Preparation and application of rotary splicing paper-based enzymolysis device

Abstract

The invention discloses preparation and application of a rotary splicing paper-based enzymolysis device, and belongs to the technical field of micro-fluidic paper chips. The method comprises the following steps: firstly, fixing nanogold on a micro-fluidic paper chip, and then assembling enzyme molecules on the surface of the nanogold modified by concanavalin A, thereby forming a directional enzyme bonding-nanogold mediated bioreactor, namely a micro-enzyme reactor. According to the micro-enzyme reactor, due to the high surface area of the nanoparticles and a directional immobilization strategy, the performance of the enzyme is expected to exceed that of a bioreactor prepared by a random bonding method. In addition, the enzyme is in butt joint with or separated from the color developing device in a rotary splicing manner, so that the operation convenience and the reusability of the enzyme are improved. The rotary splicing paper-based enzymolysis device designed by the invention is simple and convenient to operate and low in enzyme consumption, does not depend on a large-scale instrument, can be used for screening acetylcholin esterase inhibitors and tracing the activity of traditional Chinese medicinal materials, and provides a new device for drug screening.

Description

Technical Field

[0001] This invention relates to the field of microfluidic paper chip technology, and in particular to the preparation and application of a rotating splicing paper-based enzymatic hydrolysis device. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease, and patients typically experience symptoms such as memory decline, learning difficulties, mood disorders, and motor impairment. Currently, the first-line drugs for treating AD are primarily acetylcholinesterase inhibitors (AChEIs). These drugs indirectly increase the level of acetylcholine (ACh) in the brain of AD patients by inhibiting the activity of AChE in the brain, thereby improving learning and cognitive function, promoting memory recall, and slowing the progression of AD. However, these drugs have poor selectivity; while exerting their therapeutic effect, they also inhibit peripheral AChE, producing varying degrees of side effects, such as diarrhea, nausea, vomiting, and other gastrointestinal reactions. Therefore, screening for new AChEIs for the treatment of AD is particularly important.

[0003] Currently, the most commonly used method for AChEI screening is the Ellman method based on ultraviolet spectrophotometer. In addition, fluorescence spectrophotometry, radioisotope method, thin-layer chromatography, electrochemical method, high-performance liquid chromatography and capillary electrophoresis are also commonly used for enzyme inhibitor screening. However, these methods have disadvantages such as complicated operation, long time consumption and large sample volume, and are not suitable for rapid on-site screening or preliminary screening in resource-limited environments.

[0004] Microfluidic paper-based analytical devices (μPADs) are an emerging technology that uses paper as a substrate to control fluids through microchannels on the paper. The main principle is to specially treat different regions of paper fibers to give them different properties, thereby enabling the control and transport of different types and quantities of liquids. Due to its good biocompatibility, large specific surface area, ease of modification, and low cost, it shows broad application prospects in environmental monitoring, chemical detection, and biomedical diagnostics. Microfluidic paper-based analytical devices can be applied to various areas of enzyme research, such as the identification of substrates, inhibitors, and activators; enzyme-catalyzed metabolic pathways; enzyme activity assessment; and enzyme kinetics studies. However, currently, AChE microreactors are generally prepared using arbitrary bonding methods, which suffers from problems such as low AChE bonding amount and the random arrangement of AChE leading to the embedding of enzyme active sites and reduced activity. Summary of the Invention

[0005] The purpose of this invention is to provide a preparation and application of a rotating splicing paper-based enzymatic hydrolysis device to solve the problems existing in the prior art. By designing a novel rotating-sponge paper chip, after the enzymatic hydrolysis reaction is completed, the color development area only needs to be connected to the enzyme reactor, and the reaction solution can flow into the color development device under the capillary action of the paper and separate from the enzyme. No terminator needs to be added, the operation is simple, and the enzyme reactor can be reused, reducing the amount of enzyme used. This provides a new device for screening acetylcholinesterase inhibitors.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a rotating splicing paper-based enzymatic hydrolysis device, including an enzyme fixation device and a color development device, wherein the enzyme fixation device is rotatably adjustable relative to the color development device;

[0008] The enzyme immobilization device includes a disc I loaded with chitosan, gold nanoparticles, and concanavalin A, with acetylcholinesterase directionally bonded to it. A microfluidic channel I is located on the side of the disc I closest to the colorimetric device. The colorimetric device is a paper chip with at least eight independent colorimetric regions arranged circularly on the paper chip, with equidistant spacing between adjacent regions. A microfluidic channel II communicating with each independent colorimetric region is provided on the paper chip. The paper chips are evenly spaced along the circumference of the enzyme immobilization device, and each colorimetric region of the paper chip communicates with the outside through the microfluidic channel II. Spatial docking or separation of the disc I and the independent colorimetric regions is achieved through the docking or separation of the microfluidic channels I and II, enabling the reuse of the enzyme immobilization device.

[0009] Preferably, a column extends through the end of the enzyme immobilization device, and the independent color development areas are evenly spaced along the circumference of the column.

[0010] Preferably, after the enzyme immobilization device and the color development device are designed, a wax printer is used to print wax onto the qualitative filter paper, and then a hot press is used to heat the paper so that the wax melts and forms a hydrophobic barrier.

[0011] Preferably, the diameter of the disk I is 6-7 mm, and the length and width of the microfluidic channel I are set to (2-3) mm × 1 mm;

[0012] The independent color development area is a circular paper chip with a diameter of 4-5 mm, and the length and width of the microfluidic channel II are set to (6-7) mm × 1 mm.

[0013] Preferably, when the microfluidic channel I is connected to the microfluidic channel II, the enzymatic hydrolysis product is transferred from the disk I to the independent color development area under capillary action to complete the color development reaction, and the result is read by RGB colorimetry.

[0014] In the above technical solution, the enzyme immobilization device corresponds to Figure 1 Part R shown in (a) corresponds to the colorimetric device. Figure 1 Part D is shown in (a). In Part R, disk I is a 6mm diameter paper chip used to load chitosan, gold nanoparticles, concanavalin A, and directionally bind acetylcholinesterase. Another 5mm diameter paper chip (named disk II) is fixed at one end of the enzyme immobilization device by passing a stainless steel column through its center. By rotating the enzyme immobilization device around the column, disk I can be rotated so that it can connect to microfluidic channels II (1mm x 6.5mm) on different color development areas of the color development device through microfluidic channel I (length x width = 1mm x 6mm). The base of the entire enzyme immobilization device can be rectangular, with a length x width of 24mm x 10mm. The two disks are fixed to the rectangular base using a hot press (the base can be a rectangular NO.1 qualitative filter paper).

[0015] In PartD, the paper chip can be configured as a square base with a side length of 54mm. Each independent color development area is arranged in a circular shape at equal intervals on the paper chip, which facilitates docking with the enzyme immobilization device and prevents different independent color development areas from interfering with each other. Each independent color development area is a circular paper chip structure (named disk III), with a diameter of 1mm. A rectangular microfluidic channel II is set on one side of the disk III near the center of the square base. The length × width of the microfluidic channel II is 1mm × 5mm.

[0016] When using this device, rotating the column aligns disk I and the independent colorimetric zone through microfluidic channels I and II, completing the preparation of the overall rotating and splicing paper-based enzymatic digestion apparatus required for one sample colorimetric detection. After sample is added to disk I, the enzymatic digestion reaction begins. The reaction products flow sequentially through microfluidic channels I and II before entering the independent colorimetric zone. Visually, once the independent colorimetric zone is filled with the enzymatic digest, microfluidic channels I and II can be separated, thus enabling sample detection. If other samples need to be detected, the column is rotated again, aligning disk I and the remaining independent colorimetric zones through microfluidic channels I and II, thus recombining the detection process. This device allows for the simultaneous detection of multiple samples, and the enzyme reactor can be reused, saving enzyme consumption and resource utilization.

[0017] The present invention also provides a method for preparing an enzyme immobilization device, comprising the following steps:

[0018] (1) On disk I in the enzyme immobilization device, a chitosan solution is loaded and dried to introduce amino groups;

[0019] (2) Load the nano-gold solution onto the same area, incubate, and then wash;

[0020] (3) Load the same area with concanavalin A solution, incubate, and then wash;

[0021] (4) Block non-specific binding sites with bovine serum albumin solution, incubate, and then wash;

[0022] (5) Load acetylcholinesterase solution onto the same area, incubate and wash to complete directional fixation, and obtain a microenzyme reactor, which is an enzyme fixation reaction device.

[0023] Preferably, the volume ratio of the chitosan solution, the gold nanoparticle solution, the concanavalin A solution, the bovine serum albumin solution, and the acetylcholinesterase solution is 2:2:5:5:5.

[0024] Preferably, in step (2), the concentration of the gold nanoparticle solution is 5-100 pmol / L, and the incubation time is 60-110 min;

[0025] In step (3), the incubation time is 30-140 min;

[0026] In step (5), the concentration of the acetylcholinesterase solution is 0.10-10.00 mmol / L, and the incubation time is 30-60 min.

[0027] The present invention also provides the application of the aforementioned rotating splicing paper-based enzymatic hydrolysis device in screening acetylcholinesterase inhibitors or screening Alzheimer's disease drugs.

[0028] The present invention also provides the application of the aforementioned rotating splicing paper-based enzymatic hydrolysis device in the activity tracing of traditional Chinese medicinal materials.

[0029] The present invention discloses the following technical effects:

[0030] This invention proposes an innovative strategy that combines the directional immobilization of gold nanoparticles (AuNPs) with enzymes, followed by colorimetric detection to evaluate the inhibitory effects of different compounds on acetylcholinesterase (AChE). The novelty of this system lies in first immobilizing AuNPs on a microfluidic paper chip (μPAD), and then assembling enzyme molecules onto the surface of Con A-modified AuNPs. This forms a bioreactor mediated by directional enzyme bonding. The basic principle of this design is that, due to the high surface area of ​​the nanoparticles and the directional immobilization strategy, the enzyme performance is expected to surpass that of bioreactors prepared by random bonding methods. Furthermore, the rotational splicing design improves operational convenience and enhances enzyme reusability. By rotating and adjusting the position of the enzyme immobilization device (paper chip R), it can be brought into contact with or separated from the various detection areas of the colorimetric device (paper chip D), thereby enabling different inhibition rate measurements.

[0031] Compared to directed enzyme reactors that do not use AuNPs particles, AuNPs-mediated enzyme reactors are more environmentally friendly as they do not require the use of organic solvents (such as glutaraldehyde). Furthermore, this reactor exhibits stronger bioactivity, reproducibility, and stability, ensuring accurate and reliable inhibitor screening. This system is suitable for activity tracing in the isolation of traditional Chinese medicines; visual observation of color changes allows for a preliminary assessment of the necessity for further separation and purification. In addition, μPADs can be effectively used to determine the IC50 of each purified component. 50 Value. Because there are eight independent detection zones, only one paper chip D is needed to obtain the inhibitor's IC. 50 This device has the potential to revolutionize the screening and evaluation process of enzyme inhibitors, paving the way for the development of more efficient and effective drug discovery methods. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 A schematic diagram of the rotating-splitting paper-based enzymatic hydrolysis device (a) and the preparation process of the enzymatic hydrolysis reaction paper chip R (b).

[0034] Figure 2 The experimental principle (a) and preparation method (b) of the nanoparticle-paper-based enzyme reactor;

[0035] Figure 3Scanning electron microscope images of paper chips; (a) blank paper chip, (b) paper chip with AuNPs bonded, (c) paper chip with AChE bonded;

[0036] Figure 4 The effects of nano-gold bonding time (a), Con A bonding time (b), and nano-gold concentration (c) on the color development effect;

[0037] Figure 5 Results of feasibility, reproducibility, and stability determination of the microenzyme reactor; (a) enzyme bond concentration determination, (b) ATCh concentration, (c) microenzyme reactor reproducibility determination results, (d) microenzyme reactor stability determination results, (e) enzyme inhibition kinetics study results, (f) IC50 of donepezil hydrochloride. 50 Measurement results;

[0038] Figure 6 The column chromatography of crude Coptis chinensis extract (a) and the analysis process and colorimetric results of the eluent on a paper-based enzymatic hydrolysis device (b). Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] This invention prepares a novel directional immobilized AChE microenzyme reactor for screening AChEI-type anti-AD drugs. Specifically, this invention achieves directional bonding of AChE through the specific binding between concanavalin A (Con A) and AChE glycosyl groups, ensuring that the active sites of AChE are uniformly facing outwards and more regularly arranged on the reaction platform, increasing the activity and stability of AChE. Simultaneously, leveraging the large specific surface area of ​​gold nanoparticles (AuNPs), the amount of AChE bonding is increased, further enhancing the enzymatic reaction rate. To achieve enzyme reuse, this invention also designs a novel rotation-and-joint paper chip. After the enzymatic reaction, simply connect the chromogenic zone to the enzyme reactor; the reaction solution flows into the detection zone under the capillary action of the paper and separates from the enzyme, eliminating the need for a terminator and simplifying the operation. This enzyme reactor is reusable, reducing enzyme consumption. A chromogenic agent is added to the chromogenic zone for color development, and the results are recorded by taking a photo with a mobile phone. Enzyme activity is evaluated based on the colorimetric values. This analytical method does not rely on large instruments and is quick and convenient to operate. Finally, the directional immobilized AChE microreactor was used to evaluate the inhibitory activity of several AChEIs. The above technical solution is further illustrated below with specific embodiments.

[0045] (1) The main instruments and equipment involved in the following embodiments

[0046] 0.0001 ppm electronic balance (Gravimetrics AG Dietikon Switzerlang); wax spray printer (Xerox Phaser 8570, Fuji Xerox); hot press (G311, Fujian Aples Electric Co., Ltd.); ultraviolet spectrophotometer (UV-2102PCS, UNIC); scanning electron microscope (VEGA II, TESCAN, Czech Republic); pipette; Android phone (Huawei nova7, Shenzhen Zhixin New Information Technology Co., Ltd.).

[0047] (2) The main reagents and materials involved in the following examples

[0048] Acetylcholinesterase (AChE, fly head, 200 U / g, Shanghai Yuanye Biotechnology Co., Ltd.), acetylthiocholine iodide (ATCh, ≥98%, Shanghai Maclean Biochemical Co., Ltd.), chitosan (CS, ≥95.0% deacetylated, Shanghai Aladdin Reagent Co., Ltd.), concanavalin A (Con A, Jinan Biotechnology Co., Ltd., China), bovine serum albumin (BSA, ≥98%, Shanghai Aladdin Reagent Co., Ltd.), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, ≥98%, Shanghai Aladdin Reagent Co., Ltd.), tris(hydroxymethyl)aminomethane (Tris, analytical grade, Guangzhou Jinhua Chemical Reagent Co., Ltd.), donepezil hydrochloride (≥98%, Shanghai Maclean Biochemical Co., Ltd.), hydrochloric acid (analytical grade, Tianjin Hedong District Hongyan Reagent Factory), gastrodin (≥98%, Shanghai Yuanye Biotechnology Co., Ltd.), ginsenosides (≥98%, Shanghai Yuanye Biotechnology Co., Ltd.), deionized water (Xianyang Xiquan Pure Water Co., Ltd.), phosphate buffer solution (PBS, 10 mmol / L, pH 7.2~7.4, Biosharp BL302A); gold nanoparticles (AuNPs) were prepared in the laboratory (for preparation methods, please refer to the literature "Screening of Acetylcholinesterase Inhibitors by Capillary Electrophoresis with Oriented-Immobilized Enzyme Microreactors Based on Gold Nanoparticles"); NO.1 qualitative filter paper (Whatman No.1, Maidstone, England).

[0049] (3) The solution preparation methods involved in the following examples

[0050] 0.25 mg / mL CS solution: Accurately weigh 12.50 mg of CS, dissolve it in 0.05 mol / L hydrochloric acid, and dilute to 50 mL in a volumetric flask. Store under cold conditions.

[0051] AuNPs solution: Accurately pipette 0.1 mL of the prepared AuNPs stock solution, dilute with PBS buffer (10 mmol / L, pH 7.2~7.4), and bring the volume up to 10 mL in a volumetric flask. Store under cold conditions.

[0052] 20 mmol / L Tris-HCl buffer solution: Accurately weigh 60.57 mg of Tris, dissolve in deionized water, dilute to 25 mL in a volumetric flask, adjust the pH to 7.5 with 1 mol / L HCl solution, and store under cold conditions.

[0053] 10 U / mL AChE solution: Accurately weigh 50.00 mg of AChE, dissolve it in Tris-HCl (20 mmol / L, pH 7.5) buffer, aliquot it into centrifuge tubes (60 μL / tube), and store frozen.

[0054] 10 mmol / L acetylcholinesterase substrate (ATCh) solution: Accurately weigh 28.92 mg of ATCh, dissolve in deionized water, and dilute to 10 mL in a volumetric flask. Store frozen and protected from light.

[0055] 4 mg / mL DTNB solution: Accurately weigh 40.00 mg of DTNB, dissolve it in PBS buffer solution (10 mmol / L, pH 7.2~7.4) and dilute to 10 mL in a volumetric flask. Store in the refrigerator protected from light.

[0056] 1.1 mg / mL ConA solution: Accurately weigh 27.50 mg of ConA, dissolve it in PBS buffer (10 mmol / L, pH 7.2~7.4), and dilute to 25 mL in a volumetric flask. Store frozen.

[0057] 5 mg / mL BSA solution: Accurately weigh 50.00 mg of bovine serum albumin, dissolve it in PBS buffer (10 mmol / L, pH 7.2~7.4), and dilute to 10 mL in a volumetric flask. Store frozen.

[0058] 1 mg / mL Ginsenoside Stock Solution: Accurately weigh 10.00 mg of ginsenoside, dissolve in deionized water, and dilute to a 10 mL volumetric flask. Store under cold conditions.

[0059] 2 mg / mL donepezil hydrochloride stock solution: Accurately weigh 20.00 mg of donepezil, dissolve it in deionized water, and make up to 10 mL in a volumetric flask. Store in a cool, dark place.

[0060] 1 mg / mL Gastrodin stock solution: Accurately weigh 10.00 mg of gastrodin, dissolve it in deionized water, and dilute to a volumetric flask of 10 mL. Store under cold conditions.

[0061] Example 1: Preparation of a nanoparticle-paper-based enzyme reactor

[0062] 1. Paper chip fabrication

[0063] A novel rotating paper chip was designed using Inkscape-0.91 software. Figure 1 (a), by enzyme immobilization device ( Figure 1The device consists of two parts: b) and the colorimetric apparatus. The enzyme immobilization apparatus consists of a 5 mm diameter disk and a 6 mm diameter disk (with 2 mm × 1 mm channels), with the two disks 0.8 cm apart. AChE is bonded to the 6 mm diameter disk. A stainless steel column passes through the 5 mm diameter disk to fix the enzyme reactor. The position of the enzyme reactor can be rotated to connect with different colorimetric areas. This part is reusable. The colorimetric apparatus is fixed to the base by stainless steel column and consists of 8 independent colorimetric areas, each with a diameter of 5 mm, all connected to 1 mm × 6.5 mm channels. This part is not reusable; a new colorimetric apparatus must be replaced after all 8 areas have been colorimetric. After designing the paper chip, wax is printed onto NO.1 qualitative filter paper using a wax spray printer. Then, a hot press is used to heat the paper at 176.7°C for 120 s, causing the wax to melt and pass through the paper, forming a hydrophobic barrier.

[0064] 2. Preparation of nanoparticle-paper-based enzyme reactor

[0065] like Figure 2 As shown in Figure a, the detection principle of this invention is as follows: Acetylcholinesterase (AChE) can decompose acetylthiocholine iodide (ATCh) into thiocholine (TCh), and TCh can undergo a colorimetric reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The color intensity on the paper chip can be represented by RGB values ​​in Photoshop software. The RGB values ​​are linearly related to the amount of TCh generated; the more TCh generated, the larger the RGB value.

[0066] Preparation method of nanoparticle-paper-based enzyme reactor ( Figure 2 In step b), 2.0 μL of chitosan solution (0.25 mg / mL) was added to a 6 mm disc in the enzyme immobilization device and dried at room temperature for 5 min to introduce amino groups; 2.0 μL of 83.2 pmol / L AuNPs solution (particle size 12.23 ± 0.31 nm) was added and incubated with chitosan at room temperature for 60 min; the mixture was washed three times with PBS buffer (10 mmol / L, pH 7.2–7.4); 5.0 μL of Con A solution (1.1 mg / mL) was added and incubated at room temperature for 60 min to bind to AuNPs on the paper chip; 5.0 μL of BSA solution (5 mg / mL) was added and incubated at room temperature for 15 min to block non-specific binding sites; the mixture was washed three times with PBS buffer (10 mmol / L, pH 7.2–7.4); 5.0 μL of AChE solution (10 U / mL) was added and incubated at room temperature for 30 min to bind to Con A. The specific affinity between the sugar groups of A and AChE allows the enzyme to be directionally immobilized on the paper surface, thus preparing a microenzyme reactor.

[0067] This microenzyme reactor is stored frozen in a refrigerator. When needed, it can be taken out of the refrigerator and washed three times with PBS buffer solution (10 mmol / L, pH 7.2~7.4) before use.

[0068] Method for Assay of Immobilized AChE Enzyme Activity ( Figure 2 (b) Add 6.0 μL of 2.5 mmol / L LATCH solution to the microenzyme reactor and react for 6 min. Then, connect the channel of the microenzyme reactor to the channel of the colorimetric device. The hydrolysate flows into the colorimetric area of ​​the colorimetric device through capillary action on the paper. After the hydrolysate fills the colorimetric area, raise the microenzyme reactor to separate it from the colorimetric device. Add 1.2 μL of DTNB solution (4 mg / mL) to the colorimetric area and react for 40 s. Then, place the colorimetric device in a homemade photo box and take a picture using the original camera of a Huawei nova7 smartphone. Use Photoshop software to identify the RGB values ​​of the colorimetric area.

[0069] The surface morphology of blank paper chips, paper chips bonded with AuNPs, and paper chips bonded with AChE were characterized using scanning electron microscopy (SEM). The surface of the blank paper chip exhibited a distinct porous structure. Figure 3 (a) After bonding AuNPs, the porosity of the porous structure on the paper chip surface is reduced ( Figure 3 (b) After bonding AChE, the porosity of the porous structure on the paper chip surface is further reduced. Figure 3 (c) The surface AChE was successfully fixed to the paper surface.

[0070] Example 2: Effect of AuNPs bonding time on RGB color rendering intensity

[0071] AuNPs can bind to the amino groups in chitosan molecules on the paper chip surface, thus fixing them onto the paper chip surface. The reaction time directly affects the amount of AuNP bonding, thereby affecting the subsequent bonding amounts of Con A and AChE. Therefore, this embodiment examines the effect of AuNP bonding time on the RGB color rendering intensity.

[0072] Following the "Preparation Method of Nanoparticle-Paper-Based Enzyme Reactor" in Example 1, the incubation time of AuNPs solution and chitosan at room temperature was adjusted to 60 min, 70 min, 80 min, 90 min, 100 min, and 110 min, respectively. All other steps were the same as in Example 1, in order to investigate the effect of AuNPs bonding time (60-100 min) on the color intensity RGB.

[0073] The results are as follows Figure 4As shown in Figure a, the color intensity gradually increases with increasing reaction time, reaching its maximum at 80 min. Further extending the reaction time actually decreases the RGB value. This may be because as the reaction time increases, the amount of AuNPs bonded to the paper chip surface increases, leading to an increase in AChE bonds and thus a higher RGB value. However, when there are too many AuNPs bonds, they overlap, reducing the specific surface area for AuNPs to bind with Con A, thereby decreasing the amount of AChE bonds and the RGB value. Therefore, the optimal AuNPs bonding time in this invention is 80 min for the subsequent preparation of the microenzyme reactor.

[0074] Example 3

[0075] Con A binds to AuNPs on the paper chip surface via its amino groups, thus immobilizing the Con A on the paper surface. The reaction time between these two reactions affects the amount of Con A modified on the paper surface, thereby influencing the amount of AChE bonded. Therefore, this embodiment examines the effect of Con A bonding time on the RGB color rendering intensity.

[0076] Following the "Preparation Method of Nanoparticle-Paper-Based Enzyme Reactor" in Example 1, the room temperature incubation time after adding Con A solution was adjusted to 30 min, 40 min, 45 min, 60 min, 80 min, 100 min, 120 min and 140 min respectively. All other steps were the same as in Example 1, in order to investigate the effect of Con A bonding time (60-100 min) on the color intensity RGB.

[0077] The results are as follows Figure 4 As shown in Figure b, the color intensity reaches its maximum at a reaction time of 45 min, indicating that the Con A fixation amount reaches saturation at this point. Further extending the bonding time results in a decrease in color intensity. This may be because excessively long cross-linking times lead to overcrowding of Con A molecules, affecting their directional alignment and ultimately causing mutual embedding of AChE active sites and reduced activity. Therefore, the optimal Con A fixation bonding time in this invention is 45 min for subsequent microenzyme reactor preparation.

[0078] Example 4

[0079] Due to their larger specific surface area, the addition of AuNPs can increase the specific binding amount of Con A to AChE. Therefore, this embodiment investigates the effect of AuNP concentration on the RGB color rendering intensity.

[0080] Following the "Preparation Method of Nanoparticle-Paper-Based Enzyme Reactor" in Example 1, the concentration of AuNPs was adjusted to 5-60 pmol / L, and the concentration was gradually increased. Other methods and steps were the same as in Example 1 to investigate the effect of AuNPs concentration on the RGB color development intensity.

[0081] The results are as follows Figure 4 As shown in Figure c, the color intensity reaches its maximum at an AuNPs concentration of 11.7 pmol / L, indicating that AChE can achieve high-density bonding of AuNPs at this concentration. Therefore, 11.7 pmol / L AuNPs were used for the subsequent preparation of the microreactor.

[0082] Example 5

[0083] The effect of AuNPs on AChE binding capacity: Proteins with aromatic rings exhibit a UV absorption peak at 280 nm; therefore, a significant absorption peak for AChE can be observed at 280 nm. The absorbance values ​​of the elution buffer were converted into enzyme concentration using a standard curve, thereby calculating the enzyme binding capacity. Experimental results show ( Figure 5 In section a), within the concentration range of 0.04–0.2 U / mL, the absorbance (Abs) showed a good linear relationship with the AChE concentration. The fitted equation was y = 3.1080x + 0.1292 (R²). 2 It is 0.99677.

[0084] The UV absorption spectra of the enzyme eluent from the gold nanoparticle-oriented immobilized microenzyme reactor (the AuNP-added nanoparticle-paper-based enzyme reactor prepared in Example 1) and the gold nanoparticle-non-oriented immobilized microenzyme reactor (the nanoparticle-paper-based enzyme reactor prepared according to Example 1 without AuNPs) were measured using a UV spectrophotometer. AChE showed a significant absorption peak at 280 nm; the more enzyme eluted, the higher the absorbance, indicating a higher enzyme concentration in the eluent. In this invention, eight 6 mm diameter paper chips R were eluted, and the eluent was collected. The absorbance was measured using a UV spectrophotometer. The results (Table 1) show that the enzyme bonding amount per unit area after using AuNPs was 3.4 times that without AuNPs.

[0085] Table 1 Enzyme bond quantity data

[0086]

[0087] Example 6

[0088] Following the "Assay of Immobilized AChE Enzyme Activity" procedure in Example 1, the ATCh concentration was adjusted from 0.10 mmol / L to 10.00 mmol / L. Other procedures were the same as in Example 1. The effect of ATCh concentration on the colorimetric effect was then determined.

[0089] Michaelis-Menten binding constant (K) m The maximum rate (Vc) of an enzyme reaction is an important parameter reflecting the enzyme's affinity for its substrate; its value is inversely proportional to the affinity.max The Kx value reflects the enzyme's activity and is directly proportional to its activity. The Kx value of a microenzyme reactor... m and V max Determined by the Michaelis-Menten equation (3), where V represents the initial rate of the enzymatic reaction and [S] represents the substrate concentration:

[0090] ;

[0091] (Equation 1)

[0092] Fit the Michaelis-Menten curve using Origin ( Figure 5 (b) The Michaelis-Menten plot showed that the RGB colorimetric intensity increased as the ATCh concentration increased from 0.10 mmol / L to 10.00 mmol / L. Nonlinear regression using the Michaelis-Menten plot determined the Ki of the immobilized AChE. m and V max Value, K m =(0.051±0.001) mmol / L, V max =(19.37±0.243) mmol / L / min. This indicates that the microenzyme reactor prepared in this invention has high activity and strong affinity for the substrate, possibly due to the directional immobilization of Con A accelerating the diffusion of substrate and product into and out of the active site. The higher reactivity may be due to the addition of AuNPs increasing the loading of AChE, while the addition of Con A avoids the embedding of AChE active sites.

[0093] Example 7: Stability and Repeatability Study of Microenzyme Reactor

[0094] On the same day, 6.0 μL of ATCh solution (2.5 mmol / L) was added to the microenzyme reactor, and the RGB values ​​were measured five times in accordance with the "Method for Assaying Immobilized AChE Enzyme Activity" in Example 1 to evaluate the intra-day repeatability of the microenzyme reactor. 6.0 μL of ATCh solution (2.5 mmol / L) was added to the microenzyme reactor for five consecutive days to determine the inter-day repeatability of the microenzyme reactor. Different batches of microenzyme reactors were prepared, and 6.0 μL of ATCh solution (2.5 mmol / L) was added to five different batches of microenzyme reactors to determine the inter-batch repeatability of the microenzyme reactors.

[0095] Add 6.0 μL of ATCh solution (2.5 mmol / L) to the same microenzyme reactor and use it 100 times consecutively according to the "Method for Assaying Immobilized AChE Enzyme Activity" in Example 1 to examine the lifespan of the microenzyme reactor.

[0096] Add 6.0 μL of ATCh solution (2.5 mmol / L) to the newly prepared microenzyme reactor, and measure the RGB value of the color intensity according to the "Method for Assaying Immobilized AChE Enzyme Activity" in Example 1. Then place it in a refrigerator. After 40 days, take out the microenzyme reactor and measure the RGB value of the color intensity again according to the "Method for Assaying Immobilized AChE Enzyme Activity" in Example 1 to examine the storage stability of the microenzyme reactor.

[0097] The results showed that when gold nanoparticles were involved, the intra-day, inter-day, and batch-to-batch reproducibility of RGB values ​​obtained using the directional microenzyme reactor were 2.3%, 4.0%, and 1.4%, respectively, while those obtained using the non-directional microenzyme reactor were 3.3%, 7.6%, and 5.0%, respectively. The RSD of the directional microenzyme reactor was less than 5%, indicating that the method for directional binding of AChE in this experiment is reliable and stable. Furthermore, the RSD of the directional microenzyme reactor was smaller than that of the non-directional microenzyme reactor, indicating that the specific binding of Con A to AChE can improve the activity and stability of the microenzyme reactor.

[0098] Table 2. Repeatability results of the microenzyme reactor

[0099]

[0100] The same directional and non-directional microenzyme reactors were repeatedly used 100 times each (three parallel groups). With increasing number of uses, the relative activity of AChE showed a decreasing trend. Figure 5 (c) However, the relative activity of the directed microenzyme reactor was consistently greater than that of the non-directed microenzyme reactor. After 80 consecutive uses, the directed enzyme reactor retained 87.0% of its initial activity, while the non-directed microenzyme reactor still retained 79.7% of its initial activity, an improvement of 7.3% compared to the non-directed enzyme reactor. This indicates that the microenzyme reactor prepared in this invention can maintain good biological activity even after multiple reuses. Compared with free enzymes that can only be used once, this significantly reduces analytical costs. After 5 days of continuous use, the activity retention rates of the directed and non-directed enzymes were 90.2% and 81.8%, respectively. Figure 5 (d). The above results all demonstrate that the specific binding of Con A to AChE can improve the stability of the microenzyme reactor.

[0101] After the prepared microenzyme reactor was placed in a refrigerator and stored at -20℃ for 40 days, the activity of the directional microenzyme reactor could still maintain 93.3% of the initial activity, indicating that the microenzyme reactor prepared in this experiment has good stability.

[0102] Example 8: Screening and Validation of AChEI Using a Microenzyme Reactor

[0103] (1) Donepezil hydrochloride was used as a model drug. At concentrations of 0, 6 μmol / L, and 12 μmol / L, the ATCh concentration was changed (0.69 mmol / L, 2.07 mmol / L, 3.46 mmol / L, 4.84 mmol / L, 6.22 mmol / L) according to the "Method for Assaying Immobilized AChE Enzyme Activity" in Example 1. The color intensity (RGB values) was measured after reaction with the microenzyme reactor. Other steps were the same as in Example 1. A linear fit was performed using Origin software with 1 / ATCh concentration as the abscissa and 1 / RGB value as the ordinate to obtain a Lineweaver-Burk double reciprocal plot. Figure 5 (e).

[0104] 6.0 μL of ATCh solution (2.5 mmol / L) containing different concentrations of donepezil hydrochloride (1.56, 3.12, 6.25, 25, 100 nmol / L) was added dropwise to a microenzyme reactor, and the RGB values ​​of the colorimetric intensity were measured. The logarithm of the donepezil hydrochloride concentration was plotted on the x-axis, and the RGB values ​​on the y-axis, and logistic regression was performed using Origin software.

[0105] From the Lineweaver-Burk equation (Equation 2), we can see that the intercept of the line with the vertical axis represents... The intercept with the horizontal axis represents As donepezil hydrochloride concentration increases, V max Decrease, K m The unchanged result indicates that donepezil hydrochloride is a non-competitive inhibitor. Donepezil hydrochloride can bind to the inactive site of AChE, inhibiting enzyme activity, but does not affect the binding of AChE to ATCh.

[0106]

[0107] (Equation 2)

[0108] IC 50 The concentration of inhibitor required to inhibit enzyme activity by half is an important indicator of inhibitory activity. Figure 5 As shown in f, donepezil hydrochloride IC50 50 The concentration was (9.35±1.11) nmol / L. After measurement using the following UV spectrophotometric method, the IC50 of donepezil hydrochloride was obtained by logistic fitting using Origin software. 50 The concentration was (9.86±1.15) nmol / L. The results from the two methods were similar, indicating that the AChEI screening method established in this experiment is reliable. i K represents the enzyme inhibition constant, which reflects the inhibitory ability of the inhibitor. iThe smaller the IC value, the stronger the inhibitor's inhibitory effect. 50 The concentration of K is related to both the enzyme concentration and the substrate concentration used in the experiment. i The value is unaffected by these variables, and K can be calculated according to Equation 3. i It is (0.19±0.02) nmol / L.

[0109]

[0110] (Equation 3)

[0111] (2) Determination of the inhibitory activity of several AChEIs

[0112] 6.0 μL of ATCh solution (2.5 mmol / L) with or without inhibitor was added to the microenzyme reactor. The RGB values ​​of the colorimetric intensity were measured according to the "Method for Assaying Immobilized AChE Enzyme Activity" in Example 1. When AChEI is present, AChE activity is inhibited, the amount of TCh generated decreases, and the RGB value of the colorimetric result decreases. The inhibitory activity of AChEI can be calculated from the decrease in RGB value. The magnitude of the inhibitory activity can be expressed as the inhibition rate, which is calculated according to Equation 4. The logarithm of the inhibitor concentration is plotted on the x-axis, and the RGB values ​​on the y-axis. Logistic regression was performed using Origin software.

[0113]

[0114] (Equation 4)

[0115] Wherein, RGB0 represents the RGB values ​​of the microenzyme reactor with immobilized AChE reacting with an ATCh solution without inhibitors; RGB1 represents the RGB values ​​of the microenzyme reactor with immobilized AChE reacting with an ATCh solution containing inhibitors; RGB 空白0 RGB values ​​represent the reaction between a blank paper chip and an ATCh solution without inhibitors. 空白1 The RGB values ​​represent the reaction between a blank paper chip and a solution containing the inhibitor ATCh.

[0116] The inhibition rate of AChEI was determined again using the traditional ultraviolet spectrophotometry method, and the results of the two methods were compared. The specific steps are as follows: The prepared microenzyme reactor was placed in 3 mL of ATCh solution (2.5 mmol / L), and after reacting for 6 min, the microenzyme reactor was removed from the solution. 72 μL of DTNB solution (4 mg / mL) was added to the solution, and the absorbance value A was measured using an ultraviolet spectrophotometer. The inhibition rate was calculated according to Equation 5.

[0117]

[0118] (Equation 5)

[0119] Wherein, A0 is the microenzyme reactor with fixed AChE, and the RGB values ​​when it reacts with an ATCh solution without inhibitors;

[0120] A1 is a microenzyme reactor with immobilized AChE, showing the RGB values ​​during the reaction with an ATCh solution containing inhibitors; A 空白0 RGB values ​​for the reaction of a blank paper chip with an ATCh solution without inhibitors; A 空白1 The RGB values ​​represent the reaction between a blank paper chip and a solution containing the inhibitor ATCh.

[0121] The ATCh concentration was fixed at 2.5 mmol / L using the method described above. The inhibition rates of the following AChEIs were determined: gastrodin (35-150 mmol / L), ginsenoside Rg1 (30-400 mmol / L), jujuboside A (6.5-100 mmol / L), quercetin (6-100 mmol / L), emodin (25-400 mmol / L), or puerarin (12.5-400 mmol / L). Logistic regression analysis was performed using Origin software. The IC50 values ​​were then determined. 50 The values ​​were 75.19 ± 1.04 mmol / L, 106.30 ± 1.08 μmol / L, 21.28 ± 1.10 μmol / L, 28.68 ± 1.15 μmol / L, 108.80 ± 1.02 μmol / L, and 114.35 ± 1.03 μmol / L, respectively. These were compared with the IC50 obtained by the UV method. 50 The values ​​(77.90±1.05 μmol / L, 112.72±1.03 μmol / L, 16.45±1.02 μmol / L, 28.52±1.05 μmol / L, 117.44±1.06 μmol / L and 119.26±1.10 μmol / L) showed good agreement. According to the experimental results, the inhibitory trend of the several drugs on AChE was as follows: jujuboside A > quercetin > gastrodin > ginsenoside Rg1 > emodin > puerarin.

[0122] Example 9: Active tracer in the extraction of traditional Chinese medicinal materials

[0123] Preparation of crude extract of Coptis chinensis: 50g of Coptis chinensis powder was soaked in 500 mL of 0.2% sulfuric acid solution at room temperature for 24 h, and the solution was filtered and collected. The residue was processed in the same way, and the two filtrates were combined. The pH was adjusted to 7.0 with lime milk, the suspension was allowed to stand for 30 min, and the supernatant was poured off to obtain the crude extract of Coptis chinensis. Figure 6 (a)

[0124] The crude extract of Coptis chinensis was eluted with petroleum ether-ethyl acetate-methanol on a neutral alumina chromatography column. Multiple eluates were collected and added to a microenzyme reactor for enzyme activity determination. The RGB values ​​of the colorimetric intensities were measured in the microenzyme reactor according to the "Method for Assaying Immobilized AChE Enzyme Activity" in Example 1. The RGB values ​​obtained from the substrate without inhibitors were used as a reference group to determine whether the crude extract of Coptis chinensis contained AChEI active ingredient. The results are shown in Table 3. Figure 6 b. If the RGB values ​​of the crude extract of Coptis chinensis are not significantly different from those of the reference group, or the difference is too small, it indicates that the extract does not contain AChEI active ingredient or the content of active ingredient is very low, and it is not meaningful for further separation. If the RGB values ​​of the crude extract of Coptis chinensis are significantly different from those of the reference group, it indicates that the extract contains AChEI active ingredient, and the crude extract can be further separated to trace the AChEI active ingredient.

[0125] Table 3. Determination of inhibitory activity of Coptis chinensis eluent

[0126]

[0127] The eluents #3 to #7 exhibited different shades of yellow. However, in actual operation, because the enzymatic reaction was separated from the colorimetric region, the reaction solution was observed to be almost colorless after flowing from paper chip R to paper chip D, ensuring the accuracy and reliability of the experimental results. This may be the result of colored substances adhering to the surface of paper chip R.

[0128] By comparing the color intensity and the enzyme activity inhibition rate of different eluents, it was found that eluents #6 and #7 significantly reduced AChE activity, with inhibition rates of 71.8% and 49.2%, respectively, which were far higher than other eluents. This indicates that the component has the potential to inhibit AChE and is worth further separation.

[0129] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A rotary splicing paper-based enzymatic hydrolysis device, characterized in that, It includes an enzyme immobilization device and a colorimetric device, wherein the enzyme immobilization device is rotatably adjustable relative to the colorimetric device; The enzyme immobilization device includes a disc I loaded with chitosan, gold nanoparticles, and concanavalin A, with acetylcholinesterase directionally bonded to it. A microfluidic channel I is located on the side of the disc I closest to the colorimetric device. The colorimetric device is a paper chip with at least eight independent colorimetric regions arranged circularly on the paper chip, with equidistant spacing between adjacent regions. A microfluidic channel II communicating with each independent colorimetric region is provided on the paper chip. The paper chips are evenly spaced along the circumference of the enzyme immobilization device, and each colorimetric region of the paper chip communicates with the outside through the microfluidic channel II. Spatial docking or separation of the disc I and the independent colorimetric regions is achieved through the docking or separation of the microfluidic channels I and II, enabling the reuse of the enzyme immobilization device.

2. The rotary splicing paper-based enzymatic hydrolysis device as described in claim 1, characterized in that, A column extends through the end of the enzyme immobilization device, and the independent color development areas are evenly spaced along the circumference of the column.

3. The rotary splicing paper-based enzymatic hydrolysis device as described in claim 1, characterized in that, After the enzyme immobilization device and the color development device are designed, a wax printer is used to print wax onto the qualitative filter paper, and then a hot press is used to heat the paper so that the wax melts and forms a hydrophobic barrier.

4. The rotary splicing paper-based enzymatic hydrolysis device as described in claim 1, characterized in that, The diameter of the disk I is 6-7 mm, and the length and width of the microfluidic channel I are set to (2-3) mm × 1 mm; The independent color development area is a circular paper chip with a diameter of 4-5 mm, and the length and width of the microfluidic channel II are set to (6-7) mm × 1 mm.

5. The rotary splicing paper-based enzymatic hydrolysis device as described in claim 1, characterized in that, When the microfluidic channel I is connected to the microfluidic channel II, the enzymatic hydrolysis product is transferred from the disk I to the independent color development area under capillary action to complete the color development reaction, and the result is read by RGB colorimetry.

6. A method for preparing the enzyme immobilization device according to any one of claims 1-5, characterized in that, Includes the following steps: (1) On disk I in the enzyme immobilization device, a chitosan solution is loaded and dried to introduce amino groups; (2) Load the nano-gold solution onto the same area, incubate, and then wash; (3) Load the same area with concanavalin A solution, incubate, and then wash; (4) Block non-specific binding sites with bovine serum albumin solution, incubate, and then wash; (5) Load acetylcholinesterase solution onto the same area, incubate and wash to complete directional fixation, and obtain a microenzyme reactor, which is an enzyme fixation reaction device.

7. The method as described in claim 6, characterized in that, The volume ratio of the chitosan solution, the gold nanoparticle solution, the concanavalin A solution, the bovine serum albumin solution, and the acetylcholinesterase solution is 2:2:5:5:

5.

8. The method as described in claim 6, characterized in that, In step (2), the concentration of the gold nanoparticle solution is 5-100 pmol / L, and the incubation time is 60-110 min; In step (3), the incubation time is 30-140 min; In step (5), the concentration of the acetylcholinesterase solution is 0.10-10.00 mmol / L, and the incubation time is 30-60 min.

9. The use of the rotary splicing paper-based enzymatic digestion device as described in any one of claims 1-5 in screening acetylcholinesterase inhibitors or screening Alzheimer's disease drugs.

10. The application of the rotary splicing paper-based enzymatic hydrolysis device as described in any one of claims 1-5 in the activity tracing of traditional Chinese medicinal materials.

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