Preparation and application of ultrasensitive electrochemical immunosensor for specific detection of gallbladder cancer CTCs
By using SiO2 nanospheres loaded with quantum dot-modified antibodies and bismuth film-modified electrodes in the detection of gallbladder cancer cells, an electrochemical immunosensor for the specific detection of gallbladder cancer cells was constructed, which solved the problems of high misdiagnosis rate, non-specificity and poor sensitivity of existing detection methods, and achieved non-invasive and highly sensitive early diagnosis.
Patent Information
- Application Number
- CN202211460426.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-21
- Publication Date
- 2025-09-09
- Estimated Expiration
- 2042-11-21
AI Technical Summary
Existing methods for detecting gallbladder cancer have high misdiagnosis rates, lack of specificity, poor sensitivity, and are often invasive. There is a lack of highly sensitive, highly specific, low-cost, and non-invasive detection methods.
An electrochemical immunosensor for the specific detection of gallbladder cancer cells was constructed by using SiO2 nanospheres loaded with quantum dots (QDs)-modified antibodies and combining them with a bismuth film-modified glassy carbon electrode. High-sensitivity detection was achieved by targeting both EpCAM and ENPP1.
It achieves specific and highly sensitive detection of gallbladder cancer cells, provides a non-invasive means of early diagnosis, and significantly improves the specificity and sensitivity of detection.
Smart Images
Figure CN115901895B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to a preparation and application method of an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs. Background Art
[0002] Gallbladder carcinoma (GBC) is a common malignant tumor of the biliary tract characterized by its occult nature, high malignancy, invasive and metastatic potential, difficulty in early diagnosis, and poor prognosis. Common diagnostic methods for GBC include imaging, hematology, genomics, and pathological analysis. Ultrasound is the preferred imaging method, but this method has a high misdiagnosis rate. Hematology methods based on CEA and CA199 lack specificity. Genomics have poor sensitivity. Pathological analysis is the "gold standard" for diagnosis, but it is invasive. Therefore, developing a highly specific, sensitive, and non-invasive detection method is essential. With the implementation of precision medicine initiatives, research on CTCs has become increasingly popular. CTC detection is of great significance for early diagnosis, therapeutic efficacy evaluation, and prognosis monitoring. In recent years, electrochemical immunosensors have become popular in CTC research due to their advantages such as high sensitivity, high specificity, low cost, and rapid detection speed. Furthermore, quantum dots (QDs) have been widely used due to their superior electrochemical properties. Therefore, this study aims to design a quantum dot signal-responsive electrochemical immunosensor to detect CTCs in early-stage gallbladder cancer. This study could provide a new detection method for the clinical diagnosis of early-stage gallbladder cancer, with significant clinical application prospects and social value. Summary of the Invention
[0003] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to provide a preparation and application method of an ultrasensitive electrochemical immunosensor for the specific detection of gallbladder cancer CTCs with high sensitivity, high specificity, low cost and fast detection speed.
[0004] To achieve the above objectives, the present invention provides a method for preparing an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs, which is characterized by comprising the following steps: a. synthesizing SiO2 nanospheres; b. synthesizing SiO2@QD using the SiO2 nanospheres synthesized in step a; c. preparing SiO2@QD-Ab using the SiO2@QD synthesized in step b; d. constructing an immunoelectrochemical platform using the SiO2@QD-Ab synthesized in step c.
[0005] As an optimization, in step a, first add 50 mL of anhydrous ethanol, 2 mL of 28% ammonia water, and 3 mL of deionized water in sequence, stir at room temperature for 20 minutes at a speed of 400 rpm; then add 2 mL of TEOS at one time, and stir at room temperature for 4 hours; after the reaction is completed, the solution is centrifuged at 8000 rpm, washed twice with anhydrous ethanol, and finally resuspended in 10 mL of anhydrous ethanol for use and synthesized to obtain SiO2 nanospheres.
[0006] As an optimization, in step b, a positively charged polymer polyethyleneimine PEI is introduced. PEI is easily soluble in water and can self-assemble on the SiO2 surface under ultrasonic conditions to form a PEI interlayer; then, through electrostatic adsorption, carboxyl CdSe / ZnS QDs are coated on the surface of SiO2 nanospheres and SiO2@QD is synthesized.
[0007] As an optimization, in step c, 2 mL of SiO2@QD synthesized in step b was taken, centrifuged at 5500 rpm for 10 min, resuspended in 1 mL of 0.1 M MES, 100 μL of EDC and 20 μL of NHS were added, vortexed and sonicated for 15 min, the resulting solution was centrifuged at 5500 rpm for 10 min, the supernatant was discarded, resuspended in 200 μL of 0.05% PBST, 30 μL of anti-ENPP1 antibody was added, and the solution was incubated on a shaker at room temperature for 2 h; 100 μL of 5% BSA was added, and the solution was shaken and incubated at room temperature for 1 h, 4500 rpm for 10 min, the supernatant was discarded, and the solution was washed once with 0.05% PBST. Finally, the solution was resuspended in 0.2 mL of 0.05% PBST to synthesize SiO2@QD-Ab.
[0008] As an optimization, in step d, the gold electrode was first polished to a smooth mirror surface with 0.3 μm and 0.05 μm alumina powder, and then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes, and then activated with freshly prepared piranha solution for 15 minutes, and then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes; dried at room temperature and set aside; 10 μL of 200 nM SH-EpCAM aptamer was dropped on the gold electrode and incubated at 4°C overnight; the capture electrode was removed and the unbound aptamer was washed with 0.01 mM pH7.4 PBS. After natural drying, 5 μL of 0.1 M MCH was added to prevent nonspecific adsorption; washed with 0.01 mM pH7.4 PBS, and after natural drying, NOZ cells were incubated at 37°C for 60 minutes; unbound cells were washed, 5 μL of the obtained SiO2@QD-Ab was added, and incubated for 120 minutes; unbound materials were washed, and 30 μL Digest with HNO3 for 10 min, and transfer the digestion solution into 3 mL of acetic acid-acetic acid solution.
[0009] The present invention also discloses an application method of an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs, comprising constructing the above-mentioned immunoelectrochemical platform; and also comprising a bismuth film-modified glassy carbon electrode and detection: first, the glassy carbon electrode is polished into a smooth mirror surface with 0.3 μm and 0.05 μm alumina powder, then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes respectively, then activated with a freshly prepared nitric acid:acetone solution mixed in a volume ratio of 1:1 for 15 minutes, and then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes; dried at room temperature and set aside; prepared 10 mL of bismuth ion acetic acid solution with a concentration of 12.5 mg / mL; using the IT deposition method, applying a constant potential of -1.2 V, and depositing for 300 seconds; using the glassy carbon electrode modified with the bismuth film as the working electrode, Ag / AgCl as the reference electrode, and a Pt wire electrode as the auxiliary electrode, to perform SWV detection; the greater the number of cells, the more bound signal reporter molecules, and therefore the stronger the electrochemical signal peak.
[0010] As an optimization, the piranha solution was obtained by mixing 98% H2SO4 and 30% H2O2 in a volume ratio of 3:1; SH-EpCAM was activated in advance with 1 mM TCEP at room temperature for 1 h; the pH of the acetic acid-acetic acid solution was 4.5.
[0011] As an optimization, SYL3C is an aptamer targeting the EpCAM target; the Ab is an antibody targeting the ENPP1 target; and the specific materials of the SiO2 are APTES and TEOS.
[0012] In summary, in this technical solution, a thiol-modified aptamer (SYL3C) was first bound to a gold electrode using the principle of "Au-S" bonding, thereby capturing GBC cells that overexpress EpCAM. Next, SiO2 nanospheres (called SiO2@QD-Ab) were prepared, loaded with CdSe / ZnS quantum dots and modified with an anti-ENPP1 antibody that specifically targets GBC. These nanospheres can specifically target gallbladder cancer cells that overexpress ENPP1, and the presence of the quantum dots can serve as electrochemical signal probes for detection. A detection signal is only observed when cells overexpress both ENPP1 and EpCAM.
[0013] In summary, the "aptamer-cell-signaling probe" sandwich-based method for detecting gallbladder cancer cells constructed in this invention offers the advantage of being noninvasive compared to traditional GBC diagnostic methods in that it detects CTCs. Furthermore, the bismuth-film-modified detection electrode enhances sensor sensitivity. Furthermore, the dual-targeting of ENPP1 and EpCAM in the electrochemical immunoassay platform significantly improves specificity. Therefore, this dual-recognition electrochemical immunoassay platform provides a new approach for the early detection of GBC and has great potential for clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is a picture of SiO2 after successful synthesis.
[0015] Figure 2 This is a picture of PEI successfully loaded onto SiO2 nanoparticles.
[0016] Figure 3 This is a picture of the successful synthesis of SiO2@QD.
[0017] Figure 4 This is a picture of the successful synthesis of SiO2@QD-Ab (immunofluorescence).
[0018] Figure 5 Figure 2 is a picture of the capture electrode construction.
[0019] Figure 6 This is a picture of the modified bismuth film electrode.
[0020] Figure 7 This is a linear relationship diagram between electrochemical detection and cell concentration. DETAILED DESCRIPTION
[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, in the description of the present invention, the terms "upper," "lower," "left," "right," "inner," and "outer," etc., indicating directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have a specific direction, be constructed, or operate in a specific manner. Therefore, they should not be construed as limiting the present invention. The terms "first," "second," and "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.
[0022] In one embodiment, a method for preparing an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs comprises the following steps: a. synthesizing SiO2 nanospheres; b. synthesizing SiO2@QD using the SiO2 nanospheres synthesized in step a; c. preparing SiO2@QD-Ab using the SiO2@QD synthesized in step b; and d. constructing an immunoelectrochemical platform using the SiO2@QD-Ab synthesized in step c.
[0023] In this specific embodiment, in step a, 50 mL of anhydrous ethanol, 2 mL of 28% ammonia water, and 3 mL of deionized water are first added in sequence, and stirred at room temperature for 20 minutes at a speed of 400 rpm; then 2 mL of TEOS is added at one time and stirred at room temperature for 4 hours; after the reaction is completed, the solution is centrifuged at 8000 rpm, washed twice with anhydrous ethanol, and finally resuspended in 10 mL of anhydrous ethanol for use and the SiO2 nanospheres are synthesized.
[0024] In this specific embodiment, in step b, a positively charged polymer polyethyleneimine PEI is introduced. PEI is easily soluble in water and can self-assemble on the SiO2 surface under ultrasonic conditions to form a PEI interlayer; then, through electrostatic adsorption, carboxyl CdSe / ZnS QDs are coated on the surface of the SiO2 nanospheres to synthesize SiO2@QD.
[0025] In this specific embodiment, in step c, 2 mL of SiO2@QD synthesized in step b was taken, centrifuged at 5500 rpm for 10 min, resuspended in 1 mL of 0.1 M MES, 100 μL of EDC and 20 μL of NHS were added, vortexed and ultrasonicated for 15 min, and the resulting solution was centrifuged at 5500 rpm for 10 min, the supernatant was discarded, and the solution was resuspended in 200 μL of 0.05% PBST. 30 μL of anti-ENPP1 antibody was added and incubated on a shaker at room temperature for 2 h; 100 μL of 5% BSA was added, and the solution was shaken and incubated at room temperature for 1 h, 4500 rpm for 10 min, the supernatant was discarded, and the solution was washed once with 0.05% PBST. Finally, the solution was resuspended in 0.2 mL of 0.05% PBST to synthesize SiO2@QD-Ab.
[0026] Specifically, the concentration of EDC is 10 mM; the concentration of NHS is 100 mM.
[0027] In this specific embodiment, in step d, the gold electrode is first polished to a smooth mirror surface with 0.3 μm and 0.05 μm alumina powder, and then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes, and then activated with freshly prepared piranha solution for 15 minutes, and then ultrasonicated in ultrapure water, ethanol, and ultrapure water for 5 minutes; dried at room temperature for later use; 10 μL of 200 nM SH-EpCAM aptamer is dropped on the gold electrode and incubated at 4°C overnight; the capture electrode is removed, and the unbound aptamer is washed with 0.01 mM pH7.4 PBS. After natural drying, 5 μL of 0.1 M MCH is added to prevent nonspecific adsorption; washed with 0.01 mM pH7.4 PBS, and after natural drying, the NOZ cells are incubated at 37°C for 60 minutes; the unbound cells are washed, 5 μL of the obtained SiO2@QD-Ab is added, and incubated for 120 minutes; the unbound material is washed, and 30 μL of Digest with HNO3 for 10 min, and transfer the digestion solution into 3 mL of acetic acid-acetic acid solution.
[0028] Also disclosed is an application method of an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs, including constructing the above-mentioned immunoelectrochemical platform; and also including a bismuth film-modified glassy carbon electrode and detection: first, the glassy carbon electrode is polished into a smooth mirror surface with 0.3μm and 0.05μm alumina powder, then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes respectively, and then activated with a freshly prepared nitric acid: acetone solution mixed in a volume ratio of 1:1 for 15 minutes, and then continued to be ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes; dried at room temperature and set aside; prepared 10mL of bismuth ion acetic acid solution with a concentration of 12.5mg / mL; using the IT deposition method, applying a constant potential of -1.2V, and depositing for 300s; using the glassy carbon electrode modified with the bismuth film as the working electrode, Ag / AgCl as the reference electrode, and a Pt wire electrode as the auxiliary electrode to perform SWV detection; the greater the number of cells, the more signal reporter molecules are bound, and therefore the stronger the electrochemical signal peak.
[0029] In this specific embodiment, the piranha solution is prepared by mixing 98% H2SO4 and 30% H2O2 in a volume ratio of 3:1; SH-EpCAM is activated in advance with 1 mM TCEP at room temperature for 1 hour; and the pH of the acetic acid-acetic acid solution is 4.5.
[0030] In this specific embodiment, the aptamer SYL3C targets EpCAM; the Ab targets ENPP1; and the specific material of the SiO2 is APTES or TEOS.
[0031] Specifically, the manufacturer of the raw material SYL3C is Sangon Biotech (Shanghai) Co., Ltd.; the manufacturer of the raw material Ab is Abcam, Cat No. 223268, USA; the manufacturer of the raw material CdSe / ZnS QDs is Xingshuo (Suzhou), Catalog # CdSe-MPA-625; the manufacturer of the raw material SiO2 is Macklin (Shanghai) Co., Ltd.; the manufacturer of the raw material bismuth (III) nitrate pentahydrate is Macklin (Shanghai) Co., Ltd.
[0032] In summary, this technical solution first utilizes the principle of "Au-S" bonding to bind a thiol-modified aptamer (SYL3C) to a gold electrode, thereby capturing GBC cells that overexpress EpCAM. Next, SiO2 nanospheres (called SiO2@QD-Ab) loaded with CdSe / ZnS quantum dots and modified with an antibody that specifically targets GBC (anti-ENPP1 antibody) were prepared. These nanospheres can specifically target gallbladder cancer cells that overexpress ENPP1, and the presence of the quantum dots can serve as a signal probe for electrochemical signal detection. A detection signal is only observed when the cells express both ENPP1 and EpCAM.
[0033] In summary, the "aptamer-cell-signaling probe" sandwich-based method for detecting gallbladder cancer cells constructed in this invention offers the advantage of being noninvasive compared to traditional GBC diagnostic methods in that it detects CTCs. Furthermore, the bismuth-film-modified detection electrode enhances sensor sensitivity. Furthermore, the dual-targeting of ENPP1 and EpCAM in the electrochemical immunoassay platform significantly improves specificity. Therefore, this dual-recognition electrochemical immunoassay platform provides a new approach for the early detection of GBC and has great potential for clinical translation.
[0034] The specific chemical materials required are shown in Table 1:
[0035] Table 1,
[0036]
[0037] Specific preparation and testing steps and methods:
[0038] Preparation and detection steps:
[0039] A. Synthesis of SiO2 nanospheres: First, add 50 mL of anhydrous ethanol, 2 mL of 28% ammonia water, and 3 mL of deionized water, stirring at room temperature for 20 minutes at 400 rpm. Then, add 2 mL of TEOS at once and stir at room temperature for 4 hours. After the reaction is complete, centrifuge the solution at 8000 rpm, wash twice with anhydrous ethanol, and finally resuspend in 10 mL of anhydrous ethanol for later use.
[0040] B. Synthesis of SiO2@QDs: The synthesis of this composite requires the introduction of a positively charged polymer, polyethylenimine (PEI). PEI is readily soluble in water and self-assembles under ultrasound to form a PEI interlayer on the SiO2 surface. Subsequently, carboxyl-grouped CdSe / ZnS QDs are coated onto the SiO2 nanospheres through electrostatic adsorption.
[0041] C. Synthesis of SiO2@QD-Ab: Take 2mL of SiO2@QD, centrifuge at 5500rpm×10min, resuspend in 1mL of 0.1M MES, add 100μL of EDC (10mM) and 20μL of NHS (100mM), vortex mix, and sonicate for 15min. The above solution is centrifuged at 5500rpm×10min, the supernatant is discarded, and resuspended in 200μL of 0.05% PBST. 30μL of antibody (anti-ENPP1, 0.5mg / mL) is added, and the solution is incubated on a shaker at room temperature for 2h; 100μL of 5% BSA is added, and the solution is incubated at room temperature for 1h, 4500rpm×10min, the supernatant is discarded, and the solution is washed once with 0.05% PBST. Finally, the solution is resuspended in 0.2mL of 0.05% PBST.
[0042] D. Construction of immunoelectrochemical platform: First, the gold electrode was polished to a smooth mirror surface with 0.3μm and 0.05μm alumina powder, then sonicated in ultrapure water, ethanol, and ultrapure water for 5 minutes, respectively. Then, it was activated with freshly prepared piranha solution (98% H2SO4, 30% H2O2, mixed in a volume ratio of 3:1) for 15 minutes, and then sonicated in ultrapure water, ethanol, and ultrapure water for 5 minutes. The gold electrode was dried at room temperature and set aside. 10μL of 200nM SH-EpCAM aptamer was dropped on the gold electrode (pre-activated with 1mM TCEP at room temperature for 1 hour) and incubated at 4°C overnight. The capture electrode was removed and the unbound aptamer was washed with 0.01mM pH7.4 PBS. After natural drying, 5μL of 0.1M MCH was added to prevent nonspecific adsorption. 0.01mM pH7.4 After washing with PBS and drying naturally, the NOZ cells were incubated at 37°C for 60 min; unbound cells were washed, 5 μL SiO2@QD-Ab was added, and incubated for 120 min; unbound materials were washed, 30 μL HNO3 was added for digestion for 10 min, and the digestion solution was transferred to 3 mL acetic acid-acetic acid (pH 4.5) solution.
[0043] E. Bismuth Film-Modified Glassy Carbon Electrode and Detection: First, a glassy carbon electrode was polished to a smooth mirror surface using 0.3μm and 0.05μm alumina powders. The electrode was then sonicated in ultrapure water, ethanol, and ultrapure water for 5 minutes each. The electrode was then activated with freshly prepared nitric acid:acetone solution (1:1 by volume) for 15 minutes. The electrode was then sonicated in ultrapure water, ethanol, and ultrapure water for 5 minutes each. The electrode was dried at room temperature and set aside. A 10mL acetic acid solution (pH 4.5) containing 12.5mg / mL bismuth ions was prepared. The electrode was deposited using the 1 / 2-μm constant potential of -1.2V for 300 seconds. SWV detection was performed using the bismuth film-modified glassy carbon electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, and a Pt wire electrode as the counter electrode. A greater number of cells binds more reporter molecules, resulting in a stronger electrochemical signal peak.
[0044] Testing items and testing methods,
[0045] Table 2,
[0046] Test items Detection method <![CDATA[Successful synthesis of SiO2]]> SEM <![CDATA[The successful synthesis of SiO2-PEI]]> TEM <![CDATA[Successful synthesis of SiO2@QD]]> TEM <![CDATA[The successful synthesis of SiO2@QD-Ab]]> Immunofluorescence, flow cytometry The capture electrode was successfully constructed CV, EIS Bismuth film electrode successfully constructed SEM Detection of cell linearity SWV
[0047] according to Figure 1 ,Conclusion: SiO2 was successfully synthesized;
[0048] according to Figure 2 ,Conclusion: PEI was successfully loaded onto SiO2 nanoparticles;
[0049] according to Figure 3 ,Conclusion: QDs were successfully loaded onto SiO2 nanoparticles;
[0050] according to Figure 4 ,Conclusion: SiO2@QD-Ab is successfully synthesized and has targeting properties;
[0051] according to Figure 5 , a. Bare electrode, b. Aptamer, c. Aptamer + MCH, d. Aptamer + MCH + NOZ; Conclusion:
[0052] The capture electrode was successfully constructed;
[0053] according to Figure 6 ,Conclusion: The bismuth film electrode was successfully modified;
[0054] according to Figure 7 , a.1x10 1 , b.1x10 2 , c.1x10 3 , d.1x10 4 、e.1x10 5 、f.1x10 6 , Y=3.22183X-3.06759R 2=0.98859; Conclusion: The peak value of electrochemical signal is positively correlated with the number of cells.
[0055] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.
Claims
1. A method for preparing an ultrasensitive electrochemical immunosensor for specifically detecting CTCs in gallbladder cancer, characterized by: The following steps are involved: a. Synthesis of SiO2 nanospheres; b. SiO2@QD was synthesized using the SiO2 nanospheres synthesized in step a. c. SiO2@QD-Ab was prepared using the SiO2@QD synthesized in step b. d. The SiO2@QD-Ab synthesized in step c was used to construct an immunoelectrochemical platform; In step d, the gold electrode was first polished to a smooth mirror surface with 0.3 mm and 0.05 mm alumina powder, and then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 min, respectively, and then activated with freshly prepared piranha solution for 15 min, and then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 min, respectively; dried at room temperature and set aside; 10 μL of 200 nM SH-EpCAM aptamer was dropped on the gold electrode and incubated at 4°C overnight; the capture electrode was removed and the unbound aptamer was washed with 0.01 mM pH 7.4 PBS. After natural drying, 5 μL of 0.1 M MCH was added to prevent nonspecific adsorption; washed with 0.01 mM pH 7.4 PBS, and after natural drying, NOZ cells were incubated at 37°C for 60 min; unbound cells were washed, 5 μL of the obtained SiO2@QD-Ab was added, and incubated for 120 min; unbound materials were washed, and 30 μL of Digest with HNO3 for 10 min, and transfer the digestion solution to 3 mL of acetic acid-acetic acid solution; In step c, 2 mL of SiO2@QD synthesized in step b was taken, centrifuged at 5500 rpm for 10 min, resuspended in 1 mL of 0.1M MES, 100 μL of EDC and 20 μL of NHS were added, vortexed and sonicated for 15 min, and the resulting solution was centrifuged at 5500 rpm for 10 min. The supernatant was discarded and resuspended in 200 μL of 0.05% PBST. 30 μL of anti-ENPP1 antibody was added and incubated on a shaker at room temperature for 2 h; 100 μL of 5% BSA was added and incubated at room temperature for 1 h, 4500 rpm for 10 min, the supernatant was discarded, and the solution was washed once with 0.05% PBST. Finally, the solution was resuspended in 0.2 mL of 0.05% PBST to synthesize SiO2@QD-Ab.
2. The method for preparing an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs according to claim 1, characterized in that: In step a, first add 50 mL of anhydrous ethanol, 2 mL of 28% ammonia water, and 3 mL of deionized water in sequence, and stir at room temperature for 20 minutes; the rotation speed is 400 rpm; then add 2 mL of TEOS at once, and stir at room temperature for 4 hours; after the reaction is completed, the solution is centrifuged at 8000 rpm, washed twice with anhydrous ethanol, and finally resuspended in 10 mL of anhydrous ethanol for use and synthesized SiO2 nanospheres.
3. The method for preparing an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs according to claim 1, characterized in that: In step b, a positively charged polymer, polyethyleneimine (PEI), is introduced. PEI is easily soluble in water and can self-assemble on the SiO2 surface under ultrasonic conditions to form a PEI interlayer. Then, through electrostatic adsorption, carboxyl-containing CdSe / ZnS QDs are coated on the surface of SiO2 nanospheres to synthesize SiO2@QD.
4. An ultrasensitive electrochemical immunosensor for specific detection of gallbladder cancer CTCs, characterized by: The invention comprises an immunoelectrochemical platform as described in any one of claims 1 to 3; and also comprises a bismuth film-modified glassy carbon electrode and detection: first, the glassy carbon electrode is polished into a smooth mirror surface with 0.3 µm and 0.05 µm alumina powder, and then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes respectively, and then activated with a freshly prepared nitric acid: acetone solution mixed in a volume ratio of 1:1 for 15 minutes, and then ultrasonicated in ultrapure water, ethanol, and ultrapure water for 5 minutes; dried at room temperature and set aside; prepared 10 mL of 12.5 mg / mL bismuth ion acetic acid solution; using the IT deposition method, applying a constant potential of -1.2 V, and depositing for 300 seconds; using the glassy carbon electrode modified with the bismuth film as the working electrode, Ag / AgCl as the reference electrode, and a Pt wire electrode as the auxiliary electrode, for SWV detection; the more cells there are, the more signal reporter molecules are bound, and therefore the stronger the electrochemical signal peak.
5. The ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs according to claim 4, characterized in that: Piranha solution was prepared by mixing 98% H2SO4 and 30% H2O2 in a volume ratio of 3:
1. SH-EpCAM was activated in advance with 1 mM TCEP at room temperature for 1 h. The pH of the acetic acid-acetic acid solution is 4.5.