CN113008971A - Circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black - Google Patents

Circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black Download PDF

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CN113008971A
CN113008971A CN201911321158.8A CN201911321158A CN113008971A CN 113008971 A CN113008971 A CN 113008971A CN 201911321158 A CN201911321158 A CN 201911321158A CN 113008971 A CN113008971 A CN 113008971A
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circulating tumor
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冯文莉
谢国明
彭杨
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Chongqing Medical University
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Abstract

Detection and recovery of circulating tumor cells is of great clinical significance for monitoring metastasis and therapeutic effects. 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. Ketjen black is combined with gold nanoparticles for the first time and used for modifying the surface of the gold electrode, so that the conductivity is improved and the specific surface area of the electrode is increased. The PdIrBP mesoporous nanospheres and the antibody are combined together to form a signal probe which is used as a detection label to amplify a current signal. In addition, the method can be used for producing a composite materialGlycine hydrochloric acid is used as antibody eluent, and captured circulating tumor cells can be released from the electrodes and collected for further clinical study. In the range of 10/mL to 1X 106There was a good linear relationship between the electrochemical signal obtained in the/mL range and the target cell concentration, with detection limits as low as 2/mL. Therefore, the novel cell sensor model has potential clinical application value and can be used for early diagnosis and prognosis monitoring of cancer patients.

Description

Circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black
Technical Field
The invention relates to a biosensor, in particular to a biosensor for ultra-sensitive detection and nondestructive collection of circulating tumor cells based on a novel nanoparticle compound and a highly conductive carbon black material.
Background
The circulating tumor cells are closely related to the metastasis and recurrence of the tumor, so that the detection of the circulating tumor cells has important significance for the diagnosis and treatment of cancer patients, and the collection of the circulating tumor cells can be used for further research so as to judge the drug treatment effect, prognosis condition, recurrence probability and the like of the patients. The current methods for detecting circulating tumor cells mainly include methods based on cell biological properties, direct microscopic examination methods and methods based on cell physical properties, among which the methods based on cell biological properties are the most common, and among them, the method of capturing circulating tumor cells carrying corresponding antigens by using antibodies is the most widely used detection method at present. The technology can realize high-efficiency detection, but because the used antibody is single, the circulating tumor cells with epithelial cell-mesenchymal transition can not be detected, false negative results are easy to generate, and the method can not realize the lossless collection of the circulating tumor cells, so the popularization and the use of the method in clinic are limited.
In order to solve the problem, the invention introduces the electrochemical sensor to detect the circulating tumor cells, the electrochemical biosensor has higher sensitivity, good selectivity and rapid and convenient detection, so that the electrochemical biosensor has great application value in screening various genetic diseases, epidemiological investigation and early diagnosis of tumors, simultaneously, the mixed antibody is used for capturing the circulating tumor cells at different periods, the generation of false negative is reduced, and the mild antigen-antibody eluent glycine-hydrochloric acid is introduced to destroy the combination of the antibody and the cell surface antigen, thereby realizing the lossless release and collection of the cells.
With the rapid development of nanotechnology, a variety of nanomaterials are used in the construction of electrochemical sensors. Ketjen black is a highly conductive carbon black material, and has a unique branched chain structure, so that the conductive carbon black has more conductive contact points and can greatly increase the conductive performance of an electrode compared with other conductive carbon blacks. The gold nanoparticles have high stability and biocompatibility, and the effective surface area can be larger by modifying the surface of the electrode by using the gold nanoparticles, and the gold nanoparticles can be combined with more biomolecules. The PdIrBP mesoporous nanospheres have enzyme-like activity, can efficiently catalyze hydrogen peroxide to generate electrochemical signals, and can be used as signal molecules to form a signal probe together with an antibody, so that the detection sensitivity can be effectively increased.
Disclosure of Invention
The invention aims to develop a biosensor for detecting and collecting circulating tumor cells in an ultra-sensitive and high-specificity way without damage.
The specific technical scheme is as follows:
a circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black is prepared by the following steps:
(1) preparation of ketjen black 2.0mg of ketjen black was dispersed in 4.0mL of 0.5 wt.% chitosan solution by means of ultrasonic agitation to obtain a homogeneous suspension.
(2) Preparation of gold nanoparticles 100mL of 0.01% aqueous chloroauric acid solution was boiled for 10-15 minutes and then 1mL of 2.0 wt.% sodium citrate solution was added dropwise with vigorous stirring and continuous heating. The mixed solution was then stirred continuously until the color changed from yellow to deep pink, and heating was stopped. After cooling to room temperature, centrifuging for 10 minutes at the rotating speed of 11000r/min, washing and collecting the gold nanoparticles, and storing at 4 ℃.
(3) Synthesis of PdIrBP mesoporous nanospheres 30mg dioctadecyldimethylammonium chloride was added to 10.0mL deionized water and stirred to obtain a homogeneous solution. Subsequently, 1.0mL of 0.337mol/L ammonium fluoride, 1.0mL of 0.101mol/L boric acid were added to the previous solution, and then 0.8mL of 10mmol/L chloropalladate and 0.8mL of 10mmol/L chloroiridic acid as metal precursors were added. After 5 minutes of incubation, 0.8mL of ammonia (10 wt.%) was injected to adjust the pH of the solution. When the color of the mixed solution changed from brown to colorless, 1.0mL of 0.034mol/L sodium hypophosphite was mixed into the above solution, and magnetically stirred at 95 ℃ for 20 minutes. Finally, 1.0mL of freshly prepared 0.1mol/L dimethylamine borane was injected to initiate the reduction reaction and the color of the solution gradually evolved to dark brown. After 30 minutes of reaction at 95 ℃, the product was collected by centrifugation at 8000r/min for 5 minutes and washed several times with ethanol and deionized water.
(4) Preparation of Signal Probe 0.5mg of polyethylene glycol was added to 1.0mL of PdIrBP mesoporous nanospheres (1.0 mg/mL). The mixture was incubated at room temperature for 3 hours with stirring, then washed 3 times with deionized water centrifugation, and finally the precipitate was dispersed in 500 μ L of deionized water for future use. The antibody and the PdIrBP nanosphere modified by polyethylene glycol are connected by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide as coupling agents. 50 μ L of the modified PdIrBP mesoporous nanosphere prepared above was added to 450 μ L of 10mmol/L PBS at room temperature and stirred well. Then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (5. mu.L, 25mM) in water and N-hydroxysuccinimide (5. mu.L, 50mM) in water were added. After 20 minutes, the nanomaterial was washed twice with deionized water and then redispersed in 50 μ L of PBS. Subsequently, 50 μ L of antibody (20mg/mL) was added to the nanosphere suspension. After 1 hour incubation at room temperature, the reaction solution was stored in a refrigerator at 4 ℃ for future use.
(5) Construction of the biosensor gold electrodes (diameter 3mm) were polished to a mirror-like shape with 0.3 μm and 0.05 μm alumina. Then ultrasonic treatment is carried out alternately in double distilled water, ethanol and double distilled water 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, rinsed thoroughly with ultra pure water and dried in air. mu.L of the Ketjen black solution prepared in step (1) was applied to the electrode and dried at 37 ℃. And (3) dropwise adding the gold nanoparticles enriched in the step (2) onto the electrode modified with the Ketjen black by the same method. mu.L of the antibody mixture (80. mu.g/mL) was added dropwise to the modified electrode and placed at 37 ℃ for 1h with a lid. After washing with 0.01mol/L PBS buffer, 10. mu.L of 0.3% BSA was added and incubated at 37 ℃ for 1h to block non-specific binding sites. The resulting electrode was washed with PBS and stored at 4 ℃ until use.
(6) Detection and Release of circulating tumor cells 10. mu.L of circulating tumor cells were applied to the electrode prepared in step (5) and incubated at 37 ℃ for 40 minutes. Then, 10 μ L of the signal probe synthesized in step (4) is dropped onto the electrode for immunoreaction to form a double-antibody sandwich immune complex. The substances not bound to the electrodes were washed with PBS, and the DPV signals of the electrodes were detected.
After completion of the cell assay, the electrode was immersed in 1mL glycine-hydrochloric acid eluent (0.1mol/L) for 10s, and then several drops of 0.4mol/L NaOH solution were rapidly added to adjust the pH. After centrifugation at 500r/min for 5 minutes, the resulting pellet was washed 2-3 times with PBS (0.01mol/L) to obtain circulating tumor cells.
The concentration of Ketjen black used in said step (1) is preferably 0.5 mg/mL.
The concentration of the aqueous chloroauric acid solution in the step (2) is preferably 0.01%.
The concentration of the sodium citrate solution in said step (2) is preferably 2.0 wt%.
The volume of the ammonia water in the step (3) is preferably 0.8 mL.
The concentration of dimethylamine borane in the step (3) is preferably 0.1 mol/L.
The concentration of the PdIrBP mesoporous nanosphere in the step (4) is preferably 1.0 mg/mL.
The concentration of the antibody in the step (4) is preferably 20 mg/mL.
The concentration of the antibody mixture in said step (5) is preferably 80. mu.g/mL.
The incubation time of the cells in said step (6) is preferably 40 minutes.
The invention constructs a biosensor based on PdIrBP mesoporous nanosphere and Ketjen black for ultra-sensitive detection and nondestructive collection of circulating tumor cells, and the detection principle is to improve the conductivity by using a Ketjen black modified gold electrode. Then adding gold nanoparticles on the surface of the electrode for modification, thereby improving the effective surface area of the electrode and leading gold-NH to pass through2The bond binds to more capture antibody. The antibody captures the circulating tumor cells on the surface of the electrode, and then a signal probe is added to form a double-antibody sandwich structure. Circulating tumor cells were quantitatively detected by differential pulse voltammetry. Finally, the introduction of glycine-HCl buffer can destroy cell surface antigens and antigensAnd (4) combining the bodies, thereby realizing the collection of the circulating tumor cells.
The successful synthesis of various nano materials plays an important role in the preparation of the sensor, and the invention adopts a field emission scanning electron microscope, a transmission electron microscope and an atomic force microscope to carry out morphological characterization on the nano materials. As shown in fig. 1, the results of the field emission scanning electron microscope of ketjen black show that the ketjen black is uniformly distributed, and the branched structure of the ketjen black can be observed, and the unique structure provides more conductive contacts, so that the ketjen black has better conductivity compared with other carbon black materials. The transmission electron microscope result (figure 2) of the gold nanoparticles shows that the gold nanoparticles have consistent size and uniform distribution, the diameter is about 20nm, and a good foundation is laid for the combination of antibodies. FIG. 3 shows a transmission electron microscope image of PdIrBP mesoporous nanospheres, wherein the nanomaterial is spherical and has a highly branched structure, and the branches form a network similar to an organic dendritic polymer, and the network has the same size and the diameter of about 100nm and is uniformly dispersed in water. The element composition of PdIrBP mesoporous nano-particles is analyzed by an energy dispersion spectrometer. As shown in fig. 4, the nanocubes contain four elements, i.e., Pd, Ir, B, and P, further indicating the successful synthesis of the nanomaterial.
In the presence of [ Fe (CN) ] containing 0.1M KCl6]3-/4-Cyclic Voltammetry (CV) measurements were performed in solution (5mM) at a scan rate of 100mV/s to demonstrate the feasibility of the experiment with each step of modifying the working electrode. As shown in fig. 5, the current signal of the electrode was greatly increased compared to the bare electrode (curve a) after modification with KB (curve b) and AuNPs (curve c). This is due to the high specific surface area and excellent conductivity of KB and AuNPs. The change in current demonstrates the successful binding of the two nanomaterials to the electrode surface. Immobilization of Ab due to steric hindrance of antibody and blocking of BSA1After (curve d) and BSA (curve e), the current response continued to decrease, indicating Ab1As well as BSA, were successfully immobilized on the electrode surface as expected. When the MCF-7 cells were captured, the current signal was further reduced (curve f), mainly due to the reduction in the electrotransfer efficiency of the electrode interface by the electrical resistance of the cells.
The electrochemical signal generated by the hydrogen peroxide catalyzed by the PdIrBP mesoporous nanosphere can reach the peak value within a certain time, and the exploration of the proper time is helpful for capturing the maximum signal. We examined DPV signals for different reaction times and, as shown in figure 6, the highest current was obtained when the catalytic reaction time reached 40 s. Therefore, for the present study, the optimal reaction time of PdIrBP mesoporous nanospheres to catalyze hydrogen peroxide is 40 s.
The pH value of the working solution has a great influence on the bonding strength of the cells and the antibodies and the catalytic efficiency of the nano material. When the pH was adjusted from 3 to 6, the corresponding DPV signal increased rapidly, whereas when the pH exceeded 6, the DPV signal began to drop sharply (fig. 7). This is probably because both PdIrBP mesoporous nanospheres and tumor cells are more stable in a weakly acidic environment. Therefore, the optimum pH is 6.
Ab1The concentration of (b) directly affects the capture efficiency of the cells, so the experimental parameters play a very important role in the construction of the sensor. As shown in FIG. 8, the difference pulse voltammetry detected signal along with Ab1The increase in concentration increases and is greatest at 80 μ g/mL, after which the current response signal gradually decreases, probably because more antibody immobilization at the electrode interface may create more steric hindrance, thus hindering target cell recognition. Thus, Ab1The optimum concentration of (2) is 80. mu.g/mL.
The incubation time of the MCF-7 cells and the antibody also plays a decisive role in the cell capture efficiency. FIG. 9 shows the effect of incubation time on the electrochemical response of the biosensor. Within 40min, more MCF-7 cells were captured on the electrode surface with increasing incubation time, resulting in an enhanced current response. However, with further increase in incubation time, there was no significant change in the current signal. These results indicate that the optimal incubation time is 40 minutes.
The invention tests the repeatability of the sensing strategy. The method comprises the following steps: by using five freshly prepared modified electrodes at the same concentration (10)4mL and 105/mL) target cells were detected five times, Relative Standard Deviation (RSD) respectively1.06% and 1.21% (FIG. 10). This data shows that the immunosensor has excellent reproducibility.
In addition, the stability of the immunosensor was also investigated. After placing the prepared electrodes in a refrigerator at 4 ℃ for 7 days and 15 days, 95.92% and 90.3% of their initial current response were retained compared to freshly prepared electrodes, indicating better stability of the established immunosensor device.
To evaluate the analytical performance of the sensing strategy on target cells, we studied the strategy with a series of MCF-7 cells at different concentrations. As the concentration of the target cell increases, the current response increases accordingly. The calibration curve shows that the MCF-7 cell concentration is between 10/mL and 106There is good linearity between the DPV value between/mL and the logarithmic value. The linear regression equation is Y-26.226 Log [ C ═ C]+20.427, correlation coefficient (R)2) 0.9993 (FIG. 11), where Y is the peak current of the immunosensor and C is the concentration of MCF-7 cells. With a signal-to-noise ratio of 3, the minimum detection limit is estimated to be 2/mL.
To evaluate the specificity of the electrochemical immunoassay, two non-specific cell lines, HeLa cells and K562, were selected, and the specificity of the sensor was evaluated at the same concentration under optimized conditions. FIG. 12 demonstrates that the current response of non-specific cells does not change significantly at any concentration, while the electrochemical signal to MCF-7 cells is very high and increases with increasing concentration of MCF-7 cells. When these interfering cells were mixed separately with MCF-7 cells, the corresponding current response was similar to the electrochemical signal of pure MCF-7 cells at the same concentration. These results indicate that the biosensor shows excellent specificity to MCF-7 cells.
In order to verify that the strategy can efficiently release and recover circulating tumor cells, electrochemical signals generated by electrodes before and after elution are compared. As shown in FIG. 13, the current signal of the electrode (curve a) was significantly increased after the treatment with the antibody eluent, as compared to that after the capture of the target cells by the antibody (curve b). It was thus demonstrated that the cells on the electrodes could be eluted using this method. After the eluted cells were cultured for 12 hours, the morphology of the cells was observed, and as shown in FIG. 14, the cells started to grow adherently and proliferate, which proves that the elution method does not affect the activity of the cells.
In the strategy, an integrated electrochemical immunosensor for ultrasensitive detection of circulating tumor cells is developed, wherein the integrated electrochemical immunosensor takes Ketjen black/gold nanoparticles as a platform and PdIrBP mesoporous nanospheres as signal labels. The invention has the advantages that the unique functions of the Ketjen black and the gold nanoparticles greatly increase the sensitivity of the sensor by combining the Ketjen black and the gold nanoparticles; due to high catalytic activity and strong stability, the PdIrBP mesoporous nanosphere is considered as an excellent signal label, and the combination of the nanomaterials can construct an immunosensor with a wider linear range and a lower detection limit; the sensor also has enough specificity, can distinguish target cells from the actual blood sample, and can effectively resist the interference of the complex environment of the actual blood sample; and fourthly, the antibody eluent Gly-HCl can realize the efficient and nondestructive collection of the target cells, the biological activity of the released target cells is not influenced, and accurate information can be provided for determining a treatment scheme or monitoring prognosis. Therefore, the immunosensor lays a foundation for realizing accurate detection of circulating tumor cells and real liquid biopsy.
Drawings
FIG. 1 is a scanning electron microscope image of Ketjen black
FIG. 2 is a transmission electron micrograph of gold nanoparticles
FIG. 3 is a transmission electron microscope image of PdIrBP mesoporous nanospheres
FIG. 4 shows EDS elemental analysis results of PdIrBP mesoporous nanospheres
FIG. 5 is a feasibility analysis
FIG. 6 optimization of reaction time
FIG. 7 is a graph showing pH optimization of working fluid
FIG. 8 optimization of capture antibody concentration
FIG. 9 optimization of cell incubation time
FIG. 10 is a reproducibility analysis
FIG. 11 is a graph of cell concentration versus electrochemical signal
FIG. 12 is a specificity test for a sensing strategy
FIG. 13 is an analysis of the effect of cell release
FIG. 14 is a morphological diagram of recovered cells
Detailed Description
A circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black is prepared by the following steps:
(1) preparation of ketjen black 2.0mg of ketjen black was dispersed in 4.0mL of 0.5 wt% chitosan solution by means of ultrasonic agitation to obtain a uniform suspension.
(2) Preparation of gold nanoparticles 100mL of 0.01% aqueous chloroauric acid solution was boiled for 10-15 minutes, and then 1mL of 2.0 wt% sodium citrate solution was added dropwise with vigorous stirring and continuous heating. The mixed solution was then stirred continuously until the color changed from yellow to deep pink, and heating was stopped. After cooling to room temperature, centrifuging for 10 minutes at the rotating speed of 11000r/min, washing and collecting the gold nanoparticles, and storing at 4 ℃.
(3) Synthesis of PdIrBP mesoporous nanospheres 30mg dioctadecyldimethylammonium chloride was added to 10mL deionized water and stirred to obtain a homogeneous solution. Subsequently, 1.0mL of 0.337mol/L ammonium fluoride, 1.0mL of 0.101mol/L boric acid were added to the previous solution, and then 0.8mL of 10mmol/L chloropalladate and 0.8mL of 10mmol/L chloroiridic acid as metal precursors were added. After 5 minutes of incubation, 0.8mL of ammonia (10 wt.%) was injected to adjust the pH of the solution. When the color of the mixed solution changed from brown to colorless, 1.0mL of 0.034mol/L sodium hypophosphite was mixed into the above solution, and magnetically stirred at 95 ℃ for 20 minutes. Finally, 1.0mL of freshly prepared 0.1mol/L dimethylamine borane was injected to initiate the reduction reaction and the color of the solution gradually evolved to dark brown. After 30 minutes of reaction at 95 ℃, the product was collected by centrifugation at 8000r/min for 5 minutes and washed several times with ethanol and deionized water.
(4) Preparation of Signal Probe 0.5mg of polyethylene glycol was added to 1.0mL of PdIrBP mesoporous nanospheres (1.0 mg/mL). The mixture was incubated at room temperature for 3 hours with stirring, then washed 3 times with deionized water centrifugation, and finally the precipitate was dispersed in 500 μ L of deionized water for future use. The antibody and the PdIrBP nanosphere modified by polyethylene glycol are connected by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide as coupling agents. 50 μ L of the modified PdIrBP mesoporous nanosphere prepared above was added to 450 μ L of 10mmol/L PBS at room temperature and stirred well. Then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (5. mu.L, 25mM) in water and N-hydroxysuccinimide (5. mu.L, 50mM) in water were added. After 20 minutes, the nanomaterial was washed twice with deionized water and then redispersed in 50 μ L of PBS. Subsequently, 50 μ L of antibody (20mg/mL) was added to the nanosphere suspension. After 1 hour incubation at room temperature, the reaction solution was stored in a refrigerator at 4 ℃ for future use.
(5) Construction of the biosensor gold electrodes (diameter 3mm) were polished to a mirror-like shape with 0.3 and 0.05 μm alumina. Then ultrasonic treatment is carried out alternately in double distilled water, ethanol and double distilled water 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, rinsed thoroughly with ultra pure water and dried in air. mu.L of the Ketjen black solution prepared in step (1) was applied to the electrode and dried at 37 ℃. And (3) dropwise adding the gold nanoparticles enriched in the step (2) onto the electrode modified with the Ketjen black by the same method. mu.L of the antibody mixture (80. mu.g/mL) was added dropwise to the modified electrode and placed at 37 ℃ for 1h with a lid. After washing with 0.01mol/L PBS buffer, 10. mu.L of 0.3% BSA was added and incubated at 37 ℃ for 1h to block non-specific binding sites. The resulting electrode was washed with PBS and stored at 4 ℃ until use.
(6) Detection and Release of circulating tumor cells 10. mu.L of circulating tumor cells were applied to the electrode prepared in step (5) and incubated at 37 ℃ for 40 minutes. Then, 10 μ L of the signal probe synthesized in step (4) is dropped onto the electrode for immunoreaction to form a double-antibody sandwich immune complex. The substances not bound to the electrodes were washed with PBS, and the DPV signals of the electrodes were detected.
After completion of the cell assay, the electrode was immersed in 1mL glycine-hydrochloric acid eluent (0.1mol/L) for 10s, and then several drops of 0.4mol/L NaOH solution were rapidly added to adjust the pH. After centrifugation at 500r/min for 5 minutes, the resulting pellet was washed 2-3 times with PBS (0.01mol/L) to obtain circulating tumor cells.

Claims (10)

1. A circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black is prepared by the following steps:
(1) preparation of ketjen black 2.0mg of ketjen black was dispersed in 4.0mL of 0.5 wt.% chitosan solution by means of ultrasonic agitation to obtain a homogeneous suspension.
(2) Preparation of gold nanoparticles 100mL of 0.01% aqueous chloroauric acid solution was boiled for 10-15 minutes and then 1mL of 2.0 wt.% sodium citrate solution was added dropwise with vigorous stirring and continuous heating. The mixed solution was then stirred continuously until the color changed from yellow to deep pink, and heating was stopped. After cooling to room temperature, centrifuging for 10 minutes at the rotating speed of 11000r/min, washing and collecting the gold nanoparticles, and storing at 4 ℃.
(3) Synthesis of PdIrBP mesoporous nanospheres 30mg dioctadecyldimethylammonium chloride was added to 10.0mL deionized water and stirred to obtain a homogeneous solution. Subsequently, 1.0mL of 0.337mol/L ammonium fluoride, 1.0mL of 0.101mol/L boric acid were added to the previous solution, and then 0.8mL of 10mmol/L chloropalladate and 0.8mL of 10mmol/L chloroiridic acid as metal precursors were added. After 5 minutes of incubation, 0.8mL of ammonia (10 wt.%) was injected to adjust the pH of the solution. When the color of the mixed solution changed from brown to colorless, 1.0mL of 0.034mol/L sodium hypophosphite was mixed into the above solution and magnetically stirred at 95 ℃ for 20 minutes. Finally, 1.0mL of freshly prepared 0.1mol/L dimethylamine borane was injected to initiate the reduction reaction and the color of the solution gradually evolved to dark brown. After 30 minutes of reaction at 95 ℃, the product was collected by centrifugation at 8000r/min for 5 minutes and washed several times with ethanol and deionized water.
(4) Preparation of Signal Probe 0.5mg of polyethylene glycol was added to 1.0mL of PdIrBP mesoporous nanospheres (1.0 mg/mL). The mixture was incubated at room temperature for 3 hours with stirring, then washed 3 times with deionized water centrifugation, and finally the precipitate was dispersed in 500 μ L of deionized water for future use. The antibody and the PdIrBP nanosphere modified by polyethylene glycol are connected by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide as coupling agents. 50 μ L of the modified PdIrBP mesoporous nanosphere prepared above was added to 450 μ L of 10mmol/L PBS at room temperature and stirred well. Then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (5. mu.L, 25mM) in water and N-hydroxysuccinimide (5. mu.L, 50mM) in water were added. After 20 minutes, the nanomaterial was washed twice with deionized water and then redispersed in 50 μ L of PBS. Subsequently, 50 μ L of antibody (20mg/mL) was added to the nanosphere suspension. After 1 hour incubation at room temperature, the reaction solution was stored in a refrigerator at 4 ℃ for future use.
(5) Construction of the biosensor gold electrodes (diameter 3mm) were polished to a mirror-like shape with 0.3 μm and 0.05 μm alumina. Then ultrasonic treatment is carried out alternately in double distilled water, ethanol and double distilled water 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, rinsed thoroughly with ultra pure water and dried in air. mu.L of the Ketjen black solution prepared in step (1) was applied to the electrode and dried at 37 ℃. And (3) dropwise adding the gold nanoparticles enriched in the step (2) onto the electrode modified with the Ketjen black by the same method. mu.L of the antibody mixture (80. mu.g/mL) was added dropwise to the modified electrode and placed at 37 ℃ for 1h with a lid. After washing with 0.01mol/L PBS buffer, 10. mu.L of 0.3% BSA was added and incubated at 37 ℃ for 1h to block non-specific binding sites. The resulting electrode was washed with PBS and stored at 4 ℃ until use.
(6) Detection and Release of circulating tumor cells 10. mu.L of circulating tumor cells were applied to the electrode prepared in step (5) and incubated at 37 ℃ for 40 minutes. Then, 10 μ L of the signal probe synthesized in step (4) is dropped onto the electrode for immunoreaction to form a double-antibody sandwich immune complex. The substances not bound to the electrodes were washed with PBS, and the DPV signals of the electrodes were detected.
After completion of the cell assay, the electrode was immersed in 1mL glycine-hydrochloric acid eluent (0.1mol/L) for 10s, and then several drops of 0.4mol/L NaOH solution were rapidly added to adjust the pH. After centrifugation at 500r/min for 5 minutes, the resulting pellet was washed 2-3 times with PBS (0.01mol/L) to obtain circulating tumor cells.
2. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the concentration of the Ketjen black used in step (1) is preferably 0.5 mg/mL.
3. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the concentration of the chloroauric acid aqueous solution in step (2) is preferably 0.01%.
4. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the concentration of the sodium citrate solution in step (2) of the preparation method is preferably 2.0 wt.%.
5. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the volume of ammonia water in step (3) is preferably 0.8 mL.
6. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the concentration of dimethylamine borane in step (3) is preferably 0.1 mol/L.
7. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the concentration of the PdIrBP mesoporous nanospheres used in step (4) of the preparation method is preferably 1.0 mg/mL.
8. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the concentration of the antibody in step (4) is preferably 20 mg/mL.
9. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the concentration of the antibody mixture in step (5) is preferably 80 μ g/mL.
10. The circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black of claim 1, wherein the incubation time of the cells in step (6) is preferably 40 min.
CN201911321158.8A 2019-12-19 2019-12-19 Circulating tumor cell biosensor based on PdIrBP mesoporous nanospheres and Ketjen black Pending CN113008971A (en)

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Cited By (2)

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CN113848201A (en) * 2021-09-27 2021-12-28 烟台大学 Electrochemiluminescence biosensor for detecting ulinastatin
CN117269288A (en) * 2023-08-07 2023-12-22 山东师范大学 Electrochemical biosensor for GLUT1 detection and preparation method thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113848201A (en) * 2021-09-27 2021-12-28 烟台大学 Electrochemiluminescence biosensor for detecting ulinastatin
CN113848201B (en) * 2021-09-27 2024-03-22 烟台大学 Electrochemiluminescence biosensor for detecting ulinastatin
CN117269288A (en) * 2023-08-07 2023-12-22 山东师范大学 Electrochemical biosensor for GLUT1 detection and preparation method thereof
CN117269288B (en) * 2023-08-07 2024-04-19 山东师范大学 Electrochemical biosensor for GLUT1 detection and preparation method thereof

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