An ECL biosensor and its preparation and detection methods
By using PANI substrate, AuNPs, SA@Ru-M1 solution, and aptamer SYL3C on the ECL sensing platform, the problem of weak CTC detection signal was solved, achieving high sensitivity and high specificity detection of MCF-7 cells, and improving detection accuracy and anti-interference ability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-25
- Publication Date
- 2026-03-10
AI Technical Summary
Existing ECL sensing platforms suffer from extremely weak ECL signals when detecting circulating tumor cells (CTCs) due to the large spatial steric hindrance posed by the cells, which limits their application in cancer diagnosis and treatment.
Using PANI as a soft substrate, combined with AuNPs, SA@Ru-M1 solution and aptamer SYL3C, specific recognition of MUC1 protein was achieved through hybridization chain reaction (HCR), constructing an ECL biosensor to enhance cell capture and detection capabilities.
It achieves highly sensitive and specific quantitative detection of MCF-7 breast cancer cells, with a linear detection range of 102–106 cells/mL and a detection limit of up to 31 cells/mL, thus improving detection accuracy and anti-interference ability.
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Figure CN116698947B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biosensors, and particularly relates to an ECL biosensor and a preparation method and a detection method thereof. BACKGROUND
[0002] Circulating tumor cells (CTCs) are various cancer cells separated from primary tumors and migrated into peripheral blood, and are closely related to cancer metastasis, so that detection and analysis of CTCs will provide an important basis for clinical diagnosis, efficacy monitoring and related biological research of cancer. Electrochemiluminescence (ECL) combines the low background signal, strong controllability and high sensitivity of electrochemistry and chemiluminescence, and is widely used in biological fields such as immune analysis, bacterial detection, food detection and cell imaging. Because the space steric hindrance of CTCs on the electrode surface is large, the ECL signal may be extremely weak, which limits the development of ECL in the field. Therefore, developing a sensitive ECL sensing platform to realize efficient capture and detection of CTCs will play an important role in the clinical diagnosis and treatment of cancer patients. SUMMARY
[0003] The purpose of the application is to overcome the deficiencies in the prior art, provide an ECL biosensor and a preparation method and a detection method thereof, and the prepared ECL biosensor has high cell capture efficiency, high detection accuracy and strong anti-interference ability, and can realize high-sensitivity and high-specificity quantitative detection of MCF-7 cells.
[0004] The application provides the following technical solutions:
[0005] In a first aspect, a preparation method of an ECL biosensor is provided, comprising the following steps:
[0006] The pretreated ITO (indium tin oxide) electrode is placed in a mixed aqueous solution of aniline and hydrochloric acid for electrodeposition to obtain an electrode on which PANI is deposited;
[0007] Au seed solution, HAuCl4 (chloroauric acid) solution and MSA (sodium thiomalate) solution are sequentially added to water, and stirred to obtain AuNPs (gold nanoparticles) solution;
[0008] The ruthenium complex solution of anhydrous DMSO (dimethyl sulfoxide), SA (streptavidin) solution and PBS buffer solution are mixed, stirred and dialyzed overnight to obtain SA@Ru solution;
[0009] M1 chain, H1 chain and H2 chain are subjected to hybridization chain reaction (HCR), and then mixed and oscillated with the SA@Ru solution to obtain SA@Ru-M1 solution;
[0010] The AuNPs solution is dropped onto the surface of the electrode on which PANI is deposited for incubation, and then the pretreated SYL3C solution is dropped onto the surface of the electrode after cleaning for incubation, so as to obtain the aptamer-modified electrode;
[0011] The aptamer-modified electrode is soaked in the BSA solution, and then the cell to be tested is dropped onto the surface of the electrode, and after constant-temperature culture, cell fixation and permeation are performed, and finally the SA@Ru-M1 solution is dropped onto the surface of the electrode for incubation, so as to obtain the ECL biosensor.
[0012] Further, the pretreatment method of the ITO electrode comprises: sequentially ultrasonic cleaning the ITO electrode with a detergent, acetone, ethanol and water, drying the ITO electrode at 70-120℃ for 30-60 min, and then pasting an insulating tape with a pore diameter of 3-8 mm on the conductive surface of the ITO electrode for controlling the reaction area in the electrodeposition process.
[0013] Further, the electrodeposition method comprises: taking the pretreated ITO electrode as a working electrode, a Pt electrode as an auxiliary electrode, and an Ag / AgCl electrode as a reference electrode, and performing electrodeposition in aniline and hydrochloric acid mixed aqueous solution by a CHI660E electrochemical workstation by a cyclic voltammetry method, and the scanning speed is set to 100 mV / s, and scanning is performed for 4-6 cycles between 0.2-1.2V; wherein the volume of the aniline and hydrochloric acid mixed aqueous solution is 6-8 mL, and the concentration ratio of aniline to hydrochloric acid is (1:2)-(1:4).
[0014] Further, the specific preparation method of the AuNPs solution comprises:
[0015] The HAuCl4 solution with a mass fraction of 0.01-0.02% is placed in a flask, a condensation reflux device is connected, oil bath heating is performed to boiling, the sodium citrate solution with a concentration of 0.1-0.2M is added and stirred, boiling is continued, when the solution turns into wine red, heating is stopped, and stirring is performed at 25℃ for 10-20 min, so as to obtain the Au seed solution;
[0016] The Au seed solution, the HAuCl4 solution and the MSA solution with a concentration of 10mM are sequentially added into water, and stirring is performed at room temperature for 2-3h.
[0017] Further, the specific preparation method of the SA@Ru solution comprises: mixing the ruthenium complex solution of anhydrous DMSO, the SA solution and the PBS buffer solution, stirring at 4℃ for 3-6h, and dialysis overnight, so as to obtain the SA@Ru solution.
[0018] Further, the specific preparation method of the SA@Ru-M1 solution comprises the following steps: dissolving M1 chain, H1 chain and H2 chain in PBS buffer, heating at 90-95℃ for 5-10 min, and then cooling at 4℃ for 5-10 min to reduce the influence of the spatial folding of the DNA chain on the hybridization process, then oscillating at 37℃ for 1-3 h to make the HCR process fully proceed, and then oscillating with the SA@Ru solution for 0.5-1 h.
[0019] Further, the specific preparation method of the aptamer-modified electrode comprises the following steps: adding AuNPs solution to the electrode surface on which PANI is deposited, and incubating for 0.5-1 h to make the electrostatic adsorption process of the two fully act; then cleaning three times with PBS buffer with a concentration of 0.1 M; using TCEP (tris (2-carboxyethyl) phosphine) to pretreat the aptamer SYL3C solution with a concentration of 5-10 μM and a volume of 20-50 μL, wherein the mass ratio of TCEP to SYL3C is (10:1)-(50:1); adding the pretreated SYL3C solution to the cleaned electrode surface, and incubating for 6-12 h to realize the modification of the specific capture probe.
[0020] Further, the specific method for preparing the ECL biosensor by using the aptamer-modified electrode comprises the following steps: immersing the aptamer-modified electrode in BSA (bovine serum albumin) solution for 30-60 min to block the non-specific active sites; then adding the cell sample to be tested to the electrode surface, and incubating at 37℃ for 50-75 min, and then immersing in 4% paraformaldehyde fixing solution and Triton X-100 solution for 10-15 min for cell fixation and permeation, which is conducive to the improvement of the ECL signal, and finally adding 30-50 μL SA@Ru-M1 solution to the electrode surface, and incubating for 15-40 min to realize the labeling of the signal probe.
[0021] In the second aspect, an ECL biosensor prepared by using the method in the first aspect is provided.
[0022] In the third aspect, a detection method of the ECL biosensor in the second aspect is provided, which comprises the following steps:
[0023] Assembling the ECL biosensor;
[0024] Taking ITO as the working electrode, Pt wire as the auxiliary electrode, and saturated calomel as the reference electrode, immersing the assembled ECL biosensor in PBS buffer containing TprA for ECL detection to obtain the ECL signal value;
[0025] Based on the relationship curve between the ECL signal intensity value and the concentration of the cell sample to be tested, the concentration of the cell sample to be tested is calculated according to the measured ECL signal value.
[0026] Further, the concentration of the TprA is 10 mM, the PBS buffer is made of KCl, Na2HPO4 and NaH2PO4 with a concentration of 0.1 M and pH = 7.4; the ECL detection process adopts cyclic voltammetry, the scanning voltage is set to 0-1.2 V, and the high voltage of the photomultiplier tube is -800 V.
[0027] Compared with the prior art, the present application has the following beneficial effects:
[0028] (1) The ECL biosensor provided by the present application uses PANI as a soft substrate and has a reticular gap structure, which can not only accelerate the electron transfer rate and enhance the conductivity of the cell sensing interface, but also induce the adhesion and growth of cell filopodia, thereby improving the cell capture efficiency and detection accuracy;
[0029] (2) The present application uses M1 chain, H1 chain and H2 chain to carry out hybridization chain reaction, that is, the signal amplification strategy of M1 chain induced hybridization chain reaction is adopted, the prepared signal probe SA@Ru-M1 can specifically recognize cell surface MUC1 (transmembrane glycoprotein) and react with TPrA in the solution to cause ECL signal change, so that the constructed ECL biosensor realizes high sensitivity and high specificity quantitative detection of breast cancer cells MCF-7 (one kind of CTC), wherein the linear detection range of the cells is 10 2 ~ 10 6 / mL, and the detection limit can reach 31 / mL (S / N = 3);
[0030] (3) In the ECL biosensor provided by the present application, the aptamer SYL3C chain and the M1 chain are used as the capture and signal probes of cell sensing respectively, which can recognize two proteins of the same cell, thereby improving the detection accuracy and anti-interference ability of the cell sensor. DETAILED DESCRIPTION
[0031] Figure 1 Fig. 1 is a linear relationship diagram of ECL signal intensity and cell concentration logarithm in Example 1 of the present application;
[0032] Figure 2 Fig. 2 is a diagram of ECL signal change when the cell capture time is different in Example 2 of the present application;
[0033] Figure 3 Fig. 3 is a diagram of ECL signal change when the HCR reaction time is changed in Example 3 of the present application;
[0034] Figure 4 Fig. 4 is a growth situation diagram of the filopodia of the captured cells of the PANI substrate in Example 5 of the present application;
[0035] Figure 5Schematic diagram of polyacrylamide gel electrophoresis characterization of M1 chain induced hybrid chain reaction in embodiment 6 of the application;
[0036] Figure 6 ECL changes of cell sensor before and after HCR in embodiment 7 of the application;
[0037] Figure 7 Electrochemical impedance diagram of cell sensor at different stages in embodiment 8 of the application. DETAILED DESCRIPTION
[0038] The application will be further described below with reference to the drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the application, and cannot be used to limit the protection scope of the application.
[0039] Embodiment 1
[0040] Step 1, synthesis of PANI.
[0041] The ITO electrode was cleaned with a detergent, acetone, ethanol and water in sequence, and then dried at 80℃ for 30 min. An insulating tape with a pore size of 5 mm was attached to the conductive surface of the ITO electrode to control the reaction area during electrodeposition.
[0042] The pretreated ITO electrode was used as a working electrode, a Pt electrode was used as an auxiliary electrode, and an Ag / AgCl electrode was used as a reference electrode. Electrodeposition was performed in a mixed aqueous solution (6 mL) containing 0.4 M aniline and 1.2 M hydrochloric acid using a CHI660E electrochemical workstation. The cyclic voltammetry method was used, the scanning speed was set to 100 mV / s, and scanning was performed between 0.2-1.2 V for 4 cycles. After the electrode was taken out, it was rinsed to obtain the electrode with deposited PANI.
[0043] Step 2, preparation of AuNPs.
[0044] After mixing 515 μL of HAuCl4 solution (2% wt) and 99.485 mL of water, the mixture was placed in a flask, connected to a condensation reflux device, and heated to boiling in an oil bath for 5 min. Then 588 μL of 0.2 M sodium citrate solution was quickly added and stirred vigorously. Boiling was continued for 30 min. When the solution turned wine red, heating was stopped, and stirring was continued at room temperature for 15 min to obtain an Au seed solution of 18 nm. Then 350 μL of the Au seed solution, 165 μL of HAuCl4 solution (1% wt) and 240 μL of 10 mM MSA solution were added to water in sequence, and stirred at room temperature for 2 h to obtain an AuNPs solution.
[0045] Step 3, preparation of SA@Ru-M1.
[0046] The ruthenium complex solution of 500 μL anhydrous DMSO (1 mg / mL), 100 μL SA solution (1 mg / mL) and 400 μL PBS buffer were stirred at 4°C for 5 h and dialyzed overnight to obtain the SA@Ru solution.
[0047] The M1 chain, H1 chain and H2 chain were dissolved in PBS buffer (25 μL) at 2 μM, heated at 90°C for 5 min, cooled at 4°C for 5 min to reduce the influence of the spatial folding of the DNA chain on the hybridization process, then oscillated at 37°C for 2 h to fully perform the HCR process, and then mixed with 25 μL SA@Ru solution and oscillated for 1 h to obtain the SA@Ru-M1 solution for subsequent labeling of proteins.
[0048] Step 4, preparation of the aptamer-modified electrode.
[0049] 50 μL AuNPs solution was added dropwise to the PANI-deposited electrode surface and incubated for 1 h to allow the electrostatic adsorption process of the two to fully act; then the electrode was washed three times with PBS buffer with a concentration of 0.1 M. 10 mM TCEP was used to pretreat the aptamer SYL3C with a concentration of 5-10 μM and a volume of 20-50 μL to cut the S-S bond, so that it can be bonded to the electrode surface AuNPs, wherein the mass ratio of TCEP to SYL3C is 10:1. 50 μL of the pretreated SYL3C solution was added dropwise to the washed electrode surface and incubated at room temperature for 6 h to realize the modification of the specific capture probe and obtain the aptamer-modified electrode.
[0050] Step 5, assembly of the ECL biosensor.
[0051] After the aptamer-modified electrode was washed with PBS buffer, it was immersed in a BSA solution (1% wt) for 30 min to block the non-specific active sites; then 50 μL of MCF-7 cell test solution with different concentrations in the range of 10 2 ~10 6 μL / mL was added dropwise to the electrode surface, which was incubated at 37°C for 60 min, then immersed in 4% paraformaldehyde fixing solution and 0.5% Triton X-100 solution for 15 min for cell fixation and permeation, and washed with PBS buffer to remove excess impurities. Finally, 50 μL of SA@Ru-M1 solution was added dropwise to the electrode surface and incubated for 35 min to label the signal probe and complete the assembly of the ECL biosensor.
[0052] Step 6, detection of the ECL biosensor.
[0053] The ECL biosensor was placed in a PBS buffer (0.1M KCl, 0.1M Na2HPO4, 0.1M NaH2PO4, pH = 7.4) containing 10mM TprA to detect the ECL signal, and the detection process was carried out by cyclic voltammetry, with a scanning voltage of 0-1.2V and a photomultiplier high voltage of-800V. Finally, the ECL signal values corresponding to different concentrations of MCF-7 cell test solutions were obtained.
[0054] Step 7, linear relationship curve preparation.
[0055] It was found through analysis that the ECL signal intensity was proportional to the logarithmic value of the concentration of MCF-7 cells, as shown in Figure 1 , the linear relationship conforms to the formula y = 1424.778lgC-1479.136 (R 2 = 0.997), wherein the linear detection range of MCF-7 is 10 2 ~ 10 6 The detection limit is 31 / mL calculated by the signal-to-noise ratio S / N = 3.
[0056] Detection principle: PANI nanofibers with a mesh structure were electrodeposited on the surface of the ITO electrode by cyclic voltammetry, and AuNPs were electrostatically adsorbed. Since the SYL3C chain can specifically recognize the epithelial cell adhesion molecule (EpCAM), the thiol aptamer 1 chain (SYL3C) is modified on the surface of the AuNPs as a capture probe to achieve specific capture of CTCs; BSA is used to block other active sites to prevent the adsorption of non-specific molecules; then the signal probe SA@Ru-M1 is used to label the MUC1 on the surface of the cancer cells, so as to react with TPrA in the solution to generate an ECL signal. SA@Ru-M1 is connected by biotin and SA in H1 and H2 chains, and one end is the initiation chain M1 of HCR, which contains the aptamer sequence of MUC1 protein, and thus has a targeting effect. Since the surface of the metastatic breast cancer cell MCF-7 has both the above two proteins, it can be used as the target cell of the sensing platform. By capturing different concentrations of MCF-7 cells, the ECL signal produces different intensity changes, thereby realizing quantitative detection of MCF-7 cells.
[0057] Example 2
[0058] The present embodiment provides a preparation and detection method of an ECL biosensor, and the steps are as follows:
[0059] Steps 1-4 are the same as in Example 1.
[0060] Step 5, assembly of the ECL biosensor.
[0061] After the aptamer-modified electrode was washed with PBS buffer, it was immersed in a BSA solution (1% wt) for 30 min to block non-specific active sites. Then 50 μL of a cell sample to be tested with a concentration in the range of 10 2 ~10 6 The cell sample to be tested was dropped onto the electrode surface, and incubated at 37°C for 60 min. Then the cells were fixed and permeated by immersing in 4% paraformaldehyde and 0.5% Triton X-100 solution for 15 min, respectively, and washed with PBS buffer to remove excess impurities. Finally, 50 μL of SA@Ru-M1 solution was dropped onto the electrode surface and incubated for 35 min to label the signal probe, and the assembly of the ECL biosensor was completed.
[0062] Step 6, the same as in Example 1, to obtain the ECL signal value of the cell sample to be tested. Then based on the linear relationship between the ECL signal intensity and the logarithm of the MCF-7 cell concentration obtained in Step 7 of Example 1, the concentration of the cell sample to be tested was calculated according to the measured ECL signal value, to realize the quantitative detection of MCF-7.
[0063] In some other embodiments, in order to improve the detection efficiency, the SA@Ru-M1 solution and the aptamer-modified electrode can be prepared in advance according to the methods of Steps 1-4. In actual detection, only the ECL biosensor needs to be assembled according to the method of Step 5, and then the ECL signal value is detected according to the method of Step 6, and based on the relationship curve between the ECL signal intensity value and the concentration of the cell sample to be tested, the concentration of the cell sample to be tested is calculated.
[0064] Example 3
[0065] In order to realize high-sensitivity detection of MCF-7 cells by the sensing interface, this embodiment studies the effect of cell capture time on the ECL signal intensity.
[0066] The preparation and detection method of the ECL biosensor provided in this embodiment is different from that of Example 2 in that in Step 5, after 5 identical cell samples to be tested were dropped onto the electrode surface, they were incubated at 37°C for 15 min, 30 min, 45 min, 60 min, and 75 min, respectively. Finally, the ECL signal values of the samples were measured, and the results are shown in Figure 2 .
[0067] From Figure 2It is evident that cell capture time is a crucial factor affecting ECL signal intensity. As cell capture time (isotropic incubation time) increases, the ECL signal intensity gradually increases. This is attributed to the increased cell number on the electrode due to the improved capture efficiency, which in turn increases the number of protein molecules recognized by SA@Ru-M1. The maximum signal intensity is reached at a capture time of 60 min.
[0068] Example 4
[0069] To achieve high-sensitivity detection of MCF-7 cells by the sensing interface, this embodiment investigated the effect of HCR reaction time on ECL signal intensity.
[0070] The preparation and detection method of the ECL biosensor provided in this embodiment differs from that in Embodiment 2 in that: in step 3, when preparing SA@Ru-M1, six experimental groups were set up, and HCR reactions were performed at 37℃ for 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively. The ECL signal values of each sample were finally measured, and the results are as follows... Figure 3 As shown.
[0071] Depend on Figure 3 It can be seen that the HCR time can affect the hybridization of the three strands M1, H1, and H2, and thus affect the number of biotin-linked marker molecules SA@Ru-M1 in H1 and H2, leading to changes in the ECL signal. As the HCR time continues to increase, the ECL signal intensity gradually increases, reaching a high level at 120 min, and the enhancement rate thereafter weakens significantly, indicating that the HCR process is basically complete at 120 min.
[0072] Example 5
[0073] This embodiment demonstrates that PANI can induce the growth and adhesion of filamentous pseudopodia on the cell surface, thereby improving cell capture efficiency.
[0074] Cells captured on the electrode surface were treated with 50 μL of glutaraldehyde (2.5% wt) solution and fixed at 4 °C for 12 h. After washing with PBS buffer, the cells were stained with 50 μL of uranium acetate (0.5% wt) solution for 1 h. The cells were then dehydrated for 20 min by a series of different concentration gradients of ethanol (50%, 70%, 90%, 100%, 100%) and dried at 60 °C. After platinum coating the cell surface (20 s), the filopodia were observed using a Hitachi S-4800 scanning electron microscope (acceleration voltage set to 10 kV). Figure 4As shown, the cells were closely adhered to the polyaniline nanofiber, and the filamentous pseudopodia grew out of the edge with long and slender shape and large number, which were uniformly distributed around the cells. Compared with the flat surface, the polyaniline nanofiber had a complex network gap three-dimensional space structure, which could provide more aptamer functionalized capture sites and reduce the migration speed of cancer cells. The nanofiber with shape and size matching induced the extension of the filamentous pseudopodia on the cell surface, enhanced the adhesion of the cells to the nanofiber surface, and thus improved the capture efficiency of the polyaniline nanofiber as a sensing interface.
[0075] Example 6
[0076] This example analyzes the feasibility of M1 chain recognition and hybridization reaction process by polyacrylamide gel electrophoresis and imaging.
[0077] The prepared DNA chain was dropped into a newly prepared polyacrylamide gel (8% wt) groove, and then electrophoresis was carried out in 1xTBE buffer at an electric potential of 70V for 80min. After staining with ethidium bromide (EB), imaging was taken by a bio-gel imaging system. As shown in Figure 5 Lane 1-3 represent M1 chain, H1 chain and H2 chain respectively. Since the molecular weight of H1 chain and H2 chain is similar, when the two are mixed, a wide overlapping band appears in lane 6, which proves that H1 chain and H2 chain do not hybridize when simply mixed with each other. Compared with lane 5, in addition to unreacted M1 chain and H1 chain, a new bright band appears above lane 4, which proves that M1 chain only hybridizes with H1 chain and does not hybridize with H2 chain. After adding M1 chain, the new chain of M1 chain hybridizing with H1 chain in lane 7 becomes dark, and multiple bright macromolecular bands are generated, which proves that only the opening of the hairpin structure of H1 chain with M1 chain as the target recognition can induce the initiation of HCR and hybridize and amplify with H2 chain.
[0078] Example 7
[0079] This example verifies the amplification effect of HCR amplification strategy on ECL signal in cell sensing process.
[0080] The ECL test was carried out in 0.1M PBS buffer containing 10mM TPrA, and the results are shown in Figure 6 As shown, when M1 chain and H1 chain are added, a weak ECL signal appears in the cell sensor (curve a). When M1 chain, H1 chain and H2 chain are added, the ECL signal in the sensor is significantly enhanced (curve b), which further proves the progress of HCR process and is beneficial to improve the detection performance of the cell sensor.
[0081] Example 8
[0082] This example verifies the amplification effect of HCR amplification strategy on ECL signal in cell sensing process. 3- / 4-In the case of a redox probe, electrochemical impedance spectroscopy (EIS) was used to analyze the process of stepwise modification of the cell sensor on the electrode.
[0083] like Figure 7 As shown, in [Fe(CN)6] 3- / 4- In solution, the semicircular diameter of the EIS of the bare ITO electrode is relatively large (curve a, Ret = 951 Ω). After electrodepositing polyaniline on the ITO surface, the impedance decreases to 394 Ω (curve b), which is attributed to the accelerated electron transfer process due to the interstitial microstructure of the PANI nanofibers, increasing the active area of the electrode surface. After electrostatic adsorption of AuNPs by polyaniline, the impedance decreases to a minimum (curve c, Ret = 69 Ω). With sequential modification by aptamers SYL3C and BSA, the electron transfer resistance also increases significantly from 1134 Ω to 2238 Ω (curves d and e). When MCF-7 cells are captured at the biosensor interface, the semicircular diameter of the EIS increases significantly to 5705 Ω (curve f), because the cells block the active area of the electrode surface, increasing the steric hindrance and thus affecting [Fe(CN)6]. 3- / 4- The diffusion and charge transfer were then observed. Finally, the signal probe SA@Ru-M1 recognized the MUC1 protein on the target cells, further hindering charge transfer on the electrode surface and increasing the impedance to 7560 Ω (curve g). Characterization by EIS verified the success of the stepwise modification of this cell sensor and the feasibility of this approach for cell capture.
[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method of preparing an ECL biosensor, characterized by, The method comprises the following steps: The pre-processed ITO electrode is placed in a mixed aqueous solution of aniline and hydrochloric acid for electrodeposition to obtain an electrode on which PANI is deposited; Au seed solution, HAuCl4 solution and MSA solution are sequentially added to water, stirred to obtain AuNPs solution; The ruthenium complex solution of anhydrous DMSO, SA solution and PBS buffer are mixed, stirred and dialyzed overnight to obtain SA@Ru solution; The M1 chain, H1 chain and H2 chain are subjected to hybridization chain reaction, and then mixed and shaken with the SA@Ru solution to obtain SA@Ru-M1 solution; The AuNPs solution is added dropwise to the surface of the electrode on which PANI is deposited for incubation, and then the pre-processed SYL3C solution is added dropwise to the surface of the cleaned electrode for incubation to obtain an aptamer-modified electrode; The aptamer-modified electrode is immersed in BSA solution, and then the cell to be tested is added dropwise to the surface of the electrode, and after constant-temperature culture, cell fixation and permeation are performed, and finally the SA@Ru-M1 solution is added dropwise to the surface of the electrode for incubation to obtain an ECL biosensor; The specific preparation method of the SA@Ru-M1 solution comprises the following steps: the M1 chain, H1 chain and H2 chain are dissolved in PBS buffer, heated at 90-95°C for 5-10 min, cooled at 4°C for 5-10 min, and then subjected to hybridization chain reaction at 37°C for 1-3 h, and then mixed and shaken with the SA@Ru solution for 0.5-1 h; wherein the M1 chain contains the aptamer sequence of MUC1 protein.
2. The method of claim 1, wherein the ECL biosensor is prepared by the steps of: The pre-processing method of the ITO electrode comprises the following steps: the ITO electrode is sequentially cleaned by ultrasonic cleaning with a detergent, acetone, ethanol and water, and then dried at 70-120°C for 30-60 min, and then an insulating tape with a pore size of 3-8 mm is attached to the conductive surface of the ITO electrode.
3. The method of claim 1, wherein the ECL biosensor is prepared by the steps of: The method for electrodeposition comprises the following steps: the pre-processed ITO electrode is used as a working electrode, a Pt electrode is used as an auxiliary electrode, and an Ag / AgCl electrode is used as a reference electrode, a cyclic voltammetry method is adopted, and a CHI660E electrochemical workstation is used for electrodeposition in a mixed aqueous solution of aniline and hydrochloric acid, the scanning speed is set to 100 mV / s, and scanning is performed between 0.2-1.2V for 4-6 cycles; wherein the volume of the mixed aqueous solution of aniline and hydrochloric acid is 6-8 mL, and the concentration ratio of aniline to hydrochloric acid is (1:2)-(1:4).
4. The method of claim 1, wherein the ECL biosensor is prepared by, The specific preparation method of the AuNPs solution comprises the following steps: The HAuCl4 solution with a mass fraction of 0.01-0.02% is placed in a flask, a condensation reflux device is connected, oil bath heating is performed to boiling, a 0.1-0.2M sodium citrate solution is added and stirred, boiling is continued, when the solution turns into wine red, heating is stopped, and stirring is performed at 25-30°C for 10-20 min to obtain Au seed solution; The Au seed solution, HAuCl4 solution and 10mM MSA solution are sequentially added to water, and stirred at room temperature for 2-3h.
5. The method of claim 1, wherein the ECL biosensor is prepared by the steps of: The specific preparation method of the SA@Ru solution comprises the following steps: mixing a ruthenium complex solution of anhydrous DMSO, a SA solution and a PBS buffer, stirring at 4 ℃ for 3-6 h, and dialyzing overnight to obtain the SA@Ru solution.
6. The method of claim 1, wherein the ECL biosensor is prepared by, The specific preparation method of the aptamer-modified electrode comprises the following steps: adding an AuNPs solution to the surface of the PANI-deposited electrode and incubating for 0.5-1 h, then washing the electrode three times with a PBS buffer with a concentration of 0.1 M; pretreating an aptamer SYL3C solution with a concentration of 5-10 μM and a volume of 20-50 μL by using TCEP, wherein the mass ratio of TCEP to SYL3C is (10:1)-(50:1); adding the pretreated SYL3C solution to the surface of the washed electrode and incubating for 6-12 h to obtain the aptamer-modified electrode.
7. The method for preparing the ECL biosensor according to claim 1, characterized in that, The specific method for preparing the ECL biosensor by using the aptamer-modified electrode comprises the following steps: immersing the aptamer-modified electrode in a BSA solution for 30-60 min, then adding a cell sample to be tested to the surface of the electrode, incubating at 37 ℃ for 50-75 min, then immersing in a 4% paraformaldehyde fixing solution and a Triton X-100 solution for 10-15 min for cell fixation and permeation, and finally adding 30-50 μL of a SA@Ru-M1 solution to the surface of the electrode and incubating for 15-40 min to obtain the ECL biosensor.
8. An ECL biosensor prepared by using the method of any one of claims 1-7.
9. A method of detecting the ECL biosensor of claim 8, characterized by, The method comprises the following steps: Assembling the ECL biosensor; taking ITO as the working electrode, Pt wire as the auxiliary electrode and saturated calomel as the reference electrode, immersing the assembled ECL biosensor in a PBS buffer containing TprA for ECL detection to obtain an ECL signal value; calculating the concentration of the cell sample to be tested according to the measured ECL signal value based on the relationship curve between the ECL signal intensity value and the concentration of the cell sample to be tested.