CN115901895B

CN115901895B - Preparation and application methods of ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs

Info

Publication number: CN115901895B
Application number: CN202211460426.6A
Authority: CN
Inventors: 王建为; 张秀珍; 李露; 彭奇龄; 蒋宁
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2022-11-21
Filing date: 2022-11-21
Publication date: 2025-09-09
Anticipated expiration: 2042-11-21
Also published as: CN115901895A

Abstract

The present invention relates to the field of medical technology and is a method for preparing an ultrasensitive electrochemical immunosensor for specifically detecting CTCs in gallbladder cancer. The method 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. The present invention also discloses a method for applying the ultrasensitive electrochemical immunosensor for specifically detecting CTCs in gallbladder cancer. The method exhibits high sensitivity, high specificity, low cost, and rapid detection speed.

Description

Preparation and application methods of ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation and application method of an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs.

Background

Gallbladder cancer (gallbladder carcinoma, GBC) is a common malignant tumor of biliary tract system, and has the characteristics of high concealment, high malignancy, easy invasion and metastasis, difficult early diagnosis, poor prognosis and the like. Common methods for clinically diagnosing GBC include imaging, hematology, genomics, pathological analysis, and the like. The B ultrasonic detection in the imaging method is the clinical preferred method, but the method has high misdiagnosis rate, the hematology method based on CEA and CA199 has no specificity, the genomics has poor sensitivity, and the pathological analysis is the gold standard of diagnosis but belongs to the traumatic detection. Therefore, it is necessary to find a detection method with strong specificity, high sensitivity and no wound. Along with the implementation of accurate medical planning, scientists are increasingly popular in researching CTCs, and CTCs detection has great significance in aspects of early diagnosis, curative effect evaluation, prognosis monitoring and the like. In recent years, electrochemical immunosensors have been popular for use in CTCs research due to advantages of high sensitivity, high specificity, low cost, rapid detection speed, and the like. In addition, quantum Dots (QDs) are widely used because they have excellent electrochemical characteristics. Therefore, this study was designed to design a quantum dot signal responsive electrochemical immunosensor to detect early stage cholecystocarcinoma CTCs. The implementation of the research can provide a new detection method for the diagnosis of early stage cholecystocarcinoma in clinic, and has important clinical application prospect and social value.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a method for preparing and applying an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs, which has the advantages of high sensitivity, high specificity, low cost and high detection speed.

The invention provides a preparation method of a specific gall bladder cancer CTCs ultrasensitive electrochemical immunosensor, which is characterized by comprising the following steps of synthesizing a and SiO ₂ nanospheres, synthesizing SiO ₂ nanospheres synthesized in the step a to obtain SiO ₂ @QDs, c preparing SiO ₂ @QD-Ab by adopting the SiO ₂ @QDs synthesized in the step b, and d constructing an immune electrochemical platform by adopting the SiO ₂ @QD-Ab synthesized in the step c.

In the step a, 50mL of absolute ethyl alcohol, 2mL of 28% ammonia water and 3mL of deionized water are sequentially added, stirring is carried out at room temperature for 20min, the rotating speed is 400rpm, then 2mL of TEOS is added at one time, stirring is carried out at room temperature for 4h, after the reaction is finished, the solution is centrifuged at 8000rpm, the absolute ethyl alcohol is used for washing twice, and finally the solution is resuspended in 10mL of absolute ethyl alcohol for standby and synthesized to obtain the SiO ₂ nanospheres.

In the step b, an electropositive polymer polyethylenimine PEI is introduced, the PEI is easy to dissolve in water, a PEI interlayer can be formed on the surface of SiO ₂ in a self-assembly mode under the ultrasonic condition, and then carboxyl CdSe/ZnS QDs are coated on the surface of SiO ₂ nanospheres through electrostatic adsorption and are synthesized to obtain the SiO ₂ @QD.

As optimization, in step c, 2mL of SiO ₂ @QD synthesized in step b was taken, centrifuged at 5500rpm for 10min, resuspended in 1mL of 0.1M MES, added with 100. Mu.L of EDC and 20. Mu. LNHS, vortexed, sonicated for 15min, the resulting solution centrifuged at 5500rpm for 10min, the supernatant was discarded, resuspended in 200. Mu.L of 0.05% PBST, added with 30. Mu.L of anti-ENPP1 antibody, placed on a shaker for 2h incubation at room temperature, added with 100. Mu.L of 5% BSA, incubated at room temperature for 1h at 4500rpm for 10min, the supernatant was discarded, 0.05% PBST washed once, finally resuspended in 0.2mL of 0.05% PBST and synthesized to obtain SiO ₂ @QD-Ab.

In the step d, firstly, polishing and grinding a gold electrode with alumina powder with the thickness of 0.3 mu M and 0.05 mu M to form a smooth mirror surface shape, then respectively carrying out ultrasonic treatment on the gold electrode in ultrapure water, ethanol and ultrapure water for 5min, then activating the gold electrode with freshly prepared piranha solution for 15min, then continuing ultrasonic treatment on the ultrapure water, ethanol and ultrapure water for 5min, drying at room temperature for standby, dripping 10 mu L of 200nM SH-EpCAM aptamer on the gold electrode for 4 ℃ overnight incubation, taking out a capture electrode, washing the unbound aptamer with 0.01mM pH7.4 PBS, naturally drying, dripping 5 mu L of 0.1M MCH to prevent nonspecific adsorption, washing with 0.01mM pH7.4 PBS, naturally drying, incubating NOZ mu L of unbound cells for 60min, adding 5 mu L of obtained SiO ₂ @QD-120 min, washing unbound Ab material, adding 30 mu L of HNO3 for digestion for 10min, and transferring digestion solution into 3mL of acetic acid-acetic acid solution for incubation.

The invention also discloses an application method of the ultra-sensitive electrochemical immunosensor for specifically detecting the gall bladder cancer CTCs, which comprises the steps of constructing the immune electrochemical platform, further comprising a bismuth film modified glassy carbon electrode and detection, wherein the glassy carbon electrode is polished and polished into a smooth mirror surface shape by using alumina powder with the thickness of 0.3 mu m and 0.05 mu m, then the glassy carbon electrode is respectively ultrasonically treated in ultrapure water, ethanol and ultrapure water for 5min, then the freshly prepared nitric acid is mixed and activated in the volume ratio of 1:1 for 15min, then the ultrasonic treatment is continuously carried out in the ultrapure water, ethanol and ultrapure water for 5min, the room temperature is dried for standby, 10mL of bismuth ion acetic acid solution with the concentration of 12.5mg/mL is prepared, an i-t deposition method is adopted, a constant potential of-1.2V is applied, the glassy carbon electrode with the modified bismuth film is used as a working electrode, ag/AgCl is used as a reference electrode, a Pt wire electrode is used as an auxiliary electrode, and SWV detection is carried out, and the more cells are required, and the more signal reporting molecules are combined, so that the electrochemical signal peak value is stronger.

As optimization, the piranha solution is obtained by mixing 98% of H ₂SO₄ and 30% of H ₂O₂ according to a volume ratio of 3:1, SH-EpCAM is activated for 1H at room temperature by using 1mM of TCEP in advance, and the pH of acetic acid-acetic acid solution is 4.5.

As optimization, SYL3C is an aptamer aiming at an EpCAM target, ab is an antibody aiming at an ENPP1 target, and specific materials of SiO ₂ are APTES and TEOS.

In summary, in the present solution, firstly, thiol-modified aptamer (SYL 3C) is bound to gold electrode by using the principle of "Au-S" bond binding, so GBC cells highly expressing EpCAM can be captured. Then preparing CdSe/ZnS quantum dot-loaded SiO ₂ nanospheres (called SiO ₂ @QD-Ab) modified by antibody (anti-ENPP 1 anti-ibody) capable of specifically targeting GBC. The nanosphere can specifically target gallbladder cancer cells with high expression of ENPP1, and the quantum dots can be used as electrochemical signal probes for detecting electrochemical signals. Only when the cells are simultaneously expressing both ENPP1 and EpCAM are the detection signals available.

In conclusion, the aptamer-cell-signal probe sandwich type gallbladder cancer cell detection method constructed by the invention has the advantages that compared with the traditional method for diagnosing GBC, the detection sample is CTCs, so that the method has noninvasive advantages, in addition, the bismuth film modified detection electrode can enhance the sensitivity of the sensor, and meanwhile, ENPP1 and EpCAM are selected as double targets of an electrochemical immune platform, so that the specificity is remarkably improved. Therefore, the dual recognition electrochemical immune platform provides a new approach for the early detection of GBC and has good clinical transformation potential.

Drawings

FIG. 1 is a photograph of SiO ₂ after successful synthesis.

Fig. 2 is a photograph of successful loading of PEI onto SiO ₂ nanoparticles.

Fig. 3 is a picture of the successful synthesis of SiO ₂ @ QD.

FIG. 4 is a photograph of successful synthesis (immunofluorescence) of SiO ₂ @ QD-Ab.

Fig. 5 is a picture of the construction of a capture electrode.

Fig. 6 is a photograph of bismuth membrane electrode modification.

FIG. 7 is a graph of electrochemical detection versus cell concentration.

Detailed Description

The present invention will be further described with reference to the drawings and examples, and it should be noted that in the description of the present invention, the terms "upper", "lower", "left", "right", "inner", "outer", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, only for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific manner, and thus should not be construed as limiting the present invention. The terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

The embodiment of the preparation method of the ultra-sensitive electrochemical immunosensor for specifically detecting the gall bladder cancer CTCs comprises the following steps of a, synthesizing SiO ₂ nanospheres, b, synthesizing SiO ₂ nanospheres obtained through synthesis in the step a to obtain SiO ₂ @QDs, c, preparing SiO ₂ @QD-Ab through the SiO ₂ @QDs obtained through synthesis in the step b, and d, constructing an immune electrochemical platform through the SiO ₂ @QD-Ab obtained through synthesis in the step c.

In the specific embodiment, in the step a, 50mL of absolute ethyl alcohol, 2mL of 28% ammonia water and 3mL of deionized water are sequentially added, stirring is carried out at room temperature for 20min, the rotating speed is 400rpm, then 2mL of TEOS is added at one time, stirring is carried out at room temperature for 4h, after the reaction is finished, the solution is centrifuged at 8000rpm, the absolute ethyl alcohol is used for washing twice, and finally the solution is resuspended in 10mL of absolute ethyl alcohol for standby and synthesized to obtain the SiO ₂ nanospheres.

In the specific embodiment, in the step b, an electropositive polymer polyethylenimine PEI is introduced, PEI is easy to dissolve in water, a PEI interlayer can be formed on the surface of SiO ₂ in a self-assembly mode under the ultrasonic condition, and then carboxyl CdSe/ZnS QDs are coated on the surface of SiO ₂ nanospheres through electrostatic adsorption, and SiO ₂ @QD is obtained through synthesis.

In this embodiment, in step c, 2mL of the SiO ₂ @QDs synthesized in step b were centrifuged at 5500rpm for 10min, resuspended in 1mL of 0.1M MES, added with 100. Mu.L of EDC and 20. Mu. LNHS, vortexed, mixed well, sonicated for 15min, the resulting solution centrifuged at 5500rpm for 10min, the supernatant discarded, resuspended in 200. Mu.L of 0.05% PBST, added with 30. Mu.L of anti-ENPP1 antibody, incubated on an shaker at room temperature for 2h, added with 100. Mu.L of 5% BSA, incubated at room temperature for 1h at 4500rpm for 10min, the supernatant discarded, 0.05% PBST washed once, finally resuspended in 0.2mL of 0.05% PBST and synthesized to give the SiO ₂ @QD-Ab.

Specifically, EDC was 10mM and NHS was 100mM.

In the specific embodiment, in the step d, firstly, a gold electrode is polished and polished to be smooth mirror surface by using alumina powder with the thickness of 0.3 mu M and 0.05 mu M, then ultrasonic treatment is carried out on the gold electrode in ultrapure water, ethanol and ultrapure water for 5min respectively, then the gold electrode is activated by using freshly prepared piranha solution for 15min, then ultrasonic treatment is continued on the ultrapure water, ethanol and ultrapure water for 5min, room temperature drying is carried out for standby, 10 mu L of 200nM SH-EpCAM aptamer is dripped on the gold electrode, 4 ℃ is incubated overnight, a capturing electrode is taken out, the unbound aptamer is washed by using 0.01mM pH7.4 PBS, after natural drying, 5 mu L of 0.1M MCH is dripped to prevent nonspecific adsorption, 0.01mM pH7.4 PBS is used for washing, after natural drying, NOZ mu L of cells are incubated for 60min at 37 ℃,5 mu L of obtained SiO ₂ @QD-Ab is added for 120min is washed, 30 mu L of HNO3 is added for digestion for 10min, and the digestion solution is transferred into 3mL of acetic acid-acetic acid solution.

The application method of the ultra-sensitive electrochemical immunosensor for specifically detecting the gall bladder cancer CTCs comprises the steps of constructing an immune electrochemical platform, further comprising a bismuth film modified glassy carbon electrode and detection, wherein the glassy carbon electrode is polished and polished into a smooth mirror surface shape by using alumina powder with the thickness of 0.3 mu m and 0.05 mu m, then the glassy carbon electrode is respectively subjected to ultrasonic treatment in ultrapure water, ethanol and ultrapure water for 5min, then the freshly prepared nitric acid is mixed and activated for 15min by using acetone solution according to the volume ratio of 1:1, then the ultrasonic treatment is continued in the ultrapure water, ethanol and ultrapure water for 5min, drying at room temperature for standby, preparing 10mL of bismuth ion acetic acid solution with the concentration of 12.5mg/mL, applying constant potential of-1.2V by adopting an i-t deposition method, depositing 300s, taking the glassy carbon electrode with the modified bismuth film as a working electrode, ag/AgCl as a reference electrode and taking a Pt wire electrode as an auxiliary electrode, and carrying out SWV detection, and the more cells are required, so that the electrochemical signal peak value is stronger.

In this embodiment, the piranha solution is prepared by mixing 98% H ₂SO₄ and 30% H ₂O₂ in a volume ratio of 3:1, SH-EpCAM is activated in advance with 1mM TCEP at room temperature for 1H, and the pH of the acetic acid-acetic acid solution is 4.5.

In this embodiment, the aptamer SYL3C targets EpCAM, the Ab targets ENPP1, and the SiO ₂ is APTES or TEOS.

Specifically, the manufacturer of the raw material SYL3C is Abcam, cat No.223268 and USA, the manufacturer of the raw material CdSe/ZnS QDs is scintillant (Suzhou), the manufacturer of the raw material SiO ₂ is Macklin (Shanghai) and the manufacturer of the raw material bismuth (III) nitrate pentahydrate is Macklin (Shanghai) of the Co.

In summary, in the present solution, firstly, thiol-modified aptamer (SYL 3C) is bound to gold electrode by using the principle of "Au-S" bond binding, so GBC cells highly expressing EpCAM can be captured. Then preparing CdSe/ZnS quantum dot-loaded SiO ₂ nanospheres (called SiO ₂ @QD-Ab) modified by antibody (anti-ENPP 1 anti-ibody) capable of specifically targeting GBC. The nanosphere can specifically target gallbladder cancer cells with high expression of ENPP1, and the quantum dots can be used as signal probes for detecting electrochemical signals. Only when the cells express both ENPP1 and EpCAM can the detection signal be detected.

The specific required chemical materials are shown in table 1:

In the table 1 of the present invention,

The specific preparation detection steps and detection methods are as follows:

the preparation and detection steps are as follows:

the method comprises the steps of A, synthesizing SiO ₂ nanospheres, namely sequentially adding 50mL of absolute ethyl alcohol, 2mL of 28% ammonia water and 3mL of deionized water, stirring at room temperature for 20min at a rotation speed of 400rpm, adding 2mL of TEOS at one time, stirring at room temperature for 4h, centrifuging the solution at 8000rpm after the reaction is finished, washing twice by using the absolute ethyl alcohol, and finally re-suspending in 10mL of absolute ethyl alcohol for later use.

And B, synthesizing SiO ₂ @QD, namely introducing electropositive polymer Polyethylenimine (PEI) into the synthesis of the compound, wherein the PEI is easy to dissolve in water, and can self-assemble on the surface of SiO ₂ to form a PEI interlayer under the ultrasonic condition. And then coating the CdSe/ZnS QDs with carboxyl groups on the surface of the SiO ₂ nanospheres through electrostatic adsorption.

Synthesis of SiO ₂ @ QD-Ab by taking 2mLSiO ₂ @ QD, centrifuging at 5500rpm x 10min, re-suspending in 1mL of 0.1M MES, adding 100. Mu.L of EDC (10 mM) and 20. Mu. LNHS (100 mM), vortex mixing, sonicating for 15min, centrifuging the above solution at 5500rpm x 10min, discarding supernatant, re-suspending in 200. Mu.L of 0.05% PBST, adding 30. Mu.L of antibody (anti-ENPP 1,0.5 mg/mL), incubating on a shaker at room temperature for 2h, adding 100. Mu.L of 5% BSA, incubating at room temperature for 1h with shaking at 4500rpm x 10min, discarding supernatant, washing once with 0.05% PBST, and finally re-suspending in 0.2mL of 0.05% PBST.

D. The construction of an immunochemical platform comprises the steps of polishing and grinding a gold electrode with alumina powder of 0.3 μm and 0.05 μm into a smooth mirror surface shape, respectively carrying out ultrasonic treatment for 5min in ultrapure water, ethanol and ultrapure water, activating with freshly prepared piranha solution (98% H ₂SO₄,30％ H₂O₂ and mixing according to the volume ratio of 3:1) for 15min, then continuing ultrasonic treatment in ultrapure water, ethanol and ultrapure water for 5min, drying at room temperature for standby, dripping 10 mu L of 200nM SH-EpCAM aptamer on the gold electrode (1 mM TCEP is used for 1H in advance), incubating overnight at 4 ℃, taking out a capturing electrode, washing unbound ligand with 0.01mM PBS 7.4, naturally drying, dripping 5 mu L of 0.1M MCH to prevent nonspecific adsorption, washing with 0.01mM PBS 7.4, naturally drying, washing NOZ cells for 60min at 37 ℃, adding 5 mu L of SiO ₂ QD-Ab, 120min, washing unbound material, adding 30 mu L of HNO ₃, and transferring to acetic acid solution (pH: 3) for 3.4 min.

E. The bismuth film modified glassy carbon electrode and detection method comprises the steps of firstly polishing and grinding the glassy carbon electrode into a smooth mirror surface shape by using alumina powder with the concentration of 0.3 mu m and 0.05 mu m, then respectively carrying out ultrasonic treatment in ultrapure water, ethanol and ultrapure water for 5min, then activating by using freshly prepared nitric acid acetone solution (mixed according to the volume ratio of 1:1) for 15min, then continuing ultrasonic treatment in the ultrapure water, ethanol and ultrapure water for 5min, drying at room temperature for standby, preparing 10mL of bismuth ion acetic acid solution (pH 4.5) with the concentration of 12.5mg/mL, applying-1.2V constant potential by an i-t deposition method, and depositing for 300s. And (3) taking the glassy carbon electrode modified with the bismuth film as a working electrode, ag/AgCl as a reference electrode, and a Pt wire electrode as an auxiliary electrode to perform SWV detection. The greater the number of cells, the greater the number of bound signal reporters and therefore the greater the electrochemical signal peak.

A detection item and a detection method thereof,

In the table 2 of the present application,

Detecting items	Detection method
		Successful synthesis of SiO ₂	SEM
Successful synthesis of SiO ₂ -PEI	TEM
		Successful synthesis of SiO ₂ @QD	TEM
Successful synthesis of SiO ₂ @ QD-Ab	Immunofluorescence, flow
		Successful construction of Capture electrode	CV、EIS
Successful construction of bismuth membrane electrode	SEM
		Detecting cell linearity	SWV

According to FIG. 1, it is concluded that SiO ₂ was successfully synthesized;

From FIG. 2, it was concluded that PEI was successfully loaded onto SiO ₂ nanoparticles;

From fig. 3, it was concluded that QDs were successfully loaded onto SiO ₂ nanoparticles;

From FIG. 4, it was concluded that SiO ₂ @ QD-Ab was successfully synthesized and targeted;

from fig. 5, a. Bare electrode, b. Aptamer, c. Aptamer+mch, d. Aptamer+mch+ NOZ, conclusion:

Successful construction of the capture electrode;

from FIG. 6, it was concluded that bismuth membrane electrode was successfully modified;

From FIG. 7,a.1x10¹、b.1x10²、c.1x10³、d.1x10⁴、e.1x10⁵、f.1x10⁶、Y＝3.22183X-3.06759R²＝0.98859;, it was concluded that the electrochemical signal peak was positively correlated with the number of cells.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims

1. A method for preparing an ultrasensitive electrochemical immunosensor for specifically detecting gallbladder cancer CTCs, 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;

In step d, the gold electrode was first polished to a smooth mirror surface with 0.3 mm and 0.05 mm alumina powder, then sonicated in ultrapure water, ethanol, and ultrapure water for 5 min, activated with freshly prepared piranha solution for 15 min, and then sonicated in ultrapure water, ethanol, and ultrapure water for 5 min; 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; the capture electrode was washed with 0.01 mM pH 7.4 PBS, and after natural drying, NOZ cells were incubated at 37°C for 60 min; the unbound cells were washed, and 5 μL of the obtained _SiO2 @QD-Ab was added and incubated for 120 min; the unbound material was washed and 30 μL of HNO was added _3. Digest for 10 min and transfer the digestion solution into 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, 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 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, 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 rotation speed of 400 rpm; then 2 mL of TEOS is added at once 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 to synthesize _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 readily soluble in water and can self-assemble on the _SiO2 surface under ultrasonic conditions to form a PEI interlayer; then, carboxyl-containing CdSe/ZnS QDs are coated on the surface of the _SiO2 nanospheres by electrostatic adsorption to synthesize _SiO2 @QD.

4. An ultrasensitive electrochemical immunosensor for specifically detecting CTCs in gallbladder cancer, comprising: an immunoelectrochemical platform according to any one of claims 1-3; and a bismuth-film-modified glassy carbon electrode. The glassy carbon electrode is first polished to a smooth mirror surface with 0.3 µm and 0.05 µm alumina powders. The electrode is then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes, respectively. The electrode is then activated with a freshly prepared nitric acid:acetone solution (1:1 by volume) for 15 minutes. The electrode is then ultrasonically treated in ultrapure water, ethanol, and ultrapure water for 5 minutes. The electrode is then dried at room temperature and set aside. A 12.5 mg/mL bismuth ion acetate solution is prepared. An iterative deposition method is employed, applying a constant potential of -1.2 V for 300 seconds. SWV detection is 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 auxiliary electrode. A greater number of cells indicates a greater number of bound reporter molecules, resulting in a stronger electrochemical signal peak.

5. An ultrasensitive electrochemical immunosensor for specifically detecting CTCs _in gallbladder cancer according to claim 4, characterized in that: the piranha solution is prepared by mixing 98% _H2SO4 and 30% _H2O2 in a volume ratio of 3:1; the SH-EpCAM is pre-activated _with 1 mM TCEP at room temperature for 1 h;

The pH of the acetic acid-acetic acid solution is 4.5.