Choline dehydrogenase analysis sensing method based on tetrahedral programmable artificial enzyme and application of choline dehydrogenase analysis sensing method in gastric cancer

Abstract

The invention discloses a choline dehydrogenase analysis sensing method based on a tetrahedral programmable artificial enzyme and application of the choline dehydrogenase analysis sensing method in gastric cancer. According to the sensor, a Poly-A tetrahedral gold cluster is used as a catalyst, hydrogen peroxide (H2O2) is effectively promoted to be decomposed to generate hydroxyl free radicals (. OH), the free radicals oxidize o-phenylenediamine (OPD) from a colorless state to yellow oxOPD, and a fluorescence emission peak at the wavelength of 563 nm and an ultraviolet absorption peak at the wavelength of 420 nm are remarkably enhanced. Furthermore, H2O2 is generated through an enzyme reaction channel constructed by choline (Ch) and choline dehydrogenase (CHDH), and enhancement of signal peaks can also be observed at the two wavelengths, so that analysis and detection of CHDH activity are indirectly realized, and further effective screening of the CHDH inhibitor is realized. The intelligent sensor has the advantages of high accuracy, quick response, high sensitivity, excellent selectivity, low cost and the like, and shows huge application potential in biomedical research and clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to a method for preparing an intelligent sensor based on a tetrahedral programmable artificial enzyme and its application in detecting the choline dehydrogenase (CHDH) pathway in gastric cancer cells, particularly to a fluorescence and ultraviolet colorimetric sensing method based on Poly-A tetrahedral gold clusters. In view of the corresponding relationship between substrate fluorescence changes or color changes and target objects, an intelligent visual mobile phone sensor is constructed and applied to the analysis of the CHDH pathway process in gastric cancer cells. This belongs to the field of chemical biosensing in analytical chemistry. Background Art

[0002] Cell osmosis, a key pathway for material exchange, plays a crucial role in maintaining the stability of the intracellular and extracellular environments and regulating material balance. This process is primarily achieved through osmotic pressure differences and the selective permeability of the cell membrane, allowing cells to effectively regulate the concentration difference between internal and external solutes and solvents, maintaining a constant intracellular environment. The principle of cell osmosis is fundamental and crucial for maintaining normal physiological functions in organisms and is a core concept in the life sciences. Fluctuations in intracellular and extracellular osmotic pressure regulate cellular physiological states and are closely linked to the development of various diseases, highlighting the importance of osmotic pressure changes in disease prevention and treatment. In this context, the role of choline dehydrogenase (CHDH) is particularly important. CHDH is an enzyme widely present in human tissues. It catalyzes the oxidation of choline to form betaine, which participates in regulating cellular osmotic pressure. As an important cytocompatible solute, betaine helps cells cope with changes in osmotic pressure and is crucial for maintaining a stable intracellular environment and a healthy state. The differences in CHDH activity between normal cells and various cancer cells, particularly in the diagnosis of malignancies such as gastric cancer, demonstrate the potential value of CHDH testing. Despite this, current CHDH detection methods remain relatively limited and struggle to meet rapidly evolving scientific and technological demands, particularly in the areas of rapid on-site testing and intelligent healthcare. With advances in artificial intelligence (AI) technology, traditional detection methods are increasingly insufficient to meet these complex and efficient demands. Therefore, developing intelligent sensing technologies based on advanced electronic devices to improve detection sensitivity, specificity, and portability has become a key research focus. This intelligent analytical sensing approach aims to combine modern electronic technology with artificial intelligence algorithms to provide powerful support for on-site testing, while also advancing the development of intelligent healthcare to meet the growing demands of healthcare.

[0003] Gastric cancer, one of the most lethal malignancies worldwide, ranks third among the most common cancers for fatality after esophageal cancer. This high mortality rate is partly due to the lack of obvious symptoms in its early stages, resulting in most patients being diagnosed in the advanced stages. At this stage, patients may experience severe symptoms such as stomach pain, exhaustion, rapid weight loss, jaundice, and ascites, which severely impact their quality of life and shorten their survival. While Western medicine, including surgery, radiotherapy, and chemotherapy, combined with Traditional Chinese Medicine (TCM), offers a variety of treatment options for gastric cancer patients, cure rates remain low for patients in the advanced stages due to the widespread spread of cancer cells. Furthermore, the risk of recurrence and metastasis after surgery further reduces long-term survival, making early detection, diagnosis, and treatment of gastric cancer crucial for saving patients' lives. However, early screening and diagnosis of gastric cancer face numerous challenges. Firstly, because the symptoms of early gastric cancer are often subtle or lack specificity, conventional physical examinations struggle to accurately identify potential gastric cancer patients. In addition, the currently commonly used biomarkers are still limited in sensitivity and specificity, and cannot meet the accuracy requirements of early diagnosis. To address these issues, researching and developing more efficient tumor markers is an effective strategy to improve the detection rate of early gastric cancer. This requires not only a deep understanding of the molecular mechanisms of gastric cancer through basic scientific research and the search for new markers with high sensitivity and specificity, but also the use of advanced biotechnology and artificial intelligence algorithms to improve the accuracy and efficiency of screening, so as to achieve early detection, accurate diagnosis and effective treatment of gastric cancer, thereby improving patients' long-term survival rate and quality of life.

[0004] Nanoclusters, as innovative catalytic nanomaterials, have demonstrated great potential as alternatives to natural enzymes in a variety of fields. However, rationally organizing the spatial structure of their multiple subunits to engineer their catalytic properties remains a significant challenge for these nanomaterials. Against this backdrop, this patent successfully constructs a novel programmable artificial enzyme using gold nanoparticle clusters (GNCs) encoded with a tetrahedral structure based on polyadenine DNA (Poly-A). Furthermore, an innovative method for preparing a smart sensor and its application in gastric cancer cell detection were developed. First, the Poly-A tetrahedron structure was synthesized under the catalysis of terminal deoxynucleotidyl transferase (TdT). Subsequently, the Poly-A tetrahedron served as a specific anchor for the seedless nucleation and growth of gold nanoparticles. This design enabled the Poly-A tetrahedron-based high-molecular-weight GNCs to exhibit programmable peroxidase-like catalytic activity. This composite structure catalyzes hydrogen peroxide (H2O2) to generate hydroxyl radicals (·OH), which in turn oxidize colorless o-phenylenediamine (OPD) to yellow oxOPD. This study achieved effective detection of H₂O₂ using dual-mode analysis using fluorescence and UV-visible colorimetry. Furthermore, given that the enzymatic reaction pathway involving choline (Ch) and choline dehydrogenase (CHDH) is accompanied by H₂O₂ production, this method can also indirectly detect CHDH activity. Furthermore, by introducing 3,3-dimethylbutanol (DMB) and 5-ethyl, 5-isoamylbarbituric acid (Amytal) as reaction inhibitors, screening for CHDH inhibitors was completed. Based on these findings, a linear relationship between sample concentration and colorimetric values ​​was established by analyzing the RGB colorimetric values ​​of the image. This led to the construction of a smartphone-based sensor, enabling rapid and portable on-site detection. The development of this multimodal detection method provides a novel approach for analyzing CHDH in gastric cancer cells, meeting the needs of modern smart healthcare while also offering both economic and social benefits. To date, this is the first report of using fluorescence, UV-visible colorimetry, and smartphone visualization techniques for tetrahedral programmable artificial enzymes, specifically for analyzing CHDH activity in gastric cancer cells, marking a major innovation in the field of biomedicine. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing an intelligent sensor with simple operation, high sensitivity, good specificity, low cost and visual detection, and its application in CHDH biological detection in gastric cancer cells.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a choline dehydrogenase analysis and sensing method based on tetrahedral programmable artificial enzyme and its application in gastric cancer, the specific steps are as follows:

[0007] 1. Preparation of Poly-A Tetrahedral Gold Clusters

[0008] (1) DNA tetrahedron synthesis: The purchased DNA dry powder was placed in a centrifuge and centrifuged at 5000 rpm for 15 min. Then, it was diluted to 1 μM with ultrapure water according to the DNA synthesis report. All DNA sequences (S1, S2, TA-S3, TA-S4, TB-S3, and TB-S4) were mixed in equal molar amounts of four DNA chains (two combinations: S1, S2, TA-S3, and TA-S4 or S1, S2, TB-S3, and TB-S4; final concentration: 1 μM) in a buffer solution (20 mM Tris, 50 mM MgCl2, pH 8.0), reacted at 95°C for 5 min in a PCR amplification instrument, and then quickly cooled to 4°C for 30 min to prepare DNA tetrahedron A (TDNA) and DNA tetrahedron B (TDNB).

[0009] The DNA sequence is as follows:

[0010]

[0011] (2) TdT extension assay: 5 μL of 0.2 U / μL TdT, 5 μL of 5× TdT buffer (compatible with TdT, the main components are: 1 M potassium diformyl, 0.125 M tris(hydroxymethyl)aminomethane, 0.05% (V / V) polyethylene glycol octylphenyl ether, 5 mM cobalt chloride (CoCl2, pH 7.2), 10 μL of 1 μM synthetic DNA tetrahedron and 10 μL of 10 mM adenine triphosphate (dATP) + thymine triphosphate (dTTP) (90% + 10%) were mixed and incubated at 37°C for 2 h and then diluted to 100 μL with ultrapure water.

[0012] (3) Preparation of Poly-A tetrahedral gold clusters: 200 μL of 10 nM prepared DNA scaffold, 1 mL of 50 mM chloroauric acid solution (HAuCl4), and 38.8 mL of ultrapure water were mixed and incubated in a 4°C refrigerator for 5 h. Then, 10 mL of 10 mM trisodium citrate (pH 6.0) was added to the DNA mixture at 35°C under continuous stirring to obtain Poly-A tetrahedral gold clusters. The mixture was diluted half and placed in a 4°C refrigerator for later use.

[0013] 2. Preparation of Fluorescence and UV Colorimetric Dual-Mode Sensors

[0014] (1) Analysis and detection of H2O2

[0015] 10 μL of half-diluted poly-A tetrahedral gold clusters, 25 μL of 1 mM OPD, 15 μL of 1 mM H₂O₂ (final concentration 150 μM), 30 μL of acetate buffer (HAc-NaAc, 0.1 M pH = 4, prepared from 0.1312 g of sodium acetate (NaAc) and 0.83 M acetic acid (HAc)), and 20 μL of ultrapure water were added sequentially to a 200 μL centrifuge tube. The solution was thoroughly mixed, and the mixture was reacted at room temperature for 30 minutes before being transferred to a fluorescence spectrophotometer, a UV spectrophotometer, and a cuvette for analysis and testing. The results were photographed using a high-definition camera.

[0016] Based on the above steps, by changing the H2O2 concentration (fluorescence: 0, 0.001, 0.004, 0.007, 0.01, 0.04, 0.07, 0.1, 0.2, 0.4, 1, 3, 5, 10, 30, 150, 250, 400 μM; UV: 0, 0.01, 0.03, 0.07, 0.1, 0.3, 0.5, 0.7, 1, 5, 10, 20, 50, 80, 100, 150, 250, 400 μM), the above are the target substances in the system. The final concentration was set, and other steps remained unchanged. The fluorescence intensity in the wavelength range of 540-580nm and the UV absorption curve in the range of 360-520nm were measured. A linear relationship diagram of H2O2 with different concentrations was established based on the maximum fluorescence intensity at 563nm and the maximum absorbance at 420nm. This can realize fluorescence and UV dual-mode detection of H2O2 with different concentrations. By analyzing the RGB chromaticity values ​​of the colorimetric image, a linear relationship between H2O2 concentration and chromaticity value is established, and a H2O2 smartphone sensor can be constructed.

[0017] (2) Analysis and detection of CHDH

[0018] After premixing 10 μL of 10 U / mL CHDH (final concentration 1000 U / L) with 1 μL of 10 mM Ch (final concentration 100 μM) for 10 minutes, the mixed solution was quickly added with 10 μL of half-diluted Poly-A tetrahedral gold clusters, 25 μL of 1 mM OPD, 30 μL of HAc-NaAc (0.1 M pH = 4) and 24 μL of ultrapure water into a 200 μL centrifuge tube and mixed thoroughly. The mixture was reacted again at room temperature for 30 minutes. The mixture was then transferred to a fluorescence spectrophotometer, an ultraviolet spectrophotometer and a cuvette for analysis and testing, and photographed with a high-definition camera.

[0019] Based on the above steps, by changing the CHDH concentration (fluorescence: 0, 0.1, 0.2, 0.4, 0.6, 1, 2, 4, 6, 10, 30, 60, 100, 300, 600, 1000, 2000, 5000 U / L; UV: 0, 1, 2, 4, 6, 8, 10, 20, 40, 60, 100, 200, 400, 600, 800, 1000, 2000, 5000 U / L) the above is the final concentration of the target in the system. The other steps remain unchanged, measuring the fluorescence intensity within the wavelength range of 540-580nm and the UV absorption curve within the range of 360-520nm. A linear relationship diagram for different CHDH concentrations is established using the maximum fluorescence intensity at 563nm and the maximum absorbance at 420nm. This allows for dual-mode fluorescence and UV detection of CHDH at varying concentrations. By analyzing the RGB colorimetric values ​​of the colorimetric image, a linear relationship between CHDH concentration and colorimetric values ​​is established, enabling the construction of a CHDH smartphone sensor. Furthermore, the addition of DMB and Amytal to the CHDH reaction solution effectively inhibits CHDH activity, enabling the screening of small molecule inhibitors.

[0020] (3) Analysis and detection of inhibitors DMB and Amytal

[0021] In (2), 10 μL of 10 U / mL CHDH (final concentration 1000 U / L) was premixed with 4 μL of 1 μM DMB or Amytal (final concentration 20 mM) / 2 μL of 10 μM DMB or Amytal (final concentration 150 mM) for 10 min, and then 1 μL of 10 mM Ch (final concentration 100 μM) was added. The other steps remained unchanged. The mixture was then transferred to a fluorescence spectrophotometer, a UV spectrophotometer, and a cuvette for analysis and testing, and photographed using a high-definition camera.

[0022] Based on the above steps, by changing the concentration of DMB or Amytal (fluorescence: 0, 0.1, 0.2, 0.4, 0.6, 1, 2, 4, 10, 20, 100, 200mM; UV: 0, 1, 2, 4, 6, 10, 25, 50, 100, 150, 500, 1000mM), the above is the final concentration of the target in the system, and the other steps remain unchanged. The fluorescence intensity in the wavelength range of 540-580nm and the UV absorbance in the range of 360-520nm were measured, and a linear relationship diagram of different concentrations of DMB or Amytal was established with the maximum fluorescence intensity at 563nm and the maximum absorbance at 420nm. Fluorescence and UV dual-mode detection of different concentrations of DMB or Amytal can be achieved. For DMB, the half inhibitory concentration (IC 50 ) is 0.41 mM fluorescence / 3.06 mM UV; for Amytal, the half inhibitory concentration (IC 50) is 0.62mM Fluor / 4.64mM UV.

[0023] 3. Construction of mobile phone sensors

[0024] To enable on-site monitoring of changes in hydroperoxide (H2O2) and choline dehydrogenase (CHDH) concentrations, this study designed a convenient smartphone-based detection system. This system utilizes the high-performance camera of a Huawei nova 9 smartphone, combined with a custom-made darkroom device, to accurately analyze the target by capturing color changes and fluorescence intensity changes in o-phenylenediamine (OPD). The detection system's main components include: a custom-made darkroom device with a small hole on the top that matches the smartphone camera, providing a stable hold for the phone and ensuring the camera is focused on the sample; an LED light installed inside the darkroom to provide sufficient and stable illumination, ensuring consistent photography conditions; a blue light meter, which serves as a fluorescence excitation light source and plays a key role in stimulating the fluorescence signal in the sample; and a white background paper, placed inside the darkroom as a background for photography, enhancing color contrast and accuracy in photos. The prepared fluorescence and UV colorimetric sensor was placed in the darkroom, and after turning on the LED light and blue light meter, photos of the sample were taken using a Huawei nova 9 smartphone. The captured photo is then imported into free color-picking software available from a mobile app store. The software analyzes the photo and extracts the corresponding RGB color values. By calculating the ratio of red (R) to blue (B) color values, the concentrations of H2O2 and CHDH in the sample can be accurately estimated based on a pre-established standard curve. This system design not only demonstrates the potential of smartphones in modern analytical detection technologies but also provides a fast, economical, and user-friendly detection method suitable for rapid on-site testing outside the laboratory, especially in resource-limited settings.

[0025] 4. Detection of CHDH in Gastric Cancer Cells

[0026] This patent selected the gastric cancer cell line NCI-N87 as the tumor cell model for the experiment. By performing standardized cell passaging operations on the NCI-N87 cell line, effective cell proliferation and maintenance were achieved. Cell collection and subsequent cell lysis steps are continued to extract the desired protein from the obtained cell lysate. The detailed protein extraction steps have been specifically described in Example 4. The protein sample obtained using the above method was then applied to the previously described experiment (see Example 2). This experimental step is intended to evaluate the application potential of the extracted protein for the research target and further explore its effectiveness in gastric cancer cell detection and analysis. This process not only demonstrates the technical process of cell culture and protein extraction, but also provides important experimental materials for subsequent biomedical research.

[0027] Principle of the invention: The present invention is a choline dehydrogenase analysis and sensing method based on a tetrahedral programmable artificial enzyme and its application in gastric cancer. Poly-A tetrahedral gold clusters catalyze H2O2 to produce ·OH. ·OH can oxidize colorless OPD into yellow oxOPD, which has a significant fluorescence emission peak at 563nm and a UV absorption peak at 420nm. As the H2O2 concentration increases, both the fluorescence emission peak at 563nm and the UV absorption peak at 420nm are enhanced, thereby achieving visual analysis and detection of H2O2. The enzyme pathway composed of Ch and CHDH is accompanied by the production of H2O2, and the fluorescence emission peak at 563nm and the UV absorption peak at 420nm can also be detected, indirectly achieving the analysis and detection of CHDH. Finally, when the inhibitor DMB or Amytal is added, the CHDH activity is inhibited and Ch cannot be catalyzed to produce H2O2, thereby achieving the screening of CHDH inhibitors. Based on this, visual quantitative analysis of H2O2 and CHDH can be achieved, and a simple, fast, low-cost, and highly selective visual analysis method was constructed for the analysis and detection of CHDH in gastric cancer cells.

[0028] Compared with the prior art, the advantages of the present invention are: for the first time, the present invention detects H2O2 and CHDH based on the oxidation of OPD by H2O2 catalyzed by Poly-A tetrahedral gold clusters, and at the same time introduces a smartphone colorimetric device. Obviously, within a certain concentration range, the greater the H2O2 concentration, the stronger the oxidation ability of OPD, the more oxOPD produced, the darker the yellow color, and the stronger the fluorescence emission peak at 563nm and the ultraviolet absorption peak intensity at 420nm; similarly, the greater the CHDH concentration, the more H2O2 produced, the stronger the fluorescence emission peak at 563nm and the ultraviolet absorption peak intensity at 420nm, and the darker the yellow color. The experimental results show that the fluorescence emission peak intensity at 563nm and the absorbance value at 420nm are linearly related to the logarithmic values ​​of the H2O2 and CHDH concentrations within a certain range, successfully achieving the detection of the two targets and visual analysis and detection, and exploring the analysis and detection of CHDH in gastric cancer cells. Its advantages are:

[0029] (1) The target is novel. Changes in CHDH activity are closely related to the occurrence and development of many diseases. For example, in pathological conditions such as certain types of gastric diseases, nervous system diseases, and metabolic disorders, CHDH activity may change significantly. Accurate detection of CHDH activity can help with early detection, diagnosis, and disease monitoring of these diseases. Currently, there are very few reports on CHDH activity detection. Designing a new analytical sensing method for this provides new opportunities and challenges for a deeper understanding of the role of CHDH in physiological and pathological processes and the development of treatment strategies for related diseases.

[0030] (2) The method is simple and low-cost. The tetrahedral programmable artificial enzyme (Poly-A tetrahedral gold cluster) is easy to synthesize, and the main steps of the detection principle are completed under homogeneous conditions without the introduction of complex conditions. The reaction is rapid, and a fast, simple, and sensitive visual analysis and detection of the target object is achieved. By combining a smartphone with sample detection, visual analysis and on-site detection are achieved. This method is simple to operate, and the accuracy and real-time performance of the colorimetric analysis are further improved by the colorimetric analysis of digital images using a smartphone.

[0031] (3) High sensitivity. The present invention designs a method for preparing an intelligent sensor based on a tetrahedral programmable artificial enzyme, which uses a Poly-A tetrahedral gold cluster to catalyze H2O2 to produce ·OH. ·OH can oxidize colorless OPD into yellow oxOPD, which has an obvious fluorescence emission peak at 563nm, and a UV absorption peak at 420nm is detected. As the concentration of H2O2 increases, the fluorescence emission peak at 563nm and the UV absorption peak at 420nm are both enhanced, thereby realizing the visual analysis and detection of H2O2. The enzyme pathway composed of Ch and CHDH is accompanied by the production of H2O2, and can also detect the fluorescence emission peak at 563nm and the UV absorption peak at 420nm, indirectly realizing the analysis and detection of CHDH. Subsequently, four linear equations were obtained: the linear correlation equation of fluorescence intensity to H2O2 concentration is y=344.89x+1420.9, R 2 =0.9946, the detection limit is 0.29nM; the linear correlation equation of UV absorption peak intensity and H2O2 concentration is y=0.0459x+0.1306, R 2 =0.9928, the detection limit is 4.77nM; the linear correlation equation of fluorescence intensity to CHDH concentration is y=210.8x+481.4, R 2 =0.9922, the detection limit is 0.036U / L; the linear correlation equation between the UV absorption peak intensity and CHDH concentration is y=0.0282x+0.0346, R 2 =0.9971, and the detection limit is 0.51 U / L. This shows that the sensor can achieve high sensitivity detection of H2O2 and CHDH.

[0032] (4) Strong specificity. When other control substances such as glucose (GLu), dopamine (DA), citric acid (CA), ascorbic acid (AA) and uric acid (UA) are substituted in the H2O2 assay; when other control substances such as alkaline phosphatase (ALP), histone acetyltransferase (HAT), terminal deoxynucleotidyl transferase (TdT), acetylcholinesterase (AChE) and papain are substituted in the CHDH assay, these interfering substances do not interfere with the system.

[0033] (5) Strong applicability. The analysis and detection of CHDH in gastric cancer cells can be preliminarily realized. By analyzing the activity expression level of CHDH, the occurrence and development process of gastric cancer can be revealed, which is helpful for the early diagnosis and treatment monitoring of gastric cancer.

[0034] In summary, the present invention constructs a choline dehydrogenase analysis and sensing method based on tetrahedral programmable artificial enzyme and its application in gastric cancer. It has the advantages of multiple signal outputs, rapid analysis, simple operation, high sensitivity, good selectivity, low cost, etc., and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a feasibility analysis diagram of the sensor of the present invention for H2O2 and CHDH;

[0036] Figure 2 The fluorescence and ultraviolet absorption intensities of the sensor of the present invention for different concentrations of H2O2;

[0037] Figure 3 This is a linear relationship diagram of the mobile phone sensor of the present invention for different concentrations of H2O2 and RGB values;

[0038] Figure 4 The fluorescence and ultraviolet absorption intensities of the sensor of the present invention to different concentrations of CHDH;

[0039] Figure 5 This is a linear relationship diagram of the mobile phone sensor of the present invention for different concentrations of CHDH and RGB values;

[0040] Figure 6 The figure is a linear relationship diagram of the fluorescence and ultraviolet absorption intensity of the sensor of the present invention to different concentrations of DMB / Amytal;

[0041] Figure 7 This is an experimental diagram of the selectivity of the sensor of the present invention to H2O2 and CHDH;

[0042] Figure 8 This is a diagram of the analysis and detection experiment of the sensor of the present invention in gastric cancer cells;

[0043] Figure 9 This is a diagram of the analysis and detection experiment of the mobile phone sensor of the present invention in gastric cancer cells. DETAILED DESCRIPTION

[0044] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0045] Example 1 Construction of Fluorescence and UV Colorimetric Sensing Methods for Detecting H2O2 and CHDH

[0046] A. Preparation of Poly-A Tetrahedral Gold Clusters

[0047] (1) DNA tetrahedron synthesis: The purchased DNA dry powder was placed in a centrifuge and centrifuged at 5000 rpm for 15 min. Then, it was diluted to 1 μM with ultrapure water according to the DNA synthesis report. All DNA sequences (S1, S2, TA-S3, TA-S4, TB-S3, and TB-S4) were mixed in equal molar amounts of four DNA chains (two combinations: S1, S2, TA-S3, and TA-S4 or S1, S2, TB-S3, and TB-S4; final concentration: 1 μM) in a buffer solution (20 mM Tris, 50 mM MgCl2, pH 8.0), reacted at 95°C for 5 min in a PCR amplification instrument, and then quickly cooled to 4°C for 30 min to prepare DNA tetrahedron A (TDNA) and DNA tetrahedron B (TDNB).

[0048] (2) TdT extension assay: 5 μL of 0.2 U / μL TdT, 5 μL of 5× TdT buffer (available with TdT, the main components of which are: 1 M potassium diformyl, 0.125 M tris(hydroxymethyl)aminomethane, 0.05% (v / v) polyethylene glycol octylphenyl ether, 5 mM cobalt chloride (CoCl2, pH 7.2), 10 μL of 1 μM synthetic DNA tetrahedron, and 10 μL of 10 mM adenine triphosphate (dATP) + thymine triphosphate (dTTP) (90% + 10%) were mixed and incubated at 37°C for 2 h, and then diluted to 100 μL with ultrapure water.

[0049] (3) Preparation of Poly-A tetrahedral gold clusters: 200 μL of 10 nM prepared DNA scaffold, 1 mL of 50 mM chloroauric acid solution (HAuCl4), and 38.8 mL of ultrapure water were mixed and incubated in a 4°C refrigerator for 5 h. Then, 10 mL of 10 mM trisodium citrate (pH 6.0) was added to the DNA mixture at 35°C under continuous stirring to obtain Poly-A tetrahedral gold clusters. The mixture was diluted half and placed in a 4°C refrigerator for later use.

[0050] B. Preparation of Fluorescence and UV Colorimetric Sensors

[0051] a. Analysis and detection of H2O2

[0052] 10 μL of half-diluted poly-A tetrahedral gold clusters, 25 μL of 1 mM OPD, 15 μL of 1 mM H₂O₂ (final concentration 150 μM), 30 μL of acetate buffer (HAc-NaAc, 0.1 M pH = 4, prepared from 0.1312 g of sodium acetate (NaAc) and 0.83 M acetic acid (HAc)), and 20 μL of ultrapure water were added sequentially to a 200 μL centrifuge tube. The solution was thoroughly mixed, and the mixture was reacted at room temperature for 30 minutes before being transferred to a fluorescence spectrophotometer, a UV spectrophotometer, and a cuvette for analysis and testing. The results were photographed using a high-definition camera.

[0053] The fluorescence intensity of the above-mentioned sensor in the wavelength range of 540-580nm and the ultraviolet absorption curve in the range of 360-520nm are detected, as shown in FIG. Figure 1 As shown, the sensor exhibits almost no fluorescence emission or UV absorption peaks in the presence of tetrahedral programmable artificial enzyme, OPD, or H2O2 alone. When H2O2 is introduced in the presence of tetrahedral programmable artificial enzyme and OPD (curve 8), the prepared sensor exhibits a distinct fluorescence emission peak at 563nm and a distinct UV absorption peak at 420nm. The solution exhibits a colorless to yellow transition, indicating that the poly-A tetrahedral gold cluster catalyzes the oxidation of OPD by H2O2. Comparison with other samples demonstrates that this sensing method exhibits a good response to H2O2 and is suitable for H2O2 analysis and detection.

[0054] Based on the above steps, by changing the H2O2 concentration (fluorescence: 0, 0.001, 0.004, 0.007, 0.01, 0.04, 0.07, 0.1, 0.2, 0.4, 1, 3, 5, 10, 30, 150, 250, 400 μM; UV: 0, 0.01, 0.03, 0.07, 0.1, 0.3, 0.5, 0.7, 1, 5, 10, 20, 50, 80, 100, 150, 250, 400 μM), the above is the final concentration of the target in the system, and the other steps remain unchanged. The fluorescence intensity in the wavelength range of 540-580 nm and the UV absorption curve in the range of 360-520 nm were measured, and a linear relationship graph of different H2O2 concentrations was established with the maximum fluorescence intensity at 563 nm and the maximum absorbance at 420 nm. The results are as follows Figure 2 As shown in (A), the fluorescence emission peak intensity of the sensor shows a good linear relationship with the logarithmic value of the H2O2 concentration, and the yellow color deepens with the increase of the H2O2 concentration. The linear curve of the H2O2 fluorescence intensity peak is established with the maximum fluorescence intensity value at 563nm as y=344.89x+1420.9, R 2 =0.9946, the detection limit is 0.29nM. Figure 2As shown in (B), the UV absorption peak intensity of the sensor shows a good linear relationship with the logarithmic value of the H2O2 concentration. The linear curve of the H2O2 UV absorption peak is established with the maximum absorption intensity value at 420nm, y = 0.0459x + 0.1306, R 2 =0.9928, with a detection limit of 4.77nM. Fluorescence and UV detection of H2O2 at different concentrations can be achieved; the captured photos are imported into the colorimetric software of a smartphone to obtain the R, G, and B values ​​of the corresponding sample concentrations. For each sample, the R, G, and B values ​​are taken three times from different positions, and the average of the three results is finally taken. By analyzing the RGB chromaticity values ​​of the colorimetric image, such as Figure 3 As shown in the figure, a linear relationship between H2O2 concentration and colorimetric value is established (AC is fluorescence, BD is ultraviolet), and a H2O2 smartphone sensor can be constructed to realize on-site analysis of H2O2.

[0055] b. Analysis and detection of CHDH

[0056] After premixing 10 μL of 10 U / mL CHDH (final concentration 1000 U / L) with 1 μL of 10 mM Ch (final concentration 100 μM) for 10 minutes, the mixed solution was quickly added with 10 μL of half-diluted Poly-A tetrahedral gold clusters, 25 μL of 1 mM OPD, 30 μL of HAc-NaAc (0.1 M pH = 4) and 24 μL of ultrapure water into a 200 μL centrifuge tube and mixed thoroughly. The mixture was reacted again at room temperature for 30 minutes. The mixture was then transferred to a fluorescence spectrophotometer, an ultraviolet spectrophotometer and a cuvette for analysis and testing, and photographed with a high-definition camera.

[0057] The fluorescence intensity of the above-mentioned sensor in the wavelength range of 540-580nm and the ultraviolet absorption curve in the range of 360-520nm are detected, as shown in FIG. Figure 1 As shown, it can be seen that the sensor in the presence of the material, OPD, Ch, and CHDH alone has almost no fluorescence emission peak or ultraviolet absorption peak. When the reaction solution of Ch and CHDH is introduced in the presence of the material and OPD, the prepared sensor has a fluorescence emission peak at 563nm and an ultraviolet absorption peak at 420nm, and the solution (11) shows a colorless to yellow change process, indicating that the poly-A tetrahedral gold cluster catalyzes the oxidation of OPD by H2O2 produced by the enzyme pathway of Ch and CHDH. By comparison with other samples, it is shown that the sensing method has a good response to CHDH and can be used for the analysis and detection of CHDH.

[0058] Based on the above steps, by changing the CHDH concentration (fluorescence: 0, 0.1, 0.2, 0.4, 0.6, 1, 2, 4, 6, 10, 30, 60, 100, 300, 600, 1000, 2000, 5000 U / L; UV: 0, 1, 2, 4, 6, 8, 10, 20, 40, 60, 100, 200, 400, 600, 800, 1000, 2000, 5000 U / L), the above is the final concentration of the target in the system, and the other steps remain unchanged. The fluorescence intensity in the wavelength range of 540-580 nm and the UV absorption curve in the range of 360-520 nm were measured, and a linear relationship graph of different CHDH concentrations was established with the maximum fluorescence intensity at 563 nm and the maximum absorbance at 420 nm. The results are shown in FIG. Figure 4 As shown in (A), the fluorescence emission peak intensity of the sensor shows a good linear relationship with the logarithmic value of CHDH concentration, and the yellow color deepens with the increase of CHDH concentration. The linear curve of CHDH fluorescence intensity peak is established with the maximum fluorescence intensity value at 563nm as y=210.8x+481.4, R 2 =0.9922, the detection limit is 0.036U / L. Figure 4 As shown in (B), the UV absorption peak intensity of the sensor shows a good linear relationship with the logarithmic value of CHDH concentration. The linear curve of the CHDH UV absorption peak is established with the maximum absorption intensity value at 420nm as y=0.0282x+0.0346, R 2 =0.9971, and the detection limit is 0.51U / L. Fluorescence and UV detection of CHDH at different concentrations can be achieved; the taken photos are imported into the colorimetric software of the smartphone to obtain the R, G, and B values ​​of the corresponding concentration samples. The R, G, and B values ​​of each sample are taken three times from different positions, and the average of the three results is finally taken. By analyzing the RGB chromaticity value of the colorimetric image, such as Figure 5 As shown, a linear relationship between CHDH concentration and colorimetric value was established (AC for fluorescence, BD for UV), and a CHDH smartphone sensor could be constructed to realize on-site analysis of CHDH.

[0059] Example 2 Analysis and Detection of CHDH Inhibitors DMB and Amytal

[0060] The CHDH concentration in the CHDH catalytic reaction was fixed at 1000 U / L, and different concentrations of DMB or Amytal were added (fluorescence: 0, 0.1, 0.2, 0.4, 0.6, 1, 2, 4, 10, 20, 100, 200 mM; UV: 0, 1, 2, 4, 6, 10, 25, 50, 100, 150, 500, 1000 mM). The above are the final concentrations of the target in the system. The reaction was pre-incubated for 10 minutes, and the other steps remained unchanged. The results are shown in Figure 2. Figure 6As shown in Figure 2, as the inhibitor concentration increases, the solution gradually changes from yellow to colorless, and the intensity of the fluorescence emission peak and the UV absorption peak gradually decreases. For DMB, the half-inhibitory concentration (IC 50 ) is 0.41 mM fluorescence / 3.06 mM UV; for Amytal, the half inhibitory concentration (IC 50 ) is 0.62mM Fluor / 4.64mM UV.

[0061] Example 3 Specificity Detection

[0062] In order to verify the selectivity of the sensor, the sensor preparation steps of Example 1 were followed. Other reference substances such as glucose (GLu), dopamine (DA), citric acid (CA), ascorbic acid (AA) and uric acid (UA) were replaced in the H2O2 detection; other reference substances such as alkaline phosphatase (ALP), histone acetyltransferase (HAT), terminal deoxynucleotidyl transferase (TdT), acetylcholinesterase (AChE) and papain were replaced in the CHDH detection. These interfering substances did not interfere with the system. The results are shown in Figure 2. Figure 7 As shown, it can be seen that the sensor has a good response to the target and has good selectivity.

[0063] Example 4 Analysis and Detection of CHDH in Gastric Cancer Cells

[0064] The tumor cells selected in this patent are NCI-N87 cells from the gastric cancer cell line. The NCI-N87 cell line was passaged and cultured in L-15 medium containing 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin in a 37°C saturated humidity incubator containing 5% CO2. Sealed culture bottles were used during the culture process. When the fusion rate reached about 90%, digestion was performed using a digestion solution containing 0.25% trypsin and 0.02% EDTA, and the cells were passaged at a ratio of 1:3, and passaged approximately every 2-3 days. The passaged cells were collected. Before the experiment, the old culture medium was removed, the cells were washed with 1mL 1×PBS (pH=7.4), 1mL 25% Trypsin-EDTA solution was added for digestion for 1.5min, the cells were placed in the culture environment for 1min, and 2mL new culture medium was added to suspend the cells. After centrifugation at 800 g for 5 min, the cells were resuspended in 1× PBS (pH = 7.4), counted, and diluted stepwise to obtain a concentration of 500×10 4 Cells / mL sample. Then mitochondrial protein was extracted using the mitochondrial separation and protein extraction kit (PK10016), and the extracted mitochondrial protein was used for subsequent experiments. The protein extracted from the mitochondria was applied to the BCHDH in Example 1 to complete the experiment. Figure 8As shown in (A, B), as the number of NCI-N87 cells increases, the fluorescence and UV signals increase accordingly. To further prove that the captured signals are caused by CHDH, a CHDH inhibitor was introduced into the cell lysate at 10,000 cells / mL. The results are shown in Figure 8 As shown in (C, D), as the concentration of CHDH inhibitor increases, both fluorescence and UV signals decrease. Figure 8 As shown in (E, F), this patent also found that the CHDH in normal cells is more than that in gastric cancer cells, that is, the CHDH expression in gastric cancer patients is less than that in normal people, which has a certain correlation with the occurrence and development of gastric cancer. Figure 9 As shown, the mobile phone sensor is used in CHDH analysis and detection of gastric cancer cells. The output color block diagram corresponds to the bar chart below, and the change trend is consistent with Figure 8 The results are consistent, indicating that the data change trends output by mobile phone sensors and large-scale instruments are consistent, and have potential for clinical field application.

[0065] 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 choline dehydrogenase analysis and sensing method based on a tetrahedral programmable artificial enzyme and its application in gastric cancer. The mechanism is as follows: The present invention has successfully developed a choline dehydrogenase (CHDH) activity detection sensor that integrates fluorescence, ultraviolet colorimetry and smartphone multimodal sensing. The core structure of this sensor is based on encoded Poly-A tetrahedral gold nanoparticle clusters (GNCs). The programmable artificial enzyme is prepared by a unique method, demonstrating its potential for application in gastric cancer cell detection. The experiment started by synthesizing DNA tetrahedrons into Poly-A tetrahedrons through terminal deoxynucleotidyl transferase (TdT), and then binding to gold nanoparticle clusters. During this binding process, Poly-A tetrahedrons act as key positioning points for the seedless nucleation and growth of GNCs, giving Poly-A tetrahedron-based GNCs a high degree of programmability and peroxidase-like catalytic activity. This novel gold cluster structure can effectively catalyze hydrogen peroxide (H2O2) to produce hydroxyl radicals (·OH), further oxidizing o-phenylenediamine (OPD) from colorless to yellow oxOPD, and accurately detecting changes in H2O2 concentration through the dual modes of fluorescence and ultraviolet colorimetry. Thanks to the characteristic that choline (Ch) and CHDH produce H2O2 together in the enzyme reaction pathway, the sensor of the present invention can indirectly but efficiently monitor CHDH activity. In addition, by introducing DMB and Amytal as reaction inhibitors, this technology can not only detect CHDH activity, but also effectively screen inhibitors. The development of this multimodal sensor technology not only provides an innovative tool for the rapid and accurate detection of CHDH activity, but also provides an important experimental basis for the diagnosis and treatment of related diseases such as gastric cancer, showing broad application prospects.

2. The choline dehydrogenase analysis and sensing method based on tetrahedral programmable artificial enzyme and its application in gastric cancer according to claim 1, characterized in that: Changes in CHDH activity are closely associated with the development and progression of various diseases. For example, CHDH activity may vary significantly in certain pathological conditions, such as gastric diseases, neurological diseases, and metabolic disorders. Accurate detection of CHDH activity can aid in the early detection, diagnosis, and monitoring of these diseases. Currently, there are very few reports on CHDH activity detection. Designing a new analytical sensing method to better understand the role of CHDH in physiological and pathological processes and develop treatment strategies for related diseases presents new opportunities and challenges. This approach involves multimodal detection, cross-validation, and mutual confirmation of multiple parameters to avoid false positive or false negative signals and improve the accuracy of disease diagnosis.

3. A choline dehydrogenase analysis and sensing method based on a tetrahedral programmable artificial enzyme and its application in gastric cancer according to claims 1-2, characterized in that: The constructed optical sensing method can be used for the analysis and detection of different concentrations of H2O2 and CHDH. The detection limits of H2O2 are fluorescence: 0.29nM, UV: 4.77nM; the detection limits of CHDH are fluorescence: 0.036U / L, UV: 0.51U / L; it can be used for the screening of CHDH small molecule inhibitors (DMB and Amytal). For DMB, the half inhibitory concentration (IC 50 ) is 0.41 mM fluorescent / 3.06 mM UV; For Amytal, the half inhibitory concentration (IC 50 ) is 0.62mM Fluor / 4.64mM UV.

4. A choline dehydrogenase analysis and sensing method based on a tetrahedral programmable artificial enzyme and its application in gastric cancer according to claims 1-3, characterized in that: This patent designed an efficient CHDH analysis and detection method in gastric cancer cells. It was found that CHDH is highly expressed in normal cells, while CHDH is less in gastric cancer patients. This is helpful for the discovery of early markers and treatment monitoring of gastric cancer.

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