Method for simultaneously detecting bladder cancer cells in photoelectrochemical / photo-thermal dual modes

Abstract

The invention discloses a method for simultaneously detecting bladder cancer cells in photoelectrochemical / photo-thermal dual modes. The method is characterized by comprising the following steps: (1) preparing CuO-coated NPC-Apt; 2) construction of a PEC / PT dual-mode biosensing platform; and 3) PEC / PT dual-mode simultaneous detection of bladder cancer cells. The CuO-coated NPC is used as a dual-signal probe of a PEC / PT dual-mode sensing platform, and in the presence of bladder cancer cells, the CuO-coated NPC not only has photoelectrochemical signal response, but also can carry out photo-thermal imaging and temperature output analysis, so that the bladder cancer cells are simultaneously detected in dual modes. The kit has the advantages of high sensitivity, strong specificity, good accuracy and simplicity and rapidness in operation.

Description

Technical Field

[0001] The present invention relates to a method for detecting bladder cancer cells, and in particular to a method for simultaneously detecting bladder cancer cells in a photoelectrochemical / photothermal dual mode. Background Art

[0002] Bladder cancer is the most common bladder disease in the urinary system. Early diagnosis and treatment are of great significance to improving the survival rate of patients. At present, the main diagnostic methods for bladder cancer include cystoscopy, urine cytology, and imaging examinations. However, cystoscopy is invasive and may cause discomfort to patients; urine cytology has low sensitivity and is difficult to achieve early diagnosis; imaging examinations rely on professional operations, and the results are somewhat subjective. In fact, bladder tumor cells will fall off into the urine, and urine is an easily accessible non-invasive liquid biopsy sample. If the bladder tumor cells shed in the urine can be detected, it can become a more direct and representative biomarker than small molecule markers, and has good application prospects.

[0003] Currently, there are few reports on the detection methods of exfoliated bladder tumor cells in urine. Its development can draw on other tumor cell detection methods, including: single-mode detection such as electrochemistry (EC), colorimetry (CL), fluorescence (FL), surface enhanced Raman scattering (SERS), and dual-mode detection such as EC / electrochemiluminescence (ECL), FL / SERS, CL / FL, EC / CL. Compared with single-mode detection, dual-mode detection can calibrate the detection signals obtained by different modes, and has more advantages in accuracy and reliability. It is worth noting that due to the mismatch between instruments, different test systems and conditions, the response signals of the dual modes cannot often be obtained at the same time, which reduces the consistency and effectiveness of the dual-mode detection results. Therefore, it is of great research and application significance to develop a dual-mode sensing platform with simultaneous output of response signals for the detection of tumor cells.

[0004] Photoelectrochemical (PEC) technology uses light as an excitation source and photocurrent as a detection signal, and has the advantages of low background signal and high sensitivity. At the same time, photothermal (PT), as an emerging sensing technology, also uses light as an excitation source and obtains photothermal signals through temperature readings. It has the characteristics of simple operation, fast and efficient. PEC and PT technologies share the same signal input and sensing interface, and can output signals at the same time. They can be developed into a dual-mode sensing platform that outputs response signals simultaneously. At present, there are no public research reports on methods for simultaneous detection of bladder cancer cells based on photoelectrochemical / photothermal dual modes at home and abroad. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting bladder cancer cells in a photoelectrochemical / photothermal dual mode with high sensitivity, strong specificity, rapidity and convenient operation.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a method for simultaneously detecting bladder cancer cells in a photoelectrochemical / photothermal dual mode, which is not intended for diagnosis or treatment, and comprises the following steps:

[0007] (1) Preparation of CuO@NPC-Apt

[0008] a. Disperse 10-30 mg of copper oxide @ nitrogen-doped porous carbon CuO@NPC powder in 10-30 mL of ethanol / water (v / v, 19:1) mixed solution, and adjust the pH to 4.8-6.5 with acetic acid. Add 0.6-2.4 mL of 1-4 wt% 3-aminopropyltrimethoxysilane APTES, sonicate for 15-30 min, and transfer to a 70-80 ° C water bath for 0.5-2 h. Finally, centrifuge to remove excess APTES and wash with ethanol to obtain amino-functionalized CuO@NPC.

[0009] b. Take 0.5-4 mg of amino-functionalized CuO@NPC and disperse it in 0.75-3.0 mL Tris-HCl solution, then add 0.1-0.5 mL 2.5-7.5 wt% glutaraldehyde solution and shake at room temperature for 1-2 h. Subsequently, centrifuge, wash, and disperse in a Tris-HCl solution containing 0.5-1.5 mL 0.5-5 μmol / L aptamer Apt, and shake at room temperature for 1-2 h. Then, after washing the product, add 0.5-2.5 mL 0.5-2.5 wt% bovine serum albumin BSA solution and incubate at room temperature for 1-2 h to block nonspecific sites. Finally, after separation, washing, and redispersion in 0.5-2.5 mL Tris-HCl solution, a CuO@NPC-Apt dispersion is obtained.

[0010] (2) Construction of PEC / PT dual-mode biosensor platform

[0011] The indium tin oxide ITO electrode was ultrasonically treated in acetone, 1 mol / L sodium hydroxide NaOH ethanol solution, and 1 mol / L NaOH water / ethanol (v / v, 1:1) solution for 15 to 30 minutes, washed with water and dried, and then immersed in an ethanol solution containing 1 to 3 wt% APTES for 0.5 to 2 hours, washed with ethanol and dried to obtain a surface amino ITO electrode. Then, 10 to 30 μL of 2.5 to 7.5 wt% glutaraldehyde solution was added dropwise to the surface of the above electrode and incubated at room temperature for 1 to 2 hours. 10 to 30 μL of 0.5 to 5 μmol / L Apt solution was added dropwise to the surface of the electrode and incubated at room temperature for 1 to 2 hours. After blocking non-specific sites with 10 to 30 μL of 0.5 to 2.5 wt% BSA solution, an Apt / ITO electrode was obtained. The electrode needs to be washed 3 times after each modification step above.

[0012] (3) PEC / PT dual-mode simultaneous detection of bladder cancer cells

[0013] 10-30 μL of bladder cancer cell samples of different concentrations were dripped onto the Apt / ITO electrode and incubated at room temperature for 1-2 hours. Then 10-30 μL of CuO@NPC-Apt dispersion was dripped and incubated at room temperature for 1-2 hours to obtain CuO@NPC-Apt / bladder cancer cell / Apt / ITO. The electrode needs to be washed with water three times after each modification step. Using a 650nm infrared laser (1W) as the light source, an external potential of -0.3V was applied in a Tris-HCl buffer solution containing 0.1mol / LAA, and PEC / PT dual-mode simultaneous testing was performed.

[0014] Further, the synthesis method of NPC described in step (1) is as follows: 3.94g 2-methylimidazole and 1.58g Zn(OAc)2·2H2O are dissolved in 360mL anhydrous ethanol respectively. Mix rapidly under stirring, continue stirring for 5min and then stand at room temperature for 24h. After centrifugation, washing and drying at 60°C, ZIF-8 polyhedron is obtained. The ZIF-8 polyhedron is placed in a tube furnace, heated to 1000°C at a heating rate of 5°C / min in a nitrogen atmosphere and maintained for 2h, and the obtained black powder is nitrogen-doped porous carbon (NPC) polyhedron.

[0015] Further, the synthesis method of CuO@NPC described in step (1) is as follows: 25 mg NPC powder and 15 mg Cu(NO3)2·3H2O are dispersed in 5 mL ultrapure water and ultrasonicated for 1 h. Then, the dispersion is freeze-dried for 3 days to obtain Cu 2+ / NPC. Finally, Cu 2+ / NPC was placed in a tube furnace and heated to 300 °C in air at a heating rate of 5 °C / min and maintained for 1 h to obtain CuO@NPC.

[0016] Furthermore, the method for processing the bladder cancer cell samples of different concentrations in step (3) is as follows: the number of cells is counted using a cell counter, and a series of cell standard solutions are obtained by stepwise dilution, with concentrations of 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 , 5cells / mL.

[0017] Furthermore, the bladder cancer cells are selected from HT-1376, 5637, T24, 639V, TCCSUP, RT-112, SW1710 and BIU-87.

[0018] The detection principle of the present invention is as follows Figure 1 As shown in the figure, taking human bladder cancer cell HT-1376 as an example, CuO@NPC is used as a dual-signal probe for the PEC / PT dual-mode sensing platform. The aptamer Apt of HT-1376 is cross-linked to the surface of the amino-modified ITO electrode, which can specifically capture HT-1376 in urine samples. The dual-signal tag CuO@NPC-Apt modified with the aptamer can realize PEC and PT dual-mode simultaneous detection after being assembled on the electrode surface due to its specific recognition of the target HT-1376. The more HT-1376 there is, the more CuO@NPC-Apt is assembled on the electrode, and the greater the photocurrent signal and temperature signal response generated, thereby realizing the quantitative detection of HT-1376.

[0019] Compared with the prior art, the advantages of the present invention are: the present invention discloses for the first time a method for simultaneous detection of bladder cancer cells by photoelectrochemical / photothermal dual modes. With the help of the dual-signal probe CuO@NPC, in the presence of bladder cancer cells, there is not only a photoelectrochemical signal response, but also photothermal imaging and temperature output analysis, thus realizing dual-mode detection of bladder cancer cells. Of particular importance is that the dual-mode analysis of photoelectric and photothermal responses can share one excitation light source and one display output signal, greatly improving the precision and accuracy of the detection, and providing a new path for realizing sensitive, accurate, reliable and rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the principle of the photoelectrochemical / photothermal dual-mode method for simultaneous detection of bladder cancer cells of the present invention;

[0021] Figure 2 Characterization of CuO@NPC, where A: SEM image of CuO@NPC; B: XRD images of NPC and CuO@NPC; C: XPS image of CuO@NPC; D: UV-vis image of NPC and CuO@NPC;

[0022] Figure 3 It is the PEC signal response diagram corresponding to different concentrations of HT-1376;

[0023] Figure 4 is the detection sensitivity curve of the PEC sensor;

[0024] Figure 5 The photothermal response diagram and sensitivity curve corresponding to different concentrations of HT-1376;

[0025] Figure 6 Reproducibility of the PEC / PT dual-mode sensor. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to the accompanying drawings. 1. Specific embodiments

[0028] A method for simultaneous detection of bladder cancer cells by photoelectrochemical / photothermal dual modes, which is not intended for diagnosis or treatment, comprises the following steps:

[0029] (1) Preparation of CuO@NPC-Apt

[0030] a. Disperse 20 mg of copper oxide @ nitrogen-doped porous carbon CuO@NPC powder in 20 mL of ethanol / water (v / v, 19:1) mixed solution and adjust the pH to 5.0 with acetic acid. Add 1.2 mL of 2 wt% 3-aminopropyltrimethoxysilane APTES, sonicate for 20 min, and transfer to a 75 ° C water bath for 1 h. Finally, centrifuge to remove excess APTES and wash with ethanol to obtain amino-functionalized CuO@NPC.

[0031] b. Take 2 mg of amino-functionalized CuO@NPC and disperse it in 1.75 mL of Tris-HCl solution, then add 0.25 mL of 5 wt% glutaraldehyde solution and shake at room temperature for 1 hour. Subsequently, centrifuge, wash, disperse in 1 mL of 1 μmol / L Apt Tris-HCl solution, and shake at room temperature for 1 hour. Then, after washing the product, add 1 mL of 1 wt% bovine serum albumin BSA solution and incubate at room temperature for 1 hour to block nonspecific sites. Finally, after separation, washing, and redispersion in 1 mL of Tris-HCl solution, the CuO@NPC-Apt dispersion was obtained.

[0032] (2) Construction of PEC / PT dual-mode biosensor platform

[0033] The ITO electrode was ultrasonically treated in acetone, 1 mol / L NaOH ethanol solution, and 1 mol / L NaOH water / ethanol (v / v, 1:1) solution for 20 min, washed with water, dried, and immersed in an ethanol solution containing 2 wt% APTES for 1 h, washed with ethanol, and dried to obtain a surface amino-modified ITO electrode. Then, 20 μL of 5 wt% glutaraldehyde solution was added dropwise to the surface of the above electrode and incubated at room temperature for 1 h. 20 μL of 1 μmol / L Apt solution was added dropwise to the surface of the electrode and incubated at room temperature for 1 h. After blocking non-specific sites with 20 μL of 1 wt% BSA solution, an Apt / ITO electrode was obtained. The electrode needs to be washed 3 times after each modification step above.

[0034] (3) PEC / PT dual-mode simultaneous detection of bladder cancer cells

[0035] 20 μL of bladder cancer cell samples of different concentrations were added to the Apt / ITO electrode and incubated at room temperature for 1 hour. Then 20 μL of CuO@NPC-Apt dispersion was added and incubated at room temperature for 1 hour to obtain CuO@NPC-Apt / bladder cancer cell / Apt / ITO. The electrode needs to be washed 3 times after each modification step. Using a 650nm infrared laser (1W) as the light source, an external potential of -0.3V was applied in a Tris-HCl buffer solution containing 0.1mol / L AA, and PEC / PT dual-mode simultaneous testing was performed.

[0036] 2. Experimental Results Analysis

[0037] Experimental instruments: Nova NanoSEM 450 scanning electron microscope (SEM) (FEI, USA) was used to characterize the material morphology, D8Focus X-ray diffractometer (Bruker, Germany) was used to analyze the material phase structure, and K-Alpha X-ray photoelectron spectrometer (XPS) (Thermo Fisher, USA) was used to analyze the element valence and composition. UV-1800 UV-visible spectrophotometer (Shimadzu, Japan) was used to record the UV-visible spectrum. Photoelectrochemical and electrochemical tests were performed using a CHI 760E electrochemical workstation (Chenhua, China) combined with a three-electrode system: indium tin oxide (ITO) conductive glass was used as the working electrode, with a size of 2.5 cm (length) × 2.5 cm (width) × 0.1 cm (height), the electrolytic cell attached to it was 1.5 cm in diameter, a 6 mm diameter circle was used as the modified area, Ag / AgCl was used as the reference electrode, and platinum wire was used as the counter electrode. The photothermal response test was recorded using a Fotric 246M infrared thermal imager (Fei Chu Ke, China). The excitation light source used in the photoelectrochemical test and the photothermal test was a 650nm infrared laser (1W).

[0038] Cell culture: HT-1376 cells were cultured in a culture flask containing E-MEM medium (containing 10% fetal bovine serum, 100U / mL streptomycin, and 100U / mL penicillin) in a culture environment of 5% CO2 and 37°C. Afterwards, the old culture medium was discarded and the cells were rinsed with 1mL 1×PBS. Then, 1mL 0.25% trypsin digestion solution was added to the culture flask for 7min, the trypsin digestion solution was discarded and the cells were placed in a 5% CO2, 37°C culture environment for 1min, and then 2mL of new culture medium was added and the cells were repeatedly blown with a pipette to suspend the cells, and then centrifuged at 800×g for 5min to separate the cells from the culture medium.

[0039] 1. Characterization of CuO@NPC

[0040] The morphology of CuO@NPC is as follows Figure 2 As shown in A, it presents a regular polyhedral morphology of about 220nm. Figure 2 B shows that the XRD spectrum of NPC shows two broad peaks in the shape of steamed buns at 24.0° and 44.0°, indicating that the prepared NPC material is an amorphous phase. The XRD characteristic diffraction peaks of CuO in the CuO@NPC composite material match well with the CuO standard card (JCPDS 89-2529), and the other characteristic peaks are basically consistent with NPC, proving the successful preparation of CuO in NPC polyhedrons. X-ray photoelectron spectroscopy (XPS) was further used to study the elemental valence and composition of Cu in CuO@NPC, such as Figure 2 C, which proves the existence of C, N, O and Cu elements, further indicating the successful preparation of CuO@NPC. NPC and CuO@NPC were characterized by ultraviolet-visible absorption spectroscopy (UV-vis), as shown in Figure 2 As shown in D, compared with NPC, CuO@NPC shows a wider absorption range in the visible light absorption region, indicating that CuO@NPC has good light absorption ability. HT-1376 is used as the detection target and the specific embodiment method is used for detection.

[0041] 2. Sensitivity analysis

[0042] like Figure 3 As shown, the photoelectrochemical analysis detection results using the specific embodiment method show that as the concentration of HT-1376 increases, the PEC response signal increases.

[0043] like Figure 4 As shown, in 5~1.0×10 5 In the concentration range of cells / mL, the linear regression equation is y=-190.62*lgx+96.11, and the square of the correlation coefficient is R 2=0.994, where y is the photocurrent (nA) and x is the HT-1376 concentration (cells / mL). Based on the signal-to-noise ratio S / N=3, the detection limit was estimated to be 3 cells / mL.

[0044] The results of photothermal detection show that Figure 5 As shown in Figure 2, the temperature of the photothermal change (ΔT) increases with the increase of HT-1376 concentration, and the thermal imaging color changes from black to orange (inset). 5 There is a good linear relationship between ΔT and the logarithm of HT-1376 concentration in the range of cells / mL. The linear regression equation is y=1.97*lgx+1.93, and the square of the correlation coefficient is R 2 =0.994, where y is the temperature of the photothermal change (°C), and x is the concentration of HT-1376 (cells / mL). Based on the signal-to-noise ratio S / N=3, the LOD is about 5 cells / mL.

[0045] 3. Reproducibility analysis

[0046] Six modified electrodes from the same batch were used to measure 1.0×10 3 cells / mL HT-1376, the results are as follows Figure 6 As shown in Figure 3, the RSDs of PEC and PT signals were 1.1% and 1.5%, respectively. Therefore, the biosensing platform exhibited good signal stability and repeatability.

[0047] 4. Precision and accuracy

[0048] Table 1 Detection of HT-1376 in urine samples by dual-mode biosensor platform (x±s, n=5)

[0049]

[0050] The standard spike recovery experiment of HT-1376 was conducted using urine samples from healthy people. The results are shown in Table 1. As can be seen from Table 1, the recovery rate of PEC detection is 98.6% to 101.2%, with an RSD of 2.3% to 3.1%, while the recovery rate of PT detection is 98.8% to 101.8%, with an RSD of 3.0% to 4.1%. The results show that the sensor has good accuracy and precision and can be used to reliably detect actual samples.

[0051] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for simultaneous detection of bladder cancer cells by photoelectrochemical / photothermal dual modes, which is not intended for diagnosis or treatment, and is characterized in that The following steps are involved: (1) Preparation of CuO@NPC-Apt a. Disperse 10-30 mg of copper oxide @ nitrogen-doped porous carbon CuO@NPC powder in 10-30 mL of ethanol / water (v / v, 19:1) mixed solution, and adjust the pH to 4.8-6.5 with acetic acid. Add 0.6-2.4 mL of 1-4 wt% 3-aminopropyltrimethoxysilane APTES, sonicate for 15-30 min, and transfer to a 70-80 ° C water bath for 0.5-2 h. Finally, centrifuge to remove excess APTES and wash with ethanol to obtain amino-functionalized CuO@NPC. b. Take 0.5-4 mg of amino-functionalized CuO@NPC and disperse it in 0.75-3.0 mL Tris-HCl solution, then add 0.1-0.5 mL 2.5-7.5 wt% glutaraldehyde solution and shake at room temperature for 1-2 h. Subsequently, centrifuge, wash, and disperse in a Tris-HCl solution containing 0.5-1.5 mL 0.5-5 μmol / L aptamer Apt, and shake at room temperature for 1-2 h. Then, after washing the product, add 0.5-2.5 mL 0.5-2.5 wt% bovine serum albumin BSA solution and incubate at room temperature for 1-2 h to block nonspecific sites. Finally, after separation, washing, and redispersion in 0.5-2.5 mL Tris-HCl solution, a CuO@NPC-Apt dispersion is obtained. (2) Construction of PEC / PT dual-mode biosensor platform The indium tin oxide ITO electrode was ultrasonically treated in acetone, 1 mol / L sodium hydroxide NaOH ethanol solution, and 1 mol / L NaOH water / ethanol (v / v, 1:1) solution for 15 to 30 minutes, washed with water and dried, and then immersed in an ethanol solution containing 1 to 3 wt% APTES for 0.5 to 2 hours, washed with ethanol and dried to obtain a surface amino ITO electrode. Then, 10 to 30 μL of 2.5 to 7.5 wt% glutaraldehyde solution was added dropwise to the surface of the above electrode and incubated at room temperature for 1 to 2 hours. 10 to 30 μL of 0.5 to 5 μmol / LApt solution was added dropwise to the surface of the electrode and incubated at room temperature for 1 to 2 hours. After blocking non-specific sites with 10 to 30 μL of 0.5 to 2.5 wt% BSA solution, an Apt / ITO electrode was obtained. The electrode needs to be washed 3 times after each modification step above. (3) PEC / PT dual-mode simultaneous detection of bladder cancer cells 10-30 μL of bladder cancer cell samples of different concentrations were dripped onto the Apt / ITO electrode and incubated at room temperature for 1-2 hours. Then 10-30 μL of CuO@NPC-Apt dispersion was dripped and incubated at room temperature for 1-2 hours to obtain CuO@NPC-Apt / bladder cancer cell / Apt / ITO. The electrode needs to be washed 3 times after each modification step. Using a 650nm infrared laser (1W) as the light source, an external potential of -0.3V was applied in a Tris-HCl buffer solution containing 0.1mol / L AA, and PEC / PT dual-mode simultaneous testing was performed.

2. According to claim 1, a method for simultaneous photoelectrochemical / photothermal dual-mode detection of bladder cancer cells is not for the purpose of diagnosis or treatment, characterized in that The synthesis method of NPC described in step (1) is as follows: 3.94g 2-methylimidazole and 1.58g Zn(OAc)2·2H2O are dissolved in 360mL anhydrous ethanol respectively. Mix rapidly under stirring, continue stirring for 5min and then stand at room temperature for 24h. After centrifugation, washing and drying at 60°C, ZIF-8 polyhedron is obtained. The ZIF-8 polyhedron is placed in a tube furnace, heated to 1000°C at a heating rate of 5°C / min under a nitrogen atmosphere and maintained for 2h, and the obtained black powder is nitrogen-doped porous carbon (NPC) polyhedron.

3. The method of simultaneous photoelectrochemical / photothermal dual-mode detection of bladder cancer cells according to claim 1 is not for the purpose of diagnosis or treatment, and is characterized in that The synthesis method of CuO@NPC in step (1) is as follows: 25 mg NPC powder and 15 mg Cu(NO3)2·3H2O are dispersed in 5 mL ultrapure water and ultrasonicated for 1 h. Then, the dispersion is freeze-dried for 3 days to obtain Cu 2+ / NPC. Finally, Cu 2+ / NPC was placed in a tube furnace and heated to 300 °C in air at a heating rate of 5 °C / min and maintained for 1 h to obtain CuO@NPC.

4. The method of simultaneous photoelectrochemical / photothermal dual-mode detection of bladder cancer cells according to claim 1, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The method for processing the bladder cancer cell samples of different concentrations in step (3) is as follows: the number of cells is counted using a cell counter, and a series of cell standard solutions are obtained by stepwise dilution, with concentrations of 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 , 5cells / mL.

5. The method of simultaneous photoelectrochemical / photothermal dual-mode detection of bladder cancer cells according to claim 1, which is not intended for diagnosis or treatment, is characterized in that The bladder cancer cells are selected from HT-1376, 5637, T24, 639V, TCCSUP, RT-112, SW1710 and BIU-87.

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