Choline acetylase sensor constructed based on manganese dioxide nanosheet and application of choline acetylase sensor in lung cancer A549

Abstract

The invention discloses a choline acetylase (ChAT) sensor constructed on the basis of manganese dioxide nanosheets (MnO2NSs) and application of the choline acetylase (ChAT) sensor in lung cancer A549 cells. According to the method, MnO2NSs and oxidation-state TMB (oxTMB) are reduced at the same time through CoA, MnO2NSs are reduced into Mn < 2 + >, catalytic activity is lost, and TMB cannot be catalyzed to be oxidized into blue oxTMB. Along with the increase of the CoA concentration, the color of the solution becomes light, and the ultraviolet absorption peak at 652nm is reduced, so that the ultraviolet colorimetric detection of CoA is realized. In addition, the change of the color of the solution causes the change of R, G and B values, and the CoA detection by the smart phone is realized through the relationship between the B / G value and the CoA concentration. ChAT can catalyze choline and acetyl coenzyme A (Ac-CoA) to generate CoA, and detection of ChAT is indirectly realized according to signal change caused by the generated CoA. The alpha-NETA can inhibit the activity of ChAT and reduce the generation of CoA, so that the inhibition effect of the alpha-NETA is evaluated. The method is applied to analysis of ChAT content in lung cancer cells, and has the advantages of visualization, simplicity in operation, high sensitivity and good specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to a biosensing technology based on UV colorimetric and smartphone sensor, in particular to a method for preparing a UV colorimetric sensor using the oxidase-like activity of manganese dioxide nanosheet (MnO2 NSs) and the reducing property of coenzyme A (CoA), and the application of the sensor in the analysis and detection of coenzyme A (CoA), choline acetyltransferase (ChAT) and its inhibitor (alpha-NETA). By comparing the expression level of ChAT in normal lung cells and lung cancer cells, the abnormality of ChAT expression in lung cancer cells is studied. The present application belongs to the field of functional materials and biosensing technology. BACKGROUND

[0002] Lung cancer is one of the most common cancers worldwide and a leading cause of cancer-related deaths. There are about 220 million new cases and 1.79 million deaths each year. With the in-depth understanding of the biology of the disease, the application of predictive biomarkers and the improvement of treatment methods, many patients have made significant progress and improved prognosis. Lung cancer is composed of a variety of malignant tumors, mainly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Small cell lung cancer (SCLC), formerly known as oat cell cancer, is a neuroendocrine cancer, accounting for 15-20% of all lung cancer cases. Non-small cell lung cancer (NSCLC) includes lung adenocarcinoma (LAC), squamous cell carcinoma (SCC-L), large cell carcinoma (LCC) and neuroendocrine lung carcinoid.

[0003] Studies have shown that lung cancer cells express all the proteins required for the uptake of choline, including choline acetyltransferase (ChAT). Released acetylcholine (ACh) binds to nicotinic receptors (nAChR) and muscarinic receptors on lung cancer cells, promoting their proliferation, migration and invasion. Acetylcholine (ACh) is a paracrine and autocrine growth factor for lung epithelial cells, and also a regulator of airway remodeling, airway muscle contraction, mucus secretion and lung immune function. In the cytoplasm, ACh is generated from choline and acetyl-CoA (Ac-CoA) catalyzed by ChAT. In addition to being an autocrine growth factor, ACh can effectively stimulate the adhesion, migration and invasion of human lung cancer cells. The acetylcholine signaling pathway in lung cancer is modified to increase the production of growth factor ACh, which may include the up-regulation of ChAT. Numerous studies have shown that ChAT exists in patient-isolated human SCLC and NSCLC tumors, and is detected in many LAC cell lines, and ChAT expression in all LAC tumor tissues is higher than that in adjacent normal lung tissue. In addition, the expression of ChAT in human lung cancer cells is sensitive to certain small molecule mitogenic factors, and these findings confirm the mitogenic, migratory and invasive activities of acetylcholine in human lung cancer cells. Therefore, human LAC cells should express more ChAT than normal bronchial epithelial cells. By analyzing the differences in ChAT expression in human normal bronchial epithelial cells (NHBEs) and LAC cell lines, early diagnosis of lung cancer can be achieved, which has certain clinical application value. Therefore, it is necessary to construct an analysis method suitable for the detection of ChAT in cells, and then apply it to the detection of ChAT in lung cancer cells.

[0004] In addition, the reduction of ChAT expression or the destruction of its enzyme activity has been studied as a possible drug target for the treatment of human lung cancer, and the study of ChAT inhibitors is also important. Studies have shown that a-NETA is a specific ChAT inhibitor, which induces a decrease in acetylcholine secretion in human lung cancer cells by blocking ChAT activity. Traditionally, acetylcholine stimulates human lung cancer cell proliferation, induces epithelial-mesenchymal transition (EMT), migration and invasion through nAChR and muscarinic receptors. The decrease in acetylcholine levels will inhibit the above signaling pathways, and terminate the growth and survival of human lung cancer. Therefore, ChAT inhibitors may represent a new generation of drugs related to the treatment of lung cancer, and the determination of the half-inhibitory concentration (IC 50 ) of a-NETA on ChAT is crucial. At present, there are very few reports on the detection of ChAT activity analysis and the screening of its inhibitors, and it is urgent to develop a new type of analysis sensing method for the discussion of ChAT activity in cells and the screening of its inhibitors.

[0005] Nanomaterials provide an ideal platform for the construction of colorimetric sensing methods, among which manganese dioxide (MnO2) nanomaterials have attracted extensive attention due to their unique electrical, catalytic, ion exchange, and optical properties. MnO2 has abundant surface hydroxyl groups and variable manganese valence states, thus exhibiting high catalytic activity. It has excellent extinction spectra from ultraviolet light to far infrared, showing great application prospects in biomedical research. For example, the photothermal conversion properties of MnO2 make it a good candidate for targeted cancer therapy, especially its large surface area to mass ratio ensures high loading capacity for efficient drug delivery. In addition, MnO2 exhibits enzyme-like reaction characteristics and can be used as an effective nano-enzyme for biosensing. MnO2 has various structural forms, including nanofibers, nanosheets, nanoparticles, etc., among which two-dimensional nanosheets are of great interest due to their many unusual properties and good flexibility. MnO2 nanosheets are stacked by edge-shared “MnO6” octahedra and interlayer alkali ions. Compared with natural substances, this layered structure has higher chemical and thermal stability. The photophysical and photochemical properties of MnO2 nanosheets, such as excellent molar extinction coefficient and effective fluorescence quenching or absorption capacity of energy acceptance ability, make them play an important role in various optical sensing or imaging applications. Its huge surface area allows it to fully contact and react with chromogenic substrates (such as OPD, ABTS, and TMB), and due to its oxidase activity, the reaction process does not require the participation of unstable hydrogen peroxide, and it can directly catalyze the oxidation of chromogenic substrates in the presence of oxygen, thus making the signal more stable. Therefore, MnO2 nanosheets can be used as excellent materials for the construction of colorimetric sensing methods.

[0006] The present application designs a preparation method of a choline acetyltransferase sensor based on manganese dioxide nanosheets, and is applied to the detection of coenzyme A (CoA), ChAT and its inhibitor α-NETA. The method is mainly based on the oxidase activity of MnO2NSs, which oxidizes colorless TMB to bright blue oxTMB in the presence of oxygen in air, at which time an absorption peak of oxTMB is generated in the range of 480-800 nm, and the maximum absorption wavelength is at 652 nm. CoA with reducing property (because it has a thiol group) can simultaneously undergo redox reactions with strongly oxidizing MnO2NSs and oxTMB, reducing MnO2NSs to Mn 2+ , and oxTMB to TMB, so that MnO2NSs lose catalytic properties, and Mn 2+The reduction of oxTMB further reduces the absorption peak at 652 nm. With the increase of CoA concentration, the amount of MnO2 NSs decreases, the catalytic generation of oxTMB also decreases, and part of the generated oxTMB is also reduced by CoA, the absorption peak at 652 nm decreases, the blue of the solution becomes lighter, and the ultraviolet colorimetric sensing method of coenzyme A is constructed. In addition, by combining the software with the smart phone, the R, G and B values of the colorimetric sensor are extracted, and the greater the CoA concentration, the smaller the B / G value. Through the relationship between the B / G value and the CoA concentration, a smart phone sensing method for detecting CoA is constructed. Based on the reaction of ChAT catalyzing choline and Ac-CoA to generate acetylcholine and CoA, the greater the ChAT concentration, the more CoA is generated, the lighter the blue of the solution, and the smaller the absorbance value at 652 nm and the B / G value. Through the signal change caused by CoA, the detection of ChAT can be realized indirectly. Since some substances can inhibit the development of malignant tumors by inhibiting ChAT, which may be a possible way to alleviate the malignant phenotype of cancer cells, the inhibitory effect of alpha-NETA is evaluated. Finally, the prepared ultraviolet colorimetric and smart phone sensor is applied to the analysis of ChAT content in lung cancer A549. At present, there is no related report on the ultraviolet colorimetric and smart phone sensing method based on the redox reaction between MnO2 NSs and CoA. The method is used for the analysis and detection of CoA, ChAT and its inhibitor alpha-NETA, especially for the study of the change of ChAT content in the development of lung cancer, which provides a new idea for predicting the occurrence and development of lung cancer. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a choline acetyltransferase sensor based on manganese dioxide nanosheet and its application in lung cancer A549, which is simple to operate, low in cost, high in sensitivity and good in specificity.

[0008] The technical solution adopted by the present application to solve the above technical problem is as follows:

[0009] I. Preparation of MnO2 NSs

[0010] MnO2 NSs were prepared using a dissolved oxygen strategy. The specific steps are as follows: 16 mg of MnCl2·4H2O was ultrasonically dispersed into 36 mL of a water-ethanol (2:7, V / V) mixed solution, and after ensuring sufficient dispersion, the solution was adjusted to pH 12 using a 1 M NaOH solution. Then, the solution was continuously stirred in a water bath at 55°C for 1 hour to ensure the full progress of the reaction. After the reaction was completed, the generated MnO2 NSs were collected by centrifugation. The collected sample was washed with deionized water and ethanol for 3 times, respectively, to remove residual substances and impurities in the reaction. Finally, the washed MnO2 NSs were dried at 50°C overnight to obtain high-purity MnO2 NSs.

[0011] II. Biosensor preparation method

[0012] 1. Construction of CoA UV colorimetric sensor based on MnO2 NSs

[0013] 30-120 μL of acetic acid-sodium acetate buffer (HAc-NaAc, 100 mM, pH=4.0), 0-20 μL of deionized water, 0.5-20 μL of 0.5 mg / mL MnO2 NSs, 1-20 μL of 20 mM TMB and 0.1-20 μL of 1 mM CoA were sequentially added to a 96-well enzyme plate, and incubated at room temperature for 5-60 minutes. Then, the color change of the solution was photographed at a fixed height using a smartphone (iPhone 13), and the ultraviolet spectrum in the range of 480-800 nm was measured using an enzyme marker, and the absorbance change at 652 nm was recorded, thereby constructing a UV colorimetric sensor for CoA.

[0014] 2. Construction of ChAT UV colorimetric sensor based on MnO2 NSs

[0015] (1) ChAT catalyzes the production of CoA: 5-50 μL of phosphate buffer (PBS, 10 mM, pH=7.0), 0-20 μL of deionized water, 0.5-10 μL of 100 mM choline, 0.5-10 μL of 10 mM Ac-CoA and 0.1-50 μL of 0.1 mg / mL ChAT were sequentially added to a 96-well enzyme plate, and incubated in a 37°C water bath for 15-60 minutes.

[0016] (2) Oxidation reaction detection: 30-120 μL of HAc-NaAc buffer (100 mM, pH = 4.0), 0.5-20 μL of 0.5 mg / mL MnO2 NSs, 0-20 μL of deionized water and 1-20 μL of 20 mM TMB were sequentially added to the solution after (1) reaction, and incubated at room temperature for 5-60 minutes. Then the color change of the solution was photographed at a fixed height using a smart phone, and the ultraviolet spectrum in the range of 480-800 nm was measured using a microplate reader, and the absorbance change at 652 nm was recorded to construct the ultraviolet colorimetric sensor of ChAT.

[0017] 3. Construction of α-NETA ultraviolet colorimetric sensor based on MnO2 NSs

[0018] (1) ChAT activity inhibition experiment: 1-20 μg / mL of ChAT was mixed with 0.1-10 μL of 1 mM α-NETA, and added to a 96-well enzyme plate, and incubated in a 37°C water bath for 2-20 minutes.

[0019] (2) ChAT catalyzed CoA production: 5-50 μL of phosphate buffer (PBS, 10 mM, pH = 7.0), 0-20 μL of deionized water, 0.5-10 μL of 100 mM choline and 0.5-10 μL of 10 mM Ac-CoA were added to the solution after (1) reaction, and reacted at room temperature for 15-60 minutes.

[0020] (3) Then 30-120 μL of HAc-NaAc buffer (100 mM, pH = 4.0), 0.5-20 μL of 0.5 mg / mL MnO2 NSs, 0-20 μL of deionized water and 1-20 μL of 20 mM TMB were sequentially added to the solution after (2) reaction, and incubated at room temperature for 5-60 minutes, then photographed at a fixed height using a smart phone, and then the ultraviolet spectrum in the range of 480-800 nm was measured using a microplate reader.

[0021] Three, analysis and detection of CoA, ChAT and its inhibitor α-NETA by ultraviolet colorimetric sensor

[0022] 1. Analysis and detection of CoA: based on the above step two-1, by changing the concentration of CoA in step two-1 (final concentration range: 0-60 μM), keeping other steps unchanged, measuring the ultraviolet spectrum in the range of 480-800 nm using a microplate reader, and establishing a standard curve of CoA absorbance difference (ΔA) at 652 nm, thereby realizing ultraviolet analysis and detection of different concentrations of CoA. In addition, by taking colorimetric pictures of the solution, visual detection of different concentrations of CoA can be realized, and the change of solution color can be intuitively displayed.

[0023] 2. Analysis and detection of ChAT: Based on step 2-2-(1) above, by changing the concentration of ChAT in step 2-2-(1) (final concentration range: 0~20μg / mL), while keeping other steps unchanged, the ultraviolet spectrum in the range of 480~800nm ​​is measured using an ELISA reader, and a standard curve of ChAT is established using the absorbance difference (ΔA) at 652nm, thereby realizing the ultraviolet detection of different concentrations of ChAT. In addition, by taking colorimetric images, the visual detection of different concentrations of ChAT can be realized, showing the changes in solution color.

[0024] 3. Analysis and detection of α-NETA: Based on step 2-3-(1) above, by changing the concentration of α-NETA in step 2-3-(1) (final concentration range: 0~26μM), while keeping other steps unchanged, the ultraviolet spectrum in the range of 480~800nm ​​is measured using an ELISA reader, and the inhibition curve of α-NETA is established using the absorbance difference (ΔA) at 652nm, thereby realizing the ultraviolet detection of different concentrations of α-NETA, and calculating the half-inhibitory concentration (IC50) of α-NETA on ChAT. 50 Furthermore, by capturing colorimetric images, it is possible to visualize the detection of different concentrations of α-NETA and demonstrate changes in the solution color.

[0025] IV. Smartphone Sensing Devices and Operating Procedures

[0026] The smartphone sensing device includes: a smartphone (iPhone 13), a dark box, and white paper. The dark box has a built-in, stable, and sufficiently bright LED light, with a small hole at the top to match the phone's camera. The white paper serves as the background for the photo to highlight the color of the target solution. The specific operating steps are as follows: Turn on the LED light inside the dark box, ensuring stable and uniform light. Place the colorimetric sensor prepared in step three in the center of the dark box. Place the smartphone flat on top of the dark box, aligning the smartphone camera with the small hole (ensuring a consistent height for each photo). Take a colorimetric image, ensuring the image is clear and the background is white paper. Use a free colorimetric software (ColorColl) downloaded to the phone to extract the R, G, and B values ​​of the solution color in each well. Save the extracted data and analyze the relationship between the R, G, and B values ​​and the concentrations of CoA, ChAT, and α-NETA to establish a standard curve. Through these operations, the smartphone sensing device can accurately digitally record and analyze the color changes of the colorimetric sensor, thereby achieving precise detection of the concentrations of CoA, ChAT, and their inhibitor α-NETA.

[0027] Invention principle: The application is a choline acetyltransferase sensor based on manganese dioxide nanosheet and its application in lung cancer A549. Reduced coenzyme A (CoA) can simultaneously undergo redox reaction with strong oxidizing MnO2NSs and ox-TMB, making MnO2NSs reduced to Mn 2+ Thus, it loses its catalytic activity and cannot continue to oxidize colorless substrate TMB to blue. At the same time, part of the oxidized ox-TMB is also reduced by CoA. With the increase of CoA concentration, the amount of MnO2NSs in the solution decreases and ox-TMB is further reduced, the ultraviolet absorption peak at 652 nm decreases, the blue color of the solution gradually lightens, the absorbance difference (ΔA) decreases, and the B / G value also decreases. Through the relationship between ΔA and B / G value and CoA concentration, the detection of CoA concentration can be realized. ChAT can catalyze the acetyl group of Ac-CoA to transfer to choline to generate acetylcholine and CoA. With the increase of ChAT concentration, the amount of generated CoA increases, the ultraviolet absorption peak at 652 nm decreases, the blue color of the solution lightens, ΔA decreases, and B / G value decreases. Through the detection of generated CoA signal, the detection of ChAT concentration can be realized indirectly. In addition, alpha-NETA can inhibit the activity of ChAT. With the increase of the concentration of added alpha-NETA, the activity of ChAT is gradually inhibited, the generated CoA decreases, the ultraviolet absorption peak at 652 nm gradually recovers (increases), the blue color of the solution changes from light to deep, ΔA increases, and B / G value increases. In this way, the inhibitory effect of alpha-NETA on ChAT can be evaluated. Based on the above principle, the application realizes the dual-mode detection of CoA, ChAT and alpha-NETA, constructs a simple, convenient, rapid and high-sensitivity detection method, and finally applies it to the study of the relationship between the abnormality of ChAT expression in lung cancer A549 and the development of lung cancer.

[0028] Compared with the prior art, the application has the following advantages:

[0029] (1) Simple and safe material synthesis: The material synthesis method used in the application does not involve highly toxic substances, and the entire synthesis process does not require high temperature and high pressure conditions, which is safe. In addition, MnO2 nanosheet (NSs) has a large specific surface area, which can be in full contact with the substrate, which is conducive to the construction of an efficient analysis sensing method.

[0030] (2) Novel and Portable Intelligent Method: Reports on choline acetyltransferase (ChAT) activity detection are currently very rare. This invention is the first to design a smartphone sensor for ChAT activity detection based on MnO2NSs. Within a certain concentration range, the higher the CoA concentration, the less MnO2NSs in the solution and the more the oxidized ox-TMB is consumed, resulting in a lighter blue solution and a decreased B / G value. Similarly, the higher the ChAT concentration, the more CoA is produced, resulting in a lighter blue solution and a decreased B / G value. Furthermore, the higher the α-NETA concentration, the more ChAT is inhibited, leading to a gradually decreasing CoA concentration, a darker blue solution, and an increased B / G value. The results show that the B / G value has a linear relationship with CoA, ChAT, and α-NETA within a certain concentration range, thus successfully realizing smartphone-based analytical sensing of CoA, ChAT, and α-NETA.

[0031] (3) High sensitivity: The present invention is based on the ultraviolet colorimetric and smartphone sensing method designed with MnO2NSs, which reduces MnO2NSs to Mn through CoA. 2+ The changes in UV, colorimetric signals, and B / G values ​​resulted in the following linear equations: The linear correlation equation between ΔA and CoA concentration is: y = -0.006230x + 0.2538, R0 2 =0.9971, detection limit is 4.1 nM (3σ / slope); the linear correlation equation between B / G value and CoA concentration is: y = -0.000005273x + 1.082, R 2 =0.9858. The linear correlation equation between ΔA and ChAT concentration is: y = -0.01629x + 0.2399, R 2 =0.9935, detection limit is 0.046 μg / mL (3σ / slope); the linear correlation equation between B / G value and ChAT concentration is: y = -0.01375x + 1.081, R 2 =0.9870. The half-maximal inhibitory concentrations (WMCs) of α-NETA for ChAT obtained by UV colorimetry and smartphone sensing methods were 11.4 μM and 11.7 μM, respectively. This indicates that the prepared UV colorimetric and smartphone sensors can achieve high-sensitivity detection of CoA, ChAT, and α-NETA.

[0032] (3) High specificity: for CoA detection, other control substances such as potassium chloride (KCl), glycine (Gly), xanthine (GTP), sucrose (SUC), alcohol dehydrogenase (ADH), terminal transferase (TdT), lactic acid (HL), glucose oxidase (GOx), glucose dehydrogenase (GDH) have no interference with the system; for ChAT detection, other control substances such as acetylcholinesterase (AChE), uricase (UAO), cholesterol oxidase (COD), papain, lysine acetyltransferase (KAT5), glucose oxidase (GOx), xanthine oxidase (XOD), trypsin, terminal transferase (TdT) have no interference with the system.

[0033] (4) Simple operation and low cost: the smart phone is convenient to carry and can be used for on-site analysis and detection. The volume of the entire ultraviolet colorimetric sensor solution is only 100 μL, the reagent consumption is small, and the cost is low.

[0034] (5) High practical value: through the analysis of the ChAT content in normal lung cells and lung cancer A549 cells, it is found that ChAT is highly expressed in lung cancer A549 compared with normal lung cells. After treating the cells with α-NETA, the ChAT activity can be significantly inhibited, which proves the application potential of α-NETA in the occurrence and treatment of lung cancer. In addition, both human normal bronchial epithelial cells (NHBE) and human lung cancer cells (A549) are cultured in culture medium with and without nicotine (L-Nicotine). In the culture medium without L-Nicotine, the expression of ChAT in A549 is higher than that in NHBE. In the culture medium containing L-Nicotine, the ChAT content in A549 is significantly higher than that in NHBE, which shows that L-Nicotine can significantly promote the high expression of ChAT in lung cancer cells. This shows that nicotine may cause lung cancer to further develop, and tobacco is the main source of nicotine, which also proves that smoking may cause lung cancer to worsen. Therefore, by monitoring the expression level of ChAT in lung cancer A549, lung cancer can be diagnosed early and the development of lung cancer can be judged, and the introduction of inhibitors further proves the high expression of ChAT in lung cancer cells and proves that lung cancer cells will produce more ChAT under the stimulation of L-Nicotine. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Figure is the feasibility experiment diagram of the ultraviolet colorimetric sensor of the present application;

[0036] Figure 2 Figure is the standard curve diagram of ΔA value of the ultraviolet colorimetric sensor of the present application to different concentrations of CoA;

[0037] Figure 3 The standard curve of the ΔA value of the UV colorimetric sensor of the present application versus the concentration of ChAT at different concentrations;

[0038] Figure 4 The standard curve of the ΔA value of the UV colorimetric sensor of the present application versus the concentration of α-NETA at different concentrations;

[0039] Figure 5 The specific experiment graph of the UV colorimetric sensor of the present application for CoA, ChAT and α-NETA;

[0040] Figure 6 The standard curve graph of the smartphone sensor for detecting CoA, ChAT and α-NETA;

[0041] Figure 7 The comparison graph of the UV colorimetric sensor and the smartphone sensor of the present application for detecting the expression level of ChAT in normal lung cells and lung cancer cells;

[0042] Figure 8 The effect of nicotine on the expression of ChAT in normal lung cells and lung cancer cells; DETAILED DESCRIPTION

[0043] The present application will be further described in detail below with reference to the accompanying drawings.

[0044] Example 1 Preparation of MnO2 NSs

[0045] MnO2 NSs were prepared by a dissolved oxygen strategy. The specific steps are as follows: 16 mg of MnCl2·4H2O was ultrasonically dispersed into 36 mL of a water-ethanol (2:7, V / V) mixed solution, and after ensuring sufficient dispersion, 1M NaOH solution was used to adjust the solution to pH 12. Then, the solution was continuously stirred in a water bath at 55°C for 1 hour to ensure sufficient reaction. After the reaction was completed, the generated MnO2 NSs were collected by centrifugation. The collected sample was washed with deionized water and ethanol for 3 times respectively to remove residual substances and impurities in the reaction. Finally, the washed MnO2 NSs were dried at 50°C overnight to obtain high-purity MnO2 NSs.

[0046] Example 2 Preparation of buffer solution

[0047] 1. Acetic acid-sodium acetate buffer (HAc-NaAc, 100 mM, pH = 4.0)

[0048] In a 500 mL beaker, 0.6560 g of sodium acetate (NaAc) was added, followed by 50 mL of 0.83 mol / L acetic acid (HAc), and then an appropriate amount of water was added to completely dissolve it, and then transferred to a 100 mL volumetric flask, and after constant volume, it was shaken well.

[0049] 2. Phosphate buffer (PBS) (10 mM, pH = 7.0)

[0050] In a 100 mL beaker, 0.0004 mol NaH2PO4 and 0.0005 mol Na2HPO4 were added and completely dissolved in 90 mL ultrapure water, and then the solution was transferred to a 100 mL volumetric flask, and after constant volume, it was shaken well.

[0051] Example Three Biosensor Preparation Method 1, Construction of CoA UV Colorimetric Sensor Based on MnO2 NSs

[0052] In a 96-well enzyme plate, 60 μL of acetic acid-sodium acetate buffer (HAc-NaAc, 100 mM, pH = 4.0), 29.5 μL of deionized water, 2 μL of 0.5 mg / mL MnO2 NSs, 5 μL of 20 mM TMB, and 3.5 μL of 1 mM CoA were added in sequence, and incubated at room temperature for 10 minutes. Then a smartphone (iPhone 13) was used to take a photo of the color change of the solution at a fixed height, and a microplate reader was used to measure the ultraviolet spectrum in the range of 480-800 nm, and the absorbance change at 652 nm was recorded, thereby constructing a UV colorimetric sensor for CoA.

[0053] 2. Construction of ChAT UV Colorimetric Sensor Based on MnO2 NSs

[0054] (1) ChAT catalyzes the production of CoA: 15 μL of phosphate buffer (PBS, 10 mM, pH = 7.0), 3 μL of deionized water, 1 μL of 100 mM choline, 1 μL of 10 mM Ac-CoA, and 10 μL of 0.1 mg / mL ChAT were added in sequence to a 96-well enzyme plate, and incubated in a 37°C water bath for 30 minutes.

[0055] (2) Oxidation reaction detection: 60 μL of HAc-NaAc buffer (100 mM, pH = 4.0), 2 μL of 0.5 mg / mL MnO2 NSs, 3 μL of deionized water, and 5 μL of 20 mM TMB were added in sequence to the solution after (1) reaction, and incubated at room temperature for 10 minutes. Then a smartphone was used to take a photo of the color change of the solution at a fixed height, and a microplate reader was used to measure the ultraviolet spectrum in the range of 480-800 nm, and the absorbance change at 652 nm was recorded, thereby constructing a UV colorimetric sensor for ChAT.

[0056] 3. Construction of α-NETA UV Colorimetric Sensor Based on MnO2 NSs

[0057] (1) ChAT activity inhibition experiment: 10 pg / mL ChAT was mixed with 2 pL 1 mM a-NETA, and added into a 96-well enzyme plate, and incubated in a 37°C water bath for 10 minutes.

[0058] (2) ChAT catalyzed CoA production: 15 pL PBS (10 mM, pH = 7.0), 1 pL deionized water, 1 pL 100 mM choline, and 1 pL 10 mM Ac-CoA were added to the solution after (1) reaction, and reacted at room temperature for 30 minutes.

[0059] (3) Then 60 pL HAc-NaAc buffer (100 mM, pH = 4.0), 2 pL 0.5 mg / mL MnO2 NSs, 3 pL deionized water, and 5 pL 20 mM TMB were sequentially added to the solution after (2) reaction, and incubated at room temperature for 10 minutes, then photographed at a fixed height using a mobile phone, and then the ultraviolet spectrum in the range of 480-800 nm was measured using an enzyme marker.

[0060] Example Four: Analysis of the feasibility of the ultraviolet colorimetric sensor

[0061] First, the ultraviolet spectrum of the above-described ultraviolet colorimetric sensor at 480-800 nm was detected, as shown in Figure 1 A, when there were only MnO2 NSs and TMB in the solution, the solution (6) was dark blue, and the ultraviolet absorption peak at 652 nm was obvious; when CoA was added, the solution (7) became lighter blue, and the ultraviolet absorption peak at 652 nm was significantly reduced; while the MnO2 NSs-solution (1), TMB-solution (2), CoA-solution (3), TMB+CoA-solution (4), and MnO2+CoA-solution (5) were colorless, and there was almost no ultraviolet absorption peak at 652 nm, indicating that the prepared ultraviolet colorimetric sensor had good response to CoA and could be used for the detection of CoA; Subsequently, the reaction order of the reactants was investigated, and the results are shown in Figure 1 B, solution (1) was MnO2 NSs and CoA first reacted for 5 minutes, then TMB was added and reacted for another 5 minutes; solution (2) was MnO2 NSs and TMB first reacted for 5 minutes, then CoA was added and reacted for another 5 minutes; solution (3) was CoA and TMB first reacted for 5 minutes, then MnO2 NSs was added and reacted for another 5 minutes; solution (4) was MnO2 NSs, CoA and TMB reacted together for 10 minutes; it can be seen that the solution lightness order is (3)>(2)>(1)>(4), (4) is the lightest, the absorption peak is the lowest, and the reaction is the simplest, so the subsequent reaction order is (4), that is, MnO2 NSs, CoA and TMB are reacted together for 10 minutes, and then the ultraviolet absorption spectrum is measured.

[0062] Example Five UV colorimetric sensor for the analysis of CoA and ChAT and its inhibitor α-NETA

[0063] 1. By changing the CoA concentration (final concentration: 0, 0.01, 0.05, 0.15, 0.5, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60 μM) in Example Three-1, and keeping other steps unchanged, the UV absorption spectrum in the range of 480-800 nm was measured by a microplate reader, and the CoA standard curve was established at 652 nm ΔA, which realized the UV analysis of different concentrations of CoA. In addition, the visualization detection of different concentrations of CoA could be realized by colorimetric image. The results are shown in Figure 2 A, 2B, Figure 2 A is the CoA detection kinetics graph, that is, the absorbance changes with time after the addition of CoA. It can be seen that the absorbance is stable after about 10 minutes, so the optimal reaction time is 10 minutes; Figure 2 B is the CoA detection standard curve graph (obtained from the UV spectrum), and ΔA has a good linear relationship with the concentration of CoA. With the increase of CoA concentration, the color of the solution gradually changes from deep blue to light blue, and finally becomes colorless. The linear correlation equation of ΔA and CoA concentration is y = -0.006230x + 0.2538, R 2 = 0.9971, the linear range is 0-40 μM, and the detection limit is 4.1 nM (3σ / slope); which indicates that the sensor realizes the sensitive detection of CoA.

[0064] 2. By changing the ChAT concentration (final concentration: 0, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20 μg / mL) in Example Three-2-(1), and keeping other steps unchanged, the UV absorption spectrum in the range of 480-800 nm was measured by a microplate reader, and the ChAT standard curve was established by ΔA, which realized the UV analysis of different concentrations of ChAT. In addition, the visualization detection of different concentrations of ChAT could be realized by colorimetric image. The results are shown in Figure 3 A, 3B, Figure 3 A is the ChAT detection kinetics graph, that is, the absorbance changes with time after the addition of ChAT catalyzed CoA; Figure 3 B is the ChAT detection standard curve graph (obtained from the UV spectrum), and ΔA has a good linear relationship with the concentration of ChAT. With the increase of ChAT concentration, the color of the solution changes from deep blue to light blue, and finally becomes colorless. The linear correlation equation of ΔA and ChAT concentration is y = -0.01629x + 0.2399, R 2= 0.9935, linear range was 0-12 μg / mL, and the detection limit was 0.046 μg / mL (3σ / slope); it was indicated that the sensor realized high-sensitivity detection of ChAT.

[0065] 3. The other steps were unchanged, and the UV absorption spectrum in the range of 480-800 nm was measured by the microplate reader to establish the standard curve of α-NETA by ΔA, and the half-inhibitory concentration of α-NETA to ChAT was calculated, and the visual detection of different concentrations of α-NETA was realized by the colorimetric image, by changing the concentration of α-NETA (final concentration: 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 μM) in Example 3-3-(1). The results are shown in Figure 4 A, 4B, Figure 4 A is the detection kinetics graph of α-NETA, that is, after adding different concentrations of α-NETA, CoA generated by ChAT was detected to obtain the change of absorbance of the solution with time; Figure 4 B is the standard curve graph of α-NETA detection (obtained from the UV spectrum), and with the increase of the concentration of α-NETA, the color change of the solution is colorless→light blue→dark blue, and ΔA gradually increases, and the half-inhibitory concentration of α-NETA to ChAT is calculated as 11.4 μM by the inhibition curve obtained by the ultraviolet sensing method.

[0066] Example Six Specific Detection

[0067] The results are shown in Figure 5 A, 5B shows that CoA detection: other substances such as potassium chloride (KCl), glycine (Gly), xanthine (GTP), sucrose (SUC), alcohol dehydrogenase (ADH), terminal transferase (TdT), lactic acid (HL), glucose oxidase (GOx), and glucose dehydrogenase (GDH) have no interference on CoA detection; ChAT detection: other control substances such as acetylcholinesterase (AchE), uricase (UAO), cholesterol oxidase (COD), papain (Papain), lysine acetyltransferase (KAT5), glucose oxidase (GOx), xanthine oxidase (XOD), trypsin (Trypsin), and terminal transferase (TdT) have no interference on the system. It is indicated that the prepared ultraviolet colorimetric sensor has good specificity for CoA and ChAT detection.

[0068] Example Seven Smartphone Sensor Detection of CoA, ChAT and α-NETA

[0069] The LED light inside the dark box was turned on, the UV colorimetric sensor prepared in Example Five was placed in the center of the dark box, the mobile phone was placed on the top of the dark box, the camera of the smart phone was aimed at the small hole above the dark box, the photographing mode of the mobile phone was set to white balance, then the colorimetric image was taken, the R, G, B values of the color of the solution in each hole were extracted by the color extraction software-ColorColl, in order to make the data more accurate, the color was extracted at different positions, three sets of experimental data were taken for the same hole, and finally the average value of the three results was taken. According to the relationship between B / G value and CoA, ChAT and α-NETA concentration, the standard curve was established respectively. The results are as follows Figure 6 As shown in Figures 6A and 6B, with the increase of CoA and ChAT concentration, the solution gradually changed from dark blue to colorless, and the B / G value gradually decreased, and the linear correlation equations were y = -0.000005273x + 1.082, R 2 = 0.9858, the linear range was 0-40 μM; y = -0.01375x + 1.081, R 2 = 0.9870, the linear range was 0-12 μg / mL. Figure 6 As shown in Figure 6C, with the increase of α-NETA concentration, the solution gradually changed from colorless to dark blue, and the B / G value gradually increased, and the half inhibition concentration of α-NETA to ChAT calculated by the inhibition curve obtained by the smart phone sensing method was 11.7 μM. It is proved that the prepared smart phone sensor for detecting CoA, ChAT and α-NETA is feasible, and has certain practical application value.

[0070] Example Eight Analysis and Detection of ChAT Content in Lung Cancer Cell Lysate

[0071] Human lung cancer cells (A549) and human normal bronchial epithelial cells (NHBE) were obtained from Shanghai Cell Bank. The purchased A549 and NHBE were added into Dulbecco's Modified Eagle Medium (DMEM) high glucose culture medium containing 10% fetal bovine serum and 1% double antibody for culture. The culture condition was 37°C, 5% CO2, and when the cells grew to more than 80%, the culture was subcultured. That is, the culture medium in the culture bottle was removed, 5 mL PBS buffer (10 mM, pH = 7.4) was added for washing, and then 1 mL 0.25% trypsin containing EDTA was added for digestion for 3-5 minutes. After gently shaking the bottle wall, the cells were observed to fall off, 3 mL of culture medium was added to stop the digestion, and then the cells on the bottle wall were gently blown off with a pipette to completely fall off. Then the solution was sucked into a centrifuge tube and centrifuged at 1500 rpm, and the supernatant was discarded. The cells were then counted and gradually diluted to a final concentration of 1 million cells per milliliter for A549 and NHBE cells. After ultrasonic treatment for 25 minutes, they were divided and stored in a -20°C freezer. The cells were completely broken by freeze-thaw method to obtain A549 and NHBE cell lysates.

[0072] 5 mL of the obtained A549 and NHBE cell lysates were centrifuged at 9000 rpm for 30 minutes at 4°C, and the supernatant was used for subsequent experiments. The supernatant of A549 cell lysate (final cell concentration: 0, 5000, 20000, 50000, 100000 cells / mL) was extracted to replace ChAT in Example 3-2 to complete the experiment; the results are shown in Figure 7 A, Figure 7 D, the ChAT concentration increases with the increase of A549 cell number, indicating that lung cancer cells do express ChAT. In order to prove that the signal is generated by ChAT, different concentrations of ChAT inhibitor (α-NETA) were added to A549 cell lysate (100000 cells / mL) for further verification; the results are shown in Figure 7 B, 7E, with the increase of α-NETA concentration, the signal gradually recovers (increases), indicating that the ChAT activity is gradually inhibited, proving that the change of signal is indeed caused by ChAT. In addition, in order to study whether ChAT is overexpressed in liver cancer cells, the ChAT concentrations in NHBE cell lysate and A549 cell lysate with the same concentration (100000 cells / mL) were compared and analyzed; the results are shown in Figure 7C, 7F shows that the ChAT content in A549 cells is significantly higher than that in NHBE cells, and P<0.05, which is statistically significant (SPSS 26.0 statistical software is used for analysis, and when the measurement data conforms to the normal distribution and the variance is equal, the t test is used for pairwise comparison), which proves that ChAT is highly expressed in lung cancer cells compared with normal lung cells.

[0073] In addition, NHBE cells and A549 cells were cultured in serum-free medium with and without L-Nicotine [50nM, which is within the nicotine concentration range (1nM-1000nM) found in the plasma of ordinary smokers] for 36h, and then the ChAT content in the cells was detected, and the results are shown in Figure 8 A shows that 50nM of L-Nicotine slightly promotes the increase of ChAT content in lung cells, and significantly promotes the increase of ChAT content in lung cancer cells, and statistically significant differences are observed between normal lung cells and lung cancer cells cultured with and without L-Nicotine, which proves that L-Nicotine significantly promotes the increase of ChAT content; in order to further verify this hypothesis, NHBE cells and A549 cells were cultured in serum-free medium containing different concentrations of L-Nicotine (0, 1, 50, 100nM), and then the ChAT content in the cells was analyzed, and the results are shown in Figure 8 B, 8C shows that as the concentration of L-Nicotine increases, the ChAT content in NHBE cells only slightly increases, while the ChAT content in A549 cells significantly increases, which indicates that L-Nicotine significantly promotes the high expression of ChAT in lung cancer cells, and also indicates that L-Nicotine stimulation may lead to further deterioration of lung cancer. This study can be used for early diagnosis of lung cancer and has potential for clinical application.

[0074] It should be noted that the above specific examples are not limiting to the present application, and the present application is not limited to the above examples. Those skilled in the art can make changes, modifications, additions or substitutions within the essential scope of the present application, which should also be within the protection scope of the present application.​

Claims

1. A choline acetyltransferase sensor based on manganese dioxide nanosheet and its application in lung cancer A549, the mechanism is as follows: MnO2 nanosheet (MnO2NSs) with oxidase-like activity is synthesized by dissolved oxygen strategy. Under the participation of oxygen, MnO2NSs catalyzes colorless TMB to be oxidized into blue oxTMB, producing ultraviolet absorption peak with maximum absorption wavelength of 652nm. Reduced CoA reduces MnO2NSs to Mn 2+ And oxTMB is reduced to oxTMB, and MnO2NSs gradually decreases. With the increase of CoA concentration, MnO2NSs decreases, and the generated oxTMB decreases. In addition, CoA also reduces a part of oxTMB, oxTMB further decreases, the solution blue lightens, the 652nm absorption peak decreases, the ΔA value and B / G value decrease, realizing the ultraviolet colorimetric and smartphone detection of CoA. ChAT catalyzes acetyl coenzyme A (Ac-CoA) to generate acetylcholine (ACh) and CoA. With the increase of ChAT concentration, the generated CoA increases, and MnO2NSs decreases, realizing the detection of ChAT indirectly. α-NETA inhibits ChAT activity, and with the increase of α-NETA concentration, ChAT activity is gradually inhibited, the generated CoA decreases, the solution color restores blue, the 652nm absorption peak rises, the ΔA value and B / G value increase, and the inhibition effect of α-NETA is evaluated. Based on this, a simple, rapid and sensitive ultraviolet colorimetric and smartphone sensing method is constructed for the detection of CoA, ChAT and α-NETA, and is applied to the analysis of ChAT content in lung cancer A549. The method is simple and accurate, and the sensing system is stable, without involving hydrogen peroxide, and no similar report has been found so far, which provides a new idea for the study of lung cancer. 2.The choline acetylase sensor based on manganese dioxide nanosheet and its application in lung cancer A549 according to claim 1, wherein: The application provides a choline acetylase activity analysis detection method combined with a smart phone, which is extremely rare in the prior art, and a CoA, ChAT and alpha-NETA smart phone sensor is constructed by monitoring the change of the color of a solution, thereby causing the change of R, G and B values of the solution. 3.The choline acetylase sensor based on manganese dioxide nanosheet and its application in lung cancer A549 according to claims 1-2, characterized in that: The UV colorimetric and smartphone sensing method based on MnO2 nanosheets can be used for the analysis and detection of different concentrations of CoA, ChAT and a-NETA. The detection limits of CoA and ChAT are 4.1 nM and 0.046 μg / mL, respectively; the IC 50 values of a-NETA for ChAT are 11.4 μM (UV) and 11.7 μM (smartphone), respectively, and the analysis results are accurate. 4.The choline acetylase sensor based on manganese dioxide nanosheet and its application in lung cancer A549 according to claims 1-3, characterized in that: Through comparative analysis of ChAT in lung cancer A549 cells and normal lung cells, it is found that the ChAT content in lung cancer A549 cells is obviously higher than that in normal lung cells, which proves the high expression of ChAT in lung cancer A549, and with the increase of the L-Nicotine concentration, the ChAT content in NHBE cells only slightly increases, while the ChAT content in A549 cells significantly increases, which indicates that L-Nicotine significantly promotes the high expression of ChAT in lung cancer cells, and also indicates that L-Nicotine stimulation may lead to further deterioration of lung cancer, thereby providing a new idea for the occurrence and development of lung cancer.

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