Catalytic system composition for click chemical reaction, preparation method therefor, and use thereof in biological detection

Abstract

The present invention relates to the technical field of biological analysis and detection. Disclosed in the present invention are a catalytic system composition comprising a reducing sugar, 2,2'-bicinchoninic acid, and CuSO4, a preparation method therefor, a use thereof as a click chemical reaction catalyst, and a use thereof in biological detection. The preparation method for the catalytic system composition comprises the following steps: mixing 0.315-5.0 mM of a reducing sugar, 0.315-5.0 mM of 2,2'-bicinchoninic acid, and 1.25 mM of CuSO4 in a BCA detection buffer solution, incubating at 80°C for 5-60 minutes, cooling in an ice water bath, and then storing the product at room temperature for more than 10 days before use. Compared with classic click chemical reaction catalytic systems built on the basis of sodium ascorbate, the catalytic system composition of the present invention has the advantage that the catalytic system solution stored for more than 10 days contains almost no active oxygen molecules including H2O2.

Description

A catalytic system composition for click chemistry reaction, preparation method thereof, and application in biological detection

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese application No. 202311152900.3, filed September 8, 2023. Said application No. 202311152900.3 is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention belongs to the field of biological analysis and detection, and relates to a catalytic system composition comprising reducing sugar, 2,2′-bicinchoninic acid, and CuSO4, a preparation method thereof, and use thereof as a click chemistry reaction catalyst, as well as application thereof in biological detection. Background Art

[0004] Safety testing of food, health products, and cosmetics is an important part of controlling product quality. Current safety testing methods for food, health products, and cosmetics suffer from shortcomings such as high instrument dependence, complex testing methods, and low sensitivity. Therefore, the development of simple, sensitive, and efficient safety testing technologies is urgent. Click chemistry, also known as copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, has been widely used in analytical testing since its discovery in 2000. Researchers have established a series of rapid detection methods based on click chemistry, such as colorimetry, fluorescence, and electrochemical methods, and have applied them to the detection of food ingredients and additives, pesticide residues, toxins, and foodborne pathogens. The superior performance of click chemistry provides a new direction for safety testing of food, health products, and cosmetics.

[0005] However, the uncatalyzed CuAAC reaction requires high temperatures, hindering the implementation of analytical detection methods. However, using Cu(I) as a catalyst can significantly accelerate the CuAAC reaction rate at room temperature, enabling the successful development of analytical detection methods for foods, health products, and cosmetics. Generally speaking, the catalyst system for the CuAAC reaction consists of three key elements: copper ions, a reducing agent, and a Cu(I) chelating agent. The copper ions provide a source of Cu(I), the reducing agent reduces Cu(II) to Cu(I), and the Cu(I) chelating agent stabilizes Cu(I) from reacting with reactive oxygen species. Currently, the most commonly used catalyst systems for the CuAAC reaction are CuSO4, sodium ascorbate, and tri(heterocyclic)methylamine. However, catalyst systems using sodium ascorbate as a reducing agent have several limitations in biological assays that can interfere with the proper performance of analytical tests. First, in the presence of sodium ascorbate, free Cu(I) ions can promote the generation of reactive oxygen species. Reactive oxygen species can undergo side reactions in biological systems, producing various oxidative byproducts. Secondly, sodium ascorbate has a strong reducing activity, which can lead to a series of complex side reactions. For example, in certain in vitro diagnostics, such as urine tests, the presence of sodium ascorbate can lead to false-positive results. Finally, ascorbic acid's oxidation product, dehydroascorbic acid, can react with lysine and arginine residues in proteins, potentially causing cross-linking between proteins.

[0006] Reducing sugars, such as glucose, mannose, and lactose, represent a class of carbohydrates that can expose aldehyde groups in alkaline environments. These aldehyde groups can be oxidized by weak oxidants such as Cu(II) to produce acids. Reducing sugars can reduce Cu(II), such as CuSO4, to Cu(I). 2,2′-bicinchoninic acid chelates with Cu(I) to form a deep purple color with a maximum absorbance at 560 nm. Scientists have leveraged this principle to develop a 2,2′-bicinchoninic acid (BCA) assay for reducing sugars. Considering that the reaction mixture formed by the BCA assay for reducing sugars contains the three elements of a click chemistry catalyst system, the present invention optimized and prepared a catalytic system composition comprising a reducing sugar, 2,2′-bicinchoninic acid, and CuSO4, and evaluated its use as a click chemistry catalytic system and in bioassay reactions.

[0007] Summary of the Invention

[0008] A technical problem to be solved by the present invention is to provide a catalytic system composition comprising a reducing sugar, 2,2′-bicinchoninic acid and CuSO 4 , and a preparation method thereof.

[0009] Another technical problem to be solved by the present invention is to provide a catalytic system composition comprising a reducing sugar, 2,2′-bicinchoninic acid, and CuSO 4 as a catalyst for a click chemistry reaction.

[0010] Another technical problem to be solved by the present invention is to provide a click chemistry reaction catalytic system based on a catalytic system composition comprising a reducing sugar, 2,2′-bicinchoninic acid, and CuSO 4 for use in biomarker detection.

[0011] In order to solve the technical problems of the present invention, the present invention adopts the following technical solutions:

[0012] The first aspect of the present invention relates to a catalytic system composition comprising a reducing sugar, 2,2′-bicinchoninic acid, and CuSO4 mixed in a certain proportion, and the preparation method thereof is as follows:

[0013] Because catalytic efficiency depends to some extent on the concentration of the formed 2,2'-bicinchoninic acid and Cu(I) chelate, the present invention prepared a system containing varying ratios of reducing sugar, 2,2'-bicinchoninic acid, and CuSO4. The system was incubated at 80°C for 5 minutes, cooled with ice water, and the absorbance at 560nm, representing the concentration of the 2,2'-bicinchoninic acid and Cu(I) chelate, was measured. The system contained 1.25mM CuSO4 and 1.25mM 2,2'-bicinchoninic acid, and varying concentrations of reducing sugar (0, 0.315, 0.625, 1.25, 2.5, and 5.0mM) were added.

[0014] A further preferred method for preparing the composition is as follows: the content of CuSO4 in the combined system is set to 1.25 mM, the content of reducing sugar is set to 1.25 mM, and different concentrations of 2,2′-bicinchoninic acid (0.315, 0.625, 1.25, 2.5, 5.0 mM) are added.

[0015] A further preferred method for preparing the composition is as follows: the content of CuSO4, the content of 2,2'-bicinchoninic acid, and the content of reducing sugar in the combined system are set to 1.25 mM, wherein the reducing sugar is selected from, but not limited to, glucose, mannose, and lactose.

[0016] The second aspect of the present invention relates to the use of the click chemistry catalytic system composition described in the first aspect for catalyzing click chemistry reactions at room temperature. In a specific study, the efficiency of different reducing sugar / 2,2′-bicinchoninic acid / CuSO4 combination systems in catalyzing click chemistry reactions was evaluated. The effects of different concentrations of reducing sugar on the catalytic activity of the combination system were prepared and compared, that is, the catalytic efficiency of the catalytic system for click chemistry reactions with reducing sugar / 2,2′-bicinchoninic acid / CuSO4 concentrations of (1.25mM / 1.25mM / 1.25mM) and (5.0mM / 1.25mM / 1.25mM), respectively.

[0017] Further preferably, the effects of different concentrations of 2,2′-bicinchoninic acid on the catalytic activity of the combined system were prepared and compared, that is, the catalytic efficiency of the catalytic system for click chemistry with reducing sugar / 2,2′-bicinchoninic acid / CuSO4 concentrations of (1.25mM / 1.25mM / 1.25mM) and (1.25mM / 2.5mM / 1.25mM), respectively.

[0018] Further optimization was performed to compare the catalytic efficiency of the 1.25 mM reducing sugar / 1.25 mM 2,2′-bicinchoninic acid / 1.25 mM CuSO4 catalytic system for click chemistry reaction with different preparation times (0, 1, 3, 5, 10, 15, 20, 30, and 60 days).

[0019] Further optimization was performed and the H2O2 content in the 1.25mM reducing sugar / 1.25mM 2,2′-bicinchoninic acid / 1.25mM CuSO4 catalytic system with different preparation times (0, 1, 3, 5, 10, 15, 20, 30, and 60 days) was compared to evaluate whether it has advantages over the classic click chemistry catalytic system based on ascorbic acid.

[0020] A third aspect of the present invention relates to the use of the click chemistry catalytic system composition described in the first aspect for use in click chemistry-based bioassays. Specifically, this study employed a reducing sugar / 2,2′-bicinchoninic acid / CuSO₄ catalytic system to catalyze the biomarker detection reaction between cell nuclei containing EdU (5-ethynyl-2-deoxyuridine) and a fluorescent dye containing an azide.

[0021] Compared with the prior art, the present invention has the following beneficial effects: the present invention develops a novel click chemistry catalytic system composition. Compared with the classic click chemistry reaction catalytic system based on sodium ascorbate, the advantage of the catalytic system composition of the present invention is that the catalytic system solution that has been placed for more than 10 days contains almost no active oxygen molecules including H2O2. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: Absorbance-time changes of various reducing sugar / 2,2′-bicinchoninic acid / CuSO4 combination systems.

[0023] Figure 2: Absorbance values ​​of reducing sugar / 2,2′-bicinchoninic acid / CuSO4 catalytic system containing different concentrations of 2,2′-bicinchoninic acid.

[0024] Figure 3: Click chemistry reaction involving 3-azido-7-hydroxycoumarin and propargylamine hydrochloride.

[0025] Figure 4: Comparison of the activity of the glucose / 2,2′-bicinchoninic acid / CuSO4 combination system catalyzed click chemistry reactions with different ratios of glucose or 2,2′-bicinchoninic acid.

[0026] Figure 5: Comparison of the activity of the mannose / 2,2′-bicinchoninic acid / CuSO4 combination system catalyzed click chemistry reactions with different ratios of mannose or 2,2′-bicinchoninic acid.

[0027] Figure 6: Comparison of the activity of the lactose / 2,2′-bicinchoninic acid / CuSO4 combination system catalyzed click chemistry reactions with different ratios of lactose or 2,2′-bicinchoninic acid.

[0028] Figure 7: Activity of the combined system of reducing sugar / 2,2′-bicinchoninic acid / CuSO4 prepared for different days in catalyzing click chemistry reactions.

[0029] Figure 8: H2O2 content in the combined system of reducing sugar / 2,2′-bicinchoninic acid / CuSO4 at different preparation days.

[0030] Figure 9: A reducing sugar / 2,2′-bicinchoninic acid / CuSO₄ catalytic system was used to assay cell proliferation for the incorporation of azide-labeled fluorescent dye EdU. The first row shows the results using glucose, the second using mannose, and the third using lactose. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0032] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0034] Example 1: Preparation of reducing sugar / 2,2′-bicinchoninic acid / CuSO4 catalytic system and detection of absorbance at 560 nm

[0035] Experimental method: Solution A was prepared to contain 512mM sodium carbonate, 288mM sodium bicarbonate and 5.0mM 2,2′-bicinchoninic acid. Solution B was prepared to contain 5.0mM CuSO4 and 12.0mM L-serine. The BCA assay working solution was prepared by mixing solution A: solution B: PBS buffer in a volume ratio of 1:1:2. The BCA assay working solution contained 1.25mM CuSO4 and 1.25mM 2,2′-bicinchoninic acid. Different concentrations of glucose, mannose or lactose (0, 0.315, 0.625, 1.25, 2.5, 5.0mM) were added to the BCA assay working solution. Incubate at 80°C for different times (1, 3, 5, 10, 15, 20, 30, 45, 60 minutes) and immediately cool with ice water. The microplate reader ( The absorbance at 560 nm was detected in a 96-well microplate.

[0036] Experimental Results: As shown in Figure 1, upon adding glucose, mannose, or lactose to the BCA assay system, the absorbance immediately changed. The absorbance values ​​for each group peaked between 5 and 10 minutes, and further incubation resulted in a slight decrease. Addition of different concentrations of glucose, mannose, or lactose (0.315, 0.625, 1.25, 2.50, and 5.0 mM) resulted in changes in absorbance at 560 nm. When the concentration of glucose, mannose, or lactose reached 1.25 mM, further increases in glucose, mannose, or lactose had little effect on the change in absorbance. This change in absorbance indicates the successful preparation of the catalyst system.

[0037] Example 2: Preparation of Reducing Sugar / 2,2'-bicinchoninic Acid / CuSO4 Catalytic System Containing Different Concentrations of 2,2'-bicinchoninic Acid

[0038] Experimental method: Solution A was prepared to contain 512mM sodium carbonate and 288mM sodium bicarbonate. Different concentrations of 2,2′-bicinchoninic acid (0.625, 1.25, 2.50, 5.0, 10.0mM) were added to solution A to prepare solution A containing different concentrations of BCA. Solution B was prepared to contain 5.0mM CuSO4 and 12.0mM L-serine. The BCA assay working solution was prepared by mixing solution A: solution B: PBS buffer in a volume ratio of 1:1:2. The BCA assay working solution contained 1.25mM CuSO4 and different concentrations (0.315, 0.625, 1.25, 2.50, 5.0mM) of 2,2′-bicinchoninic acid. Glucose, mannose or lactose was added to the BCA assay working solution to a final concentration of 1.25mM. Incubate at 80°C for 5min and immediately cool with ice water. The microplate reader ( The absorbance at 560 nm was detected in a 96-well microplate.

[0039] Experimental Results: As shown in Figure 2, a system containing 2,2′-bicinchoninic acid (BCA) at various concentrations (0.315, 0.625, 1.25, 2.50, and 5.0 mM) was incubated at 80°C for 5 minutes, and the absorbance was measured. The results show that the absorbance of the system increased rapidly as the BCA concentration increased from 0.315 to 2.5 mM, reaching peak levels at BCA concentrations between 2.5 and 5.0 mM. At this peak, the ratio of CuSO₄ to BCA was approximately 1:2. This suggests that two BCA molecules can chelate one Cu(I) molecule for the formation of a BCA-Cu(I) chelate complex. Therefore, a 1:2 CuSO₄ to BCA ratio favors the formation of high-concentration 2,2′-bicinchoninic acid-Cu(I) chelates. Similar results were observed using glucose, mannose, or lactose as reducing sugars.

[0040] Example 3: Activity Evaluation of Various Reducing Sugar / 2,2′-bicinchoninic Acid / CuSO4 Combination Systems Catalyzing Click Chemistry Reactions

[0041] Experimental method: 3-azido-7-hydroxycoumarin (0.2 μL, 50 mM DMSO stock solution), propargylamine hydrochloride (1.0 μL, 50 mM water stock solution) and the prepared reducing sugar / 2,2′-bicinchoninic acid / CuSO4 catalytic system (40 μL) were added to PBS buffer (160 μL, pH 7.2), and the fluorescence intensity (E x =395nm, E m=470nm). The fluorescence enhancement factor, F / F0, was calculated, where F0 and F are the fluorescence intensities before and after the CuAAC reaction, respectively. In a control experiment, only 3-azido-7-hydroxycoumarin (azide) was added to the PBS buffer without propargylamine hydrochloride (alkynyl) to evaluate the effect of the catalyst system on the azide group. In the present invention, catalytic systems with reducing sugar / 2,2′-bicinchoninic acid / CuSO4 concentrations of (1.25mM / 1.25mM / 1.25mM) and (5.0mM / 1.25mM / 1.25mM), respectively, were prepared, and their catalytic activity for the CuAAC reaction was evaluated. Similarly, catalytic systems with reducing sugar / 2,2′-bicinchoninic acid / CuSO₄ concentrations of (1.25mM / 1.25mM / 1.25mM) and (1.25mM / 2.5mM / 1.25mM) were prepared and their catalytic activity for the CuAAC reaction was evaluated. The reducing sugars used in the experiments were glucose, mannose, or lactose.

[0042] Experimental Results: As shown in Figure 3, the cycloaddition reaction between coumarin azide and alkyne is often used to evaluate the efficiency of catalyst systems in CuAAC reactions. Coumarin azide is weakly fluorescent, but the cycloaddition reaction produces a highly fluorescent triazole. The increase in fluorescence intensity can indicate the reaction rate.

[0043] As shown in Figure 4, the experimental results show that the reaction solution containing only azide showed essentially no fluorescence change. However, in the CuAAC reaction solution containing both azide and alkyne groups, the addition of the glucose / 2,2′-bicinchoninic acid / CuSO₄ system promoted changes in fluorescence intensity. Both 1.25 mM and 5.0 mM glucose systems exhibited strong CuAAC catalytic activity, with the 1.25 mM glucose system exhibiting slightly higher activity than the 5.0 mM glucose system. Similarly, 1.25 mM and 2.5 mM BCA systems were both able to catalyze click chemistry reactions. 1.25 mM BCA exhibited strong catalytic activity, while 2.5 mM BCA exhibited relatively weak catalytic activity. The high concentration of chelating agent in the glucose / 2,2′-bicinchoninic acid / CuSO₄ system was detrimental to the catalytic activity of the click chemistry reaction.

[0044] As shown in Figure 5, the experimental results show that the reaction solution containing only azide showed essentially no fluorescence change. However, in the CuAAC reaction solution containing both azide and alkyne groups, the addition of the mannose / 2,2′-bicinchoninic acid / CuSO₄ system promoted changes in fluorescence intensity. Both the 1.25 mM and 5.0 mM mannose catalytic systems exhibited strong CuAAC activity, with the 1.25 mM mannose system showing slightly higher activity than the 5.0 mM mannose system. Similarly, the 1.25 mM and 2.5 mM BCA catalytic systems were both able to catalyze click chemistry reactions. 1.25 mM BCA exhibited strong catalytic activity, while 2.5 mM BCA exhibited relatively weak catalytic activity. The high concentration of chelating agent in the mannose / 2,2′-bicinchoninic acid / CuSO₄ system was detrimental to the catalytic activity of the click chemistry reaction.

[0045] As shown in Figure 6, the experimental results show that the reaction solution containing only azide exhibited essentially no fluorescence change. However, the addition of the lactose / 2,2′-bicinchoninic acid / CuSO₄ system to the CuAAC reaction solution containing both azide and alkyne groups promoted changes in fluorescence intensity. Both 1.25 mM and 5.0 mM lactose catalytic systems exhibited strong CuAAC activity, with the 1.25 mM lactose system exhibiting slightly higher activity than the 5.0 mM lactose system. Similarly, 1.25 mM and 2.5 mM BCA catalytic systems were able to catalyze click chemistry reactions. 1.25 mM BCA exhibited strong catalytic activity, while 2.5 mM BCA exhibited relatively weak activity. The high concentration of chelating agent in the lactose / 2,2′-bicinchoninic acid / CuSO₄ system was detrimental to the catalytic activity of the click chemistry reaction.

[0046] Example 4: Evaluation of the activity of reducing sugar / 2,2′-bicinchoninic acid / CuSO4 combined systems prepared on different days in catalyzing click chemistry reactions

[0047] Experimental Methods: Solution A was prepared to contain 512 mM sodium carbonate, 288 mM sodium bicarbonate, and 5.0 mM 2,2′-bicinchoninic acid. Solution B was prepared to contain 5.0 mM CuSO₄ and 12.0 mM L-serine. The BCA working solution was prepared by mixing Solution A:Solution B:PBS buffer in a 1:1:2 volume ratio. The BCA working solution contained 1.25 mM CuSO₄ and 1.25 mM 2,2′-bicinchoninic acid. 1.25 mM glucose, mannose, or lactose was added to the BCA working solution. The samples were incubated at 80°C for 5 minutes, cooled on ice, and then stored at room temperature for different days (0, 1, 3, 5, 10, 15, 20, 30, and 60 days). The catalytic activity of the samples in the CuAAC reaction was evaluated.

[0048] Experimental Results: As shown in Figure 7, the freshly prepared (0-day) glucose / 2,2′-bicinchoninic acid / CuSO4 catalytic system exhibited weak CuAAC catalytic activity, with a F / F0 change of 1.92 within 10 minutes. After aging at room temperature, the activity of the catalytic system gradually increased. The catalytic activity remained relatively weak from 1 to 10 days, with a F / F0 change of 2.03-3.05 within 10 minutes. Starting at 10 days, the activity of the catalytic system began to increase rapidly, reaching very strong catalytic activity from 15 to 60 days, with a F / F0 change of 3.79-10.76 within 10 minutes.

[0049] The freshly prepared (day 0) mannose / 2,2′-bicinchoninic acid / CuSO4 system exhibited weak CuAAC activity, with a F / F0 change of 1.63 within 10 minutes. After aging at room temperature, the activity of the system gradually increased. From day 1 to 10, the catalytic activity remained relatively weak, with a F / F0 change of 1.70 to 3.57 within 10 minutes. Starting at day 10, the activity of the system began to increase rapidly, reaching very strong activity between days 15 and 60, with a F / F0 change of 3.45 to 13.64 within 10 minutes.

[0050] The freshly prepared lactose / 2,2′-bicinchoninic acid / CuSO4 system (i.e., day 0) exhibited weak CuAAC activity, with a F / F0 change of 1.61 within 10 minutes. After aging at room temperature, the activity of the system gradually increased. From day 1 to 10, the catalytic activity remained relatively weak, with a F / F0 change of 1.87 to 2.40 within 10 minutes. Starting at day 10, the activity of the system began to increase rapidly, reaching very strong activity between days 15 and 60, with a F / F0 change of 2.57 to 7.59 within 10 minutes.

[0051] Example 5: H2O2 Content in Various Reducing Sugar / 2,2′-bicinchoninic Acid / CuSO4 Combination Systems

[0052] Experimental method: Using Amplex TM The Red Hydrogen Peroxide / Peroxidase Assay Kit was used to measure the H2O2 content in various reducing sugar / 2,2′-bicinchoninic acid / CuSO4 systems. Working solutions were prepared according to the manufacturer's instructions. To 50 μL of the working solution, 50 μL of PBS buffer containing a 10% reducing sugar / 2,2′-bicinchoninic acid / CuSO4 (1.25 mM / 1.25 mM / 1.25 mM) catalytic system prepared for different days (0, 1, 3, 5, 10, 15, 20, 30, and 60 days) was added. After incubation for 30 minutes, the H2O2 content in the system was measured. PBS buffer containing a 10% sodium ascorbate / THTPA / CuSO4 (1.25 mM / 1.25 mM / 1.25 mM) catalytic system and PBS buffer were also measured as controls. The reducing sugars used in the experiments were glucose, mannose, or lactose.

[0053] Experimental Results: As shown in Figure 8, the H2O2 content in various solutions was measured. The experimental results show that consistent experimental results were obtained using three reducing sugars: glucose, mannose, and lactose.

[0054] A freshly prepared (i.e., day 0) 5% glucose / 2,2′-bicinchoninic acid / CuSO4 system contained 0.485 μM H2O2. However, after aging at room temperature, the H2O2 content gradually decreased, reaching a value (0.085-0.089 μM) similar to that measured in PBS buffer (0.173 μM), essentially indicating the absence of significant H2O2 and other reactive oxygen species. Notably, as shown in Figure 7, the catalytic activity of the glucose / 2,2′-bicinchoninic acid / CuSO4 system significantly increased after aging for more than 10 days, making it more suitable as a catalyst for click chemistry reactions.

[0055] A freshly prepared (i.e., day 0) 5% mannose / 2,2′-bicinchoninic acid / CuSO4 system contained 0.394 μM H2O2. However, after aging at room temperature, the H2O2 content gradually decreased. After 10 days, the H2O2 levels in the 10-, 15-, 30-, and 60-day systems (0.086-0.089 μM) were similar to those in PBS buffer (0.173 μM), indicating the absence of significant H2O2 and other reactive oxygen species. Notably, as shown in Figure 7, the catalytic activity of the mannose / 2,2′-bicinchoninic acid / CuSO4 system significantly increased after aging for more than 10 days, making it more suitable as a catalyst for click chemistry reactions.

[0056] A freshly prepared (i.e., day 0) 5% lactose / 2,2′-bicinchoninic acid / CuSO4 system contains 0.490 μM H2O2. However, after aging at room temperature, the H2O2 content gradually decreases. After 10 days, the H2O2 content (0.098-0.200 μM) is similar to the H2O2 content measured in PBS buffer (0.173 μM), essentially indicating the absence of significant H2O2 and other reactive oxygen species. Notably, as shown in Figure 7, the catalytic activity of the lactose / 2,2′-bicinchoninic acid / CuSO4 system significantly increases after aging for more than 10 days, making it more suitable as a catalyst for click chemistry reactions.

[0057] In contrast, the catalytic system containing 5% sodium ascorbate / THTPA / CuSO4 (1.25mM / 1.25mM / 1.25mM) contained 1.051μM H2O2. As is well known, ascorbic acid-based catalytic systems need to be prepared immediately before use, and their catalytic activity in the CuAAC reaction will decrease significantly in a short period of time.

[0058] Example 6: Application of the Reducing Sugar / 2,2′-bicinchoninic Acid / CuSO4 Catalytic System in Cell Proliferation Assays Incorporating EdU with Azide-Containing Fluorescent Dyes

[0059] Experimental Methods: MCF-7 cells (purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured and incubated overnight with 10 μM EdU. After elution with PBS, the cells were fixed with 4% paraformaldehyde for 15 minutes, and then permeabilized with 1% Triton X-100 for another 15 minutes. A 20% volume glucose / 2,2′-bicinchoninic acid / CuSO₄ catalytic system was added to PBS buffer, and a fluorescent dye (FAM azide, 6-isomer) was added to the system to a final concentration of 250 μM. The cells were incubated for 30 minutes. After washing with PBS, the cells were observed under a fluorescence microscope. Glucose, mannose, or lactose were used as the reducing sugar for the experiments.

[0060] Experimental Results: Similar labeling detection results were obtained using glucose, mannose, or lactose as reducing sugars in the experiment. After 30 minutes of catalysis using the reducing sugar / 2,2′-bicinchoninic acid / CuSO4 catalytic system, the cell nuclei were observed to be fluorescently labeled, as shown in the image on the left in Figure 9. The fluorescence intensity of the cell nuclei indicates the difference in EdU incorporation into the cell nuclei. The image on the right in Figure 9 also shows the superposition effect of the fluorescently labeled cell nuclei and the cells under visible light. These results indicate that the reducing sugar / 2,2′-bicinchoninic acid / CuSO4 catalytic system can successfully catalyze the fluorescent dye labeling of EdU-incorporated cell nuclei.

[0061] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A catalytic system composition for click chemistry reaction, characterized in that: The catalytic system composition contains a combination of reducing sugar, 2,2′-bicinchoninic acid (BCA) and CuSO 4 .

2. The catalytic system composition for click chemistry reaction according to claim 1, characterized in that: The reducing sugar represents a type of carbohydrate that can expose an aldehyde group in an alkaline environment, and the reducing sugar is selected from one or more of the following: glucose, mannose, lactose, maltose or fructose.

3. A method for preparing a catalytic system composition for click chemistry reaction according to claim 1 or claim 2, characterized in that: The preparation method comprises the following steps: mixing 0.315-5.0 mM reducing sugar in a BCA detection buffer containing 1.25 mM CuSO4 and 0.315-5.0 mM 2,2′-bicinchoninic acid, incubating at 80° C. for 5-60 minutes, cooling with ice water, storing at room temperature and using.

4. The method for preparing a catalytic system composition for click chemistry reaction according to claim 3, characterized in that: The preparation method comprises the following steps: mixing 1.25 mM reducing sugar in a BCA detection buffer containing 1.25 mM CuSO4 and 1.25 mM 2,2′-bicinchoninic acid, incubating at 80° C. for 5 minutes, cooling with ice water, storing at room temperature and using.

5. The method for preparing a catalytic system composition for click chemistry reaction according to claim 4, characterized in that: The preparation method comprises the following steps: mixing 1.25 mM reducing sugar in a BCA detection buffer containing 1.25 mM CuSO4 and 1.25 mM 2,2′-bicinchoninic acid, incubating at 80° C. for 5 minutes, cooling with ice water, and placing at room temperature for more than 10 days before use.

6. The method for preparing a catalytic system composition for click chemistry reaction according to claim 5, characterized in that: The preparation method comprises the following steps: mixing 1.25 mM reducing sugar in a BCA detection buffer containing 1.25 mM CuSO4 and 1.25 mM 2,2'-bicinchoninic acid, incubating at 80°C for 5 minutes, cooling with ice water, placing at room temperature for 10-60 days and then using.

7. The method for preparing the click chemistry catalytic system composition according to claim 6, characterized in that: The preparation method comprises the following steps: (1) preparing solution A and solution B, wherein solution A contains 512 mM sodium carbonate, 288 mM sodium bicarbonate and 5.0 mM 2,2′-bicinchoninic acid, and solution B contains 5.0 mM CuSO4 and 12.0 mM L-serine; (2) preparing a BCA detection buffer: the BCA detection buffer is prepared by mixing solution A: solution B: PBS buffer in a volume ratio of 1:1:2; (3) preparing a catalytic system composition for a click chemistry reaction: adding reducing sugar to the BCA detection buffer to a final concentration of 1.25 mM, incubating at 80°C for 5 minutes, cooling with ice water, and placing at room temperature for 10-60 days before use.

8. Use of a catalytic system composition for click chemistry reaction according to claim 1 or claim 2 for catalyzing click chemistry reaction at room temperature, characterized in that: The click chemistry reaction is a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.

9. Use of the catalytic system composition for click chemistry reaction according to claim 1 or claim 2 in biological detection based on click chemistry reaction.

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