Preparation method of intelligent sensor based on bimetallic nano-enzyme and application of intelligent sensor in breast cancer CTCs

Abstract

The invention discloses a preparation method of an intelligent sensor based on bimetallic nano-enzyme and application of the intelligent sensor in breast cancer CTCs, in the presence of CoPTA / Fc-9 nano-enzyme, the added S < 2-> can compete with TMB for H2O2, along with the increase of the concentration of the S < 2->, the ultraviolet absorption peak at 652 nm is reduced, and the color of the solution becomes light from blue, so that the visual analysis and detection of the S < 2-> are realized. When Zn < 2 + > is introduced, the amount of free S < 2-> is reduced, a change process from light color to blue color is presented, and the absorbance at 652 nm is increased. Next, due to the strong affinity of Zn < 2 + > to phosphate, Zn < 2 + > can be combined with phosphate ions which are a product of ALP dephosphorylation reaction, so that the amount of free S < 2-> is increased, and the solution becomes light from blue; when the inhibitor is added, the dephosphorylation reaction of ATP cannot be catalyzed, and screening of the ALP inhibitor is achieved. The method has the advantages of high accuracy, rapidness, high sensitivity, high selectivity, low cost and the like, successfully realizes detection of a target object, and can be applied to ALP-mediated dephosphorylation research in CTCs.

Description

Technical Field

[0001] The present invention relates to a preparation method of an intelligent sensor based on a bimetallic nanozyme and its application in the dephosphorylation detection of circulating tumor cells (CTCs) in breast cancer, in particular to a UV colorimetric sensing method based on a CoPTA / Fc-9 bimetallic nanozyme. An intelligent visual mobile phone sensor is constructed in view of the corresponding relationship between the substrate color change or absorbance magnitude and the target, and it is applied to the analysis of the dephosphorylation process in the circulating tumor cells of breast cancer patients, belonging to the field of chemical biosensing in analytical chemistry. Background Art

[0002] Post-translational modification of proteins is ubiquitous in eukaryotes and prokaryotes, plays an important regulatory role in the stability and activity of proteins, and is one of the key links in the biological functions of proteins. Phosphorylation modification mainly occurs on the side chains of amino acids such as histidine, aspartic acid, serine, threonine, tyrosine, and arginine, and realizes the regulatory function by forming simple ester bonds, phosphonamide P-N bonds, or chemical bonds such as mixed anhydrides or acyl phosphates. The phosphorylation process mainly involves the phosphorylation reaction of kinases and the dephosphorylation reaction of phosphatases to achieve dynamic regulation functions. Alkaline phosphatase (ALP) is an important phosphohydrolase in human body fluids and tissues and plays an important role in the diagnosis of various diseases, including prostate cancer, bone cancer, diabetes, and liver diseases. These diseases are affected by the activity of ALP, and ALP participates in multiple biological processes, including metabolism, signal transduction, molecular transport, and the expression of genetic information. ALP can catalyze the hydrolysis and transphosphorylation reactions of various phosphate compounds. Among them, adenosine triphosphate (ATP) is one of the substrates for the dephosphorylation reaction of ALP. This reaction can remove the 5'-phosphate group in ATP and convert it into adenosine. At present, there are various ALP detection methods. Common basic detection means include surface-enhanced Raman scattering, electrochemical methods, photoelectrochemical sensing, fluorescence, and colorimetric detection. However, with the development of artificial intelligence technology, these basic detection methods can no longer meet the needs of the times and technology. Therefore, it is necessary to combine electronic devices to design intelligent and smart analytical sensing methods to meet the requirements of on-site detection and intelligent medicine.

[0003] Breast cancer is one of the most common malignant tumors in women, with an incidence rate accounting for about 7-10% of all malignant tumors in the body. It has become the most common type among female tumors and is the second leading cause of tumor-related deaths. Currently, surgical treatment remains the preferred treatment option for breast cancer patients. The surgical scope mainly covers the tumor and the surrounding tissues within a range of 1-2 cm, which is applicable to early breast cancer, commonly seen in stage II, where the maximum diameter of the tumor is ≤3 cm. However, some breast cancer patients still face the risk of recurrence after treatment. This may be related to the fact that the early symptoms of breast cancer are not obvious, resulting in most patients being in the locally advanced stage when they are diagnosed. This is because the sensitivity and selectivity of the currently commonly used tumor markers for stage detection have certain limitations. Traditional imaging examinations (such as ultrasound and magnetic resonance imaging, etc.) are also difficult to detect small metastatic lesions in the early stage of solid organs and peritoneal metastases, thus unable to achieve early monitoring and missing the best treatment opportunity. Therefore, reducing the postoperative recurrence rate, improving the long-term survival rate, and detecting postoperative recurrence and metastasis early have become urgent problems to be solved. In 1869, Ashworth first proposed the concept of circulating tumor cells (CTCs). CTCs are defined as tumor cells that enter the peripheral blood circulation from solid tumors or metastatic lesions spontaneously or through diagnostic and treatment procedures. Several CTCs can aggregate together to form circulating tumor microthrombi, which have high invasiveness and are considered important risk signs for breast cancer metastasis. However, CTCs may have real-time changes in the number decline at the individual level, so the detection that only analyzes the number of CTCs cannot meet the long-term detection and treatment requirements of breast cancer. The epigenetic research of CTCs is still an area that has not been deeply developed, but it has great potential. More and more evidence shows that epigenetics, especially phosphorylation modification, plays an important role in various cellular mechanisms. Revealing the phosphorylation profile of CTCs is crucial for understanding the tumor metastasis mechanism and is helpful for the detection and treatment of breast cancer.

[0004] In view of the advantages of ultra-high environmental stability, controllable structure and composition, tunable catalytic activity, and good biocompatibility of nanozymes, the present invention designs a preparation method of an intelligent sensor based on CoPTA / Fc-9 bimetallic nanozyme and its application in the dephosphorylation of CTCs. First, using ferrocene formic acid as a doping source, introducing it into the metal framework material, making the lamellar material break into small particles, increasing the specific surface area and active sites, and synthesizing CoPTA / Fc-9 bimetallic nanozyme, which can catalyze hydrogen peroxide (H 2 O 2 ) to generate hydroxyl radicals (·OH), oxidizing colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxTMB. This patent discovers that sulfide ions (S 2- ) can compete with TMB for H 2 O2 , resulting in the fading of the blue solution, and zinc ions (Zn 2+ ) can combine with free S 2- , making S 2- immobilized and unable to interfere with the above catalytic reaction. This patent utilizes the interaction relationship between S 2- and Zn 2+ to design a sensing switch, successfully realizing the analysis and detection of S 2- and Zn 2+ that play important roles in human physiological processes. Additionally, since ALP can catalyze the dephosphorylation process of ATP, generating a large amount of phosphate ions, phosphate ions have a strong affinity for Zn 2+ , and phosphate ions can inhibit the ALP catalytic reaction to a certain extent. Along with their continuous combination, the reaction proceeds continuously in the forward direction; while sodium vanadate (Na 3 VO 4 ) can hinder the occurrence of the reaction, thereby realizing the detection of ALP activity and the screening of inhibitors. Based on this, using the RGB chromaticity values of images, a linear relationship between the sample concentration and the chromaticity value is established, and a smartphone sensor is constructed to achieve rapid on-site detection; finally, the phosphorylation process affects the occurrence and development of cancer. This patent explores the dephosphorylation in breast cancer CTCs. After the CTCs are extracted and subjected to cell passage culture, the cells are lysed and the protein content is extracted. It is found that the degree of dephosphorylation reaction participated by ALP in the lysates with different cell numbers is different. The results show that this method is expected to be applied to the exploration of phosphorylation in breast cancer CTCs. So far, no visualization detection of S 2- and Zn 2+ based on CoPTA / Fc-9 bimetallic nanozymes, and no application cases in the analysis and detection of ALP-mediated dephosphorylation in breast cancer CTCs have been found. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of an intelligent sensor with simple operation, high sensitivity, good specificity, fast detection speed, and visualization detection, and its application in breast cancer CTCs.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A preparation method of an intelligent sensor based on bimetallic nanozymes and its application in breast cancer CTCs, the specific steps are as follows:

[0007] (1) Preparation of CoPTA / Fc-9

[0008] Dissolve 0.2 - 0.5 g of terephthalic acid (1 - 3 mmol) and 0.02 - 0.07 g of ferrocene carboxylic acid (Fc - COOH, 0.2 - 0.5 mmol) in 5 - 10 mL of N,N - dimethylformamide (DMF), and stir for 10 - 30 min until fully dissolved to obtain solution A; then dissolve 0.3 - 0.6 g of cobalt nitrate hexahydrate (1 - 3 mmol) in 5 - 10 mL of DMF to obtain solution B; then slowly add solution B to solution A under vigorous stirring. After stirring the mixture for 10 - 30 min, place it in a 30 mL high - temperature reaction kettle with a polytetrafluoroethylene liner, react at 100 °C for 5 - 15 h. After natural cooling to room temperature, centrifuge to collect the solid (3900 rpm), then wash it alternately with 1 - 3 mL of DMF and absolute ethanol 3 times, and dry it at 60 °C for 5 - 15 h to obtain the CoPTA / Fc - 9 solid powder. Redisperse it in ultrapure water to 0.1 - 0.5 mg / mL for standby.

[0009] (2) Preparation of ultraviolet colorimetric sensor

[0010] a.S 2- Analysis and detection

[0011] Add 5 - 10 μL of 0.1 mg / mL CoPTA / Fc - 9 aqueous solution (final concentration 0.005 - 0.01 mg / mL), 10 - 30 μL of 1 mM TMB (final concentration 0.1 - 0.3 mM), 10 - 30 μL of 1 mM H 2 O 2 (final concentration 0.1 - 0.3 mM), 1 - 5 μL of 1 mM Na 2 S (final concentration 10 - 50 μM), 10 - 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 10 - 30 μL of ultrapure water into a 200 μL centrifuge tube in sequence. Mix the solution well, then react the above - mentioned mixture at room temperature for 10 - 20 min, and then transfer it to an ultraviolet spectrophotometer and a cuvette for analysis and testing, and take pictures with a high - definition camera.

[0012] Based on the above steps, by changing the concentration of S 2- (final concentration: 0, 0.001, 0.003, 0.005, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 1000, 10000 μM), with other steps unchanged, measure the ultraviolet absorption peak intensity in the wavelength range of 500 - 800 nm, and establish a linear relationship diagram of different concentrations of S 2- at the maximum absorbance value at 652 nm, and the determination of different concentrations of S can be realized2- UV detection; by analyzing the RGB chromaticity values of colorimetric pictures, establish the 2- linear relationship between the S 2- concentration and the chromaticity value, and a smartphone sensor for S can be constructed.

[0013] b. Analysis and detection of Zn 2+ Analysis and detection

[0014] Mix 1 - 5 μL of 10 mM Na 2 S (final concentration 100 - 500 μM) with 1 - 5 μL of 10 mM ZnCl 2 (final concentration 100 - 500 μM) for 5 - 10 min, then successively add the mixed solution to 5 - 10 μL of 0.1 mg / mL CoPTA / Fc - 9 aqueous solution (final concentration 0.005 - 0.01 mg / mL), 10 - 30 μL of 1 mM TMB (final concentration 0.1 - 0.3 mM), 10 - 30 μL of 1 mM H 2 O 2 (final concentration 0.1 - 0.3 mM), 10 - 30 μL of HAc - NaAc (0.1 M pH = 4) and 10 - 30 μL of ultrapure water into a 200 μL centrifuge tube, add and mix well, react at room temperature for another 10 - 20 min, then transfer the above - mentioned mixed solution to an ultraviolet spectrophotometer and a cuvette for analysis and testing, and take pictures with a high - definition camera.

[0015] Based on the above steps, by changing the Zn 2+ concentration (final concentration: 0, 0.001, 0.003, 0.007, 0.01, 0.03, 0.07, 0.1, 0.3, 0.7, 1, 5, 10, 30, 70, 100, 1000, 10000 μM), with other steps unchanged, measure the intensity of the ultraviolet absorption peak in the wavelength range of 500 - 800 nm, and establish a linear relationship graph of different concentrations of Zn 2+ to achieve the UV detection of different concentrations of Zn 2+ ; by analyzing the RGB chromaticity values of colorimetric pictures, establish the linear relationship between the Zn 2+ concentration and the chromaticity value, and a smartphone sensor for Zn can be constructed. 2+

[0016] c. Analysis and detection of ALP

[0017] Mix 1 - 5 μL of 1 mM ATP (final concentration 10 - 50 μM) with 5 - 15 μL of 500 mU / L ALP (final concentration 25 - 75 mU / L) (added to 0.1 M Tris - HCl buffer solution (pH = 8.2, containing 0.1 M NaCl and 2 mM MgCl 2 ), then add 1 - 5 μL of 10 mM ZnCl 2 (final concentration 100 - 500 μM), and react for 10 - 20 min after mixing at 37 °C. Then mix with 1 - 5 μL of 10 mM Na 2 S (final concentration 100 - 500 μM) and react for 5 - 10 min. Next, successively add the mixed solution to 5 - 10 μL of 0.1 mg / mL CoPTA / Fc - 9 aqueous solution (final concentration 0.005 - 0.01 mg / mL), 10 - 30 μL of 1 mM TMB (final concentration 0.1 - 0.3 mM), 10 - 30 μL of 1 mM H 2 O 2 (final concentration 0.1 - 0.3 mM), 10 - 30 μL of HAc - NaAc (0.1 M pH = 4) and 10 - 30 μL of ultrapure water into a 200 μL centrifuge tube, mix well, react again at room temperature for 10 - 20 min, then transfer the above - mentioned mixed solution to a UV - visible spectrophotometer and a cuvette for analysis and testing, and take pictures with a high - definition camera.

[0018] Based on the above steps, by changing the ALP concentration (final concentrations: 0, 0.01, 0.1, 0.3, 1, 3, 10, 30, 100, 320, 1000, 3200, 10000, 16000, 80000, 5×10 5 , 10 6 , 5×10 6 mU / L), with other steps unchanged, measure the intensity of the UV absorption peak in the wavelength range of 500 - 800 nm, establish a linear relationship graph of ALP at different concentrations with the maximum absorbance value at 652 nm, and the UV detection of ALP at different concentrations can be achieved; by analyzing the RGB chromaticity values of the colorimetric pictures, establish a linear relationship between the ALP concentration and the chromaticity value, and the visual detection of ALP at different concentrations can be achieved. In addition, adding sodium vanadate (Na 3 VO 4 ) to the ALP reaction solution can better inhibit the ALP activity, thus realizing the screening of small - molecule inhibitors.

[0019] (3) Construction of the mobile phone sensor

[0020] The mobile phone model is Huawei nova 9, and the photographing device assembly includes: a camera obscura (with a small hole at the top, just matching the smartphone camera), an LED light (ensuring sufficient and stable light source inside the camera obscura), and a white paper (serving as the photographing background). By using the smartphone to capture in real time the color change of TMB caused by the change of S 2- 、Zn 2+ and the concentration change of ALP, thus realizing the real-time dynamic monitoring of the target object. Put the ultraviolet colorimetric sensor prepared in (2) into the built camera obscura, turn on the LED light, take a photo with the mobile phone, and then import the obtained photo into the free color picker software downloaded from the mobile phone application store to obtain the corresponding R, G, B values. According to the ratio of B to R and S 2- 、Zn 2+ and the relationship of the ALP concentration, establish the standard curve respectively.

[0021] (4) Detection of the dephosphorylation process in CTCs

[0022] Capture CTCs in the blood of breast cancer patients by magnetic separation method, and perform cell passage on all the collected cells. Collect and lyse the cells after passage, and then extract proteins from the cell lysate (for specific steps, see Example 4). Apply the extracted proteins to the experiment completed in (2).

[0023] Principle of the invention: The present invention is a preparation method of an intelligent sensor based on bimetallic nanozyme and its application in breast cancer CTCs. CoPTA / Fc-9 nanozyme catalyzes H 2 O 2 to generate hydroxyl radicals (·OH), and ·OH can oxidize colorless TMB into blue oxTMB, which has an obvious ultraviolet absorption peak at 652 nm. The addition of S 2- can compete with TMB for H 2 O 2 , resulting in the fading of the blue solution. As the concentration of S 2- increases, the ultraviolet absorption peak at 652 nm decreases and the solution color changes from blue to light, thus realizing the visual analysis detection of S 2- . When Zn 2+ is introduced, since Zn 2+ combines with S 2- to form ZnS, reducing the amount of free S 2- , thus resulting in the reduction of S 2 O 2 competing with TMB for H 2- . A change process of the light color returning to blue is presented, and the absorbance of the solution at 652 nm increases. Based on this, the visual analysis detection of Zn 2+ is realized. Then, due to Zn 2+The strong affinity for phosphate can bind to the phosphate ions, the products of the ALP dephosphorylation reaction, making the dephosphorylation process more complete. At the same time, it also reduces the amount of free Zn 2+ and increases the amount of free S 2- . As a result, the solution will become lighter in color and the absorbance at 652 nm will decrease. Finally, when an inhibitor is added, since the activity of ALP is inhibited and it cannot catalyze the dephosphorylation reaction of ATP, the screening of ALP inhibitors can be achieved. Based on this, the visualization and quantitative analysis of S 2- , Zn 2+ and ALP can be realized, and a simple, rapid, highly sensitive, highly selective and label-free visualization analysis method is constructed for the study of dephosphorylation in breast cancer CTCs.

[0024] Compared with the prior art, the advantages of the present invention are as follows: For the first time, the present invention is based on the dual-metal nanozyme catalyzing H 2 O 2 to oxidize TMB for detecting S 2- , Zn 2+ and ALP, and at the same time, a smartphone colorimetric device is introduced. Obviously, within a certain concentration range, the greater the concentration of S 2- , the greater the competition with TMB, the less oxTMB is produced, the lighter the blue color, and the lower the absorbance value at 652 nm; similarly, the greater the concentration of Zn 2+ , the more S 2- is consumed, and the higher the absorbance value at 652 nm and the darker the blue color; when the ALP dephosphorylation reaction is introduced, the free Zn 2+ is consumed, and the free S 2- in the solution increases, and the absorbance value at 652 nm decreases and the blue color becomes lighter. The experimental results show that the magnitude of the absorbance value at 652 nm has a linear relationship with the logarithm of the concentrations of S 2- , Zn 2+ and ALP within a certain range, successfully realizing the detection of three target substances and visualization analysis detection, and exploring the relationship between phosphorylation in CTCs and the occurrence and development of breast cancer. Its advantages are as follows:

[0025] (1) Construction of a smartphone sensor. Combining the smartphone with sample detection realizes visualization analysis and on-site detection. The operation is simple, and the digital image colorimetric analysis method based on the smartphone further improves the accuracy and timeliness of the colorimetric analysis method.

[0026] (2) The method is simple and the cost is low. The synthesis of the CoPTA / Fc-9 material is simple, the detection process is realized in a homogeneous phase, without introducing some extreme conditions, the operation is simple, the reaction speed is fast, and the rapid, simple and sensitive visualization analysis detection of the target substance is realized.

[0027] (3) High sensitivity. The present invention designs a preparation method of an intelligent sensor based on bimetallic nanozymes. By using the CoPTA / Fc-9 material, it can catalyze H 2 O 2 to oxidize TMB into blue oxTMB, which has an obvious ultraviolet absorption peak at 652 nm. Based on the fact that S 2- can compete with TMB for H 2 O 2 , the addition of S 2- causes the blue solution to fade, thus realizing the analysis and detection of S 2- . Then, Zn 2+ is used to remove S 2- from the solution, and the solution will turn blue again, thereby indirectly realizing the analysis and detection of Zn 2+ . Based on the strong affinity of Zn 2+ for phosphate, it is further utilized to accelerate the dephosphorylation reaction of ALP and ATP, and the activity detection of ALP can also be indirectly realized. Subsequently, three linear equations are obtained: the linear correlation equation between the intensity of the ultraviolet absorption peak and the concentration of S 2- is y = -0.0624x + 0.2295, R 2 = 0.9977, and the detection limit is 0.28 nM; the linear correlation equation between the intensity of the ultraviolet absorption peak and the concentration of Zn 2+ is y = 0.0523x + 0.2667, R 2 = 0.9977, and the detection limit is 0.31 nM; the linear correlation equation between the intensity of the ultraviolet absorption peak and the concentration of ALP is y = -0.0331x + 0.2919, R 2 = 0.9972, and the detection limit is 0.0035 mU / L. It shows that the sensor can achieve high-sensitivity detection of S 2- , Zn 2+ and ALP.

[0028] (4) High specificity. For the detection of S 2- : Other control substances such as fluoride ions (F - ), bromide ions (Br - ), iodide ions (I - ), chloride ions (Cl - ) and hydroxide ions (OH - ) have no interference on the system; for the detection of Zn 2+ : Other control substances such as nickel ions (Ni 2 + ), iron ions (Fe 3+ ), manganese ions (Mn 2+ ), cobalt ions (Co 2+ ) and chromium ions (Cr 3+) For the detection of ALP: other reference substances such as histone acetyltransferase (HAT), terminal deoxynucleotidyl transferase (TdT), acetylcholinesterase (AChE), papain, and cholesterol oxidase (ChOx); these interfering substances have no interference on the system.

[0029] (5) Strong applicability. Zn 2+ There is a substance that can promote the dephosphorylation reaction of ALP, so it is more specific for the dynamic phosphorylation; finally, this method is used for the analysis and detection of dephosphorylation in breast cancer CTCs to explore the relationship between the phosphorylation process and the occurrence and development of breast cancer.

[0030] In summary, the present invention constructs a preparation method of an intelligent sensor based on a bimetallic nanozyme and its application in breast cancer CTCs, which has the advantages of dual signal output, rapid analysis, simple operation, high sensitivity, good selectivity, low cost, etc., and has good application prospects. Brief Description of the Drawings

[0031] Figure 1 It is the feasibility analysis diagram of S 2- , Zn 2+ and ALP for the sensor of the present invention;

[0032] Figure 2 It is the linear relationship diagram of the ultraviolet absorption intensity and RGB value of the sensor of the present invention for different concentrations of S 2- ;

[0033] Figure 3 It is the linear relationship diagram of the ultraviolet absorption intensity and RGB value of the sensor of the present invention for different concentrations of Zn 2+ ;

[0034] Figure 4 It is the linear relationship diagram of the ultraviolet absorption intensity and RGB value of the sensor of the present invention for different concentrations of ALP;

[0035] Figure 5 It is the linear relationship diagram of the ultraviolet absorption intensity of the sensor of the present invention for different concentrations of Na 3 VO 4 ;

[0036] Figure 6 It is the selectivity experiment diagram of S 2- , Zn 2+ and ALP for the sensor of the present invention;

[0037] Figure 7 It is the analysis and detection experiment diagram of the sensor of the present invention in breast cancer CTCs. Detailed Embodiments

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

[0039] Example 1 Construction of an ultraviolet and smartphone colorimetric sensing method for detecting ALP

[0040] A. Preparation of CoPTA / Fc-9

[0041] 0.3323 g of terephthalic acid (2 mmol) and 0.0368 g of ferrocene carboxylic acid (Fc-COOH, 0.22 mmol) were dissolved in 10 mL of N,N-dimethylformamide (DMF), and stirred for 25 min until completely dissolved to obtain solution A; then 0.582 g of cobalt nitrate hexahydrate (2 mmol) was dissolved in 10 mL of DMF to obtain solution B; then solution B was slowly added to solution A under vigorous stirring. After the mixture was stirred for 15 min, it was placed in a 30 mL high-temperature reaction kettle with a polytetrafluoroethylene liner and reacted at 100 °C for 10 h. After naturally cooling to room temperature, the solid was collected by centrifugation (3900 rpm), and then washed alternately with 2 mL of DMF and absolute ethanol 3 times, and dried at 60 °C for 12 h to obtain CoPTA / Fc-9 solid powder, which was redispersed in ultrapure water to 0.1 mg / mL and reserved.

[0042] B. Preparation of ultraviolet colorimetric sensor

[0043] a. Analysis and detection of S 2-

[0044] 10 μL of 0.1 mg / mL CoPTA / Fc-9 aqueous solution (final concentration 0.01 mg / mL), 25 μL of 1 mM TMB (final concentration 0.25 mM), 25 μL of 1 mM H 2 O 2 (final concentration 0.25 mM), 3 μL of 1 mM Na 2 S (final concentration 30 μM), 20 μL of acetate buffer (HAc-NaAc, 0.1 M pH = 4) and 17 μL of ultrapure water were successively added to a 200 μL centrifuge tube. The solution was thoroughly mixed, and then the above mixture was reacted at room temperature for 20 min and then transferred to an ultraviolet spectrophotometer and a cuvette for analysis and testing, and photographed with a high-definition camera.

[0045] The ultraviolet absorption curve of the above-mentioned sensor in the range of 500 - 800 nm was detected. As shown in Figure 1 A, it can be seen that the sensor has almost no ultraviolet absorption peak when CoPTA / Fc-9, S 2- and H 2 O 2 exist alone. When CoPTA / Fc-9 and H 2 O2 When, the prepared sensor has an obvious ultraviolet absorption peak at 652 nm, and the solution (2) shows a color change process from colorless to blue, indicating that CoPTA / Fc-9 catalyzes the oxidation of H 2 O 2 of TMB. After further adding S 2- , the ultraviolet absorption peak value at 652 nm weakens, and the solution (1) shows a color change process from blue to colorless, indicating that S 2- competes with TMB for H 2 O 2 . By comparing with other samples (3 - 15), it shows that this sensing method has a good response to S 2- and can be used for the analysis and detection of S 2- .

[0046] Based on the above steps, by changing the concentration of S 2- (final concentrations: 0, 0.001, 0.003, 0.005, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 3, 5, 10, 30, 50, 100, 150, 1000, 10000 μM), with other steps unchanged, measure the ultraviolet absorption peak intensity in the wavelength range of 500 - 800 nm. As shown in Figure 2 A, the logarithm of the ultraviolet absorption peak intensity of the sensor shows a good linear relationship with the concentration of S 2- , and as the concentration of S 2- increases, the blue color gradually fades. Taking the absorbance peak value at 652 nm to establish a linear curve for different concentrations of S 2- as y = -0.0624x + 0.2295, R 2 = 0.9977, and the detection limit is 0.28 nM; import the taken photos into the color picker software of the smartphone to obtain the R, G, B values of the samples corresponding to different concentrations. Take the R, G, B values three times from different positions for each sample, and finally take the average of the three results. By analyzing the RGB chromaticity values of the colorimetric pictures, as shown in Figure 2 B, establish a linear relationship between the concentration of S 2- and the chromaticity value, and a smartphone sensor for S 2- can be constructed.

[0047] b. Analysis and detection of Zn 2+

[0048] In the above step a, 1 μL of 10 mM Na 2 S (final concentration 100 μM) and 1 μL of 10 mM ZnCl 2 ​After mixing (final concentration 100 μM) and reacting for 10 min, successively add the mixed solution to 10 μL of 0.1 mg / mL CoPTA / Fc-9 aqueous solution (final concentration 0.01 mg / mL), 25 μL of 1 mM TMB (final concentration 0.25 mM), 25 μL of 1 mM H 2 O 2 (final concentration 0.25 mM), 20 μL of HAc-NaAc (0.1 M pH = 4) and 18 μL of ultrapure water to a 200 μL centrifuge tube, add and mix well, react again at room temperature for 20 min, then transfer the above mixed solution to a UV-visible spectrophotometer and a cuvette for analysis and testing, and take pictures with a high-definition camera.

[0049] ATP is the substrate for the dephosphorylation reaction of ALP, which can induce the aggregation of AuNPs, while the product adenosine triggers the redispersion of AuNPs, as Figure 1 shown in B. Using AuNPs (1) as a colorimetric indicator, and due to the formation of the Zn 2+ -phosphate complex, the dephosphorylation reaction can be further accelerated. It can be seen that after adding Zn 2+ , the AuNPs aggregate (2). As the concentration of Zn 2+ increases, the color of the solution changes from gray to red, accompanied by a blue shift of the UV absorption peak (3 - 5).

[0050] Detect the UV absorption curve of the above-mentioned sensor in the range of 500 - 800 nm, as Figure 1 shown in C. It can be seen that after adding Zn 2+ , the UV absorption peak value increases again, and the solution (2) changes from colorless to blue, indicating that the free S 2- decreases, and the UV absorption peak value at 652 nm increases. By comparing with other samples (4 - 7), it shows that this sensing method has a good response to Zn 2+ and can be used for the analysis and detection of Zn 2+ .

[0051] Based on the above steps, by varying the Zn 2+ concentration (final concentrations: 0, 0.001, 0.003, 0.007, 0.01, 0.03, 0.07, 0.1, 0.3, 0.7, 1, 5, 10, 30, 70, 100, 1000, 10000 μM), with other steps unchanged, measure the UV absorption peak intensity in the wavelength range of 500 - 800 nm. As Figure 3 shown in A, the UV absorption peak intensity of the sensor shows a good linear relationship with the logarithm of the Zn 2+ concentration, and as the Zn 2+With the increase in concentration, the blue color deepens continuously. A linear curve of different concentrations of Zn was established based on the absorbance peak at 652 nm. 2+ is y = 0.0523x + 0.2667, with R 2 = 0.9977 and a detection limit of 0.31 nM. The taken photos were imported into the color picker software of the smartphone to obtain the R, G, B values of the samples corresponding to different concentrations. Three R, G, B values were taken from different positions for each sample, and finally the average of the three results was taken. By analyzing the RGB chromaticity values of the colorimetric pictures, as Figure 3 shown in Fig. B, a linear relationship between the Zn 2+ concentration and the chromaticity value was established, and a Zn 2+ smartphone sensor could be constructed.

[0052] c. Analysis and detection of ALP

[0053] In the above step b, 1 μL of 10 mM ZnCl 2 (final concentration 100 μM), 1 μL of 1 mM ATP (final concentration 10 μM), and 10 μL of 500 mU / L ALP (final concentration 50 mU / L) (prepared with 0.1 M tris(hydroxymethyl)aminomethane buffer solution (Tris-HCl, pH = 8.2, containing 0.1 M NaCl and 2 mM MgCl 2 )) were mixed at 37 °C and reacted for 10 min, then mixed with 1 μL of 10 mM Na 2 S (final concentration 100 μM) and reacted for 10 min. Then, in sequence, this mixed solution was mixed with 10 μL of 0.1 mg / mL CoPTA / Fc-9 aqueous solution (final concentration 0.01 mg / mL), 25 μL of 1 mM TMB (final concentration 0.25 mM), 25 μL of 1 mM H 2 O 2 (final concentration 0.25 mM), 20 μL of HAc-NaAc (0.1 M pH = 4), and 7 μL of ultrapure water were added to a 200 μL centrifuge tube, mixed well, and reacted at room temperature for another 20 min. Then, the above mixed solution was transferred to a UV-visible spectrophotometer and a cuvette for analysis and testing, and a high-definition camera was used to take pictures.

[0054] The UV absorption curve of the above-mentioned sensor in the range of 500 - 800 nm was detected. As Figure 1 shown in Fig. D, it can be seen that after the addition of ALP, ATP, and Zn 2+ to the reaction, the solution (2) changed from blue to colorless again, and the UV absorption peak at 652 nm decreased, indicating that the free Zn 2+ decreased, resulting in a decrease in the free S 2-Increase. By comparing with other samples (3 - 4), it shows that this sensing method has a good response to ALP and can be used for the analysis and detection of ALP.

[0055] Based on the above steps, by changing the ALP concentration (final concentration: 0, 0.01, 0.1, 0.3, 1, 3, 10, 30, 100, 320, 1000, 3200, 10000, 16000, 80000, 5×10 5 , 10 6 , 5×10 6 mU / L), with other steps unchanged, measure the intensity of the ultraviolet absorption peak in the wavelength range of 500 - 800 nm. As Figure 4 shown in Figure A, the intensity of the ultraviolet absorption peak of the sensor shows a good linear relationship with the logarithm of the ALP concentration. And as the ALP concentration increases, the blue color deepens continuously. Taking the absorbance peak value at 652 nm to establish the linear curve of different concentrations of ALP as y = -0.0331x + 0.2919, R 2 = 0.9972, and the detection limit is 0.0035 mU / L; Import the taken photos into the color picking software of the smartphone to obtain the R, G, B values of the samples corresponding to different concentrations. Take the R, G, B values three times from different positions for each sample, and finally take the average of the three results. By analyzing the RGB chromaticity values of the colorimetric pictures, as Figure 4 shown in Figure B, establish the linear relationship between the ALP concentration and the chromaticity value, and an ALP smartphone sensor can be constructed.

[0056] Example 2 Analysis and detection of ALP inhibitor Na 3 VO 4

[0057] Fix the ALP concentration in Example 1c at 5×10 5 mU / L, and add different concentrations of Na 3 VO 4 respectively, with the final concentration being: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 μM, and other steps unchanged. The results are as Figure 5 shown. As the concentration of Na 3 VO 4 increases, the absorbance at 652 nm increases and the solution color deepens. The half - inhibitory concentration (IC 50 ) of Na 3 VO 4 is 9.72 μM.

[0058] Example 3 Specific detection

[0059] To verify the selectivity of the sensor, according to the sensor preparation steps in Example 1 above, in S 2- ​During the detection, replacing other reference substances such as fluoride ion (F - ), bromide ion (Br - ), iodide ion (I - ), chloride ion (Cl - ) and hydroxide ion (OH - ) has no interference on the system; during the Zn 2+ detection, replacing other reference substances such as nickel ion (Ni 2+ ), iron ion (Fe 3+ ), manganese ion (Mn 2 + ), cobalt ion (Co 2+ ) and chromium ion (Cr 3+ ); during the ALP detection, replacing other reference substances such as histone acetyltransferase (HAT), terminal deoxynucleotidyl transferase (TdT), acetylcholinesterase (AChE), papain (Papain) and cholesterol oxidase (ChOx). The results are as shown in Figure 6 A, Figure 6 B, Figure 6 C. It can be seen that the sensor has good responses to the three target substances (S 2- , Zn 2+ and ALP) respectively and has good selectivity.

[0060] Example 4 Detection of the Dephosphorylation Process in Breast Cancer CTCs

[0061] The DNA sequences used are shown in the following table:

[0062]

[0063] A. Enrichment of Circulating Tumor Cells

[0064] I. Preparation of magnetic beads: Weigh 1.89 g of FeCl 3 ·6H 2 O, 0.7 g of TSC and 0.5 g of PEG, add 70 mL of EG to completely dissolve them. This process can be accelerated by means of ultrasonic wave and heating (40 - 50 °C). After the solution becomes clear and transparent, add 6.96 g of NaAc·3H 2 O to the above solution to completely dissolve it, and continuously stir at room temperature for 2 h. Subsequently, transfer the stirred clear and transparent solution to a 100 mL polytetrafluoroethylene high-pressure reactor, seal it, and react at 200 °C for 10 h. Wait for the reactor to cool naturally to room temperature, collect the black solution, wash the black product 4 times with ethanol and water respectively, and store it at 4 °C for later use.

[0065] II. Preparation of gold nanoparticle solution (AuNPs): Immerse glassware such as flasks that directly contact the reaction solution in aqua regia for 30 min, then repeatedly rinse with deionized water multiple times to remove surface carboxyl groups, and then place in a drying oven to dry. Add 150 mL of 2.2 mM sodium citrate solution and 1 mL of 25 nM chloroauric acid solution to the flask, turn on the reflux condenser, turn on magnetic stirring, heat to boiling, and then heat for about 10 min until the solution turns wine red to obtain a gold nanoparticle solution with a particle size of about 10 nm.

[0066] III. Gold nanoparticle coating: Take 20 mg of the magnetic beads prepared above, suspend them in 100 mL of ethanol-water (4:1, V / V), sonicate for 10 min to completely disperse them, add 1 mL of AuNP, and stir and react for 4 h.

[0067] IV. DNA modification: Take 1 mL of the above magnetic beads, sonicate and then add 100 μL of 10 μM DNA1 and react at 37 °C for 1 h to bind DNA to the magnetic beads coated with gold nanoparticles through Au-S.

[0068] V. Antibody modification: The circulating tumor cells selected in this patent are breast cancer cells. Add 100 μL of antibody CD44 to the magnetic beads modified with DNA above, react overnight at 4 °C, perform magnetic separation, wash 3 times with PBS, and store at 4 °C for later use.

[0069] The blood samples of tumor patients selected in this patent are from the sample library of Ningbo Second Hospital. Mix the blood samples of 100 breast cancer patients, centrifuge at 1500 r / min for 10 min, discard the supernatant, and then add 10 mL of phosphate buffer (PBS) for dilution. Mix the prepared magnetic beads with 1 mL of the diluted sample in a 1.5 mL EP tube, incubate at room temperature for a period of time, enrich on a magnetic stand for 5 min, wash 3 times with PBS, and finally resuspend with PBS. After shearing the captured cells with exonuclease (Exo I), separate the magnetic beads, passage the obtained breast cancer CTCs, and culture them in L-15 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin in an incubator containing 5% CO 2Cultivate in a 37°C saturated humidity incubator, and use a sealed culture flask during the cultivation process. When the fusion rate reaches about 90%, digest with a digestive solution containing 0.25% trypsin and 0.02% EDTA, passage at a ratio of 1:3, and passage once every 2 - 3 days. Collect the passaged cells, use a nuclear extraction kit (ActiveMotif, Carlsbad, CA) to prepare whole cell extracts, wash the cells twice with 8 mL of frozen PBS / phosphatase inhibitor buffer (0.8 mL of PBS at pH 7.4, 6.8 mL of deionized water, 0.4 mL of phosphatase inhibitor), add cell lysis buffer RIPA (1% Triton X - 100, 50 mM Tris - HCl pH 7.4, 150 mM NaCl, 100 mM NaF, 1 mM Na 3 VO 4 , 10 mM EDTA, 1 mM PMSF, 2 μg / mL Aprotinin), collect in a 1.5 mL EP tube, lyse on ice for 30 min, after the suspension is incubated on ice for 30 min, pipette up and down to mix for 3 min every 2 min. Centrifuge the suspension in a microcentrifuge pre - cooled to 4°C at 14000g for 20 min. Transfer the supernatant containing the nuclear protein extract to a pre - cooled microcentrifuge tube for subsequent experiments.

[0070] B. Analysis of ALP activity in breast cancer CTCs

[0071] Apply the protein extracted from the cell lysate to replace ALP in Example 1c or Example 2 to complete the experiment, and the ALP concentration is 5×10 5 mU / L. As shown in Figure 7 A, as the number of breast cancer CTCs cells increases, the ultraviolet signal decreases. To further prove that the captured signal is caused by ALP, introduce an ALP inhibitor into the cell lysate at 10000 cells / mL, and the results are as shown in Figure 7 B. As the concentration of the ALP inhibitor increases, the ultraviolet signal increases; as shown in Figure 7 C, this patent also finds that ALP in normal cells is less than that in breast cancer CTCs cells, and the addition of the inhibitor makes the ultraviolet signal increase significantly, indicating that the ALP inhibitor Na 3 VO 4 can better inhibit ALP activity, indicating that the ALP content in breast cancer patients will increase, and there is a correlation with the occurrence and development of breast cancer. It shows that the prepared sensor can be used for the detection of ALP activity in breast cancer cells, has clinical application potential, and provides a new idea for exploring the correlation between phosphorylation and the occurrence and development of breast cancer.

[0072] It should also be noted that the above specific embodiments are not limitations on the present invention, and the present invention is not limited to the above examples. Those skilled in the relevant art of this technical field who make changes, modifications, additions, or substitutions within the scope of the essence of the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a smart sensor based on bimetallic nanozymes and its application in breast cancer CTCs, the mechanism of which is as follows: The present invention is the first to design a smart sensor based on CoPTA / Fc-9 nanozymes catalyzing H 2 O 2 The alkaline phosphatase UV and smartphone colorimetric dual-mode sensor was constructed by oxidizing TMB. First, the synthesized CoPTA / Fc-9 nanozyme catalyzed H 2 O 2 Hydroxyl radicals (·OH) are generated, which can oxidize colorless TMB into blue oxTMB, which has a clear ultraviolet absorption peak at 652nm, thus constructing a simple analytical detection system. 2- The addition of H 2 O 2 , with S 2- With the increase of concentration, the UV absorption peak at 652nm decreases and the color of the solution changes from blue to light, thus achieving S 2- Visual analysis and detection. 2+ When Zn 2+ With S 2- Combined to form ZnS, reducing free S 2- The amount of H 2 O 2 S 2- The color of Zn decreased, and the color changed from light to blue, and the absorbance at 652nm increased. Based on this, the Zn 2+ Visual analysis and detection. Next, due to Zn 2+ It has a strong affinity for phosphate and can bind to phosphate ions, the product of ALP dephosphorylation reaction, while also reducing free Zn 2+ The amount of free S 2- As the amount of α-phosphorylation increases, the solution changes from blue to light blue, and the absorbance at 652 nm decreases. Finally, when the inhibitor is added, the ALP activity is inhibited and cannot catalyze the dephosphorylation reaction of ATP, thus achieving the screening of ALP inhibitors.

2. A method for preparing an intelligent sensor based on bimetallic nanozyme and its application in breast cancer CTCs according to claim 1, characterized in that: The development of a single-mode analysis method is extended to a multi-mode method, with cross-validation and mutual confirmation of multiple parameters. The multi-mode analysis program can exclude false positives or false negatives that may occur due to a single detection method, and can improve the accuracy of disease diagnosis; A smartphone sensing device is redesigned, and quantitative detection of the target substance is achieved through colorimetric analysis according to the difference in the ultraviolet absorption intensity of the analyzed solution.

3. A method for preparing an intelligent sensor based on bimetallic nanozyme and its application in breast cancer CTCs 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 S 2- , Zn 2+ and ALP. The detection limit of S 2- is 0.28 nM, the detection limit of Zn 2+ is 0.31 nM, and the detection limit of ALP is 0.0035 mU / L. It can be used for the screening of small molecule inhibitors of ALP (Na 3 VO 4 ), and the IC 50 is 9.72 μM.

4. A method for preparing an intelligent sensor based on bimetallic nanozyme and its application in breast cancer CTCs according to claims 1-3, characterized in that: This patent designs an efficient method for sorting and enriching circulating tumor cells (CTCs) of breast cancer, and conducts subculture, initially realizing the dephosphorylation analysis of ALP in the extracted proteins of CTCs, which can make up for the important information that is masked and missed due to the sampling of population cells in breast cancer tissues.

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