A method for synthesizing HRP catalyzed polyacrylamide gel based on gold nanocluster monitoring

Abstract

The application provides a synthesis method of HRP catalytic polyacrylamide gel based on gold nanocluster monitoring, which comprises the following steps: taking acrylamide and N, N'-methylene bisacrylamide as monomers, and synthesizing polyacrylamide gel under the action of a ternary initiation system of HRP / H2O2 / ACAC. The aggregation-induced emission (AIE) phenomenon of BSA-stabilized gold nanoclusters (BSA-Au NCs) is used to monitor the polymerization reaction of the system. The application not only innovatively uses the environment-friendly ternary initiation system of HRP / H2O2 / ACAC to replace the TEMED / APS system to initiate Acr-Bis polymerization, and overcomes the potential toxicity of TEMED / APS, but also innovatively uses the AIE characteristics of BSA-Au NCs to realize real-time monitoring of monomer polymerization reaction.

Description

Technical Field

[0001] This invention relates to the field of polyacrylamide synthesis technology, and in particular to a method for synthesizing HRP-catalyzed polyacrylamide gel based on gold nanoclusters monitoring. Background Technology

[0002] Polyacrylamide gel electrophoresis (PAGE) is an electrophoretic method using polyacrylamide gels as carriers, providing a universal, mild, and high-resolution approach for the analysis and characterization of proteins and nucleic acids. Polyacrylamide gels are three-dimensional network gels formed by the polymerization and cross-linking of acrylamide and N,N'-methylenebisacrylamide (Acr-Bis) under the catalysis of N,N,N',N'-tetramethylethylenediamine / ammonium persulfate (TEMED / APS). However, the potential toxicity of APS and TEMED limits their application in the biological field. For example, APS can produce a variety of skin and respiratory reactions, including allergic eczema-like contact dermatitis, irritant dermatitis, localized edema, generalized urticaria, rhinitis, asthma, and syncope. Furthermore, TEMED irritates the skin, eyes, and respiratory tract, and also exhibits strong neurotoxicity by inhibiting acetylcholinesterase and thus having a toxic effect on the brain. Therefore, exploring a safe gel polymerization catalytic system is extremely important.

[0003] Enzyme-mediated free radical polymerization was first reported by Palavano in 1951. Enzymatic polymerization is a convenient and environmentally friendly strategy for preparing nanocomposite hydrogels with high mechanical strength. Horseradish peroxidase (HRP) is a very popular and efficient enzyme that has been extensively studied in the preparation of hydrogels. In 1992, Derango et al. first demonstrated the potential use of HRP and other oxidases as catalysts for the free radical polymerization of vinyl monomers. In 2013, Su et al. reported the polymerization of hydrogels initiated by ACAC radicals generated from the HRP / H2O2 / ACAC ternary system. In this ternary system, HRP catalyzes the production of OH· from H2O2 to initiate the polymerization of acrylamide monomers, further oxidizing ACAC to ACAC·. This ternary system catalyzes the polymerization of Acr-Bis to generate polyacrylamide gel, but whether the polyacrylamide gel formed by this catalysis possesses the same electrophoretic properties as polyacrylamide gels formed by traditional TEMED / APS catalysis has not yet been reported.

[0004] The diffusion of monomers and catalysts plays a crucial role in polymerization kinetics. In-situ monitoring of the polymerization process helps us fully understand the differences in monomer polymerization initiated by two systems. However, existing monitoring methods for free radical polymerization systems require specific instruments, such as rheometers, viscometers, or nuclear magnetic resonance (NMR) spectrometers. Fluorescence, due to its high sensitivity, is an ideal tool for this field. Since most polymers exhibit very weak or no fluorescence, external fluorescent dyes are needed to label the polymers. For example, Academician Tang Benzhong's team used covalently modified tetraphenylethylene (TPE, a luminol with aggregation-induced emission (AIE) activity) to monitor the polymerization of various monomers. Due to the restriction of intramolecular motion (RIM), the fluorescence of AIE luminol is enhanced in the aggregated state. Therefore, the fluorescence of AIE luminol is highly sensitive to environmental changes, making it suitable as a probe to sense viscosity changes during polymerization. However, TPE has poor water solubility and cannot be applied to Acr-Bis polymerization systems. The AIE properties of multi-ligand-stabilized gold nanoclusters (Au NCs) with excellent water solubility are often used in the field of biosensors. Among them, BSA-stabilized Au NCs (BSA-Au NCs) have advantages such as simple synthesis, low toxicity, biocompatibility, and excellent photostability. However, no studies have been reported on monitoring the polymerization reaction of BSA-Au NCs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for synthesizing HRP-catalyzed polyacrylamide gels based on gold nanoclusters monitoring. This method solves the problem of potential toxicity of APS and TEMED in existing technologies.

[0006] In a first aspect, the present invention provides a method for synthesizing HRP-catalyzed polyacrylamide gel based on gold nanocluster monitoring, comprising the following steps: using acrylamide and N,N'-methylenebisacrylamide as monomers, synthesizing polyacrylamide gel under the action of an HRP / H2O2 / ACAC ternary initiation system, and finally applying the synthesized polyacrylamide gel to electrophoresis.

[0007] In one embodiment of the present invention, the following steps are included:

[0008] S1 mixes acrylamide and N,N'-methylenebisacrylamide premix with water to obtain a mixed solution;

[0009] S2 was reacted by adding an HRP / H2O2 / ACAC ternary initiation system to a mixed solution to obtain a polyacrylamide gel.

[0010] In one specific embodiment of the present invention, in step S1, the volume ratio of acrylamide and N,N'-methylenebisacrylamide premix to water is 3:5;

[0011] The ratio of acrylamide to N,N'-methylenebisacrylamide in the premix is ​​29:1.

[0012] In one specific embodiment of the present invention, in step S2, the concentration of ACAC is 4.88-29.25 mg / mL; the concentration of HRP is 0.02-0.1 mg / mL; and the concentration of H2O2 is 0.5-1.0 mM.

[0013] Preferably, the reaction time is 10-40 min.

[0014] In one specific embodiment of the present invention, BSA-Au NCs are added in step S1;

[0015] Preferably, the volume ratio of BSA-Au NCs to water is 2:11.

[0016] In one specific embodiment of the present invention, the preparation method of BSA-Au NCs includes: adding tetrachloroauric acid to a BSA solution for reaction, adding an alkaline solution and stirring the reaction, incubating, and filtering.

[0017] Preferably, the alkaline solution is NaOH; the reaction time is 3-7 min; the incubation temperature is 38℃; and the incubation time is 12 h.

[0018] In a second aspect, the present invention provides a polyacrylamide gel prepared by the above-described synthesis method.

[0019] In a second aspect, the present invention provides the application of the above-described polyacrylamide gel in protein and nucleic acid electrophoresis.

[0020] The technical principle of this invention is as follows:

[0021] Typically, Au NCs are embedded in nanomaterials or microporous nanomaterials such as metal-organic frameworks to achieve AIE luminescence through spatial confinement effects. This inspired us to induce the AIE effect by doping Au NCs into polyacrylamide gels. Encouragingly, the fluorescence intensity of BSA-AuNCs catalyzed by both catalytic systems in polyacrylamide gels was significantly enhanced compared to that of free BSA-Au NCs in Acr-Bis solution, indicating that the fluorescence properties of the composites originate from BSA-Au NCs.

[0022] The invention proposes using an HRP / H2O2 / ACAC ternary system to replace TEMED / APS for catalyzing Acr-Bis polymerization. It has been verified that gels catalyzed by the HRP / H2O2 / ACAC ternary system can be used for functional experiments such as protein and nucleic acid electrophoresis, demonstrating the application potential of polyacrylamide gels catalyzed by the HRP / H2O2 / ACAC system in electrophoresis.

[0023] Furthermore, the AIE properties of BSA-Au NCs were used for kinetic monitoring of the Acr-Bis monomer polymerization reaction. It was found that HRP / H2O2 / ACAC could initiate monomer polymerization within 10 min. As expected, HRP-catalyzed polyacrylamide gels remained suitable for protein and nucleic acid electrophoresis.

[0024] Ultimately, these findings provide new insights into the monitoring of polymerization systems and offer advanced strategies for exploring safe catalytic systems for gel polymerization.

[0025] The present invention has the following beneficial effects:

[0026] (1) This invention innovatively uses an environmentally friendly HRP / H2O2 / ACAC ternary initiation system to replace the TEMED / APS system to initiate Acr-Bis polymerization.

[0027] (2) This invention innovatively proposes to utilize the AIE properties of BSA-Au NCs to monitor the polymerization reaction of monomers in real time. The AIE effect of BSA-Au NCs in the gel can be used as a probe to sense the viscosity change during polymerization. At the same time, the formation of polyacrylamide gel using this enzyme initiation system can replace the highly toxic TEMED / APS initiation system, and nucleic acid and protein electrophoresis can still be performed. Attached Figure Description

[0028] Figure 1 A shows the fluorescence spectrum of BSA-Au NCs, with photographs of BSA-Au NCs taken under sunlight (a) and ultraviolet light ((365nm, b)). Figure 1 B represents the UV-Vis absorption spectrum of BSA-Au NCs (red line) and the UV-Vis absorption spectrum of BSA (black line); Figure 1 C is an HRTEM image of BSA-Au NCs in aqueous solution; Figure 1 D is the fluorescence decay curve of the BSA-Au NCs solution.

[0029] Figure 2 A represents the fluorescence spectra of polyacrylamide gel containing BSA-Au NCs before and after polymerization under the catalysis of TEMED / APS or HRP / H2O2 / ACAC initiation systems; Figure 2 B is a schematic diagram of the AIE effect of BSA-Au NCs in polyacrylamide gel; Figure 2 C represents the fluorescence kinetics of the TEMED / APS-catalyzed BSA-Au NCs polymerization system; Figure 2 D represents the monomer conversion rate of the TEMED / APS-initiated polymerization system at different time points; Figure 2 E represents the exponential relationship between the conversion rate and fluorescence intensity of TEMED / APS catalysis; Figure 2 F represents the fluorescence kinetics of the HRP / H2O2 / ACAC-catalyzed BSA-Au NCs polymerization system; Figure 2 G represents the monomer conversion rate of the HRP / H2O2 / ACAC-initiated polymerization system at different time points; Figure 2 H represents the conversion rate of HRP / H2O2 / ACAC catalysis, which is exponentially related to PL intensity;

[0030] Figure 3 A shows the response photos of the polymerization system under different electrophoretic conditions (pH, SDS, and TBE); 3B shows the response photos of the polymerization system to different concentrations of ACAC; 3C shows the response photos of the polymerization system to different concentrations of HRP; 3D shows the response photos of the polymerization system to different concentrations of H2O2.

[0031] Figure 4 Functional validation of HRP / H2O2 / ACAC-catalyzed polyacrylamide gel electrophoresis: Electrophoresis was performed using TEMED / APS (left) and HRP / H2O2 / ACAC (right) catalyzed 10% polyacrylamide gels to analyze DNA marker (A) and protein marker (B) at different concentrations; C shows the Western blot analysis of β-actin separated by the two polyacrylamide gel electrophoresis methods.

[0032] Figure 5 A schematic diagram of the HRP / H2O2 / ACAC ternary initiation system catalyzing the polymerization of Acr-Bis and the AIE phenomenon of BSA-Au NCs induced by the polymerization of Acr-Bis;

[0033] Figure 6 HRTEM image of BSA-Au NCs embedded in polyacrylamide gel (1%). Detailed Implementation

[0034] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] Reagents used: 30% acrylamide (acrylamide / bisacrylamide = 29:1), ammonium persulfate (APS), and 10% sodium dodecyl sulfate (SDS) were purchased from Wuhan Saiwei Biotechnology Co., Ltd. 1M Tris-HCl (pH 8.8) and 1M Tris-HCl (pH 6.8) were purchased from Beijing Labgic Technology Co., Ltd. Chloroauric acid (HAuCl4) and sodium hydroxide (NaOH) were provided by Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin (BSA) was purchased from Dalian Meilun Biotechnology Co., Ltd. Horseradish peroxidase (HRP, Rz>3.0) was purchased from Shanghai Sangon Biotech Co., Ltd. N,N,N',N'-Tetramethylethylenediamine (TEMED) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Methanol was purchased from Chongqing Chuandong Chemical (Group) Co., Ltd. Coomassie Brilliant Blue staining solution and the corresponding destaining solution were purchased from Beijing Leijie Biotechnology Co., Ltd. Mouse anti-human β-actin antibody (mAbcam8226) and goat anti-mouse HRP-IgG antibody (ab205719) were purchased from Abcam (Shanghai, China). Multicolor pre-stained protein ladder and Goldview were purchased from Epizyme Biomedical Technology Co., Ltd. (Shanghai). DL500 DNA Marker was purchased from TAKARA. BSA-Au NCs (bovine serum albumin (BSA) protected gold nanoclusters) could be purchased from Qiyue Biotechnology or prepared according to the literature (Protein-directed synthesis of highly fluorescent gold nanooclusters. J Am Chem Soc. 2009, 131(3): 888-889.).

[0036] Example 1: Preparation of BSA-Au NCs

[0037] BSA-AuNCs exhibiting red light emission and high quantum yield were prepared by in-situ stabilization and reduction of gold precursors using BSA. Aqueous solutions of HAuCl4 (170 μL, 29.4 mM) were added to a BSA solution (500 μL, 50 mg / mL), and the reaction was carried out with vigorous stirring for 3 min. Then, 50 μL of NaOH (1 M) solution was added, and the reaction was carried out with vigorous stirring for 7 min. Finally, the reaction was incubated at 38 °C for 12 h to complete the reaction. The prepared BSA-Au NCs were then filtered through a 3.5 kDa dialysis membrane. The size, morphology, and optical properties of the synthesized BSA-Au NCs were characterized by HRTEM, UV-Vis absorption spectroscopy, and laser emission spectroscopy. Finally, the prepared BSA-Au NCs were stored at 4 °C for further use.

[0038] like Figure 1 As shown in Figure A, the synthesized BSA-Au NCs solution is dark brown and emits red light under ultraviolet light (365 nm) irradiation. Figure 1 A shows the fluorescence spectrum of BSA-Au NCs. The prepared BSA-Au NCs exhibited dual emission wavelengths of 437 nm and 667 nm under 368 nm excitation. UV-Vis absorption spectroscopy results showed that the synthesized BSA-Au NCs had no absorption peak at 520 nm, indicating that Au NCs were formed rather than gold nanoparticles. Figure 1 B). HRTEM images show that BSA-Au NCs have a very uniform pattern typical of Au NCs ( Figure 1 C), with an average diameter of 2 ± 0.6 nm. The above experiments demonstrate that BSA-Au NCs were successfully synthesized. Figure 1 Figure D shows the fluorescence decay curve of the BSA-Au NCs solution at 670 nm, and the quantum yield of the BSA-Au NCs solution in the 400–700 nm range (excited at 380 nm). The average fluorescence lifetime of BSA-Au NCs was determined to be 1.42 μs, and the photoluminescent quantum yield (PLQY) was approximately 10.57%, consistent with previous findings.

[0039] Example 2: Monitoring the polymerization reaction of Acr-Bis by fluorescence kinetics

[0040] Acr-Bis (330 μL), H2O (550 μL), and BSA-Au NCs (100 μL) were mixed under vigorous vortexing to obtain a homogeneous solution. Then, an initiation system of HRP (1 μL, 100 μg / mL) / H2O2 (1 μL, 1M) / ACAC (20 μL) or TEMED (1 μL) / APS (10 μL) was added to the above mixed solution. After mixing, 200 μL of the mixture was immediately added to a 96-well plate for detection. The excitation wavelength was 370 nm, and the emission wavelength was 430 nm. The mechanism of HRP / H2O2 / ACAC initiating the polymerization of ACR-BIS monomers is as follows: Figure 5 As shown.

[0041] Compared with the fluorescence intensity of free BSA-Au NCs in Acr-Bis solution, the fluorescence intensity of BSA-Au NCs catalyzed by both catalytic systems in polyacrylamide gel was significantly enhanced. Figure 2 A) indicates that the fluorescence properties of the complex originate from BSA-Au NCs. Meanwhile, Figure 6 HRTEM image of BSA-Au NCs embedded in a 1% polyacrylamide gel. BSA-Au NCs are mainly aggregated in the hydrogel. Figure 2B shows the AIE effect of BSA-Au NCs in polyacrylamide gel. Therefore, the AIE effect caused by the local viscosity increase of AuNCs can be considered for monitoring the polymerization reaction of monomers. Figure 2 C and 2F show the fluorescence kinetics of Acr-Bis polymerization catalyzed by TEMED / APS or HRP / H2O2 / ACAC, respectively. To elucidate the relationship between fluorescence intensity and monomer conversion, [the following parameters were used]. 1 1H NMR spectroscopy characterized the two initiator systems ( Figure 2 The monomer conversion rate of the D and G catalytic polymerization system changes with time. Notably, we found that the monomer conversion rate is related to the PL intensity (…). Figure 2 The exponential relationship between E and H indicates that fluorescence intensity and monomer conversion rate show similar trends. These results suggest that changes in the fluorescence intensity of BSA-AuNCs can reflect the degree of polymerization of the polyacrylamide gel. These results further confirm that the fluorescence intensity gradually increases as the polyacrylamide gel solidifies, validating that the AIE effect of BSA-AuNCs in the gel can serve as a probe to sense changes in viscosity during polymerization.

[0042] Example 2: Optimization of Acr-Bis reaction conditions

[0043] Before conducting the electrophoresis experiment, we need to investigate whether some components in nucleic acid and protein electrophoresis will affect the HRP-catalyzed polymerization of Acr-Bis.

[0044] like Figure 3 As shown in Figure A, compared to the control group (black line), all curves increased and gradually reached equilibrium, indicating that changing the pH or buffer solution of the reaction system, or adding SDS, did not affect the polymerization reaction. HRP can initiate ACR polymerization by catalyzing the oxidation of ACAC by H2O2. Therefore, the concentrations of ACAC, HRP, and H2O2 were optimized to explore the appropriate solidification time. Figure 3 (B~D). For example... Figure 3 As shown in Figure B, with a fixed HRP concentration of 0.1 mg / mL and a fixed H2O2 concentration of 1.0 mM, the ACAC concentration in the range of 4.88–29.25 mg / mL can induce polymerization in the reaction system. Therefore, 4.88 mg / mL was selected as the optimal ACAC concentration for further optimization experiments. Figure 3 C shows the effect of HRP on Acr-Bis polymerization. When the HRP concentration is too low... Figure 3C shows the effect of HRP on the polymerization of Acr-Bis. When the HRP concentration is too low, it cannot promote the increase of fluorescence intensity (HRP = 0.01 mg / mL), indicating that low concentrations of HRP are insufficient to promote the complete polymerization of Acr-Bis. Therefore, 0.02 mg / mL HRP was selected for further optimization. Figure 3 D depicts the effect of H2O2 on gel polymerization. The fluorescence intensity of the system containing a low concentration of H2O2 (0.1 mM) increases relatively slowly, indicating that the polymerization of Acr-Bis is incomplete. Therefore, we chose 0.5 mM H2O2 so that the monomers could polymerize in about 10 minutes in subsequent experiments.

[0045] Example 3: Non-denaturing polyacrylamide gel electrophoresis of nucleic acids (natural-PAGE)

[0046] First, 10% natural polyacrylamide gels catalyzed by TEMED / APS or HRP / ACAC / H2O2 were prepared. Then, DNA markers of different concentrations were added to the lanes, and electrophoresis was performed at a constant voltage of 110V for 45 min in 1×TBE buffer×(triboric acid, EDTA, pH 8.3). The gel results were recorded using a gel imaging system. Next, the gels were stained with GelRed nucleic acid dye for 20 min and imaged using the gel imaging system.

[0047] Since HRP can catalyze the polymerization of Acr-Bis monomers, the ability of polyacrylamide gels to perform protein and nucleic acid electrophoresis requires further verification. Nucleic acid electrophoresis, protein electrophoresis, and Western blotting were performed on polyacrylamide gels catalyzed by the two different catalytic systems. The nucleic acid electrophoresis results for both gels are shown below. Figure 4 A. Similar to polyacrylamide gels prepared by conventional methods (left), gels induced by the HRP / H2O2 / CAC system can also clearly distinguish DNA chains of different molecular weights (right). The success of nucleic acid electrophoresis prompted us to further verify the protein electrophoresis function of gels prepared by HRP / H2O2 / ACAC.

[0048] Example 4 Polyacrylamide gel electrophoresis of proteins

[0049] First, SDS-polyacrylamide gels (10% separating gel, 5% stacking gel) catalyzed by TEMED / APS or HRP / H2O2 / ACAC catalytic systems were prepared. Then, different concentrations of protein markers were added to the lanes in Tris-glycine running buffer for electrophoresis. The gels were first electrophoresed at a constant voltage of 80V for 30 min, then at a constant voltage of 120V for 60 min, and then stained with Coomassie Brilliant Blue. Subsequently, the washing steps were repeated multiple times with destaining solution. Finally, gel images were acquired using a Huawei P40 gel imaging system.

[0050] like Figure 4 As shown in Figure B, electrophoresis was performed using protein markers of different concentrations as templates. As expected, the enzyme-initiated polyacrylamide gel also distinguished proteins of different molecular weights. Compared to the control group ( Figure 4 Unlike the left side of B, HRP-catalyzed gels are better able to distinguish large molecular weight proteins at the same electrophoresis time, indicating that the pore size of enzyme-catalyzed polyacrylamide gels is different from that of hydrogels prepared by TEMED / APS.

[0051] Example 5: Protein Blotting

[0052] First, total protein was extracted from MDA-MB-231 cells (human breast cancer MDA-MB-231 cell line). β-actin was used as a template for Western blotting (WB). Then, stacking gels (boiling gels) and separating gels catalyzed by APS / TEMED and HRP / H2O2 / ACAC were prepared, respectively. Before electrophoresis, protein samples were heated in a boiling water bath for 10 minutes in sample buffer (1× loading buffer). Afterward, protein samples were loaded into individual wells and subjected to electrophoresis. The separated proteins were then imprinted onto a PVDF membrane by electrotransfer, followed by incubation with a primary antibody (mouse anti-human β-actin antibody) and then with a secondary antibody (goat anti-mouse IgG antibody). Finally, the signal was detected using a ChampChemi imaging system (Beijing, China).

[0053] like Figure 4 As shown in Figure C, β-actin was selected as the detection template. Cellular proteins were separated by two types of gel electrophoresis and then transferred to a PVDF membrane for autoradiography. The experimental results showed that the β-actin band was clear.

[0054] The above results indicate that HRP / H2O2 / ACAC can completely replace the highly toxic TEMED / APS system as the initiation system for catalyzing the formation of polyacrylamide gels.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for monitoring the synthesis of HRP catalyzed polyacrylamide gel based on gold nanoclusters, characterized by: Includes the following steps: Polyacrylamide gel was synthesized using acrylamide and N,N'-methylenebisacrylamide as monomers under the action of an HRP / H2O2 / ACAC ternary initiation system; the specific steps are as follows: S1 mixes acrylamide and N,N'-methylenebisacrylamide premix, water, and BSA-AuNCs to obtain a mixed solution; S2 was reacted by adding an HRP / H2O2 / ACAC ternary initiation system to a mixed solution to obtain a polyacrylamide gel; the ACAC concentration was 4.88~29.25 mg / mL; the HRP concentration was 0.02-0.1 mg / mL; and the H2O2 concentration was 0.5-1.0 mM. The preparation method of BSA-AuNCs in S1 includes: adding tetrachloroauric acid to a BSA solution for reaction, adding an alkaline solution and stirring the reaction, incubating, and filtering.

2. The method for synthesis of HRP catalyzed polyacrylamide gel based on gold nanoclusters monitoring according to claim 1, characterized in that: In step S1, the volume ratio of acrylamide and N,N'-methylenebisacrylamide premix to water is 3:5; the ratio of acrylamide to N,N'-methylenebisacrylamide in the premix is ​​29:

1.

3. The method for synthesis of HRP catalyzed polyacrylamide gel based on gold nanoclusters monitoring according to claim 2, characterized in that: The reaction time is 10-40 min.

4. The method for synthesizing HRP-catalyzed polyacrylamide gel based on gold nanoclusters as described in claim 1, characterized in that: The alkaline solution is NaOH; the reaction time is 3-7 min; the incubation temperature is 38℃; and the incubation time is 12 h.

5. The method for synthesis of HRP catalyzed polyacrylamide gel based on gold nanoclusters monitoring according to claim 1, characterized in that: BSA - Au NCs: Water volume ratio is 2:

11.

6. The polyacrylamide gel prepared by the synthesis method according to any one of claims 1-5.

7. The application of the polyacrylamide gel according to claim 6 in protein and nucleic acid electrophoresis.

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