Histone acetylated transferase ultraviolet colorimetric sensor based on Ag < + >-TMB system and intracellular biological application thereof

Abstract

The invention discloses an innovative histone acetylation transferase ultraviolet colorimetric sensor which is based on a silver ion (Ag < + >) and 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) system. Under the action of Ag < + >, TMB is oxidized into a blue oxidized form (oxTMB), and shows a strong ultraviolet absorption peak at the wavelength of 652nm, so that the visual quantitative detection of Ag < + > is realized. When coenzyme A (CoA) is added to form a CoA-Ag < + > coordination polymer, or through the catalytic action of histone acetylated transferase (HAT p300), the concentration of free Ag < + > is reduced, the oxidation effect on TMB is correspondingly inhibited, the color of a solution is changed from blue to colorless, and then reduction of absorbance is observed at 652 nm. The process further analyzes and detects HAT p300 in breast cancer cells, compared with normal cells and high expression of HAT p300 in cancer cells, a simple, convenient, rapid, high-sensitivity and high-selectivity unmarked visual analysis strategy is provided, and the method is used for researching the histone acetylation process and has important scientific research and application value.

Description

Technical Field

[0001] This invention focuses on the development of a UV colorimetric sensor and its wide application, especially by utilizing Ag + -TMB system has established an innovative histone acetyltransferase (HAT) UV colorimetric sensor. This sensor is designed to perform a variety of bioanalytical detection, including silver ion (Ag + ), coenzyme A (CoA), histone acetyltransferase (HAT p300), and small molecule inhibitors. Its uniqueness lies in its ability to analyze and detect HAT p300 activity in breast cancer MCF-7 cells under experimental conditions and compare it with the acetylation status in normal cells. This invention belongs to the field of analytical chemistry and chemical biosensor technology, providing a novel and effective tool for the detection of acetylation-related biomarkers and cancer diagnosis. Background Art

[0002] Post-translational modifications (PTMs) of proteins play a crucial role in enhancing the diversity of protein functions. By promoting protein subcellular localization, interactions, activity, and stability, PTMs enable proteins to respond precisely and dynamically to various internal and external stimuli. These modifications include key biological processes such as acetylation, methylation, phosphorylation, and ADP-ribosylation. Protein acetylation, in particular, plays a key role in disease prevention and treatment, cell biology research, and the development of clinical therapeutic strategies. However, despite its widely recognized importance, research on protein acetylation remains in its infancy, leaving a vast space for exploration. Among the numerous PTMs, histone acetyltransferases (HATs) have garnered significant attention. Through their unique modification mechanisms, HATs regulate diverse biological processes, including DNA damage repair, tumor progression, embryonic development, and chromosomal stability, and are crucial for maintaining normal biological function. Therefore, monitoring HAT activity and potential inhibitors are of significant importance in preventing and treating diseases caused by aberrant HAT activity. Although various techniques for detecting HAT activity have been developed, including fluorescence, electrochemistry, and electrochemiluminescence, these methods are still limited by several factors, such as the need for rapid visualization. Against this backdrop, the development of a rapid and visual analytical technique for monitoring histone acetyltransferase activity is of great scientific and practical value. Such technological advancements will not only deepen our understanding of protein acetylation and its role in biological processes, but also provide new strategies for disease prevention and treatment.

[0003] Silver ions (Ag + ), an important heavy metal ion, is widely used in cosmetics, building materials and medical products due to its significant antibacterial properties.+ Its high toxicity to bacteria, viruses, algae, and fungi means that excessive intake can lead to serious health problems such as cytotoxicity, organ failure, and mitochondrial dysfunction. Therefore, it is extremely urgent and important to develop a simple, rapid, highly sensitive, and specific visual detection method for accurately measuring Ag+ content in the fields of medical care and environmental monitoring.

[0004] The present invention innovatively develops a Ag-based + and 3,3',5,5'-tetramethylbenzidine (TMB) system for histone acetyltransferase (HAT p300) UV colorimetric sensor and its application in biology. + Induce TMB to oxidize into a blue oxidized state (oxTMB), which exhibits a significant absorption peak in the ultraviolet region at 652nm. By monitoring the intensity of this absorption peak and the change in TMB color, this system can achieve the purpose of Ag + Rapid visual analysis of concentration. In addition, this sensor utilizes Ag + The special affinity between Ag and thiol makes + Can bind to coenzyme A (CoA). This action results in free Ag + The amount of CoA decreases, thereby weakening its oxidative effect on TMB. As the concentration of CoA increases, the intensity of the ultraviolet absorption peak at 652nm decreases, and the color of TMB gradually changes from blue to colorless, providing a simple method for the rapid visualization of CoA. Furthermore, under the action of histone acetyltransferase HAT p300, this system can catalyze the transfer of acetyl from acetyl-CoA to substrate polypeptides to generate a certain amount of CoA. Using the established CoA visualization detection method, a UV colorimetric sensor for HAT p300 activity was constructed, which achieved a simple and rapid detection of its activity, and finally realized the study of the acetylation status in breast cancer cells MCF7. The development of this sensor does not require the preparation of nanomaterials or complex labeling steps, is simple to operate, and has broad application prospects. So far, the Ag-based + This is the first report on the construction of a histone acetyltransferase UV colorimetric sensor using the -TMB system and its intracellular biological application. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a high sensitivity, high specificity, fast detection speed, accurate and reliable results, low cost, and visual + -TMB system histone acetyltransferase UV colorimetric sensor and its intracellular biological applications.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: based on Ag+ -TMB system histone acetyltransferase UV colorimetric sensor and its intracellular biological application, the specific steps are as follows:

[0007] (1) Based on Ag + -TMB system-based UV colorimetric sensor for histone acetyltransferase

[0008] A.Ag + -TMB system

[0009] Add 100-500 μL of 0.1 M, pH 4.0 acetate buffer (HAc-NaAc), 10-50 μL of 0.1-3 mM AgNO₃, and 10-50 μL of 1-6 mM TMB to a 0.5 mL centrifuge tube. Mix thoroughly, incubate at 35°C for 5-30 minutes, and transfer to a microplate reader for analysis or photograph with a high-definition camera.

[0010] B. Based on Ag + -TMB system to construct CoA UV colorimetric sensor

[0011] Add 10-50 μL of 10 mM phosphate buffer (pH 7.0), 10-50 μL of 0.1-3 mM AgNO₃, and 10-50 μL of 100-1000 μM CoA to a 0.5 mL centrifuge tube. Shake the tube at 30°C on a thermostatic magnetic stirrer (500 rpm) for 5-15 minutes. Then, add 100-500 μL of 0.1 M HAc-NaAc (pH 4.0) and 10-50 μL of 1-6 mM TMB. Mix the solution thoroughly and incubate at 35°C for 5-30 minutes. Transfer the tube to a microplate reader for analysis or photograph with a high-definition camera.

[0012] C. Based on Ag + -TMB system construction HAT p300 UV colorimetric sensor

[0013] A HAT reaction solution with a total volume of 10-50 μL was prepared as follows: 1-5 μL, 2-10 μg / mL HAT p300, 1-10 μL, 1 mM substrate peptide, 0.5-5 μL, 2-20 mM Ac-CoA, 1-10 μL, H2O, and 2-50 μL, 10 mM, pH 7.0, mixed and reacted at 30°C for 50-100 min.

[0014] Add 10-50 μL of 10 mM phosphate buffer (pH 7.0), 10-50 μL of 0.1-3 mM AgNO₃, and 10-50 μL of the above HAT reaction solution to a 0.5 mL centrifuge tube. Shake the mixture at 30°C on a thermostatic magnetic stirrer (500 rpm) for approximately 5-15 minutes. Then, add 100-500 μL of HAc-NaAc (0.1 M, pH 4.0) and 10-50 μL of 1-6 mM TMB. Mix the solution thoroughly and incubate at 35°C for 5-30 minutes. Transfer the sample to a microplate reader for analysis or photograph with a high-definition camera.

[0015] (2) Application: Ag + , CoA and HAT p300 analysis

[0016] Based on the above step A, by changing the AgNO3 concentration in step A (final concentration: 0-400 μM), the other steps remain unchanged, and measuring the UV absorbance in the wavelength range of 500-800 nm, the maximum UV absorbance at 652 nm is used to establish the AgNO3 + Standard curve can be used to analyze different concentrations of Ag + UV detection; different concentrations of Ag can be achieved through colorimetric images + Visual detection.

[0017] Based on the above step B, by changing the CoA concentration in step A (final concentration: 0-300 μM), while keeping the other steps unchanged, measuring the UV absorbance in the wavelength range of 500-800 nm, and establishing a CoA standard curve with the maximum UV absorbance value at 652 nm, UV detection of different concentrations of CoA can be achieved; visual detection of different concentrations of CoA can be achieved through colorimetric images.

[0018] Based on the above step C, by changing the HAT concentration in step C (final concentration: 0-100 ng / mL), while keeping the other steps unchanged, the UV absorbance in the wavelength range of 500-800 nm was measured, and a HAT p300 standard curve was established with the maximum UV absorbance value at 652 nm. UV detection of different concentrations of HAT p300 can be achieved; visual detection of different concentrations of HATp300 can be achieved through colorimetric images.

[0019] Principle of invention: The present invention develops a Ag-based + -TMB system histone acetyltransferase (HAT) UV colorimetric sensor and its application principle in cells. The core mechanism is to use silver ions (Ag +) oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) and converts it into blue oxidized TMB (oxTMB) with a strong ultraviolet absorption peak (652nm). As the concentration of Ag+ increases, the absorption peak at 652nm increases, achieving visual detection of Ag+. When coenzyme A (CoA) is added to the system, the thiol groups on it react with Ag+. + By forming Ag-S bonds, the two adjacent Ag + The metal aurophilic interaction occurs between them, forming CoA-Ag + Coordination polymer. This process reduces the amount of free Ag+, thereby reducing the oxidation of TMB, causing the solution to gradually change from blue to colorless, and reducing the absorbance at 652nm, thereby achieving visual detection of CoA. Furthermore, the present invention, by introducing histone acetyltransferase (HAT p300) and acetyl-CoA, can promote the transfer of the acetyl group of acetyl-CoA to the lysine residue of histone, producing CoA and acetylated peptides. As the concentration of HAT p300 increases, more CoA is produced, resulting in free Ag+. + The decrease in the absorbance of the solution at 652 nm decreases, showing a change from blue to colorless. On the contrary, acetyl-CoA that does not participate in the acetyl transfer reaction cannot bind to Ag due to the blocking effect of the acetyl group on the sulfhydryl group. + The reaction causes TMB to change from colorless to blue. Subsequently, inhibitor experiments and intracellular acetylation analysis experiments were designed, and it was found that the content of HAT p300 was high in breast cancer cells compared with normal cells. In summary, the present invention provides a simple, rapid, highly sensitive, highly selective and label-free visualization analysis method, which is suitable for studying the histone acetylation process and can achieve Ag + Visual quantitative analysis of , CoA and HATp300.

[0020] Compared with the prior art, the advantages of the present invention are: the present invention is the first to be based on Ag + -TMB system has constructed a histone acetyltransferase UV colorimetric sensor. Obviously, within a certain concentration range, Ag + The higher the concentration, the stronger the oxidation ability of TMB, the more oxTMB is produced, the darker the blue color is, and the stronger the absorption peak intensity at 652nm is. Similarly, the higher the concentration of CoA (HAT p300), the more Ag is consumed. + The more, the weaker the absorption peak intensity at 652nm and the lighter the blue. The experimental results show that the magnitude of the absorption peak intensity at 652nm is related to Ag + The concentrations of CoA and HAT p300 showed a linear relationship within a certain range, successfully achieving visual analysis and detection of the three targets. Its advantages are:

[0021] (1) The method is simple. The main processes of the detection principle are all implemented in a homogeneous phase, which improves the reaction speed, reduces the complexity of the operation, and realizes a fast, simple, and sensitive visual analysis and detection of the target.

[0022] (2) High sensitivity. The present invention is based on Ag + -TMB system to prepare UV colorimetric sensors using Ag + It can induce the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to blue 3,3',5,5'-tetramethylbenzidine (oxTMB), which has a significant absorption peak at 652nm. + The coordination binding between them regulates free Ag + concentration, and three linear equations were obtained: UV absorption peak intensity versus Ag + The concentration linear correlation equation is y = 0.0044C Ag+ +0.0427, R 2 =0.9908, the detection limit is 0.16nM; the linear correlation equation of the UV absorption peak intensity and CoA concentration is y=-0.187lgC CoA +0.4503, R 2 =0.9977, the detection limit is 3.7nM; the linear correlation equation of the UV absorption peak intensity and HAT p300 concentration is y=-0.29lgC p300 +0.53, R 2 =0.9985, the detection limit is 0.02ng / mL. This shows that the sensor can detect Ag + , CoA and HAT p300 to achieve high-sensitivity detection.

[0023] (3) Strong specificity. + Detection: Other reference substances such as magnesium ions (Mg 2+ ), chromium ions (Cr 3+ ), nickel ions (Ni 2+ ), lead ions (Pb 2+ ), manganese ions (Mn 2+ ), cadmium ions (Cd 2+ ), copper ions (Cu 2+ ), potassium ion (K + ), cobalt ions (Co 2 + ), calcium ions (Ca 2+ ), sodium ion (Na + ) and zinc ions (Zn 2+) had no interference on the system; for CoA detection: other control substances such as tyrosine (Tyr), glucose (Glu), adenosine triphosphate (ATP), adenosine diphosphate (ADP) and reduced coenzyme I (NADH) had no interference on the system; for HAT p300 detection: other control substances such as terminal transferase (TdT), papain (Papain), glucose oxidase (GOx), lysozyme (LZM), alkaline phosphatase (ALP) and uracil-DNA glycosylase (UDG) had no interference on the system.

[0024] (4) High accuracy. The recovery rate is between 90% and 110%.

[0025] (5) Low cost. The present invention can realize the Ag + , CoA and HAT concentrations are accurately detected, which is suitable for the low-cost requirements in industrialization.

[0026] (6) Strong applicability. The sensor has mild reaction conditions and fast reaction speed; it has stable performance and is suitable for practical applications in biosensing and ecological environments. This patent extracts active proteins from different cell lines and proves that the cells contain HATp300, and the content in breast cancer cells is higher than that in normal cells.

[0027] In summary, the present invention is based on Ag + -TMB system to construct a histone acetyltransferase UV colorimetric sensor for Ag + The visual analysis and detection of Ag in the sample to be tested has the advantages of high sensitivity, strong specificity, simple operation, accurate results, rapid analysis and low cost. + , CoA and HAT p300 visual quantitative analysis, no similar work has been found, and it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a feasibility experiment diagram of the colorimetric sensor of the present invention;

[0029] Figure 2 The sensor of the present invention reacts to different concentrations of Ag + Calibration curve of UV absorption peak intensity versus concentration;

[0030] Figure 3 The calibration curve of the sensor of the present invention is a graph showing the UV absorption peak intensity versus the logarithm of the concentration of CoA at different concentrations;

[0031] Figure 4 The figure is a calibration curve of the sensor of the present invention showing the UV absorption peak intensity versus the logarithm of the concentration of HAT p300 at different concentrations;

[0032] Figure 5 The calibration curve of the sensor of the present invention is a graph showing the ultraviolet absorption peak intensity versus the logarithm of the concentration of anacardic acid and C646 at different concentrations;

[0033] Figure 6 The sensor of the present invention is Ag + Specificity experimental diagram;

[0034] Figure 7 This is an experimental diagram showing the specificity of the sensor of the present invention for CoA;

[0035] Figure 8 This is an experimental diagram showing the specificity of the sensor of the present invention for HAT p300;

[0036] Figure 9 This is a diagram of an experiment using the sensor of the present invention to analyze the acetylation status in cells. DETAILED DESCRIPTION

[0037] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0038] Example 1 Based on Ag + -TMB system-based UV colorimetric sensor for histone acetyltransferase

[0039] A.Ag + -TMB system

[0040] Add 160 μL of 0.1 M, pH 4.0 acetate buffer (HAc-NaAc), 20 μL of 1.5 mM AgNO₃, and 20 μL of 3 mM TMB to a 0.5 mL centrifuge tube, for a total of 200 μL. Then, mix the solution thoroughly, incubate at 35°C for 10 minutes, and transfer to a microplate reader for analysis or photograph with a high-definition camera.

[0041] B. Based on Ag + -TMB system to construct CoA UV colorimetric sensor

[0042] Add 20 μL of 10 mM phosphate buffer (pH 7.0), 20 μL of 1.5 mM AgNO₃, and 20 μL of 500 μM CoA to a 0.5 mL centrifuge tube. Shake at 30°C on a thermostatic magnetic stirrer (500 rpm) for approximately 8 minutes. Then, add 120 μL of HAc-NaAc (0.1 M, pH 4.0) and 20 μL of 3 mM TMB, for a total of 200 μL. Mix the solution thoroughly and incubate at 35°C for 10 minutes. Transfer the sample to a microplate reader for analysis or photograph with a high-definition camera.

[0043] C. Based on Ag +-TMB system construction HAT p300 UV colorimetric sensor

[0044] The steps for preparing a HAT reaction solution with a total volume of 20 μL are as follows: 2.5 μL of 4 μg / mL HAT p300 (final concentration: 50 ng / mL), 4 μL of 1 mM substrate peptide (final concentration: 20 μM), 1 μL of 10 mM Ac-CoA (final concentration: 50 μM), 2.5 μL of H2O, and 10 μL of phosphate buffer (10 mM, pH 7.0), mix well, and react at 30°C for 80 min.

[0045] Add 20 μL of 10 mM phosphate buffer (pH 7.0), 20 μL of 1.5 mM AgNO₃, and 20 μL of the above HAT reaction solution to a 0.5 mL centrifuge tube. Shake the mixture at 30°C on a thermostatic magnetic stirrer (500 rpm) for approximately 8 minutes. Then, add 120 μL of HAc-NaAc (0.1 M, pH 4.0) and 20 μL of 3 mM TMB, for a total of 200 μL. Mix the solution thoroughly and incubate at 35°C for 10 minutes. Transfer the sample to a microplate reader for analysis or photograph with a high-definition camera.

[0046] The UV absorption curve of the above sensor in the range of 500-800 nm is detected. Figure 1 As shown, it can be seen that in Ag + The sensor with TMB and CoA alone has almost no absorption peak. + When the prepared sensor has an obvious UV absorption value at 652nm, and the solution (1) shows a change process from colorless to blue, indicating that Ag + After further addition of CoA, the UV absorption value at 652nm weakened, and the solution (2) showed a change from blue to colorless, indicating that CoA combined with a large amount of Ag. + By comparing with other samples (3-7), it is shown that this sensing method is effective for Ag + and CoA have good response and can be used for Ag + and CoA analysis.

[0047] Example 2Ag + , CoA and HAT p300 analysis

[0048] Based on the steps in Example 1, by changing the Ag in Example 1A +concentration (final concentration: 0, 0.001, 0.01, 0.05, 0.1, 1.0, 5.0, 10, 30, 50, 80, 120, 150, 200, 260, 300, 350, 400 μM), other steps remain unchanged, and the Ag + The results are as follows. Figure 2 As shown, the UV absorption peak intensity of the sensor is + The concentration of Ag + As the concentration increases, the blue color deepens. The UV absorption peak intensity of the sensor is + The concentration linear correlation equation is, y = 0.0044C Ag+ +0.0427, R 2 =0.9908, the linear range is 0.001-300 μM, and the detection limit is 0.16 nM; Based on the steps in Example 1 above, by changing the CoA concentration in Example 1B (final concentration: 0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 20, 30, 50, 100, 150, 200, 300 μM), the detection of CoA can be achieved. The results are as follows Figure 3 As shown, the UV absorption peak intensity of the sensor shows a good linear relationship with the logarithm of CoA concentration, and as the CoA concentration increases, the blue color gradually fades. The linear correlation equation of the UV absorption peak intensity of the sensor with CoA concentration is y = -0.187logC CoA +0.4503, R 2 =0.9977, the linear range is 0.01-150 μM, and the detection limit is 3.7 nM; based on the steps in Example 1, the detection of HAT p300 can be achieved by changing the concentration of HAT p300 in Example 1C (final concentration: 0, 0.1, 0.2, 0.3, 0.5, 0.8, 1, 2, 3, 5, 10, 20, 30, 50, 80, 100 ng / mL). The results are as follows Figure 4 As shown, the UV absorption peak intensity of the sensor shows a good linear relationship with the logarithmic value of the HAT p300 concentration, and as the HAT p300 concentration increases, the blue color gradually fades. The linear correlation equation of the UV absorption peak intensity of the sensor with the HAT p300 concentration is y = -0.29logC p300 +0.53, R 2 =0.9985, the linear range is 0.1~50ng / mL, and the detection limit is 0.02ng / mL, which shows that the sensor can detect Ag + , CoA and HAT p300 with high sensitivity.

[0049] Example 3 Detection of HAT p300 Inhibitors Anacardic Acid and C646

[0050] Based on Example 1, the concentration of HAT p300 was set to 50 ng / mL, and different concentrations of inhibitors were added in sequence. Specifically, the concentrations of anacardic acid were: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 200, 300 μM; the concentrations of C646 were: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 0.8, 1, 2, 5, 10, 20, 30, 50, 100, 200 μM. These concentrations of inhibitors were then used to prepare the sensor. The UV absorption peak intensity of the sensor was recorded using a microplate reader. The results showed that Figure 5 As can be seen, as the concentration of the inhibitors anacardic acid and C646 increases, the intensity of the UV absorption peak increases accordingly, manifested by a deepening of the blue color. This indicates that anacardic acid and C646 have a stronger inhibitory effect on HAT p300 activity. Specifically, the half-inhibitory concentrations (IC50) of anacardic acid and C646 are 3.87 μM and 1.09 μM, respectively.

[0051] Example 4 Specificity Detection

[0052] like Figure 6 、 7 As shown in Figure 8, compared with other small molecules and protease substances, the sensor is more sensitive to three targets (Ag + , CoA and HAT p300) respectively have good responses and excellent selectivity.

[0053] Example 5 Analysis and detection of intracellular acetylation status

[0054] In this example, we successfully extracted proteins from breast cancer cells MCF-7 using a nuclear protein extraction kit, and used these proteins in the HAT p300 activity and inhibitor detection experiments described previously. This process does not involve the use of commercial HAT p300, but directly applies MCF-7 cells in cell culture. First, MCF-7 cells were dispersed in a sterile phosphate buffered saline (PBS) with a pH of 7.0 and 0.2M, and a series of cell suspensions of different concentrations were prepared, with concentrations of 0, 1000, 3000, 5000, 10000, and 20000 cells / ml (cells / mL), and then the active protein was extracted using a kit. The experimental results showed that as the number of cells increased, the content of HAT p300 protein also increased, proving that HAT p300 does exist in MCF-7 cells ( Figure 9A). To ensure that the signal changes were caused by the extracted HAT p300 protein, we designed an experiment using specific inhibitors. After extracting the protein at a concentration of 20,000 cells / mL, we numbered the protein samples and added different concentrations of anacardic acid (10μM and 300μM, marked as low and high, respectively) and C646 (20μM and 200μM, marked as low and high, respectively), two known inhibitors of HAT p300, to four parallel samples. The experimental results showed ( Figure 9 B), both inhibitors effectively inhibited HAT p300 activity, and the inhibitory effects showed a consistent trend, thus verifying that the signal changes were indeed caused by HAT p300. Further, by adding normal cell extract protein as a control group, it was found that the signal changes of MCF-7 cancer cells were significantly greater than those of normal cells ( Figure 9 C), indicating that the HAT p300 content in MCF-7 cells is higher than that in normal cells. This discovery provides a new perspective for exploring the role of acetylation factors in cancer development and progression, and contributes to a deeper understanding of the causes and progression of cancer.

[0055] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions, or substitutions made by those skilled in the art within the spirit and scope of the present invention should also fall within the scope of protection of the present invention.

Claims

1. An Ag-based + -TMB system histone acetyltransferase UV colorimetric sensor and its intracellular biological application, characterized by: The present invention pioneered a method based on Ag + -TMB system histone acetyltransferase (HAT p300) UV colorimetric sensor. + ) induces the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to a blue oxidized state (oxTMB), generating a specific UV absorption peak at 652nm, achieving Ag + Rapid visual detection of concentration. + Binding to coenzyme A (CoA) reduces free Ag + , which weakens the oxidation of TMB, resulting in a decrease in the absorption peak at 652nm and a change in the color of TMB from blue to colorless, thus providing a method for rapid detection of CoA. By monitoring the acetyl transfer reaction catalyzed by HAT p300, a simple and rapid visual analysis of HAT p300 activity is further achieved without the need for nanomaterial preparation or labeling. The simplicity and efficiency of this method have opened up new avenues for histone acetylation research and related applications.

2. According to claim 1, a Ag-based + -TMB system histone acetyltransferase UV colorimetric sensor and its intracellular biological application, characterized by: The present invention combines ultraviolet spectroscopy, colorimetry and Ag + -TMB system, a novel Ag-targeting + The research team developed a sensitive, rapid, label-free chemical biosensor for , CoA, and HAT p300, which enables efficient analysis and detection of these molecules. The sensor has mild reaction conditions and fast response speed, and exhibits good stability and applicability, making it suitable for practical applications in biosensing and life sciences.

3. According to claim 1-2, a Ag-based + -TMB system histone acetyltransferase UV colorimetric sensor and its intracellular biological application, characterized by: Based on Ag + -The UV colorimetric sensing method constructed by the TMB system can be used for different concentrations of Ag + , CoA and HAT p300 analysis, Ag + The detection limit is 0.16nM, the detection limit of CoA is 3.7nM, and the detection limit of HAT p300 is 0.02ng / mL; it can be used for the screening of HAT p300 small molecule inhibitors (anacardic acid and C646), IC 50 3.87 μM and 1.09 μM respectively.

4. According to claim 1-3, a Ag-based + -TMB system histone acetyltransferase UV colorimetric sensor and its intracellular biological application, characterized by: By extracting and analyzing active proteins in breast cancer cells MCF7, this patent not only proves the existence of HAT p300 in cells, but also finds that the HAT p300 content in breast cancer cells is significantly higher than that in normal cells, providing a new research direction for a deeper understanding of the progression mechanism of breast cancer.

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