A preparation method of a nanomaterial for detecting alpha-glucosidase activity

Abstract

The application discloses a preparation method of Mo-based nanomaterial for detecting alpha-glucosidase (alpha-Glu), which comprises the following steps: firstly, synthesizing MoO3 by taking MoS2 and H2O2 as raw materials; and then bonding a small molecule containing a mercapto group with Mo atoms in the MoO3 as a ligand to prepare non-stoichiometric molybdenum oxide MoO 3‑x The material has a characteristic localized surface plasmon resonance (LSPR) effect, shows excellent optical properties, and presents a typical blue solution. In addition, the material has sensitive response characteristics to H2O2, and can be used for H2O2 detection and H2O2 generation related enzyme cascade analysis. Based on the above-mentioned blue color and H2O2 response characteristics, colorimetric analysis of alpha-Glu and its inhibitors can be further realized, and excellent sensitivity and selectivity are achieved.

Description

Technical Field

[0001] This invention belongs to the field of nanoenzyme biosensing, specifically relating to a method for preparing nanomaterials for detecting α-glucosidase activity. Background Technology

[0002] Diabetes mellitus is a complex and prevalent disease characterized by postprandial hyperglycemia. It can be classified into three main types (type 1, type 2, and gestational diabetes mellitus), with type 2 diabetes mellitus (T2DM) accounting for 90% of all diabetes cases. Research has found that alpha-glucosidase (α-Glu) is a hydrolytic enzyme distributed along the brush membrane of intestinal epithelial cells. It hydrolyzes a series of oligosaccharides by catalyzing the cleavage of α-1,4-glucanopyranoside bonds. α-1,4-glucanopyranoside bonds play a crucial role in carbohydrate digestion and are key enzymes in controlling blood glucose levels. Alpha-glucosidase inhibitors (AGIs) are effective oral hypoglycemic agents. Unlike other classes of hypoglycemic drugs that improve blood glucose by promoting insulin secretion, AGIs treat type 2 diabetes by inhibiting carbohydrate digestion and delaying glucose absorption to lower blood glucose. Therefore, with the rising incidence of type 2 diabetes, developing efficient methods for monitoring α-Glu activity and screening AGIs is of great significance for finding new hypoglycemic drugs for type 2 diabetes and for the clinical medical field.

[0003] Although some methods for detecting α-Glu have been reported in the prior art, most of these methods rely on nanozymes with peroxidase (POD)-like or oxidase (OX)-like activities. These methods still have some limitations on bioanalytical platforms, such as requirements for chromogenic substrates (TMB, ABTS, or OPD) or harsh acidic detection environments, which leads to problems such as complicated detection steps, long detection time, and poor sensitivity of the sensing system.

[0004] Therefore, establishing a simple, highly sensitive, and highly selective method is of great value for disease diagnosis and biomedical research.

[0005] Plasma nanomaterials exhibit strong light-material interactions due to localized surface plasmon resonance (LSPR) excitation. LSPR excitation occurs when the valence electron oscillation frequency of the nanomaterial matches the incident light frequency. Molybdenum oxide (MoO3) is a stable n-type semiconductor material with a two-dimensional covalently bonded oxide layer structure, making it a good host for various dopants. By introducing equivalent heteroatoms (hydrogen atoms) or oxygen vacancies, reaching the carrier concentration threshold for LSPR generation, the LSPR characteristics of MoO3 become apparent. By reducing MoO3 nanomaterials at room temperature with a reducing agent, oxygen-vacancy-rich MoO exhibiting LSPR properties can be easily obtained. 3-x MoO 3-xNanomaterials exhibit strong absorption in the visible to near-infrared region, which can overlap with the red region of visible light to produce a deep blue color. They are highly reactive and easily oxidized by oxidants, leading to the loss of LSPR (Laminated Light Peroxide Reduction). Based on this, oxygen-rich vacancy MoO₂, possessing a typical deep blue color and excellent hydrogen peroxide response, is... 3-x It can be used to construct an enzyme cascade reaction platform to achieve excellent sensing of α-Glu and AGIs. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a method for detecting α-glucosidase activity, addressing the problems of complex detection steps, long detection time, and poor sensitivity of the sensing system in the existing technologies. This method utilizes molybdenum-based nanomaterials MoO2 exhibiting localized surface plasmon resonance (LSPR) effects. 3-x As a detection reagent, MoO 3-x The excellent optical properties enable a sensitive response to H2O2, allowing for applications in H2O2 detection and the analysis of enzyme cascade reactions related to H2O2 formation. Based on these characteristics of blue color and H2O2 response, colorimetric analysis of α-Glu and its inhibitors can be further achieved with excellent sensitivity and selectivity. This invention also reveals that by using a self-made hydrogel kit and capturing images of the kit with a smartphone, analysis can be performed using applications such as ImageJ. The results obtained by this sensor are consistent with those obtained by laboratory microbial plate readers.

[0007] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing molybdenum-based nanomaterials, characterized in that the preparation method includes at least the following steps:

[0009] Step S1: Prepare a molybdenum disulfide dispersion, add hydrogen peroxide solution to the dispersion, and obtain molybdenum trioxide after sufficient reaction;

[0010] Step S2: Reduce the molybdenum trioxide obtained in step S1 to obtain the non-stoichiometric Mo-based nanomaterial MoO. 3-x .

[0011] Preferably, in step S1, the molybdenum disulfide dispersion is prepared by dispersing molybdenum disulfide in a beaker containing deionized water and stirring until homogeneous.

[0012] Preferably, in step S1, the concentration of the molybdenum disulfide dispersion is 5–50 mM.

[0013] Preferably, in step S1, the mass fraction of hydrogen peroxide is 10% to 30%, and the volume is 1 to 10 mL.

[0014] Preferably, step S1 further includes heating the solution obtained after the reaction to evaporate and remove hydrogen sulfide and the remaining hydrogen peroxide.

[0015] Preferably, in step S2, the reducing agent is one or more of ascorbic acid, N-acetyl-L-cysteine, dithiothreitol, sodium borohydride, and hydrazine hydrate.

[0016] Preferably, in step S2, the reducing agent is N-acetyl-L-cysteine.

[0017] Secondly, the present invention also provides a non-stoichiometric Mo-based nanomaterial MoO prepared by the above method. 3-x .

[0018] Thirdly, the present invention also provides a method for preparing non-stoichiometric Mo-based nanomaterials MoO. 3-x Detection of α-glucosidase.

[0019] Furthermore, α-glucosidase, p-nitrophenyl-α-D-glucopyranoside, and glucose oxidase were incubated in a water bath, and then molybdenum-based nanomaterials were added.

[0020] Furthermore, a gel kit was prepared by mixing molybdenum-based nanomaterials with agarose; α-glucosidase, p-nitrophenyl-α-D-glucopyranoside, and glucose oxidase were incubated in a water bath; and then the above solution was mixed with the gel kit.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] 1. This invention employs a simple and rapid room-temperature solution method to prepare non-stoichiometric MoO2. 3-x Because its LSPR band absorption overlaps with the visible red region, it appears as a deep blue color, which can be used to visually detect the oxidant H2O2, and can be further extended to enzyme cascade biosensing related to H2O2 generation.

[0023] 2. This invention enables colorimetric analysis of α-Glu activity and its inhibitors, and has excellent sensitivity and selectivity.

[0024] 3. This invention utilizes a self-made hydrogel reagent kit. Images of the kit captured by a smartphone can be analyzed using applications such as ImageJ. The results obtained by this sensor are consistent with those obtained by a laboratory microbial plate reader. Attached Figure Description

[0025] Figure 1The target product MoO prepared in Examples 1-5 3-x UV-vis map

[0026] Figure 2 The target product MoO prepared in Example 1 3-x TEM image

[0027] Figure 3 The target product MoO prepared in Example 1 3-x XPS total spectrum

[0028] Figure 4 The target product MoO prepared in Example 1 3-x and FT-IR spectrum

[0029] Figure 5 The target product MoO prepared in Example 1 3-x UV spectra of H2O2 at different concentrations

[0030] Figure 6 The target product MoO prepared in Example 1 3-x UV spectra of α-Glu with different activities

[0031] Figure 7 The target product MoO prepared in Example 1 3-x Linear relationship between α-Glu and different activities

[0032] Figure 8 The target product MoO prepared in Example 1 3-x Grayscale image of hydrogel reagent kit

[0033] Figure 9 The target product MoO prepared in Example 1 3-x Linear relationship between hydrogel kit and different concentrations of α-Glu Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0035] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0036] I. Preparation of Examples

[0037] Example 1

[0038] Step S1: Weigh 80 mg of MoS2 and disperse it in a beaker containing 47.5 mL of deionized water. Stir and mix thoroughly. Add 3.75 mL of H2O2 solution to allow it to react completely. Stir at room temperature until the solution turns pale yellow. You can repeat the addition of H2O2 and stirring to obtain a yellow solution. Heat to 80 °C to evaporate and remove hydrogen sulfide and the remaining H2O2. After heating for 3-4 hours, control the final volume of the solution to 50 mL to obtain a MoO3 solution.

[0039] Step S2: Add 10 mL of the MoO3 solution obtained in Step S1 to a centrifuge tube, then add 40 mg of N-acetyl-L-cysteine ​​(NAC), shake well, let stand, and react overnight to obtain non-stoichiometric Mo-based nanomaterials, denoted as NAC-MoO. 3-x .

[0040] Example 2

[0041] The difference from Example 1 is that 5 mg of dithiothreitol (DTT) was added in step S2 to obtain a non-stoichiometric Mo-based nanomaterial, denoted as DTT-MoO. 3-x .

[0042] Example 3

[0043] The difference from Example 1 is that 5 mg of ascorbic acid (AA) was added in step S2 to obtain a non-stoichiometric Mo-based nanomaterial, denoted as AA-MoO. 3-x .

[0044] Example 4

[0045] The difference from Example 1 is that 5 mg of hydrazine hydrate (N2H4) was added in step S2 to obtain a non-stoichiometric Mo-based nanomaterial, denoted as N2H4-MoO. 3-x .

[0046] Example 5

[0047] The difference from Example 1 is that 0.5 mg of sodium borohydride (NaBH4) was added in step S2 to obtain a non-stoichiometric Mo-based nanomaterial, denoted as NaBH4-MoO. 3-x .

[0048] II. Sample Evaluation Method and Evaluation Results of the Examples

[0049] Figure 1The figures show the UV absorption spectra of Mo-based nanomaterials prepared in different embodiments. It can be seen from the figures that when the reducing agent is NAC, it exhibits a better UV absorption peak, i.e., better LSPR performance. Therefore, in the following experiments, we will use the NAC-MoO obtained in Example 1. 3-x Further testing will be conducted.

[0050] The NAC-MoO prepared in Example 1 3-x TEM, XPS, and FT-IR tests were performed separately.

[0051] Figure 2 The prepared sample is NAC-MoO 3-x TEM image. From Figure 2 As can be seen from the sample NAC-MoO 3-x The particle size is around 5nm, and the particle size is uniformly dispersed.

[0052] from Figure 3 XPS spectroscopy revealed the presence of C, N, O, S, and Mo elements in the sample. Furthermore, combined with... Figure 4 NAC and NAC-MoO 3-x The FT-IR clearly shows that the -SH bonds in the NAC disappear, indicating that the -SH bonds in the NAC form Mo-S bonds with Mo. Furthermore, from... Figure 4 The graph also shows Mo-O-Mo and Mo=O bonds, indicating that NAC-MoO 3-x There are unsaturated oxygen vacancies on the surface.

[0053] NAC-MoO 3-x Response to different H2O2 concentrations

[0054] Add 100 μL of the NAC-MoO sample prepared in Example 1 to a 2 mL centrifuge tube. 3-x 1800 μL of Tri-HCl buffer solution (pH = 7) was added, followed by the addition of 100 μL of H₂O₂ of different concentrations, and the UV absorption spectra were recorded. For example... Figure 5 As shown, the absorbance gradually decreases as the H2O2 concentration increases in the range of 0.1-1 mM.

[0055] Exploring NAC-MoO 3-x Response to different α-Glu concentrations

[0056] Add 100 μL of α-Glu at different concentrations, 300 μL of p-nitrophenyl-α-D-glucopyranoside (20 mM), 1400 μL of Tri-HCl buffer solution (pH = 7), and 100 μL of glucose oxidase (1 mg / mL) to a 2 mL centrifuge tube. Incubate in a 37 °C water bath for 120 min. Then add 100 μL of the sample NAC-MoO prepared in Example 1. 3-x Record the UV absorption spectrum after holding for 10 minutes. Figure 6 As shown, as the α-Glu activity increases in the range of 0.01-0.2 U / mL, the absorbance gradually decreases, and the linear relationship is as follows: Figure 7 As shown, R can be obtained through calculation. 2 =0.99. The blank sample was scanned 20 times at 770 nm, and the average deviation was 2.58543 × 10⁻⁶. -4 Further calculations revealed a detection limit of 0.08 U / L, indicating that it exhibits excellent sensitivity for detecting α-Glu activity.

[0057] Exploring NAC-MoO 3-x Response of the gel kit to different α-Glu concentrations

[0058] Add 100 mg agarose and 10 mL deionized water to a beaker, heat and stir at 90 °C for 30 min, and take 2 mL of the NAC-MoO2 prepared in Example 1. 3-x Add the above solution and stir vigorously for 2 minutes. Add 60 μL of the above solution to a 96-well plate and cool to room temperature to form NAC-MoO₂. 3-x The gel electrophoresis kit was prepared and stored at 4°C for later use. In a 2 mL centrifuge tube, 100 μL of α-Glu at different concentrations, 300 μL of p-nitrophenyl-α-D-glucopyranoside (20 mM), 100 μL of buffer solution (pH = 7), and 100 μL of glucose oxidase (1 mg / mL) were added and incubated in a 37°C water bath for 120 min. 100 μL of the above solution was then added to the gel electrophoresis kit. After 30 min, the sample was photographed with a smartphone, and the grayscale was analyzed using ImageJ software. The grayscale image is shown below. Figure 8 As shown, as the α-Glu activity increases in the range of 0.04-0.5 U / mL, the absorbance gradually decreases, and the linear relationship is as follows: Figure 9 As shown, R can be obtained through calculation. 2 =0.99. This method demonstrates its potential for on-site quantitative detection.

[0059] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. Use of a molybdenum-based nanomaterial for detecting α-glucosidase, characterized in that, The detection method is: incubating alpha-glucosidase, p-nitrophenyl-alpha-D-glucopyranoside and glucose oxidase in a water bath, then adding molybdenum-based nanomaterial MoO 3-x ; the particle size of the molybdenum-based nanomaterial is 1-10 nm, and the preparation method of the molybdenum-based nanomaterial comprises the following steps: Step S1: preparing a molybdenum disulfide dispersion liquid, adding a hydrogen peroxide solution into the dispersion liquid, and obtaining molybdenum trioxide after sufficient reaction; Step S2: reducing the molybdenum trioxide obtained in step S1 to obtain a non-stoichiometric Mo-based nanomaterial MoO 3-x .

2. Use according to claim 1, characterized in that: In step S1, the method for preparing the molybdenum disulfide dispersion liquid is: dispersing molybdenum disulfide in a beaker containing deionized water, and stirring uniformly.

3. Use according to claim 1, characterized in that: In step S1, the concentration of the molybdenum disulfide dispersion liquid is 5-50 mM.

4. Use according to claim 1, characterized in that: In step S1, the solution obtained after the reaction is further heated to evaporate and remove hydrogen sulfide and residual hydrogen peroxide.

5. The use according to claim 1, characterized in that: In step S2, the reducing agent used for the reduction is one or more of ascorbic acid, N-acetyl-L-cysteine, dithiothreitol, sodium borohydride, and hydrazine hydrate.

6. Use according to claim 1, characterized in that: The molybdenum-based nanomaterial is mixed with agarose to prepare a gel reagent kit; α-glucosidase, p-nitrophenyl-α-D-glucopyranoside, and glucose oxidase are incubated in a water bath; and the above solution is mixed with the gel reagent kit.

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