A nanocomposite catalyst and a one-step method for determining glycated albumin

Abstract

The present invention provides a nanocomposite catalyst, and the preparation process of the nanocomposite catalyst is as follows: first, peroxidase nanomimetic enzyme is subjected to aldehyde modification to obtain CHO-nanozyme, and then the CHO-nanozyme is coupled with a protease and a ketoamine oxidase to obtain a nanocomposite catalyst. The present invention also provides a one-step method for determining glycated albumin, comprising the following steps: adding the aforementioned nanocomposite catalyst to a glycated albumin test solution, then adding a TMB solution to react for a period of time, observing the results, and the color of the solution after the reaction turns blue, indicating the presence of glycated albumin, and the blue color deepens as the concentration of glycated albumin in the glycated albumin test solution increases. The present invention uses a composite catalyst that integrates the triple functions of protease, ketoamine oxidase, and peroxidase, and detects glycated albumin in a one-step cascade reaction, which can simplify the operation and improve the detection efficiency and accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of nanocatalytic materials and biomedicine, and particularly relates to a nanocomposite catalyst and a one-step method for determining glycated albumin. Background Art

[0002] Glycated albumin (Glycated Albumin) is a product formed by the non-enzymatic glycation reaction between glucose and serum albumin in the blood. It effectively reflects the average blood glucose level over the past two to three weeks and is unaffected by factors such as individual hemoglobin metabolism disorders, serum total protein and albumin concentrations, creatinine, uric acid, and bilirubin. The results are accurate and reliable, helping medical professionals gain a comprehensive and precise understanding of the patient's true condition. Glycated albumin is of great value in the screening and diagnosis of diseases such as diabetes, nephrotic syndrome, cardiovascular and cerebrovascular diseases, and pancreatic cancer, especially in the observation and treatment efficacy monitoring of clinically diagnosed diabetic patients.

[0003] Currently, the main methods for detecting glycated albumin are: high performance liquid chromatography and ketoamine oxidase method.

[0004] The high-performance liquid chromatography method has accurate test results, but it requires professional and expensive high-performance liquid chromatography instruments and professional technicians, and is not suitable for self-examination in primary hospitals and outpatient patients.

[0005] The ketoamine oxidase method combines biocatalysis with a colorimetric reaction to detect glycated albumin. It offers high specificity and sensitivity, excellent precision, strong anti-interference ability, a wide linear range, and minimal equipment and personnel requirements, making it an ideal method for detecting glycated albumin. However, the ketoamine oxidase method requires three enzymes—protease, ketoamine oxidase, and peroxidase—to catalyze three tandem reactions, making the process cumbersome and time-consuming, exceeding 3 hours. The ketoamine oxidase method involves the following steps: 1) the protease reacts with glycated albumin to release glycated amino acids; 2) the glycated amino acids, catalyzed by ketoamine oxidase, specifically oxidize the ketoamine bond, releasing H₂O₂; and 3) the H₂O₂ reacts with the substrate under the action of peroxidase to produce a colorimetric reaction, allowing the quantitative determination of glycated albumin via color change. These three tandem reactions are closely linked, and even the slightest error in any of these steps can compromise the accuracy of the assay. Furthermore, the three enzymes used in the ketoamine oxidase method are essentially proteins, resulting in poor stability (susceptibility to loss of activity due to acid, alkali, heat, organic solvents, and other factors), high cost, and difficulty in storage, limiting their application in clinical testing of glycated albumin. Rapid, convenient, and accurate detection of glycated albumin has become a technical challenge that urgently needs to be addressed.

[0006] Therefore, a new method for rapid, convenient and accurate determination of glycated albumin is needed in this field. Summary of the Invention

[0007] The purpose of the present invention is to provide a nanocomposite catalyst and a one-step method for determining glycated albumin, so as to solve the problem of how to detect glycated albumin quickly, conveniently and accurately raised in the background art.

[0008] The present invention provides a nanocomposite catalyst, and the preparation process of the nanocomposite catalyst is as follows:

[0009] The peroxidase nanomimetic enzyme was first modified by aldehyde group to obtain CHO-nanozyme, and then the CHO-nanozyme was coupled with protease and ketoamine oxidase to obtain a nanocomposite catalyst.

[0010] In one specific embodiment, the peroxidase nanomimetic enzyme comprises one or more of nano-Fe₃O₄, nano-CoFe₃O₄, nano-Co₃O₄, nano-CeO₂, nano-MnO, nano-MoS₂, and nano-WS₂. This peroxidase nanomimetic enzyme is a novel artificial enzyme with catalytic functions similar to those of natural peroxidase, capable of catalyzing H₂O₂ to produce reactive oxygen free radicals. Furthermore, it possesses small particle size, strong loading capacity, excellent stability, adjustable catalytic activity, good biocompatibility, low cost, and ease of modification and industrial production, meeting the prerequisites for serving as an excellent carrier.

[0011] In a specific embodiment, the particle size of the peroxidase nanomimetic enzyme is 1 to 1000 nm.

[0012] In a specific embodiment, the process of the peroxidase nanomimetic enzyme aldehyde modification is as follows:

[0013] The peroxidase nanomimetic enzyme was added to the chitosan solution and mixed thoroughly. Then, sodium polyphosphate solution was added and reacted at room temperature for a period of time. The chitosan-modified peroxidase nanomimetic enzyme was separated and collected. After repeated washing with ultrapure water and glacial acetic acid solution, it was redispersed in PBS buffer. Then, sodium periodate solution was added for reaction. After washing again, the CHO-nanozyme was collected.

[0014] In a specific embodiment, the mass percentage concentration of the chitosan solution is 0.05-0.2%; the mass percentage concentration of the sodium polyphosphate solution is 0.5-5.0%; the mass percentage concentration of the glacial acetic acid solution is 0.5-1.5%; the mass percentage concentration of the sodium periodate solution is greater than or equal to 1.0%; and the pH value of the PBS buffer solution is 1.5-6.5.

[0015] In a specific embodiment, the process of coupling CHO-nanozyme with protease and ketoamine oxidase to obtain a nanocomposite catalyst is as follows:

[0016] The CHO-nanozyme is completely dispersed in water, and then a protease solution and a ketoamine oxidase solution are added respectively; after mixing, coupling is performed, and after the coupling is completed, separation and washing are performed to obtain a nanocomposite catalyst.

[0017] In a specific embodiment, the coupling reaction temperature is controlled at 0-8°C; the coupling reaction time is 1-4 hours; the mass percentage concentration of the protease solution is 0.1-1.00%; the mass percentage concentration of the ketoamine oxidase solution is 0.1-1.00%.

[0018] The present invention also provides a one-step method for determining glycated albumin, comprising the following steps:

[0019] The aforementioned nanocomposite catalyst was added to the glycated albumin test solution, and then TMB solution was added to react for a period of time. The results were observed. After the reaction, the color of the solution turned blue, indicating the presence of glycated albumin, and the blue color became darker as the concentration of glycated albumin in the glycated albumin test solution increased.

[0020] In a specific embodiment, the TMB solution is a 1-500 μmol / L TMB solution.

[0021] In a specific embodiment, the TMB solution is a 50-300 μmol / L TMB solution. If the TMB solution concentration is less than 50 μmol / L, it is difficult to distinguish the color difference with the naked eye, and precision instruments are required. If the TMB solution concentration is greater than 300 μmol / L, the color of the generated oxTMB is too dark or even precipitates, which is not conducive to observation and measurement.

[0022] The beneficial effects of the present invention include:

[0023] 1. High-performance liquid chromatography requires specialized, expensive high-performance liquid chromatographs and professional technicians, making it unsuitable for self-examination in primary care hospitals or outside hospitals. The ketoamine oxidase method is cumbersome and time-consuming, and the required reagents are expensive, unstable, and difficult to store. The present invention uses a composite catalyst that combines the triple functions of protease, ketoamine oxidase, and peroxidase to detect glycated albumin in a single step through a cascade reaction, simplifying the operation and improving detection efficiency and accuracy.

[0024] 2. Using peroxidase nano-mimic enzyme to replace peroxidase improves the stability of the catalyst and reduces the cost.

[0025] The composite catalyst prepared by the peroxidase nano-mimetic enzyme loaded with protease and ketoamine oxidase has the functions of three catalysts: protease, ketoamine oxidase and peroxidase-like enzymes. It does not require the use of expensive instruments and materials such as high-performance liquid chromatography and peroxidase. In addition, the detection can be achieved in one step by simply adding the sample to be tested. The operation is convenient and the results are accurate. It is particularly suitable for testing in emergency departments, grassroots hospitals and homes.

[0026] 3. The peroxidase nanomimetic enzymes used in the present invention are mainly metal oxide nanoparticles such as nano-Fe3O4, CoFe3O4, Co3O4, CeO2, MnO and nanoparticles such as MoS2, WS2. These nanomaterials are cheap, simple to prepare, have good biocompatibility, and are also easy to industrialize.

[0027] In addition to the above-described objects, features and advantages, the present invention has other objects, features and advantages. The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 This is a transmission electron microscope image of the nano-Fe3O4 selected in Example 3;

[0030] Figure 2 This is a transmission electron microscopy image of the CHO-nanozyme prepared in Example 3;

[0031] Figure 3 This is the electrophoresis diagram of the nanocomposite catalyst prepared in Example 3;

[0032] Figure 4 This is the result of one-step determination of glycated albumin using nanocomposite catalyst;

[0033] Figure 5 This is the glycated albumin test result diagram for control group 1;

[0034] Figure 6 This is the result of glycated albumin determination for control group 2;

[0035] Figure 7 This is the result of the one-step determination of the limit concentration of glycated albumin using the nanocomposite catalyst;

[0036] Figure 8 Schematic diagram of the one-step determination of glycated albumin using integrated nanocomposite catalysts in a tandem catalytic process. DETAILED DESCRIPTION

[0037] The embodiments of the present invention are described in detail below with reference to the accompanying drawings. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] Example 1

[0039] 1) Aldehyde modification of peroxidase nanomimetic enzyme

[0040] 10mg of nano-MnO was added to 10mL of a 0.1% chitosan solution by mass. After thorough mixing, 1mL of a 0.5% sodium polyphosphate solution was added and the mixture was allowed to react at room temperature for 30 minutes. The chitosan-modified nanozyme was then isolated and collected. After repeated washing with ultrapure water and 1% glacial acetic acid, the mixture was dispersed in 10mL of PBS buffer (pH 2.0). Then, 1mL of a 1.0% sodium periodate solution was added and the mixture was allowed to react at room temperature for 3.5 hours. After further washing, the CHO nanozyme was collected. CHO nanozyme is an aldehyde-modified peroxidase nanomimetic enzyme.

[0041] 2) Preparation of nanocomposite catalysts

[0042] The CHO nanozyme was completely dispersed in 10 mL of water. Then, 1 mL of a 0.10% (by weight) protease solution and 1 mL of a 0.10% (by weight) ketoamine oxidase solution were added. After mixing, the mixture was coupled at 4°C for 1.0 h. After completion of the coupling, the nanocomposite catalyst was isolated and washed.

[0043] Example 2

[0044] 1) Nanozyme aldehyde modification

[0045] 10mg of nano-Co₃O₄ was added to 10mL of a 0.1% chitosan solution by mass. After thorough mixing, 1mL of a 1.0% sodium polyphosphate solution was added and the mixture was allowed to react at room temperature for 30 minutes. The chitosan-modified nanozyme was then isolated and collected. After repeated washing with ultrapure water and 1% glacial acetic acid, the mixture was dispersed in 10mL of PBS buffer (pH 3.0). Then, 1mL of a 2.0% sodium periodate solution was added and the mixture was allowed to react at room temperature for 3.5 hours. The CHO nanozyme was then washed again and collected.

[0046] 2) Preparation of composite catalyst

[0047] The CHO nanozyme was completely dispersed in 10 mL of water, followed by the addition of 1 mL of a 0.25% by weight protease solution and 1 mL of a 0.25% by weight ketoamine oxidase solution. After mixing, the mixture was coupled at 4°C for 1.5 hours. After completion of the coupling, the nanocomposite catalyst was isolated and washed.

[0048] Example 3

[0049] 1) Nanozyme aldehyde modification

[0050] 10mg of nano-Fe₃O₄ was added to 10mL of a 0.1% chitosan solution and mixed thoroughly. Then, 1mL of a 5.0% sodium polyphosphate solution was added and the mixture was allowed to react at room temperature for 30 minutes. The chitosan-modified nanozyme was isolated and collected. After repeated washing with ultrapure water and 1% glacial acetic acid, the mixture was dispersed in 10mL of PBS buffer (pH 4.0). Then, 1mL of a 3.0% sodium periodate solution was added and the mixture was allowed to react at room temperature for 1.5 hours. The CHO nanozyme was then washed again and collected.

[0051] 2) Preparation of composite catalyst

[0052] The CHO nanozyme was completely dispersed in 10 mL of water. Then, 1 mL of a 0.50% by weight protease solution and 1 mL of a 0.50% by weight ketoamine oxidase solution were added. After mixing, the mixture was coupled at 4°C for 2.0 hours. After completion of the coupling, the nanocomposite catalyst was isolated and washed.

[0053] Example 4

[0054] 1) Nanozyme aldehyde modification

[0055] 10mg of nano-CeO2 was added to 10mL of a 0.1% chitosan solution and thoroughly mixed. Then, 1mL of a 5.0% sodium polyphosphate solution was added and the mixture was allowed to react at room temperature for 30 minutes. The chitosan-modified nanozyme was isolated and collected. After repeated washing with ultrapure water and 1% glacial acetic acid, the mixture was dispersed in 10mL of PBS buffer (pH 5.0). Then, 1mL of a 5.0% sodium periodate solution was added and the mixture was allowed to react at room temperature for 3.5 hours. The CHO nanozyme was collected after further washing.

[0056] 2) Preparation of composite catalyst

[0057] The CHO nanozyme was completely dispersed in 10 mL of water. Then, 1 mL of a 0.75% (by weight) protease solution and 1 mL of a 0.75% (by weight) ketoamine oxidase solution were added. After mixing, the mixture was coupled at 4°C for 2.0 hours. After completion of the coupling, the nanocomposite catalyst was isolated and washed.

[0058] Example 5

[0059] 1) Nanozyme aldehyde modification

[0060] 10mg of nano-MoS2 was added to 10mL of a 0.1% chitosan solution and thoroughly mixed. Then, 1mL of a 5.0% sodium polyphosphate solution was added and the mixture was allowed to react at room temperature for 30 minutes. The chitosan-modified nanozyme was isolated and collected. After repeated washing with ultrapure water and 1% glacial acetic acid, the mixture was dispersed in 10mL of PBS buffer (pH 6.0). Then, 1mL of a saturated sodium periodate solution was added and the mixture was allowed to react at room temperature for 3.5 hours. The CHO nanozyme was collected after further washing.

[0061] 2) Preparation of composite catalyst

[0062] The CHO nanozyme was completely dispersed in 10 mL of water. Then, 1 mL of a 1.00% (by weight) protease solution and 1 mL of a 1.00% (by weight) ketoamine oxidase solution were added. After mixing, the mixture was coupled at 4°C for 3 hours. After completion, the nanocomposite catalyst was isolated and washed.

[0063] Example 6

[0064] One-step determination of glycated albumin using nanocomposite catalyst

[0065] The nanocomposite catalyst prepared in Example 3 was selected as the experimental group, and the glycated albumin determination kit (ketoamine oxidase method) was selected as the control group. The amount of reagent added in the control group was carried out according to the instructions, and the catalyst was added in the following ways:

[0066] In control group 1, protease, ketoamine oxidase, and peroxidase were added sequentially at 2-min intervals;

[0067] In control group 2, all protease, ketoamine oxidase, and peroxidase were added initially.

[0068] 5 μg of the nanocomposite catalyst was added to 1.0 mL of glycated albumin solutions with respective mass percentage concentrations of 10.0%, 8.0%, 6.0%, 4.0%, and 2.0%. Subsequently, 100 μL of a 100 μmol / L TMB solution was added to the reaction system. The reaction was allowed to react for 10 minutes, and the results were observed. The results clearly show that after 10 minutes, the glycated albumin concentrations in all five groups measured using the nanocomposite catalyst prepared in the present invention turned blue, and the color darkened significantly with increasing glycated albumin concentration. In contrast, the two control groups showed no significant color change.

[0069] Example 7

[0070] One-step determination of limiting concentration of glycated albumin using nanocomposite catalyst

[0071] Furthermore, the nanocomposite catalyst prepared in Example 3 was used to determine the limiting concentration of glycated albumin in a one-step method. Specifically, 5 μg of the nanocomposite catalyst was added to 1.0 mL of a 0.05% by mass glycated albumin solution. Subsequently, 100 μL of a 100 μmol / L TMB solution was added to the reaction system. The reaction was allowed to react for 10 minutes, and the results were observed.

[0072] The results show that the experimental composition is light blue. When 1 μL of the reaction solution is dropped onto a piece of white paper, a clear blue mark can be seen on the paper. This indicates that the nanocomposite catalyst prepared by the present invention has high sensitivity for detecting glycated albumin, with a detection limit of 0.05%.

[0073] The nanocomposite catalyst of the present invention has the following advantages: (1) it has the catalytic properties of protease, ketoamine oxidase and peroxidase, and can catalyze an integrated tandem reaction. The intermediate products such as glycosylated amino acids and H2O2 generated during the reaction quickly enter the next reaction through an in-situ reaction. The entire tandem reaction can be completed within 30 minutes, reducing the diffusion distance and steric hindrance that need to be overcome for the release of intermediate products and entry into the next reaction in conventional tandem reactions, thereby improving the reaction efficiency and detection sensitivity; (2) the small particle size can improve the dispersibility and specific surface area of ​​the catalyst, increase the effective collision probability with the substrate molecules, and improve the reaction speed; (3) the steric hindrance of the carrier and the shielding effect on the free enzyme are reduced, thereby preventing the catalytic activity from decreasing; (4) the spatial structure and molecular conformation of the biological enzyme are regulated, making the conformation more solid and improving the stability.

[0074] The one-step method for determining glycated albumin in the present invention realizes rapid, convenient and accurate detection of glycated albumin, can shorten detection time, simplify operation steps, improve detection accuracy and reduce detection costs, and has great market value.

[0075] The detection principle of nanocomposite catalyst is:

[0076] When the sample contains glycated albumin, it first undergoes hydrolysis with the protease on the nanocomposite catalyst. The resulting glycated amino acids rapidly react with the ketoamine oxidase on the nanocomposite catalyst in situ to produce H₂O₂. Catalyzed by the peroxidase nanomimetic enzyme on the nanocomposite catalyst, H₂O₂ rapidly reacts with TMB in situ to form the blue oxidized product, oxTMB. After the reaction, the absorbance is measured to achieve quantitative detection of glycated albumin.

[0077] The reaction mechanism of the one-step determination of glycated albumin by integrated tandem catalysis of nanocomposite catalysts is as follows:

[0078]

[0079] The present invention provides a nanocomposite catalyst for in vitro detection of glycated albumin and a new detection method for glycated albumin, which shortens detection time, simplifies operation steps, improves detection accuracy and reduces detection costs.

[0080] The nanocomposite catalyst of the present invention combines the catalytic properties of three catalysts: protease, ketoamine oxidase, and peroxidase nanomimetic enzyme. It can catalyze the rapid reaction of glycated albumin with TMB through an integrated tandem reaction, generating the blue product oxTMB within 30 minutes. The absorbance is then measured to quantitatively determine glycated albumin. The nanocomposite catalyst of the present invention can effectively improve the efficiency and sensitivity of detection, enabling one-step detection of glycated albumin.

[0081] The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, several simple deductions and substitutions can be made without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

Claims

1. A nanocomposite catalyst, characterized in that: The preparation process of the nanocomposite catalyst is as follows: First, the peroxidase nanomimetic enzyme was modified with aldehyde groups to obtain CHO-nanozyme, and then the CHO-nanozyme was coupled with protease and ketoamine oxidase to obtain a nanocomposite catalyst; The process of the peroxidase nanomimetic enzyme aldehyde modification is as follows: The peroxidase nanomimetic enzyme was added to the chitosan solution and mixed thoroughly, and then the sodium polyphosphate solution was added. The mixture was reacted at room temperature for a period of time, and the chitosan-modified peroxidase nanomimetic enzyme was separated and collected. The chitosan-modified peroxidase nanomimetic enzyme was repeatedly washed with ultrapure water and glacial acetic acid solution and then redispersed in PBS buffer. Then, sodium periodate solution was added to react; the CHO-nanozyme was collected after washing again; The mass percentage concentration of the chitosan solution is 0.05-0.2%; the mass percentage concentration of the sodium polyphosphate solution is 0.5-5.0%; the mass percentage concentration of the glacial acetic acid solution is 0.5-1.5%; the mass percentage concentration of the sodium periodate solution is greater than or equal to 1.0%; the pH value of the PBS buffer solution is 1.5-6.5; The process of coupling CHO-nanozyme with protease and ketoamine oxidase to obtain nanocomposite catalyst is as follows: The CHO-nanozyme is completely dispersed in water, and then a protease solution and a ketoamine oxidase solution are added respectively; after mixing, coupling is performed, and after the coupling is completed, separation and washing are performed to obtain a nanocomposite catalyst; The temperature of the coupling reaction is controlled at 0-8°C; the time of the coupling reaction is 1-4 hours; the mass percentage concentration of the protease solution is 0.1-1.00%; the mass percentage concentration of the ketoamine oxidase solution is 0.1-1.00%.

2. The nanocomposite catalyst according to claim 1, characterized in that The peroxidase nano-mimetic enzyme includes one or more of nano-Fe3O4, nano-CoFe3O4, nano-Co3O4, nano-CeO2, nano-MnO, nano-MoS2 and nano-WS2.

3. The nanocomposite catalyst according to claim 1, characterized in that The particle size of the peroxidase nanomimetic enzyme is 1 to 1000 nm.

Images