A circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjenblack

Abstract

The detection and recovery of circulating tumor cells are of great clinical significance for monitoring metastasis and treatment efficacy. In this work, we developed an integrated electrochemical sensing model based on nanomaterials to achieve highly sensitive detection and non-destructive collection of circulating tumor cells. Vulcan XC-72 was combined with gold nanoparticles for the first time and used to modify the surface of the gold electrode, thus improving the conductivity and increasing the specific surface area of the electrode. PdIrBP mesoporous nanospheres were conjugated with antibodies to form signal probes, which were used as detection tags to amplify the current signal. In addition, glycine hydrochloride was used as the antibody eluent to release and collect the captured circulating tumor cells from the electrode for further clinical research. A good linear relationship was obtained between the electrochemical signal and the target cell concentration in the range from 10 / mL to 1×10<supgt;6< / supgt; / mL, with a detection limit as low as 2 / mL. Therefore, this novel cell sensor model has potential clinical application value and can be used for early diagnosis and prognosis monitoring of cancer patients.

Description

Technical Field

[0001] The present invention relates to a biosensor, and particularly to a biosensor for ultrasensitive detection and non-destructive collection of circulating tumor cells based on a novel nanoparticle composite and a highly conductive carbon black material. Background Art

[0002] Circulating tumor cells are closely related to the metastasis and recurrence of tumors. Therefore, the detection of circulating tumor cells is of great significance for the diagnosis and treatment of cancer patients. At the same time, the collection of circulating tumor cells can be used for further research to judge the drug efficacy, prognosis and recurrence probability of patients. Currently, the methods for detecting circulating tumor cells mainly include methods based on cell biological properties, direct microscopy methods and methods based on cell physical properties. Among these methods, the methods based on cell biological properties are the most common. Among them, using antibodies to capture circulating tumor cells carrying corresponding antigens is the most widely used detection method at present. Using this technology can achieve efficient detection. However, due to the single antibody used, circulating tumor cells that have undergone epithelial-mesenchymal transition cannot be detected, and false negative results are likely to occur. Moreover, this method cannot achieve non-destructive collection of circulating tumor cells. Therefore, the popularization and use of this method in clinical practice have been restricted.

[0003] To solve this problem, the present invention introduces an electrochemical sensor to detect circulating tumor cells. Electrochemical biosensors have high sensitivity, good selectivity and rapid and convenient detection, which makes them have great application value in various genetic disease screenings, epidemiological investigations and early diagnosis of tumors. At the same time, a mixed antibody is used to capture circulating tumor cells in different periods, reducing the generation of false negatives, and a mild antigen-antibody eluent of glycine-hydrochloric acid is introduced to destroy the binding between the antibody and the cell surface antigen, realizing non-destructive release and collection of cells.

[0004] With the rapid development of nanotechnology, a variety of nanomaterials have been used in the construction of electrochemical sensors. Ketjen black is a highly conductive carbon black material. Due to its unique branched structure, compared with other conductive carbon blacks, it has more conductive contact points, which can greatly increase the conductivity of the electrode. Gold nanoparticles have high stability and biocompatibility. Modifying the electrode surface with them can make the effective surface area larger and can combine with more biomolecules. PdIrBP mesoporous nanospheres have enzyme-like activity and can efficiently catalyze hydrogen peroxide to generate electrochemical signals. Using them as signal molecules to form signal probes with antibodies can effectively increase the detection sensitivity. Summary of the Invention

[0005] The object of the present invention is to develop a biosensor for ultrasensitive and highly specific detection of circulating tumor cells and non-destructive collection thereof.

[0006] The specific technical solution is as follows:

[0007] A circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black, and its preparation method includes the following steps:

[0008] (1) Preparation of Ketjen black: With the aid of ultrasonic stirring, 2.0 mg of Ketjen black is dispersed in 4.0 mL of 0.5 wt.% chitosan solution to obtain a uniform suspension.

[0009] (2) Preparation of gold nanoparticles: 100 mL of 0.01% aqueous chloroauric acid solution is boiled for 10 - 15 minutes, and then 1 mL of 2.0 wt.% sodium citrate solution is added dropwise under vigorous stirring and continuous heating. Then the mixed solution is continuously stirred until the color changes from yellow to dark pink, and the heating is stopped. After cooling to room temperature, it is centrifuged at 11000 r / min for 10 minutes, the gold nanoparticles are washed and collected, and stored at 4°C.

[0010] (3) Synthesis of PdIrBP mesoporous nanospheres: 30 mg of dioctadecyldimethylammonium chloride is added to 10.0 mL of deionized water and stirred to obtain a uniform solution. Subsequently, 1.0 mL of 0.337 mol / L ammonium fluoride and 1.0 mL of 0.101 mol / L boric acid are added to the previous solution, and then the metal precursors 0.8 mL of 10 mmol / L chloropalladic acid and 0.8 mL of 10 mmol / L chloroiridic acid are added. After incubating for 5 minutes, 0.8 mL of ammonia water (10 wt.%) is injected to adjust the pH of the solution. When the color of the mixed solution changes from brownish-yellow to colorless, 1.0 mL of 0.034 mol / L sodium hypophosphite is mixed into the above solution, and magnetically stirred at 95°C for 20 minutes. Finally, 1.0 mL of freshly prepared 0.1 mol / L dimethylamine borane is injected to initiate the reduction reaction, and the color of the solution gradually turns dark brown. After reacting at 95°C for 30 minutes, the product is collected by centrifuging at 8000 r / min for 5 minutes, and washed several times with ethanol and deionized water.

[0011] (4) Preparation of signal probes: Add 0.5 mg of polyethylene glycol to 1.0 mL of PdIrBP mesoporous nanospheres (1.0 mg / mL). Stir and incubate the mixture at room temperature for 3 hours, then centrifuge and wash it 3 times with deionized water. Finally, disperse the precipitate in 500 μL of deionized water for future use. Antibodies and polyethylene glycol-modified PdIrBP nanospheres are linked by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide as coupling agents. Add 50 μL of the above-prepared modified PdIrBP mesoporous nanospheres to 450 μL of 10 mmol / L PBS at room temperature and stir well. Subsequently, add an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (5 μL, 25 mM) and an aqueous solution of N-hydroxysuccinimide (5 μL, 50 mM). After 20 minutes, wash the nanomaterials twice with deionized water and then redisperse them in 50 μL of PBS. Subsequently, add 50 μL of antibodies (20 mg / mL) to the nanosphere suspension. After incubating at room temperature for 1 hour, store the reaction solution in a refrigerator at 4 °C for future use.

[0012] (5) Construction of biosensor: Polish the gold electrode (diameter 3 mm) to a mirror-like state with 0.3 μm and 0.05 μm alumina. Subsequently, ultrasonically treat it for 10 minutes alternately in double-distilled water, ethanol, and double-distilled water. After drying at room temperature, activate the electrode in freshly prepared piranha solution (98% sulfuric acid: 30% hydrogen peroxide, volume ratio 3:1) for 5 minutes, thoroughly rinse it with ultrapure water, and dry it in air. Add 10 μL of the Ketjen black solution prepared in step (1) to the electrode and dry it at 37 °C. Dropwise add the gold nanoparticles enriched in step (2) onto the electrode modified with Ketjen black in the same way. Drop 10 μL of the antibody mixture (80 μg / mL) onto the modified electrode, cover it, and place it at 37 °C for 1 h. After rinsing with 0.01 mol / L PBS buffer, add 10 μL of 0.3% BSA and incubate it at 37 °C for 1 h to block non-specific binding sites. Wash the resulting electrode with PBS and store it at 4 °C for later use.

[0013] (6) Detection and release of circulating tumor cells: Add 10 μL of circulating tumor cells to the electrode prepared in step (5) and incubate it at 37 °C for 40 minutes. Then drop 10 μL of the signal probe synthesized in step (4) onto the electrode for immunoreaction to form a sandwich immunorecomplex. Wash away the substances not bound to the electrode with PBS and detect the DPV signal of the electrode.

[0014] After the cell detection is completed, immerse the electrode in 1 mL of glycine-hydrochloric acid eluent (0.1 mol / L) for 10 s, and then quickly add a few drops of 0.4 mol / L NaOH solution to adjust the pH value. After centrifuging at 500 r / min for 5 minutes, wash the obtained precipitate with PBS (0.01 mol / L) 2-3 times to obtain circulating tumor cells.

[0015] The concentration of Vulcan XC-72 used in the step (1) is preferably 0.5 mg / mL.

[0016] The concentration of chloroauric acid aqueous solution in the step (2) is preferably 0.01%.

[0017] The concentration of sodium citrate solution in the step (2) is preferably 2.0 wt%.

[0018] The volume of ammonia water in the step (3) is preferably 0.8 mL.

[0019] The concentration of dimethylamine borane in the step (3) is preferably 0.1 mol / L.

[0020] The concentration of PdIrBP mesoporous nanospheres in the step (4) is preferably 1.0 mg / mL.

[0021] The concentration of the antibody in the step (4) is preferably 20 mg / mL.

[0022] The concentration of the antibody mixture in the step (5) is preferably 80 μg / mL.

[0023] The time for cell incubation in the step (6) is preferably 40 minutes.

[0024] The present invention constructs a biosensor based on PdIrBP mesoporous nanospheres and Vulcan XC-72 for ultrasensitive detection and non-destructive collection of circulating tumor cells. The detection principle is to use Vulcan XC-72 modified gold electrode to improve the conductivity. Then, gold nanoparticles are added to modify the electrode surface, so as to improve the effective surface area of the electrode, and through the Au-NH 2 bond to bind with more capture antibodies. The antibody captures the circulating tumor cells on the electrode surface, and then a signal probe is added to form a sandwich structure. The circulating tumor cells are quantitatively detected by differential pulse voltammetry. Finally, the introduction of glycine hydrochloride buffer can disrupt the binding between the antigen and antibody on the cell surface, so as to realize the collection of circulating tumor cells.

[0025] The successful synthesis of various nanomaterials plays a crucial role in the preparation of this sensor. The present invention uses field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy to perform morphological characterization on it. As shown in the appendix Figure 1As shown, the field emission scanning electron microscopy results of Ketjen black show that Ketjen black is evenly distributed, and the branched structure of Ketjen black can be observed. This unique structure provides more conductive contact points. Therefore, compared with other carbon black materials, Ketjen black has better conductivity. The transmission electron microscopy results of gold nanoparticles (attached Figure 2 ) show that the gold nanoparticles are of uniform size and evenly distributed, with a diameter of about 20 nm, laying a good foundation for the binding of antibodies. Attached Figure 3 shows the transmission electron microscopy image of PdIrBP mesoporous nanospheres. The nanomaterial is spherical and has a highly branched structure. These branches form a network similar to organic dendrimers, with a uniform size and a diameter of about 100 nm, and are evenly dispersed in water. An energy dispersive spectrometer is used to analyze the elemental composition of PdIrBP mesoporous nanospheres. As attached Figure 4 shows, this nanocube contains four elements, Pd, Ir, B, and P, further indicating the successful synthesis of this nanomaterial.

[0026] In a [Fe(CN) 6 3- / 4- (5 mM) solution containing 0.1 M KCl, cyclic voltammetry (CV) detection is carried out at a scanning rate of 100 mV / s to prove the feasibility of the experiment by verifying each step of modifying the working electrode. As attached Figure 5 shows, after modification with KB (curve b) and AuNPs (curve c), the current signal of the electrode increases significantly compared to the bare electrode (curve a). This is attributed to the high specific surface area and excellent conductivity of KB and AuNPs. The change in current proves that these two nanomaterials are successfully bound to the electrode surface. Due to the steric effect of antibodies and the blocking effect of BSA, after immobilizing Ab 1 (curve d) and BSA (curve e), the current response continues to decline, indicating that Ab 1 and BSA are also successfully immobilized on the electrode surface as expected. When MCF-7 cells are captured, the current signal further decreases (curve f), which is mainly due to the fact that the resistance of the cells reduces the electrotransfer efficiency at the electrode interface.

[0027] The electrochemical signal generated by PdIrBP mesoporous nanospheres catalyzing hydrogen peroxide will reach a peak within a certain time. Exploring this appropriate time helps to capture the maximum signal. We detected the DPV signals at different reaction times. As attached Figure 6 shows, when the catalytic reaction time reaches 40 s, the highest current can be obtained. Therefore, for this study, the optimal reaction time for PdIrBP mesoporous nanospheres to catalyze hydrogen peroxide is 40 s.

[0028] ​The pH value of the working solution has a great influence on the binding strength between cells and antibodies as well as the catalytic efficiency of nanomaterials. When the pH value is adjusted from 3 to 6, the corresponding DPV signal increases rapidly, while when the pH value exceeds 6, the DPV signal begins to decrease sharply (attached Figure 7 ). This may be because both PdIrBP mesoporous nanospheres and tumor cells are more stable in a weakly acidic environment. Therefore, the optimal pH value is 6.

[0029] Ab 1 concentration directly affects the cell capture efficiency, so this experimental parameter plays a very important role in the construction of this sensor. As shown in the attachment Figure 8 , the differential pulse voltammetry detection signal increases with the increase of Ab 1 concentration and reaches the maximum at 80 μg / mL. After that, the current response signal gradually decreases. The reason for this phenomenon may be that more antibodies immobilized on the electrode interface may generate greater steric hindrance, thus hindering the recognition of target cells. Therefore, the optimal concentration of Ab 1 is 80 μg / mL.

[0030] The incubation time of MCF-7 cells and antibodies also plays a decisive role in cell capture efficiency. The attachment Figure 9 shows the influence of incubation time on the electrochemical response of the biosensor. Within 40 min, with the increase of incubation time, more MCF-7 cells will be captured on the electrode surface, resulting in an enhanced current response. However, with the further increase of incubation time, the current signal does not change significantly. These results indicate that the optimal incubation time is 40 minutes.

[0031] The present invention tested the repeatability of this sensing strategy. The method is as follows: Five freshly prepared modified electrodes were used to detect target cells five times at the same concentrations (10 4 / mL and 10 5 / mL), and the relative standard deviations (RSDs) were 1.06% and 1.21% respectively (attached Figure 10 ). This data indicates that this immunosensor has excellent repeatability.

[0032] In addition, the stability of this immunosensor was also studied. After the prepared electrodes were placed in a refrigerator at 4 °C for 7 days and 15 days, 95.92% and 90.3% of their initial current responses were retained compared with freshly prepared electrodes, indicating that the established immunosensor has good stability.

[0033] To evaluate the analytical performance of this sensing strategy towards target cells, we studied this strategy using a series of MCF-7 cells at different concentrations. When the concentration of target cells increased, the current response increased correspondingly. The calibration curve showed a good linearity between the DPV values and the logarithm values when the concentration of MCF-7 cells ranged from 10 / mL to 10 6 / mL. The linear regression equation was Y = 26.226 Log[C] + 20.427, and the correlation coefficient (R 2 ) was 0.9993 (see appendix Figure 11 ), where Y was the peak current of the immunosensor and C was the concentration of MCF-7 cells. The lowest detection limit was estimated to be 2 / mL at a signal-to-noise ratio of 3.

[0034] To evaluate the specificity of the electrochemical immunoassay, two non-specific cell lines, HeLa cells and K562, were selected to evaluate the specificity of the sensor at the same concentration under optimized conditions. See appendix Figure 12 . It was demonstrated that there was no significant change in the current response of non-specific cells at any concentration, while the electrochemical signal for MCF-7 cells was very high and this signal increased with the increase in the concentration of MCF-7 cells. When these interfering cells were mixed with MCF-7 cells respectively, the corresponding current response was similar to the electrochemical signal of pure MCF-7 cells at the same concentration. These results indicated that this biosensor showed excellent specificity towards MCF-7 cells.

[0035] To verify that this strategy could efficiently release and recover circulating tumor cells, we compared the electrochemical signals generated by the electrodes before and after elution. As shown in appendix Figure 13 , compared with after antibody capturing of target cells (curve b), the current signal of the electrode (curve a) increased significantly after treatment with the antibody eluent. This demonstrated that the cells on the electrode could be eluted using this method. After culturing the eluted cells for 12 hours, the cell morphology was observed. As shown in appendix Figure 14 , the cells had started to adhere and proliferate, indicating that this elution method did not affect the cell viability.

[0036] In this strategy, we developed an integrated electrochemical immunosensor for ultrasensitive detection of circulating tumor cells, which uses Ketjenblack / gold nanoparticles as a platform and PdIrBP mesoporous nanospheres as signal tags. The advantages of the present invention are as follows: ① The unique functions of Ketjenblack and gold nanoparticles enable their combination to greatly increase the sensitivity of the sensor; ② Due to their high catalytic activity and strong stability, PdIrBP mesoporous nanospheres are considered excellent signal tags, and the combination of these nanomaterials enables the construction of an immunosensor with a wide linear range and a low detection limit; ③ The invented sensor also has sufficient specificity to distinguish target cells from actual blood samples and can effectively resist interference from the complex environment of actual blood samples; ④ Through the antibody eluent Gly-HCl, efficient and non-destructive collection of target cells can be achieved, and the biological activity of the released target cells is not affected at all, which can provide accurate information for determining treatment plans or monitoring prognoses. Therefore, this immunosensor represents a further step towards achieving precise detection of circulating tumor cells and also lays a foundation for truly realizing liquid biopsy. Description of the Drawings

[0037] Figure 1 It is a field emission scanning electron microscope image of Ketjenblack

[0038] Figure 2 It is a transmission electron microscope image of gold nanoparticles

[0039] Figure 3 It is a transmission electron microscope image of PdIrBP mesoporous nanospheres

[0040] Figure 4 It is the EDS elemental analysis result of PdIrBP mesoporous nanospheres

[0041] Figure 5 It is the feasibility analysis

[0042] Figure 6 It is the optimization of the reaction time

[0043] Figure 7 It is the optimization of the pH value of the working solution

[0044] Figure 8 It is the optimization of the capture antibody concentration

[0045] Figure 9 It is the optimization of the cell incubation time

[0046] Figure 10 It is the repeatability analysis

[0047] Figure 11 It is the relationship between cell concentration and electrochemical signal

[0048] Figure 12 It is the specificity test of the sensing strategy

[0049] Figure 13 Effect analysis of cell release

[0050] Figure 14 Morphological diagram of recycled cells Specific implementation manner

[0051] A circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black, and its preparation method includes the following steps:

[0052] (1) Preparation of Ketjen black 2.0 mg of Ketjen black is dispersed in 4.0 mL of 0.5 wt% chitosan solution by means of ultrasonic stirring to obtain a uniform suspension.

[0053] (2) Preparation of gold nanoparticles 100 mL of 0.01% aqueous chloroauric acid solution is boiled for 10 - 15 minutes, and then 1 mL of 2.0 wt% sodium citrate solution is added dropwise under vigorous stirring and continuous heating. Then the mixed solution is continuously stirred until the color changes from yellow to dark pink, and the heating is stopped. After cooling to room temperature, it is centrifuged at 11000 r / min for 10 minutes, the gold nanoparticles are washed and collected, and stored at 4°C.

[0054] (3) Synthesis of PdIrBP mesoporous nanospheres 30 mg of dioctadecyldimethylammonium chloride is added to 10 mL of deionized water and stirred to obtain a uniform solution. Subsequently, 1.0 mL of 0.337 mol / L ammonium fluoride and 1.0 mL of 0.101 mol / L boric acid are added to the previous solution, and then 0.8 mL of 10 mmol / L chloropalladic acid and 0.8 mL of 10 mmol / L chloroiridic acid as metal precursors are added. After incubating for 5 minutes, 0.8 mL of ammonia water (10 wt.%) is injected to adjust the pH of the solution. When the color of the mixed solution changes from brownish yellow to colorless, 1.0 mL of 0.034 mol / L sodium hypophosphite is mixed into the above solution, and magnetically stirred at 95°C for 20 minutes. Finally, 1.0 mL of freshly prepared 0.1 mol / L dimethylamine borane is injected to initiate the reduction reaction, and the color of the solution gradually changes to dark brown. After reacting at 95°C for 30 minutes, the product is collected by centrifuging at 8000 r / min for 5 minutes, and washed several times with ethanol and deionized water.

[0055] (4) Preparation of signal probes: 0.5 mg of polyethylene glycol was added to 1.0 mL of PdIrBP mesoporous nanospheres (1.0 mg / mL). The mixture was stirred and incubated at room temperature for 3 hours, then centrifuged and washed 3 times with deionized water. Finally, the precipitate was dispersed in 500 μL of deionized water for future use. Antibodies and polyethylene glycol-modified PdIrBP nanospheres were linked using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide as coupling agents. 50 μL of the above-prepared modified PdIrBP mesoporous nanospheres was added to 450 μL of 10 mmol / L PBS at room temperature and stirred well. Subsequently, an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (5 μL, 25 mM) and an aqueous solution of N-hydroxysuccinimide (5 μL, 50 mM) were added. After 20 minutes, the nanomaterials were washed twice with deionized water and then redispersed in 50 μL of PBS. Subsequently, 50 μL of antibodies (20 mg / mL) was added to the nanosphere suspension. After incubation at room temperature for 1 hour, the reaction solution was stored in a refrigerator at 4 °C for future use.

[0056] (5) Construction of biosensor: The gold electrode (3 mm in diameter) was polished to a mirror-like state with 0.3 and 0.05 μm alumina. Subsequently, it was ultrasonically treated in double-distilled water, ethanol, and double-distilled water alternately for 10 minutes. After drying at room temperature, the electrode was activated in freshly prepared piranha solution (98% sulfuric acid: 30% hydrogen peroxide, volume ratio 3:1) for 5 minutes, thoroughly rinsed with ultrapure water, and dried in air. 10 μL of the Ketjenblack solution prepared in step (1) was added to the electrode and dried at 37 °C. In the same way, the gold nanoparticles enriched in step (2) were dropped onto the electrode modified with Ketjenblack. 10 μL of the antibody mixture (80 μg / mL) was dropped onto the modified electrode, covered, and placed at 37 °C for 1 h. After rinsing with 0.01 mol / L PBS buffer, 10 μL of 0.3% BSA was added and incubated at 37 °C for 1 h to block non-specific binding sites. The resulting electrode was washed with PBS and stored at 4 °C for later use.

[0057] (6) Detection and release of circulating tumor cells: 10 μL of circulating tumor cells was added to the electrode prepared in step (5) and incubated at 37 °C for 40 minutes. Then, 10 μL of the signal probe synthesized in step (4) was dropped onto the electrode for immunoreaction to form a double-antibody sandwich immune complex. The substances not bound to the electrode were washed away with PBS, and the DPV signal of the electrode was detected.

[0058] After the cell detection is completed, immerse the electrode in 1 mL of glycine-hydrochloric acid eluent (0.1 mol / L) for 10 s, and then quickly add a few drops of 0.4 mol / L NaOH solution to adjust the pH value. After centrifuging at 500 r / min for 5 minutes, wash the obtained precipitate with PBS (0.01 mol / L) 2-3 times to obtain circulating tumor cells.

Claims

1. Preparation method of a circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black, characterized in that, it comprises the following steps: (1) Preparation of Ketjen black: 2.0 mg of Ketjen black is dispersed in 4.0 mL of 0.5 wt.% chitosan solution by means of ultrasonic stirring to obtain a uniform suspension; (2) Preparation of gold nanoparticles: 100 mL of 0.01% chloroauric acid aqueous solution is boiled for 10 - 15 minutes, then 1 mL of 2.0 wt.% sodium citrate solution is added dropwise under vigorous stirring and continuous heating, and then the above mixed solution is continuously stirred until the color changes from yellow to dark pink, and the heating is stopped; after cooling to room temperature, it is centrifuged at 11000 r / min for 10 minutes, the gold nanoparticles are collected by washing with deionized water, and stored at 4°C; (3) Synthesis of PdIrBP mesoporous nanospheres: 30 mg of dioctadecyldimethylammonium chloride is added to 10.0 mL of deionized water and stirred to obtain a uniform solution; subsequently, 1.0 mL of 0.337 mol / L ammonium fluoride and 1.0 mL of 0.101 mol / L boric acid are added to the previous solution, and then 0.8 mL of 10 mmol / L chloropalladic acid and 0.8 mL of 10 mmol / L chloroiridic acid as metal precursors are added; after incubation for 5 minutes, 0.8 mL of ammonia water (10 wt.%) is injected to adjust the pH of the solution; when the color of the mixed solution changes from brownish yellow to colorless, 1.0 mL of 0.034 mol / L sodium dihydrogen phosphate is mixed into the above solution, and magnetically stirred at 95°C for 20 minutes; finally, 1.0 mL of freshly prepared 0.1 mol / L dimethylamine borane is injected to initiate the reduction reaction, and the color of the solution gradually changes to dark brown; after reacting at 95°C for 30 minutes, the product is collected by centrifuging at 8000 r / min for 5 minutes, and washed several times with ethanol and deionized water; (4) Preparation of signal probe: Add 0.5 mg of polyethylene glycol to 1.0 mL of PdIrBP mesoporous nanospheres (1.0 mg / mL). Stir and incubate the mixture at room temperature for 3 hours, then centrifuge and wash it 3 times with deionized water. Finally, disperse the precipitate in 500 μL of deionized water for future use. The antibody and polyethylene glycol-modified PdIrBP nanospheres are linked by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide as coupling agents. Add 50 μL of the above-prepared modified PdIrBP mesoporous nanospheres to 450 μL of 10 mmol / L PBS at room temperature and stir well. Subsequently, add an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (5 μL, 25 mM) and an aqueous solution of N-hydroxysuccinimide (5 μL, 50 mM). After 20 minutes, wash the nanomaterials twice with deionized water and then redisperse them in 50 μL of PBS. Subsequently, add 50 μL of the antibody (20 mg / mL) to the nanosphere suspension. After incubating at room temperature for 1 hour, store the reaction solution in a refrigerator at 4 °C for future use. (5) Construction of biosensor: Polish the gold electrode (diameter 3 mm) to a mirror-like state with 0.3 μm and 0.05 μm alumina, and then ultrasonically treat it alternately in double-distilled water, ethanol, and double-distilled water for 10 minutes. After drying at room temperature, activate the electrode in freshly prepared piranha solution (98% sulfuric acid: 30% hydrogen peroxide, volume ratio 3:1) for 5 minutes, thoroughly rinse it with ultrapure water, and dry it in air. Add 10 μL of the Ketjen black solution prepared in step (1) to the electrode and dry it at 37 °C. Dropwise add the gold nanoparticles enriched in step (2) onto the electrode modified with Ketjen black in the same way. Then, add 10 μL of the antibody mixture (80 μg / mL) dropwise onto the modified electrode, cover it, and place it at 37 °C for 1 h. After rinsing with 0.01 mol / L PBS buffer, add 10 μL of 0.3% BSA and incubate it at 37 °C for 1 h to block non-specific binding sites. Wash the obtained electrode with PBS and store it at 4 °C for later use. (6) Detection and release of circulating tumor cells: Add 10 μL of circulating tumor cells to the electrode prepared in step (5) and incubate it at 37 °C for 40 minutes. Then, drop 10 μL of the signal probe synthesized in step (4) onto the electrode for immunoreaction to form a sandwich immunorecomplex. Wash away the substances not bound to the electrode with PBS and detect the DPV signal of the electrode. After the cell detection is completed, immerse the electrode in 1 mL of glycine-hydrochloric acid eluent (0.1 mol / L) for 10 s, and then quickly add a few drops of 0.4 mol / L NaOH solution to adjust the pH value. After centrifuging at 500 r / min for 5 minutes, wash the obtained precipitate 2 - 3 times with PBS (0.01 mol / L) to obtain circulating tumor cells.

2. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the concentration of the Ketjen black used in step (1) is 0.5 mg / mL.

3. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the concentration of the chloroauric acid aqueous solution in step (2) is 0.01%.

4. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the concentration of the sodium citrate solution in step (2) is 2.0 wt.%.

5. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the volume of the ammonia water in step (3) is 0.8 mL (10 wt.%).

6. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the concentration of the dimethylamine borane in step (3) is 0.1 mol / L.

7. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the concentration of the PdIrBP mesoporous nanospheres used in step (4) is 1.0 mg / mL.

8. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the concentration of the antibody in step (4) is 20 mg / mL.

9. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the concentration of the antibody mixture in step (5) is 80 μg / mL.

10. The preparation method of the circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black as claimed in claim 1, characterized in that, the cell incubation time in step (6) is 40 minutes.

Images