan or seaweed polysaccharide in 1 mL of ultrapure water to prepare a pullulan or seaweed polysaccharide solution with a concentration of 6~12wt% for later use;

[0040] Step S2-3, Modification solution: Mix the above pullulan polysaccharide or seaweed polysaccharide solution, XOD@Si solution and NBT solution evenly in a volume ratio of 2:3:5;

[0041] Step S2-4, Specific modification process: Cut the nitrocellulose NC membrane into 1 cm * 1 cm squares as the paper base of the chip, add 2 μL of modification solution to the NC membrane, and dry at room temperature for 15 min to complete the modification; In the same way, a maximum of 3 detection sites can be modified on a single paper base; Unless otherwise specified, the paper base modification method in subsequent experiments shall be carried out in accordance with this step.

[0042] Step S3: Paper-based chip modification and packaging

[0043] Pre-packaging of paper-based chips: Five finished paper-based chips are assembled into a 6 cm*10 cm aluminum foil vacuum packaging bag, and vacuum-sealed using a vacuum machine.

[0044] A paper-based enzyme biosensor for hypoxanthine detection was prepared by the method described above.

[0045] Plotting the standard curve for hypoxanthine

[0046] Under optimized conditions, we applied this method to the detection of hypoxanthine. The concentration gradient of hypoxanthine was set to 0.01-5.0 mM. The results were scanned, and images were acquired using a Canon scanner. A circular region (35 x 35 mm wide) was selected at the detection location using ImageJ software. The cropped image was then analyzed to obtain the average intensity of the RGB channels. The Euclidean distance (D) was calculated using the RGB values.

[0047]

[0048] The results were plotted as a standard curve, and the detection limit and linear range were calculated.

[0049] The experimental results were obtained by collecting images using mobile phones of different brands and processing the results to calculate the detection limit and linear range.

[0050] Comparison of color development on paper-based chips at different enzyme concentrations

[0051] A concentration gradient of XOD@Si enzyme and a concentration gradient of XOD enzyme were set up from 6 U / mL to 18 U / mL. The concentrations of pullulan or seaweed polysaccharide were consistently maintained at 6–12 wt%, and the concentration of NBT was consistently maintained at 4–8 mg / mL. A paper-based biosensor was prepared, and 100 μL of a 1.25 mM hypoxanthine solution was added to the paper-based chip. The image was scanned using a scanner, and color intensity analysis was performed.

[0052] Storage stability test

[0053] Several paper-based sensors were prepared and placed in light-proof sealed bags. Vacuum sealing and heat sealing were performed using a vacuum sealer. The sensors were stored in groups of three at room temperature. Every so often, 1.25 mM hypoxanthine was added to each sensor. After the color development stabilized, the paper-based chips were scanned and the data were processed.

[0054] Research Results

[0055] 1. Standard Curve

[0056] The prepared paper-based chip was used to detect hypoxanthine at concentrations ranging from 0.01 mM to 5.0 mM, with water as a blank control. The experimental results are shown in the figure. The color of the paper-based chip gradually deepened and reached its maximum within 10 minutes, allowing for easy preliminary signal interpretation by the naked eye. The color signal was acquired using a Canon scanner, and the average RGB values ​​of the captured color area were read using ImageJ software. It can be seen that the color intensity increases with increasing hypoxanthine concentration; however, the increase in color intensity gradually leveled off after the hypoxanthine concentration reached 0.625 mM. Specifically, the standard curve showed good linearity (R² = 0.992) when the hypoxanthine concentration was between 0.0195 and 0.625 mM, and the calculated detection limit was 6.9 μM, indicating a low detection limit.

[0057] 2. Color development of paper-based biosensors at different enzyme concentrations

[0058] A major advantage of the paper-based biosensor developed using this method is its ability to save on enzyme dosage. By setting XOD@Si enzyme concentration gradients and XOD enzyme concentration gradients of 6 U / mL–18 U / mL, and controlling the PuL concentration at 8 wt% and the NBT concentration at 8 mg / mL, the paper-based biosensor was prepared. 100 μL of a 1.25 mM hypoxanthine solution was added to the paper-based chip, and the image was scanned and color intensity analyzed. The experimental results are shown in the figure. Under each enzyme concentration gradient, the color development level of the paper-based biosensor based on the biomimetic immobilization strategy was higher than that of the natural enzyme paper-based biosensor. Specifically, when the XOD@Si concentration was 12 U / mL, the color development level of the paper-based biosensor based on the biomimetic immobilization strategy was twice that of the natural enzyme paper-based biosensor, demonstrating a significant effect in saving enzyme dosage.

[0059] 3. Storage stability

[0060] A major advantage of the paper-based biosensor developed using this method is its excellent room-temperature storage stability. Several paper-based sensors were prepared and placed in light-proof sealed bags. These bags were then vacuum-sealed and heat-sealed, and stored in groups of three at room temperature. Periodically, 1.25 mM hypoxanthine was added to each sensor. After the color development stabilized, the paper-based chip was scanned and data processed. The experimental results are as follows: after 7 days of room-temperature storage, the silicon enzyme paper-based biosensor still maintained 85% of its color intensity; after 21 days of room-temperature storage, the silicon enzyme paper-based biosensor still maintained 67% of its color intensity.

[0061] In the description of this invention, the term "a plurality of" refers to two or more. Unless otherwise explicitly defined, the terms "upper," "lower," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. The terms "connection," "installation," "fixing," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection or an indirect connection through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0062] In the description of this specification, the terms "one embodiment," "some embodiments," "specific embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0063] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., 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 method for preparing a paper-based enzyme biosensor for hypoxanthine detection, characterized in that... Specifically, it includes the following steps: Step S1, Paper-based chip fabrication: The enzyme solution, silicon precursor solution, and mineralization system solution required for the synthesis of xanthine oxidase silicon enzyme specifically include the following steps: Step S1-1: Preparation of xanthine oxidase (XOD) solution: Dissolve XOD in ultrapure water to prepare xanthine oxidase aqueous solution; Step S1-2, Preparation of silicon precursor solution: Take 10 μL of tetraethyl orthosilicate (TEOS), propyltriethoxysilane (TEOPS), and (3-aminopropyl)triethoxysilane (APTES) in a volume ratio of 4:2:4, dissolve them in 10 mL of ultrapure water, and vortex mix. Step S1-3, Preparation of mineralization system solution: Dissolve xanthine oxidase aqueous solution in silicon precursor solution to form a mixture; place the mixture at 4℃ for 6 hours to obtain silicon enzyme XOD@Si solution; Step S2, Colorimetric System Configuration Step S2-1, Preparation of colorimetric solution: Weigh nitrotetrazolium chloride (NBT) powder, dissolve it in ultrapure water, and prepare an NBT solution for later use; Step S2-2, Polysaccharide Immobilization System Solution: Accurately weigh pullulan, dissolve it in 1 mL of ultrapure water, and prepare a pullulan solution for later use; Step S2-3, Modification solution: Mix the pullulan polysaccharide solution, XOD@Si solution and NBT solution in a volume ratio of 2:3:5 until homogeneous; Step S2-4, Specific modification process: Cut the nitrocellulose NC membrane into 1 cm * 1 cm squares as the paper base of the chip, add 2 μL of modification solution to the NC membrane, and dry at room temperature for 15 min to complete the modification; following the same method, a maximum of 3 detection sites can be modified on a single paper base; Step S3: Paper-based chip modification and packaging Pre-packaging of paper-based chips: Five finished paper-based chips are assembled into a 6 cm*10 cm aluminum foil vacuum packaging bag, and vacuum-sealed using a vacuum machine.

2. The method for preparing a paper-based enzyme biosensor for hypoxanthine detection according to claim 1, characterized in that... In step S1-1, the concentration of xanthine oxidase aqueous solution is 6~10 mg / mL.

3. The method for preparing a paper-based enzyme biosensor for hypoxanthine detection according to claim 1, characterized in that... In steps S1-3, 9 μL of 10 mg / mL xanthine oxidase aqueous solution is dissolved in 250 μL of silicon precursor solution to form a mixture.

4. The method for preparing a paper-based enzyme biosensor for hypoxanthine detection according to claim 1, characterized in that... In step S2-1, nitrotetrazolium chloride (NBT) powder is dissolved in 3 mL of ultrapure water to prepare an NBT solution with a concentration of 4-8 mg / mL.

5. The method for preparing a paper-based enzyme biosensor for hypoxanthine detection according to claim 1, characterized in that... In step S2-2, pullulan is dissolved in 1 mL of ultrapure water to prepare a pullulan solution with a concentration of 6-12 wt%.

6. A paper-based enzyme biosensor for hypoxanthine detection, characterized in that, It is prepared by the method described in any one of claims 1 to 5.

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