Multi-mode visual sensor for activity analysis of histone acetyltransferase in circulating tumor cells

Abstract

The invention discloses a multi-mode visual sensor for activity analysis of histone acetyltransferase in circulating tumor cells, which is characterized in that in the presence of Cu (II), OPD is oxidized into OPDox, the fluorescence intensity or the ultraviolet absorbance value is increased, the solution presents macroscopic yellow under natural light, and the fluorescence intensity or the ultraviolet absorbance value is increased. The red fluorescent powder presents orange visible to naked eyes under a blue light instrument, and is integrated into a smart phone through the change process of the color from colorless to yellow, so that intelligent field detection is realized; based on the interaction of Cu (II) and CoA and the fact that CoA is a KAT Tip60 acetylation product, the fluorescence intensity or the ultraviolet absorbance is reduced, the sensor not only can realize the analysis and detection of Cu (II) and CoA, but also can realize the multi-mode visual detection of KAT Tip60. Finally, esophageal cancer circulating tumor cells are taken as a sample, and intracellular KAT Tip60 activity monitoring is realized. The novel multi-mode visual colorimetric sensor is constructed for the first time and used for histone acetylation biological process analysis, and scientific significance and social benefits are achieved.

Description

Technical Field

[0001] The present invention relates to the research and application of a Cu(II)-OPD-mediated multimodal visualization sensor, and in particular to an in-depth discussion of a visualization analysis and detection method for "histone acetylation" in protein post-translational modification, thereby achieving sensitive selective detection of Cu(II), coenzyme A, histone acetyltransferase and its inhibitors. Ultimately, the visualization sensor is applied to the analysis of histone acetyltransferase activity in circulating tumor cells, belonging to the field of chemical biosensing in analytical chemistry. Background Art

[0002] Cancer is a highly lethal and metastatic malignancy. By the time any symptoms appear, cancer is often already in its advanced stages, accompanied by macroscopic and microscopic metastases. Currently, tissue biopsy is the standard for cancer diagnosis, but it has limitations, such as tumor heterogeneity and site-specificity. This limits the usefulness of information collected from tissue biopsies for cancer diagnosis and treatment. Liquid biopsies extract biomolecule samples from tumors that circulate in body fluids. Interestingly, circulating tumor cells (CTCs) in the blood, as early events in carcinogenesis, are associated with the development of metastatic cancer and poor prognosis, offering new avenues for their biochemical detection and treatment. However, CTCs in the blood are extremely rare, and CTCs are easily disrupted during flow or collection. Therefore, assessing cancer progression based on CTC counts has significant limitations. In summary, the search for new targeted cancer therapies remains a pressing issue, and integrating the specialized biological processes of CTCs with cancer diagnosis and treatment offers a unique and promising approach.

[0003] Protein post-translational modifications (PTMs) play a key role in eukaryotic transcription and in regulating chromatin dynamics. Common PTMs include acetylation, methylation, ubiquitination, and phosphorylation. Histone acetylation is a crucial modification of gene transcription, regulated by histone acetyltransferases (KATs). KAT dysfunction is associated with diseases such as inflammatory processes, Huntington's disease, heart disease, diabetes, AIDS, and cancer. Detecting KAT activity and screening its inhibitors will facilitate biochemical studies of gene transcription and the development of anticancer drugs. Various techniques have been developed to detect KAT activity, including radioassays, fluorescence, mass spectrometry, electrochemistry, and electrochemiluminescence. While these methods can successfully analyze KAT activity, they have drawbacks that need to be overcome. Radioassays require radiolabeled acetyl-CoA, which increases operational complexity and is environmentally harmful. Fluorescence and mass spectrometry techniques require expensive and bulky instrumentation and cumbersome experimental procedures. Electrochemical analysis is susceptible to environmental influences and can result in unstable detection and analysis. Therefore, it is of great significance to develop a simple, highly sensitive, low-cost and visual detection method for monitoring KAT activity.

[0004] The present invention discloses a multimodal visualization sensor for analyzing histone acetyltransferase activity in circulating tumor cells. First, in the presence of Cu(II), OPD is oxidized to OPDox, and the solution exhibits a yellow color visible to the naked eye under natural light. The color change from colorless to yellow enables ultraviolet visualization analysis of Cu(II). Second, OPDox is placed in a blue light meter, where a distinct fluorescence phenomenon is observed. Through the fluorescence phenomenon, it is observed that the fluorescence phenomenon increases with increasing Cu(II) concentration, based on which fluorescence visualization analysis of Cu(II) can be achieved. Subsequently, by tracking the changes in OPD, a smartphone image is obtained, and the average pixel intensity of its red (R), green (G), and blue (B) channels is extracted using a MATLAB algorithm. A linear relationship is established with G / B as the ordinate and Cu(II) concentration as the abscissa, thus enabling portable on-site detection of smartphones. Due to the interaction between Cu(Ⅱ) and CoA to form a CoA-Cu(Ⅱ) coordination polymer, Cu(Ⅱ) in the solution is consumed, the redox effect on OPD is weakened, the ultraviolet absorbance / fluorescence intensity decreases, and as the CoA concentration increases, the color changes from yellow to colorless, successfully realizing the multimodal analysis and detection of CoA; In addition, this patent uses KAT Tip60 as the analysis target. In the catalytic acetylation reaction, the acetyl group on acetyl-CoA is transferred to the lysine residue of the substrate peptide, producing a large number of CoA molecules. Therefore, this method can be used for indirect analysis and detection of KAT activity, and can also be used for screening KAT small molecule inhibitors. Based on this, the patent of this invention realizes the detection and analysis of Cu(Ⅱ), CoA, KAT and its inhibitors, constructs a new multimodal visualization analysis and detection method for histone acetylation, and ultimately applies it to the analysis of histone acetyltransferase activity in esophageal cancer CTCs, which is of great significance in the fields of chemistry, biology, and medicine. So far, no method for preparing Cu(Ⅱ)-OPD-mediated histone acetyltransferase multimodal visualization sensor has been discovered. It has been applied for the first time to analyze the histone acetyltransferase activity in esophageal cancer CTCs, providing a new direction for the early diagnosis and treatment of clinical esophageal cancer, which has both scientific significance and social value. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a multimodal visual sensor for analyzing histone acetyltransferase activity in circulating tumor cells with good specificity, high sensitivity, simple operation, low cost and visual discernibility.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a multimodal visual sensor for analyzing histone acetyltransferase activity in circulating tumor cells, the specific steps of which are as follows:

[0007] (1) Establishment of Cu(Ⅱ)-OPD multi-mode visual analysis and detection method

[0008] Take 40mM 1~10μL OPD and 500μM 1~20μL Cu(Ⅱ) and add them to 50~100μL 0.1M The final volume of each test sample was maintained at 100 μL in pH 4.5 acetate buffer; the mixed solution was vortexed to mix evenly, and then the mixed solution was placed in a constant temperature and humidity incubator at 37°C for 30 minutes. After being taken out and cooled to room temperature, the following three operations were performed: ① Fluorescence detection was performed with the following parameters: voltage, 600 V, excitation wavelength, 414 nm, emission wavelength, 560 nm, slit width, 10 nm, wavelength range, 500-650 nm; ② The ultraviolet absorption curve was tested using a microplate reader with a wavelength setting of 350-550 nm; ③ The smartphone image was then obtained by tracking the changes in OPD, and the average pixel intensities of the red (R), green (G), and blue (B) channels were extracted using the MATLAB algorithm. A linear relationship was established with G / B as the vertical axis and Cu(Ⅱ) concentration as the horizontal axis, realizing portable on-site detection of smartphones.

[0009] (2) Establishment of CoA multimodal visualization analysis and detection method

[0010] Take 1-10 μL of 10-200 μM CuSO4·5H2O and 1-10 μL of 10-500 μM CoA, and add them to 1-15 μL of PBS (10 mM pH 7.0) buffer. Place the mixture on a constant temperature magnetic stirrer at 30°C and incubate with slow stirring for 1-20 minutes. Then add 1-10 μL of 40 mM OPD. Then transfer the above solution to a test dish, add 60-80 μL of 0.1 M pH 4.5 acetate buffer, wait for 10 minutes at room temperature, perform UV / fluorescence testing, and complete the mobile phone data matching.

[0011] (3) Establishment of KAT multi-mode visual colorimetric analysis detection method

[0012] (a) KAT analysis: 1-10 μL of 5-10000 ng / mL KAT, 1-10 μL of 50-200 μM substrate peptide, and 1-10 μL of 100-500 μM Ac-CoA were added to 5-15 μL of 10 mM PBS (pH 7.0) buffer, and then stirred at 30°C for 10-100 min. Subsequently, 1-10 μL of 10-200 μM CuSO4·5H2O was mixed with the KAT reaction solution, and the mixture was incubated at 30°C for 1-20 min under slow stirring on a constant temperature magnetic stirrer. 1-10 μL of 40 mM OPD was then added. The solution was then transferred to a test dish, and 60-70 μL of 0.1 M pH 7.0 was added. 4.5 Acetate buffer, wait for 10 minutes at room temperature, then perform UV / fluorescence test and complete the matching of mobile phone data.

[0013] (b) KAT inhibitor analysis: 1-10 μL of 5-10000 ng / mL KAT, 1-10 μL of 10-2000 μM MG149 or 1-10 μL of 10-1000 μM NU9056 were sequentially taken and mixed for 2 min. Then, 1-10 μL of 50-200 μM substrate peptide (RGKGGKGLGKGGAKA) and 1-10 μL of 100-500 μM Ac-CoA were added to 5-15 μL PBS (10 mM pH 5.0). 7.0) buffer, then placed on a constant temperature magnetic stirrer and reacted at 30°C with stirring for 10-100 minutes. Subsequently, 1-10 μL of 10-200 μM CuSO4·5H2O was mixed with the KAT reaction solution. The mixture was placed on a constant temperature magnetic stirrer at 30°C with slow stirring for 1-20 minutes. Then, 1-10 μL of 40 mM OPD was added. The solution was then transferred to a test dish, and 50-80 μL of 0.1 M pH 4.5 acetate buffer was added. After waiting at room temperature for 10 minutes, UV / fluorescence testing was performed to complete the mobile phone data matching.

[0014] Multi-mode visual colorimetric analysis of different concentrations of Cu(Ⅱ):

[0015] Fluorescence: In step (1), different concentrations of Cu(II) were added (final concentrations: 0, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 80 μM), while the other experimental steps in step (1) remained unchanged. Based on this, a series of Cu(II) fluorescence sensors were obtained and applied to the fluorescence sensing analysis of Cu(II).

[0016] UV: In step (1), different concentrations of Cu(II) were added (final concentrations: 0, 0.01, 0.02, 0.04, 0.06, 0.1, 0.3, 0.5, 1, 3, 5, 10, 20, 40, 60, 100 μM), while the other experimental steps in step (1) remained unchanged. Based on this, a series of Cu(II) UV sensors were obtained and applied to the UV sensing analysis of Cu(II).

[0017] Multi-mode visual colorimetric analysis of different CoA concentrations:

[0018] Fluorescence: In the above step (2), different concentrations of CoA were added (final concentrations: 0, 0.001, 0.002, 0.004, 0.01, 0.04, 0.07, 0.1, 0.4, 1, 4, 10, 20, 40, 70, 100 μM), and the other experimental steps in step (2) remained unchanged. Based on this, a series of CoA fluorescence sensors were obtained and applied to the fluorescence sensing analysis of CoA.

[0019] UV: In the above step (2), different concentrations of CoA were added (final concentrations: 0, 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 0.6, 1, 2, 4, 10, 20, 40, 60, 100 μM), and the other experimental steps in step (2) remained unchanged. Based on this, a series of CoA UV sensors were obtained and applied to the UV sensing analysis of CoA.

[0020] Multi-mode visual colorimetric analysis of different concentrations of KAT Tip60:

[0021] Fluorescence: In the above step (3a), different concentrations of KAT were added (final concentrations: 0, 0.01, 0.02, 0.04, 0.07, 0.1, 0.2, 0.4, 0.7, 2, 4, 7, 10, 20, 40, 70 ng / mL), and the other experimental steps in step (3a) remained unchanged. Based on this, a series of KAT fluorescent sensors were obtained and applied to the fluorescence sensing analysis of KAT.

[0022] UV: In the above step (3a), different concentrations of KAT were added (final concentrations: 0, 0.1, 0.2, 0.4, 0.7, 1, 2, 4, 7, 20, 40, 70, 100, 200, 350, 500 ng / mL), and the other experimental steps in step (3a) remained unchanged. Based on this, a series of KAT UV sensors were obtained and applied to the UV sensing analysis of KAT.

[0023] Multi-mode visual colorimetric assay for different concentrations of KAT inhibitors:

[0024] Fluorescence: In the above step (3b), KAT was first incubated with different concentrations of MG149 (final concentration: 0, 1, 2, 3, 5, 10, 20, 30, 50, 80, 100, 200, 500, 1000, 2000, 5000 μM) and different concentrations of NU9056 (final concentration: 0, 0.1, 0.15, 0.3, 0.5, 0.7, 1, 1.5, 2, 3, 5, 7, 10, 20, 30, 50 μM) for 10 min. The other experimental steps in step (3b) remained unchanged. Based on this, a series of MG149 and NU9056 fluorescence sensors were obtained and applied to the fluorescence sensing analysis of MG149 and NU9056.

[0025] UV: In the above step (3b), KAT was first incubated with different concentrations of MG149 (final concentration: 0, 1, 2, 4, 6, 10, 20, 40, 60, 80, 100, 200, 400, 1000, 2000, 3000 μM) and different concentrations of NU9056 (final concentration: 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.5, 3, 5, 10, 20, 50, 100, 200, 500 μM) for 10 min. The other experimental steps in step (3b) remained unchanged. Based on this, a series of MG149 and NU9056 UV sensors were obtained and applied to the UV sensing analysis of MG149 and NU9056.

[0026] Principle of the invention: The present invention designs a multimodal visual sensor for analyzing histone acetyltransferase activity in circulating tumor cells. First, under pH 4.5 conditions, OPD can be oxidized by metal ions Cu(Ⅱ) to generate OPDox. At this time, the ultraviolet absorption intensity increases (fluorescence intensity increases), and the color of the solution changes from colorless to yellow. Secondly, the special binding effect between the thiol group of CoA and Cu(Ⅱ) causes the Cu(Ⅱ) in the system to change from a free state to a complex state, resulting in the inhibition of the OPD oxidation process. As the CoA concentration continues to increase, the ultraviolet absorption (fluorescence) intensity continues to decrease, and the color of the solution changes from yellow to colorless. In the presence of KAT, acetyl-CoA acts as an acetyl donor, and the acetyl group is transferred to the lysine residue of the substrate peptide to generate the product CoA and acetylated polypeptide. Based on this, the activity detection of KAT can be indirectly realized. The small molecules MG149 and NU9056 have an inhibitory effect on the enzyme-catalyzed reaction dominated by KAT, so the analysis and detection of the small molecules MG149 and NU9056 can also be realized. Using ultraviolet (fluorescence) spectrophotometry and multi-angle photography, a multimodal visualization analysis method was established for the correlation of absorbance with Cu(II), CoA, KAT, and its inhibitors. Based on this detection principle, a simple, highly sensitive, low-cost, and visually detectable visualization sensor was constructed. Subsequently, smartphone images were obtained by tracking changes in optical density (OPD). The average pixel intensities of the red (R), green (G), and blue (B) channels were extracted using a MATLAB algorithm. A linear relationship was established with G / B as the ordinate and Cu(II) concentration as the abscissa, enabling portable, on-site detection using a smartphone. The prepared sensor was ultimately used to monitor KAT activity in CTCs of esophageal cancer, potentially facilitating early detection and treatment of esophageal cancer in clinical practice.

[0027] Compared with existing technologies, the present invention has the following advantages: The present invention constructs a multimodal visual sensor for analyzing histone acetyltransferase activity in circulating tumor cells. First, under acidic conditions, the substrate OPD is oxidized by the heavy metal ion Cu(II) to OPDox. The UV absorption (fluorescence) intensity increases with increasing Cu(II) concentration, and the solution color gradually changes from colorless to yellow. Second, as CoA binds to Cu(II) to form a coordination polymer, the Cu(II) content in the solution decreases, weakening the oxidative effect on OPD. The UV absorption (fluorescence) intensity decreases with increasing CoA concentration, and the solution color gradually changes from yellow to colorless. Obviously, within a certain concentration range, the higher the CoA concentration, the more Cu(II) is bound in the solution, the less OPD is oxidized, the lighter the yellow color of the solution, and the smaller the UV absorption (fluorescence) value. Based on this phenomenon, the optimal reaction ratio of CoA and Cu(II) is determined within a certain concentration range. CoA is the product of the KAT-catalyzed acetylation reaction. Based on the relationship between CoA and KAT, KAT activity analysis is achieved through indirect quantification of CoA. The small molecule inhibitors MG149 and NU9056 have an inhibitory effect on the catalytic reaction dominated by KAT. This inhibitory effect can lead to a further increase in the UV absorption (fluorescence) intensity. The experimental results show that the absorbance (fluorescence) value is linearly related to the concentration of Cu(II), CoA, KAT and its inhibitor within a certain range, achieving visual analysis and detection of these four targets. Its advantages are:

[0028] (1) Dual-mode visualization analysis method for the target KAT Tip60. Currently, most analytical detection methods have been developed to achieve histone acetylation analysis, but there is no dual-mode visualization analysis method for the target KAT Tip60. This patent is based on the oxidation of OPD by Cu(Ⅱ), presenting two optical signal output modes, based on which a Cu(Ⅱ)-OPD-mediated dual-mode visualization analysis method for KAT Tip60 is established.

[0029] (2) High sensitivity. The present invention is the first to use the Cu(Ⅱ)-OPD-mediated visualization analysis method for the detection of Cu(Ⅱ), CoA, KAT and its inhibitors, with good selectivity and high sensitivity. Most importantly, the present invention constructs a dual-mode visualization sensor for KAT based on the Cu(Ⅱ)-OPD-mediated visualization analysis method, with a detection limit of 0.062 ng / mL, a detection limit of 0.0041 ng / mL (fluorescence) and a detection limit of 0.062 ng / mL (ultraviolet), indicating that the sensor can achieve high-sensitivity detection of KAT Tip60.

[0030] (3) Smartphone preparation and easy operation. The present invention obtains smartphone images by tracking the changes in OPD, extracts the average pixel intensity of its red (R), green (G), and blue (B) channels using the MATLAB algorithm, and establishes a linear relationship with G / B as the vertical coordinate and KATTip60 concentration as the horizontal coordinate, thus realizing portable on-site detection of KATTip60 on smartphones.

[0031] (4) Preparation and detection methods require little reagent and are low in cost. The present invention does not require the involvement of nanomaterials, but only requires OPD as a substrate and can detect KAT Tip60 through Cu(II) regulation.

[0032] In summary, the present invention constructs a Cu(II)-OPD-mediated multimodal visualization analysis and detection method for histone acetyltransferase and applies it to the detection of Cu(II), CoA, KAT and their inhibitors. It has the advantages of simple operation, good stability, low cost, and can be distinguished by the naked eye. It can realize the detection of lower concentrations of Cu(II), CoA, KAT and their inhibitors, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The experimental diagrams show the feasibility analysis of the sensor of the present invention for Cu(II) detection and its response to different concentrations of Cu(II);

[0034] Figure 2 The experimental diagrams show the feasibility analysis of the sensor of the present invention for CoA detection and its response to different concentrations of CoA;

[0035] Figure 3 The experimental diagrams show the feasibility analysis of the sensor of the present invention for detecting KAT Tip60 and its response to different concentrations of KAT Tip60;

[0036] Figure 4 The experimental diagrams show the feasibility analysis of the sensor of the present invention for detecting MG149 and NU9056 and its response to different concentrations of MG149 and NU9056;

[0037] Figure 5 This is a diagram of an experiment using the sensor of the present invention to monitor KAT Tip60 activity in esophageal cancer CTCs. DETAILED DESCRIPTION

[0038] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0039] Example 1 Establishment of a multi-mode visual analysis and detection method and analysis and detection of Cu(II) at different concentrations

[0040] Take 5 μL of 40mM OPD (final concentration: 2mM) and 4 μL of 500μM Cu(Ⅱ) (final concentration: 20μM) and add them to 91 μL of 0.1M pH 4.5 acetate buffer, maintaining the final volume of each test sample at 100 μL; the above mixed solution was vortexed to mix, and then the mixed solution was placed in a constant temperature and humidity incubator at 37°C for 30 minutes, taken out and cooled to room temperature, and the following three operations were performed: ① Fluorescence detection was performed, and the parameters were set as follows: voltage, 600 V, excitation wavelength, 414 nm, emission wavelength, 560 nm, slit width, 10 nm, wavelength range, 500-650 nm; ② The ultraviolet absorption curve was tested using an enzyme marker, and the wavelength was set: 350-550 nm; ③ Then, the smartphone image was obtained by tracking the changes in OPD, and the average pixel intensities of the red (R), green (G), and blue (B) channels were extracted using the MATLAB algorithm. A linear relationship with G / B as the vertical coordinate and Cu (Ⅱ) concentration as the horizontal coordinate was established, realizing portable on-site detection of smartphones.

[0041] In order to verify its feasibility, a series of experiments were designed, marked as Figure 1 , where FL represents fluorescence intensity, ΔFL represents fluorescence intensity difference, Wavelength represents wavelength, Abs represents absorbance value, ΔAbs represents absorbance difference, Smartphone represents smartphone, G / B represents the ratio of green to blue, and logX represents the logarithm of target X. Figure 1 As shown in A, in the presence of Cu(Ⅱ), a large fluorescence signal value is presented at around 560nm, and as the concentration of Cu(Ⅱ) increases, the fluorescence intensity increases continuously ( Figure 1 B), and in the range of 0.001 to 20 μM, the fluorescence intensity difference was linearly related to the logarithm of Cu(Ⅱ) concentration ( Figure 1 C), the detection limit is 0.34nM (S / N=3). In the inserted figure, it shows a change process from colorless to orange under blue light, which can be clearly observed by the naked eye, indicating obvious visualization; at the same time, Figure 1 As shown in D, in the presence of Cu(Ⅱ), a large UV absorption signal value is presented at around 448nm, and as the concentration of Cu(Ⅱ) increases, the UV absorption value increases continuously ( Figure 1 E), and in the range of 0.01 to 40 μM, the absorbance difference was linearly related to the logarithm of Cu(Ⅱ) concentration ( Figure 1 F), with a detection limit of 5.6nM (S / N=3). In the inserted figure, the naked eye can see a change from colorless to yellow, indicating obvious visualization; then based on the visualization picture, this patent integrated it into the mobile phone ( Figure 1G), with G / B as the ordinate and the logarithm of Cu(Ⅱ) concentration as the abscissa, a standard curve is formed, and the color blocks are collected in the red-green-blue RGB channels, such as Figure 1 As shown in Figures 1H and 1I, two good linear equations are obtained, and the linear curve and color block in the smartphone can be used to realize the visual analysis and detection of Cu(Ⅱ).

[0042] Example 2 CoA multi-mode visual analysis and detection

[0043] 4 μL of 100 μM CuSO4·5H2O (final concentration: 20 μM) and 2 μL of 500 μM CoA (final concentration: 50 μM) were added to 14 μL of PBS (10 mM pH 7.0) buffer. The mixture was incubated on a thermostatic magnetic stirrer at 30°C with slow stirring for 12 min. 5 μL of 40 mM OPD (final concentration: 2 mM) was then added. The solution was then transferred to a test dish, 75 μL of 0.1 M pH 4.5 acetate buffer was added, and the test was performed after waiting for 10 min at room temperature. Other steps were the same as in Example 1.

[0044] A multi-mode visual analysis and detection method for CoA was established based on the catalytic effect of Cu(II) on OPD, where FL represents fluorescence intensity, ΔFL represents fluorescence intensity difference, Wavelength represents wavelength, Abs represents absorbance value, ΔAbs represents absorbance difference, Smartphone represents smartphone, G / B represents the ratio of green to blue, and logX represents the logarithm of target X. Figure 2 As shown in A and 2B, this patent found that the addition of CoA can hinder this catalytic reaction (curves 1, 2, 3, 4, 7), but CoA must be mixed with Cu (II) before reacting with OPD (curve 4). If the addition order is changed, the catalytic effect of Cu (II) on OPD cannot be hindered (curves 5, 6). In the inserted figure, No. 4 (light yellow) and No. 7 (bright yellow or orange) form a sharp contrast, indicating that the addition of CoA hinders this catalytic reaction; then, through the designed smartphone ( Figure 2 C) verified this result, and Figure 2 A and Figure 2 B corresponds.

[0045] In addition, if Figure 2 As shown in D, as the concentration of CoA increases, the fluorescence intensity at 560 nm decreases continuously, and in the range of 0.001 to 40 μM, the fluorescence intensity difference is linearly related to the logarithm of the CoA concentration ( Figure 2E), the detection limit is 0.32nM (S / N=3). In the inset figure, it shows a change process from orange to colorless under blue light, which can be clearly observed by the naked eye, indicating obvious visualization; at the same time, Figure 2 As shown in G, as the concentration of CoA increases, the UV absorption signal at 448 nm decreases continuously, and in the range of 0.01 to 40 μM, the absorbance difference is linearly related to the logarithm of the CoA concentration ( Figure 2 H), the detection limit is 4.7nM (S / N=3). In the inserted figure, the naked eye can see a change from yellow to colorless, which shows obvious visualization. Then, based on the visualization picture, this patent integrates it into the mobile phone with G / B as the vertical axis and the logarithm of CoA concentration as the horizontal axis to form a standard curve, and collects color blocks in red-green-blue RGB channels, such as Figure 2 As shown in Figures F and 2I, two good linear equations are obtained, and the linear curve and color block in the smartphone can be used to realize CoA visualization analysis and detection.

[0046] Example 3 Multimodal Visual Analysis and Detection of KAT Tip60 and Its Inhibitors

[0047] (a) KAT analysis: 2 μL of 2000 ng / mL KAT (final concentration: 200 ng / mL), 2 μL of 200 μM substrate peptide (final concentration: 20 μM), and 2 μL of 500 μM Ac-CoA (final concentration: 50 μM) were added to 14 μL of PBS (10 mM pH 7.0) buffer, and then stirred at 30°C for 90 min. Subsequently, 2 μL of 200 μM CuSO4·5H2O (final concentration: 20 μM) was mixed with the KAT reaction solution, and the mixture was incubated at 30°C for 5 min with slow stirring on a constant temperature magnetic stirrer. 5 μL of 40 mM OPD (final concentration: 2 mM) was then added. The solution was then transferred to a test dish, and 73 μL of 0.1 M pH 4.5 acetate buffer was added. The mixture was allowed to react at room temperature for 10 min before testing. Other procedures were the same as those in Examples 1 and 2.

[0048] (b) KAT inhibitor analysis: 2 μL of 2000 ng / mL KAT (final concentration: 200 ng / mL), 2 μL of 2000 μM MG149 (final concentration: 200 μM), or 2 μL of 1000 μM NU9056 (final concentration: 100 μM) were taken in sequence and mixed for 2 min. Then, 2 μL of 200 μM substrate peptide (final concentration: 20 μM) and 2 μL of 500 μM Ac-CoA (final concentration: 50 μM) were added to 12 μL PBS (10 mM pH 7.0) buffer. The mixture was then placed on a thermostatic magnetic stirrer and stirred at 30°C for 90 min. Subsequently, 2 μL of 200 μM CuSO4·5H2O (final concentration: 20 μM) was mixed with the KAT reaction solution, and the mixture was incubated on a thermostatic magnetic stirrer at 30°C with slow stirring for 5 minutes. Then, 5 μL of 40 mM OPD (final concentration: 2 mM) was added. The solution was then transferred to a test dish, and 73 μL of 0.1 M pH 4.5 acetate buffer was added. The test was performed after waiting for 10 minutes at room temperature. Other steps were the same as those in Examples 1 and 2.

[0049] KAT Tip60 analysis and detection has inherent weaknesses and cannot achieve signal amplification like DNA, which brings great difficulties to analysis and detection. Currently, KAT Tip60 is related to many diseases in clinical medicine and therefore has great scientific research value. CoA is the product of the acetylation catalytic reaction of KAT Tip60. This patent realizes KAT Tip60 analysis and detection through the analysis and detection of CoA. The process only involves OPD and Cu(II), which is low cost and simple steps. The specific results are as follows Figure 3 As shown in the figure, FL represents fluorescence intensity, ΔFL represents fluorescence intensity difference, Wavelength represents wavelength, Abs represents absorbance value, ΔAbs represents absorbance difference, Smartphone represents smart phone, G / B represents the ratio of green to blue, logX represents the logarithm of target X, F / F0 represents the ratio (final fluorescence intensity / initial fluorescence intensity), Ω / Ω0 represents the ratio (final electrochemical impedance value / initial electrochemical impedance value), GO represents graphene, antibody represents acetyl antibody, Peptide represents substrate peptide, before acetylation represents before acetylation, after acetylation represents after acetylation, and Relativeactivity represents the relative activity of the enzyme.

[0050] like Figure 3As shown in A, among them, 1. Ac-CoA+Peptide; 2. Tip60+Peptide; 3. Tip60+Ac-CoA; 4. Tip60+Ac-CoA+Peptide, only No. 4 showed obvious inhibition of catalysis, indicating that CoA was produced in the Tip60-catalyzed reaction. This system can be used to monitor Tip60 activity. In order to prove the occurrence of acetylation reaction, this patent set up two groups of experiments: the first group, such as Figure 3 As shown in Figure B, FITC (fluorescein) molecules are added to the C-terminus of the substrate peptide, and the peptide can be well adsorbed on the GO surface. The acetylated peptide can hinder the cleavage of carboxypeptidase, while the unacetylated peptide is cleaved by carboxypeptidase. Therefore, compared with the unacetylated peptide, the fluorescence intensity of the acetylated peptide decreases with the increase of GO, that is, F / F0 becomes smaller and smaller, indicating the occurrence of the acetylation reaction catalyzed by Tip60; the second group, such as Figure 3 As shown in Figure C, by adding cysteine to the C-terminus of the substrate peptide, the peptide can be better adsorbed to the gold electrode surface. The acetylated peptide can adsorb more acetyl antibodies, increasing the impedance. The unacetylated peptide cannot adsorb acetyl antibodies and has very low impedance. Therefore, compared with the unacetylated peptide, the impedance value of the acetylated peptide increases with the increase of acetyl antibodies, that is, Ω / Ω0 becomes larger and larger, indicating the occurrence of the acetylation reaction catalyzed by Tip60.

[0051] In addition, if Figure 3 As shown in Figure 4, as the concentration of Tip60 increases, the fluorescence intensity at 560 nm decreases continuously, and in the range of 0.01 ng / mL to 20 ng / mL, the fluorescence intensity difference is linearly related to the logarithm of the Tip60 concentration ( Figure 3 E), the detection limit is 0.0041 ng / mL (S / N=3). In the inserted figure, it shows a change process from orange to colorless under blue light, which can be clearly observed by the naked eye, indicating obvious visualization; at the same time, Figure 3 As shown in Figure 2, as the concentration of Tip60 increases, the UV absorption signal at 448 nm decreases continuously, and in the range of 0.1 to 200 ng / mL, the absorbance difference is linearly related to the logarithm of the Tip60 concentration ( Figure 3 H), the detection limit is 0.062ng / mL (S / N=3). In the inserted figure, the naked eye can see a change from yellow to colorless, which shows obvious visualization. Then, based on the visualization picture, this patent integrates it into the mobile phone with G / B as the vertical axis and the logarithm of Tip60 concentration as the horizontal axis to form a standard curve, and collects color blocks in red-green-blue RGB channels, such as Figure 3 As shown in Figures F and 3I, two good linear equations are obtained, and the linear curve and color block in the smartphone can be used to realize Tip60 visual analysis and detection.

[0052] In order to verify the selectivity, anti-interference and repeatability of the prepared sensor, the Tip60 sensor was prepared according to the above steps, and the KAT reaction solution was replaced by biomolecules with the same concentration as KAT to participate in the reaction, such as protein kinase A (PKA), acetylcholinesterase (AChE), choline oxidase (ChOx), alkaline phosphatase (ALP), methylase (MeT), glucose oxidase (GOx), etc. Figure 4 As shown in A, the detection sensor has good specificity for KAT Tip60; secondly, the KAT reaction solution is superimposed with biological molecules of the same concentration as KAT to participate in the reaction, such as Figure 4 As shown in B, the detection sensor has good anti-interference performance for KAT Tip60; the repeatability of this patent has also been explored, such as Figure 4 As shown in Figure C, the deviation of the final results of five parallel tests was less than 3%, showing good repeatability.

[0053] In order to verify the screening ability of the prepared sensor for Tip60 inhibitors, different concentrations of MG149 and NU9056 were added, and the inhibitors were pre-mixed with Tip60 for 10 minutes. The reaction volume of the solution was 100 μL, and other steps remained unchanged. Based on this, a series of MG149 and NU9056 sensors were obtained. Figure 4 As shown in Figures 4D and 4G, the fluorescence intensity and absorbance did not decrease with the addition of the two inhibitors, indicating that the inhibitors effectively inhibited the activity of Tip60. Figure 4 As shown in E, 4F, 4H, and 4I, with the increase of MG149 and NU9056 concentrations, the relative activity of the enzyme decreased, so the inhibitory effect became more and more obvious. By calculating the IC 50 They are: 56.23μM (fluorescence, 4E) and 2.47μM (fluorescence, 4F), 52.51μM (ultraviolet, 4H) and 1.62μM (ultraviolet, 4I), and the color changes of the inserted figures are from colorless to orange or bright yellow, which can realize the screening of KAT inhibitors.

[0054] Example 4 Multimodal Visual Analysis and Detection of KAT Tip60 in Circulating Tumor Cells of Esophageal Cancer

[0055] The samples in the experiment were collected from Ningbo Second Hospital. The samples were taken out from the sample library, separated and enriched, and then subcultured in the laboratory. The nuclear protein was then extracted using a kit and used in the above Example 3. The specific process is as follows: Figure 5As shown in A, Enrichment and sorting means enrichment and sorting, Cell culture and extraction means cell subculture, Analysis means analysis and detection, C means substance concentration, Normal Cells means normal cells, CTCs means circulating tumor cells, No Glucose means no glucose, Under Glucose means with glucose, Samples means the number of samples, and G / B means the ratio of green to blue.

[0056] like Figure 5 As shown in Figure B, cells were first prepared into suspensions of 0, 2000, 5000, 10000, 15000, and 20000 cells / mL, and then protein extraction was performed. As can be seen from the figure, as the number of cells increases, the G / B value continues to decrease, indicating that the extracted protein contains a certain amount of acetylase (inferred due to the enzyme substrate specificity). Subsequently, using 20000 cells / mL as a template, a certain amount of inhibitor was added. It was found that after the addition of the inhibitor, the G / B value increased ( Figure 5 C), indicating that the inhibitor can inhibit the activity of the extracted protein, proving the existence of KAT Tip60 from the perspective of inhibitor. Figure 5 As shown in Figure D, this patent sets up four groups of sample cells. Groups one and two are cultured in a glucose-free environment, and groups three and four are cultured in a glucose environment. Groups one and three are normal cells, and groups two and four are CTCs. As can be seen from the figure, in an environment with glucose, histone acetylation expression is upregulated, and in an environment without glucose, histone acetylation expression is downregulated, and the degree of acetylation in normal cells is significantly lower than that in CTCs, where P<0.05 is statistically significant. Currently, it has been found that the degree of histone acetylation in cells in some major diseases is disordered clinically, and the results of this patent are consistent with this, indicating that the multimodal visualization sensor developed by this patent helps to explore and reveal the occurrence and development process of major clinical diseases, and has both scientific significance and social benefits.

[0057] It should also be noted that the above specific embodiments do not limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art may make changes, modifications, additions or substitutions within the spirit of the present invention, which shall also fall within the scope of protection of the claims of the present invention.

Claims

1. A multimodal visualization sensor for analyzing histone acetyltransferase activity in circulating tumor cells, the mechanism of which is as follows: First, in the presence of Cu(II), OPD is oxidized to OPDox, and the solution appears yellow under natural light, visible to the naked eye. The color change from colorless to yellow enables ultraviolet visualization analysis of Cu(II); Second, OPDox is placed in a blue light meter, where obvious fluorescence is observed. The fluorescence phenomenon is observed to increase with increasing Cu(II) concentration, and based on this fluorescence visualization analysis of Cu(II) can be achieved; Subsequently, a smartphone image is obtained by tracking the changes in OPD, and the average pixel intensities of its red (R), green (G), and blue (B) channels are extracted using a MATLAB algorithm. A linear relationship is established with G / B as the ordinate and Cu(II) concentration as the abscissa, thus enabling portable on-site detection of smartphones. Due to the interaction between Cu(Ⅱ) and CoA to form a CoA-Cu(Ⅱ) coordination polymer, Cu(Ⅱ) in the solution is consumed, the redox effect on OPD is weakened, the ultraviolet absorbance / fluorescence intensity decreases, and as the CoA concentration increases, the color changes from yellow to colorless, successfully realizing the multimodal analysis and detection of CoA; In addition, this patent uses KAT Tip60 as the analysis target. In the catalytic acetylation reaction, the acetyl group on acetyl-CoA is transferred to the lysine residue of the substrate peptide, producing a large number of CoA molecules. Therefore, this method can be used for indirect analysis and detection of KAT activity, and can also be used for screening KAT small molecule inhibitors. Based on this, the patent of this invention realizes the detection and analysis of Cu(Ⅱ), CoA, KAT and its inhibitors, constructs a new multimodal visualization analysis and detection method for histone acetylation, and ultimately applies it to the analysis of histone acetyltransferase activity in esophageal cancer CTCs, which is of great significance in the fields of chemistry, biology, and medicine. So far, no method for preparing Cu(Ⅱ)-OPD-mediated histone acetyltransferase multimodal visualization sensor has been discovered, which provides a new direction for the early diagnosis and treatment of clinical esophageal cancer, with both scientific significance and social value.

2. The multimodal visual sensor for analyzing histone acetyltransferase activity in circulating tumor cells according to claim 1, characterized in that: For the first time, a multimodal visual sensor for histone acetyltransferase activity analysis was constructed, involving visualization and quantitative analysis methods such as fluorescence, UV, and smartphones. In-depth discussions were conducted using KAT Tip60 as a template, with detection limits of 0.0041 ng / mL (fluorescence) and 0.062 ng / mL (UV). Smartphones provide a new direction for on-site visualization analysis and detection in clinical practice.

3. A multimodal visual sensor for analyzing histone acetyltransferase activity in circulating tumor cells according to claims 1-2, characterized in that: The constructed multimodal visualization sensor was applied to the analysis of histone acetyltransferase activity in esophageal cancer CTCs. This patent set up four groups of sample cells: groups one and two were cultured in a glucose-free environment, and groups three and four were cultured in a glucose-containing environment. Groups one and three were normal cells, and groups two and four were CTCs. As can be seen from the figure, in the presence of glucose, histone acetylation expression was upregulated, while in the absence of glucose, histone acetylation expression was downregulated, and the degree of acetylation in normal cells was significantly lower than that in CTCs, with P < 0.05, which was statistically significant. The multimodal visualization sensor developed by this patent helps to explore and reveal the occurrence and development process of major clinical diseases, and has both scientific significance and social benefits.

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