Preparation and application of sandwich type ZnNi-MOF electrochemical immunosensor
By using sandwich ZnNi-MOF structure and CeO2-Pt composite materials in electrochemical immunosensors, a high-sensitivity immunosensing platform was constructed, which solved the problem of insufficient detection sensitivity of CA15-3 in the prior art, and achieved high sensitivity analysis of breast cancer markers.
Patent Information
- Application Number
- CN202411664377.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-02-28
AI Technical Summary
Existing electrochemical immunosensors are insufficient in detecting CA15-3, an important biomarker of breast cancer, and it is difficult to meet the needs of early diagnosis.
A sandwich ZnNi-MOF electrochemical immunosensor was used, and the CeO2-Pt composite material was used as a co-reaction promoter and ZnNi-MOF/PEI-L012@Ab2 as a self-enhanced signal probe was used to build an "signal on" immune sensing platform.
The detection sensitivity to CA15-3 is significantly improved, with the detection limit as low as 5.75×10-5U m-1, and has good selectivity and stability.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of electrochemical immunosensors, in particular to the preparation and application of a sandwich ZnNi-MOF electrochemical immunosensor. Background Art
[0002] Breast cancer is the most common malignant tumor in women worldwide, seriously threatening women's life and health. Early detection, diagnosis and treatment are the key to improving the survival rate of breast cancer patients. CA15-3 is an important biomarker for breast cancer diagnosis and monitoring, but its concentration is often low in early breast cancer patients, making it difficult to analyze and detect.
[0003] Electrochemiluminescence (ECL) technology has the advantages of high sensitivity, low background signal, simple operation, and easy automation. It is a sensor platform suitable for biomarker detection. Electrochemical immunosensors combine immunological technology with electrochemical analysis technology to achieve quantitative analysis of target analytes. Currently, this technology has been applied to the detection of a variety of analytes, such as biotoxins, antibiotics, pathogenic bacteria, pesticide residues, cancer markers, etc. How to further improve the sensitivity of electrochemical immunosensors in detecting important biomarkers of breast cancer is of great significance for the early diagnosis of breast cancer. Summary of the invention
[0004] The present invention aims to provide a preparation and application of a sandwich ZnNi-MOF electrochemical immunosensor to further improve the sensitivity of the electrochemical immunosensor in detecting important biomarkers of breast cancer.
[0005] In order to solve the above technical problems, the specific scheme adopted by the present invention is: a method for preparing a sandwich ZnNi-MOF electrochemical immunosensor, wherein a biotin Ab1 solution is dripped onto a CeO2-Pt / streptavidin-modified GCE, incubated, and then sequentially soaked in BSA and incubated with CA15-3. Finally, the obtained electrode is incubated with a ZnNi-MOF / PEI-L012@Ab2 bioconjugate to obtain the sandwich ZnNi-MOF electrochemical immunosensor.
[0006] As a further optimization of the above technical solution: the specific steps of CeO2-Pt / streptavidin modification of GCE are: CeO2-Pt composite material is dispersed in ultrapure water, then streptavidin is added, mixed evenly, dripped on GCE, and dried.
[0007] As a further optimization of the above technical solution: CeO2 powder is added to ethylene glycol, ultrasonically treated, and NaOH ethylene glycol solution and H2PtCl6·6H2O ethylene glycol solution are added in sequence under magnetic stirring, reacted at 180°C for 2h, cooled and centrifuged to collect the obtained product, and dried to obtain a CeO2-Pt composite material.
[0008] As a further optimization of the above technical solution: the preparation method of CeO2 powder is as follows: Ce(NO3)3·6H2O is dissolved in water, NaOH solution is added dropwise under magnetic stirring, the mixture is reacted at 100°C for 24 hours, and the product is collected by centrifugation after cooling to obtain CeO2 powder.
[0009] As a further optimization of the above technical scheme: the preparation method of ZnNi-MOF / PEI-L012@Ab2 bioconjugate is: activate ZnNi-MOF / PEI-L012 in GA solution, centrifuge and wash to obtain activated ZnNi-MOF / PEI-L012, covalently couple the activated ZnNi-MOF / PEI-L012 solution with Ab2, add BSA, centrifuge and wash to obtain ZnNi-MOF / PEI-L012@Ab2 bioconjugate.
[0010] As a further optimization of the above technical solution: ZnNi-MOF powder and PEI solution are mixed, ultrasonically treated, and centrifuged to obtain PEI-coated ZnNi-MOF; GA solution and L012 solution are added to the PEI-coated ZnNi-MOF, ultrasonically treated, stirred, and centrifuged to obtain ZnNi-MOF / PEI-L012.
[0011] As a further optimization of the above technical scheme: Zn(NO3)2·6H2O, Ni(acac)2, PVP and NH2-BDC were dissolved in a solvent mixture consisting of ethanol, DMF and DMAC, reacted at 150°C for 4 hours, and the obtained product was collected by centrifugation after cooling to obtain ZnNi-MOF.
[0012] A sandwich ZnNi-MOF electrochemical immunosensor is prepared by the above-mentioned preparation method.
[0013] Application of a sandwich ZnNi-MOF electrochemical immunosensor in the analysis and detection of breast cancer biomarkers.
[0014] As a further optimization of the above technical solution: the breast cancer biomarker is CA15-3.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] The present invention utilizes a sandwich ECL immunosensor to achieve the "signal on" measurement of CA15-3, and the sensor is composed of ZnNi-MOF / PEI-L012 as a self-enhanced signal probe and a CeO2-Pt composite material as a co-reaction promoter. The polyethyleneimine (PEI) used is a known cationic polymer with a high density of amino groups and is used as an effective co-reactant for luminol. Further modification of ZnNi-MOF with PEI will increase the luminol loading capacity, thereby obtaining a self-enhanced ECL emitter with excellent ECL efficiency. The present invention also designs PEI-coated ZnNi-MOF hollow nanospheres as electroactive carriers for loading ultra-high amounts of L012 (luminol analogs) and detection antibody (Ab2) modification. The hollow structure gives ZnNi-MOF a higher surface-to-volume ratio, larger void space, and abundant active sites. The synthesized CeO2-Pt composite is an excellent co-reaction promoter, indicating that the ECL efficiency of L012 is significantly improved and provides a bio-friendly substrate for further immobilization of the capture antibody (Ab1). A "signal-on" immunosensing platform was constructed using the combination of ZnNi-MOF / PEI-L012@Ab2 (ECL probe) and CeO2-Pt@Ab1 (recognition unit), which can be used to specifically detect the breast cancer marker CA15-3 with high sensitivity and a detection limit as low as 5.75×10 -5 U m -1 .
[0017] The hollow bimetallic ZnNi-MOF material of the present invention has obvious crystal pores, a higher surface-to-volume ratio and abundant active sites. In addition, the synergistic effect between heterogeneous metals improves the electrochemical and electrocatalytic activity of ZnNi-MOF. The ZnNi-MOF modified with polyethyleneimine (PEI) greatly enhances the loading capacity of the luminescent material, thereby having excellent ECL efficiency and self-enhanced ECL emitter. In addition, the combination of CeO2 and Pt further enhances the electrocatalytic performance of CeO2 as a co-reaction promoter with high catalytic efficiency, thereby producing a stable and enhanced ECL intensity.
[0018] The present invention adopts PEI-coated ZnNi-MOF hollow nanospheres as electroactive carriers to load ultra-high amounts of L012 (luminol analogues), thereby realizing electrochemiluminescence resonance energy transfer (ECL-RET) in metal organic frameworks. Based on this energy transfer mechanism, a sandwich-type electrochemical immunosensor platform based on hollow sphere structure ZnNi-MOF / PEI-L012 emitters and reaction facilitators is constructed, thereby realizing high-sensitivity analysis and detection of breast cancer marker CA15-3. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1Microstructures of prepared ZnNi-MOF and CeO2-Pt: (A) TEM image of ZnNi-MOF; (B) TEM image of CeO2-Pt; (C) HAADF-STEM image of ZnNi-MOF and corresponding EDS element mapping (Zn is yellow, Ni is pink, C is red, N is green, and O is cyan);
[0020] Figure 2 Middle: (A) UV-visible spectra of ZnNi-MOF, ZnNi-MOF / PEI, ZnNi-MOF / PEI-L012 and L012; (B) FT-IR spectra of ZnNi-MOF, ZnNi-MOF / PEI and ZnNi-MOF / PEI-l012;
[0021] Figure 3 Middle: (A) Electrochemical performance of the electrode assembly process: EIS curves of GCE (a), CeO2-Pt / streptavidin (b), CeO2-Pt / streptavidin / biotin Ab1 (c), CeO2-Pt / streptavidin / biotin Ab1 / BSA (d), CeO2-Pt / streptavidin / biotin Ab1 / BSA / CA15-3 (e), CeO2-Pt / streptavidin / biotin Ab1 / BSA / CA 15-3 / ZnNi-MOF / PEI-L012@Ab2(f);(B) ECL curves of GCE in different test solutions; L012(a), L012+PEI solution(b), L012+H2O2 solution(c), L012+PEI+H2O2 solution(d);(C) ECL response of CeO2@Ab1 treated immunosensor(a) and ECL response of CeO2-Pt@Ab1 treated immunosensor(b);
[0022] Figure 4 Middle: (A) Different concentrations of CA15-3 (ah: 0.0005, 0.001, 0.005, 0.01, 0.1, 1, 10 and 50 U mL -1 ) in the presence of 1U mL -1 Stability evaluation of the ECL immunosensor for CA15-3 under 10 consecutive scanning cycles;
[0023] Figure 5 Middle: Performance of the immunosensor and the optimal choice of concentrations of L012 (A), H2O2 (B), ZnNi-MOF / PEI-L012@Ab2 (C), and CeO2-Pt@Ab1 (D);
[0024] Figure 6 Schematic diagram of the process of the preparation method of the present invention: (A) Preparation process of ZnNi-MOF / PEI-L012@Ab2ECL signal probe; (B) Synthesis process of CeO2-Pt / streptavidin; (C) ECL sensing detection of CA15-3 based on ZnNi-MOF / PEI-L012 nanoprobe. DETAILED DESCRIPTION
[0025] The technical solution of the present invention is further elaborated in detail below in conjunction with specific embodiments. The parts of the present invention that are not described and disclosed in detail in the following embodiments should be understood as the prior art known or should be known to those skilled in the art.
[0026] The present invention discloses a method for preparing a sandwich ZnNi-MOF electrochemical immunosensor, which is used for analyzing and detecting breast cancer marker CA15-3. The specific preparation method is as follows:
[0027] Preparation of CeO2 nanorods:
[0028] CeO2 nanorods were prepared by hydrothermal method. In this process, a 50mL round-bottom flask was first prepared, 0.672gCe(NO3)3·6H2O was dissolved in 10mL of water and placed in it, and 20mL (10M) NaOH solution was slowly dripped into the flask under vigorous magnetic stirring (600rpm). After stirring the above solution for 20 minutes, a white slurry was obtained, which was transferred to a polytetrafluoroethylene liner and heated to 100℃ for 24h. After cooling, the product was collected by centrifugation, washed several times with deionized water, and finally dried at 120℃ for 12h to obtain yellow CeO2 nanorod powder.
[0029] Preparation of CeO2-Pt composite materials:
[0030] 0.05g of the prepared CeO2 powder was added to a beaker containing 25mL of ethylene glycol. After ultrasonication for 30min, a magnet was added and 0.25mL of 1M NaOH ethylene glycol solution and 1mL of 30mM H2PtCl6·6H2O ethylene glycol solution were added to the dispersion in sequence under magnetic stirring, and stirring was continued for 30min. Then, the mixture was placed in an oil bath at 180°C for 2h. The color of the solution was observed to gradually change from light yellow to gray, indicating the formation of Pt particles. After cooling to room temperature naturally, the CeO2 nanorods (CeO2-Pt) coated with Pt nanoparticles were collected by centrifugation and washed with water several times. Finally, the mixture was dried overnight at 60°C to obtain the synthesized CeO2-Pt.
[0031] Preparation of bimetallic ZnNi-MOF:
[0032] ZnNi-MOF nanospheres with hollow structures were synthesized by a one-pot hydrothermal method. 26 mg Zn(NO3)2·6H2O, 40 mg Ni(acac)2, 1.4261 g PVP and 15 mg NH2-BDC were dissolved in a solvent mixture consisting of 9.6 mL ethanol, 8 mL DMF and 8 mL DMAC. The mixture was stirred until the solid was completely dissolved and then reacted at 150 °C for 4 h. After cooling, the obtained product was collected by centrifugation, washed three times with ethanol, and finally dried in vacuum at 70 °C for 12 h.
[0033] Preparation of ZnNi-MOF / PEI-L012:
[0034] Take a 50mL glass bottle and mix 8mg ZnNi-MOF powder and 25mL 1% PEI solution in it. Ultrasonicate the mixture for 20min to fully disperse the ZnNi-MOF nanospheres in the PEI solution. Then stir at 25°C for 4h. The PEI-coated ZnNi-MOF product can be obtained by centrifugation, which is washed several times with deionized water. Then 1.25mL, 12.5% GA solution and 3.75mL 1.6mM L012 solution are added to the above precipitate as crosslinkers, ultrasonicate for 10min and stir for 12h, and the product is suspended in 2mL deionized water after centrifugal washing.
[0035] Preparation of ZnNi-MOF / PEI-L012@Ab2 bioconjugate:
[0036] In order to bind with the antibody, ZnNi-MOF / PEI-L012 was activated in 2.5% GA solution at 25 °C for 1 h to activate the amino groups on its surface, and ZnNi-MOF / PEI-L012 was obtained after centrifugation and washing. Then, 0.5 mL of the activated ZnNi-MOF / PEI-L012 solution was mixed with 0.5 mL of 10 μg mL -1 Ab2 was covalently coupled at 4°C for 10 h. 0.5 mL of 1% BSA was added and stirred at 37°C for 1 h to block nonspecific binding sites. After centrifugation and washing, the Ab2-immobilized ZnNi-MOF / PEI-L012 bioconjugate was finally dispersed in 2 mL of PBS.
[0037] Preparation of sandwich ECL immunosensor:
[0038] Before each modification, the GCE was polished with alumina powders of different sizes. Then, 6.0 mg of CeO2-Pt powder was ultrasonically dispersed in 2 mL of ultrapure water. 0.3 mg mL -12 mL of streptavidin was stirred for 2 h, 5 μL of the mixed solution was dropped onto clean GCE and dried at 4 °C overnight, followed by the addition of 5 μL of biotin Ab1 (10 μg mL -1 ) solution was incubated on CeO2-Pt / streptavidin-modified GCE for 1 h, and the excess biotin Ab1 was washed off with PBS buffer. In order to prevent nonspecific adsorption of biomolecules, the prepared GCE was soaked in 1% BSA for 3 h, and the Ab1-loaded electrode was incubated with different concentrations of CA15-3. Finally, the obtained electrode was incubated with ZnNi-MOF / PEI-L012@Ab2 bioconjugate for 1 h, and then rinsed with PBS for use.
[0039] Next, the performance of some intermediate products in the preparation process and the performance of the prepared sandwich ECL immunosensor were tested in combination with the specific preparation process:
[0040] Preparation and characterization of CeO2-Pt:
[0041] CeO2 nanorods were prepared by hydrothermal method. In this process, a 50mL round-bottom flask was prepared, 0.672g Ce(NO3)3·6H2O was dissolved in 10mL water, and 20mlL (10M) NaOH solution was slowly dripped into the flask under vigorous magnetic stirring (600rpm). The above solution was stirred for 20min to obtain a white slurry, which was transferred to a polytetrafluoroethylene liner and heated to 100℃ for 24h. After cooling, the product was collected by centrifugation, washed several times with deionized water, and finally dried at 120℃ for 12h to obtain yellow CeO2 nanorod powder. Then, 0.05 g of the prepared CeO2 powder was added to a beaker containing 25 mL of ethylene glycol. After ultrasonication for 30 minutes, a magnet was added and 0.25 mL of 1 M NaOH ethylene glycol solution and 1 mL of 30 mM H2PtCl6·6H2O ethylene glycol solution were added to the above dispersion in sequence under magnetic stirring, and stirring was continued for 30 minutes. Then, the mixture was placed in an oil bath at 180 ° C for 2 hours, and the color of the solution was observed to gradually change from light yellow to gray, indicating the formation of Pt particles. After cooling naturally to room temperature, the CeO2 nanorods (CeO2-Pt) coated with Pt nanoparticles were collected by centrifugation and washed with water several times. Finally, it was dried overnight at 60 ° C to obtain the synthesized CeO2-Pt.
[0042] The TEM image of the prepared CeO2 is shown in Figure 1 As shown in A. CeO2 was observed to be in the shape of nanorods. Pt nanoparticles were deposited on the surface of CeO2 nanorods by hydrothermal method. Figure 1As shown in B, monodispersed Pt nanoparticles with a diameter of 5 nm are distributed on the CeO2 nanorods, indicating that the CeO2-Pt nanocomposite material was successfully prepared.
[0043] Preparation and characterization of ZnNi-MOF / PEI-L012:
[0044] First, bimetallic MOF was synthesized: ZnNi-MOF nanospheres with hollow structure were synthesized by a one-pot hydrothermal method. 26mg Zn(NO3)2·6H2O, 40mg Ni(acac)2, 1.4261g PVP and 15mg NH2-BDC were dissolved in a solvent mixture consisting of 9.6mL ethanol, 8mL DMF and 8mL DMAC. The mixture was stirred until the solid was completely dissolved and then reacted at 150℃ for 4h. After cooling, the obtained product was collected by centrifugation, washed three times with ethanol, and finally vacuum dried at 70℃ for 12h. Then a 50mL glass bottle was taken and 8mg ZnNi-MOF powder and 25mL 1% PEI solution were mixed therein. The mixture was ultrasonically treated for 20min to fully disperse the ZnNi-MOF nanospheres in the PEI solution. Then it was stirred at 25℃ for 4h. The PEI-coated ZnNi-MOF product can be obtained by centrifugation, which is washed several times with deionized water. Then, 1.25 mL of 12.5% GA solution and 3.75 mL of 1.6 mM L012 solution were added to the above precipitate as cross-linking agents, ultrasonicated for 10 min and then stirred for 12 h. After centrifugal washing, the product was suspended in 2 mL of deionized water.
[0045] The UV-visible spectra of the prepared ZnNi-MOF, ZnNi-MOF / PEI and ZnNi-MOF / PEI-L012 are shown in Figure 2 As shown in A. The characteristic ligand metal charge transfer band of ZnNi-MOF is located at a wavelength of 324nm, and the characteristic absorption peaks of L012 are at 326nm and 400nm. Due to the poor light absorption performance of PEI, there is no obvious change in the ultraviolet absorption spectrum compared with ZnNi-MOF and ZnNi-MOF / PEI. After modification with L012, the two ultraviolet characteristic absorption peaks of ZnNi-MOF / PEI-L012 showed a red shift, indicating that L012 was successfully combined with ZnNi-MOF / PEI. In addition, the substance was measured by FT-IR, and the results showed that ( Figure 2 B) Due to the CH bending vibration in L012 benzene, the composite material has a characteristic peak at 690cm-1, which once again proves the successful composite between the two.
[0046] Assembly of the sandwich immunosensor:
[0047] The cyclic voltammetry (CV) method was used to detect the stepwise assembly process of the immunosensor. A typical three-electrode system was adopted, and EIS experiments were carried out in an electrolyte solution of 0.1 M KCl + 5.0 mM [Fe(CN)6] 3- / 4- As can be seen from Figure (3A, line a), the unmodified GCE has a small charge transfer resistance and good electrochemical activity in the electrolyte solution. EIS is an effective method for evaluating charge transport properties, which also indicates that the charge transfer speed at the electrode interface is faster. It can be seen from the figure that the semicircle diameter of the materials is CeO2-Pt / streptavidin / biotin Ab1 < CeO2-Pt / streptavidin / biotin Ab1 / BSA < CeO2-Pt / streptavidin / biotin Ab1 / BSA / CA15-3 < CeO2-Pt / streptavidin / biotin Ab1 / BSA / CA15-3 / ZnNi-MOF / PEI-L012@Ab2. This shows that the surface resistance of the electrode increases with the increase in the number of modification layers. The test EIS results are consistent with the CV test results, verifying the success of the construction.
[0048] <ECL performance of the sandwich immunosensor>
[0049] Under the optimal test conditions (L012 doping amount: 1.2 mM; H2O2 concentration: 5 mM; ZnNi-MOF / PEI-L012@Ab2 concentration: 1 mg mL -1 ; CeO2-Pt@Ab1 concentration: 1.5 mg mL -1 ), a "signal-on" immunoassay platform based on the double-antibody sandwich strategy was used to detect the CA15-3 biomarker. Figure 4 Figure A shows the effect of different concentrations of CA15-3 on the ECL intensity of the immunosensor. The ECL response signal of the immunosensor gradually increases with the increase in the concentration of CA15-3. When CA15-3 is present in the test solution, ZnNi-MOF / PEI-L012@Ab2 and CeO2-Pt@Ab1 will approach each other, resulting in the "signal-on" ECL emission of ZnNi-MOF / PEI-L012@Ab2. Therefore, the change in the ECL signal (I) is positively correlated with the logarithm of the CA15-3 concentration (lg c, U mL -1 ). In the range of 0.0005 - 50 U mL -1 , the fitted linear equation is I = 861.3lg c + 3748.3, and the correlation coefficient is 0.9976 (R 2 = 0.9976), as shown in Figure 4 Figure B. The detection limit (LOD) of this sandwich ECL immunosensor for detecting CA15-3 is as low as 5.75×10 -5 U mL -1This indicates that the sensor has good analytical performance and can be used to measure CA15-3.
[0050] In addition, in order to verify the relative selectivity and stability of the sandwich ECL immunosensor in detecting CA15-3, three interfering proteins: CEA, AFP and PSA were selected for detection, such as Figure 4 C indicates that even if the concentration of these proteins is as high as 100 ng mL -1 The corresponding ECL intensity is the same as the blank sample ECL intensity. -1 ) When the target antigen is mixed with other antigens, the ECL response to the antigen mixture remains almost unchanged compared to the response to pure CA15-3. The results show that the immunosensor has a high selectivity for CA15-3. In view of the high complexity of the physiological detection environment, the stability of the immunosensor functionalized with CA15-3 antigen was tested. Figure 4 D records the ECL to 1U mL -1 Time response of CA15-3. After 10 consecutive scans, the ECL intensity did not change significantly. The relative standard deviation (RSD) was 1.39%, which proved that the immunosensor had good stability and could be used to detect CA15-3 in human serum samples.
[0051] <Optimization of experimental conditions>
[0052] In order to obtain sufficient L012 doping in ZnNi-MOF / PEI composites, optimizing relevant parameters is beneficial to improving analytical sensitivity. Figure 5 A shows the effect of doping L012 concentration (0.3-1.8mmol / L) on the ECL response of the ZnNi-MOF / PEI-L012 emitter. When the L012 doping concentration in the ZnNi-MOF / PEI composite material varies in the range of 0.3-1.2mmol / L, the ECL signal continues to enhance. When the L012 doping concentration is greater than 1.2mmol / L, the ECL signal intensity of ZnNi-MOF / PEI-L012 remains basically unchanged. Therefore, the optimal doping concentration of L012 in the ZnNi-MOF / PEI composite material was determined to be 1.2mmol / L. In addition, the H2O2 concentration has a significant effect on the ECL efficiency and sensitivity of ZnNi-MOF / PEI-L012. For the ZnNi-MOF / PEI-L012 emitter, the ECL emission intensity significantly improved with the increase of H2O2 concentration (from 0.5mmol / L to 5mmol / L, Figure 5B). When the H2O2 concentration exceeded 5mmol / L, the ECL intensity of ZnNi-MOF / PEI-L012 did not increase further, so 5mmol / L was selected as the optimal H2O2 concentration. In addition to the L012 doping concentration and H2O2 concentration in the ZnNi-MOF / PEI composite material, the concentration of ZnNi-MOF / PEI-L012@Ab2 is also a key parameter affecting the performance of the immunosensor. With the increase of the ZnNi-MOF / PEI-L012@Ab2 concentration, the ECL emission intensity of the immunosensor first increased and then decreased. At a concentration of 1mg mL -1 The maximum value is reached when Figure 5 C). The results show that the optimal concentration of ZnNi-MOF / PEI-L012@Ab2 is 1 mg mL -1 .
[0053] In addition, an immunosensor with a suitable CeO2-Pt@Ab1 concentration is also important for building an efficient test system. If the CeO2-Pt@Ab1 concentration is too dilute, its amplification effect on the ECL of the ZnNi-MOF / PEI-L012@Ab2 probe is weak, while a high CeO2-Pt@Ab1 concentration will hinder the interfacial electron transfer process, resulting in a weakened ECL signal. In the designed "signal-on" immunoassay, the ECL signal is significantly enhanced with the increase of CeO2-Pt@Ab1 concentration ( Figure 5 D). When the concentration of CeO2-Pt@Ab1 is 1.5 mg mL -1 When the ECL intensity value reaches the maximum value, 1.5 mg mL -1 of CeO2-Pt@Ab1 as the optimal concentration.
[0054] <Detection of CA15-3 in actual samples>
[0055] In order to evaluate the feasibility of sandwich immunosensor, the standard addition method was used to detect CA15-3 in human serum samples. A series of standard concentrations of CA15-3 (1, 5, and 10 U mL -1 ) was injected into serum samples of 3 patients, and the ECL signals were recorded to evaluate the quantitative performance of the immunosensor. The corresponding results are shown in Table 1. The prepared sandwich immunosensor had a good recovery rate of 98.2-101.6% and a low RSD value of 1.9-3.1%. These results obtained in serum samples indicate that the developed sandwich immunosensor platform has high practicality in the identification of breast cancer marker CA15-3.
[0056] Table 1 Samples <![CDATA[Added(U mL -1 )]]> <![CDATA[Found(U mL -1 )]]> RSD(%) Recovery(%) 1 1.00 0.982 3.1 98.2 2 5.00 4.97 2.5 99.4 3 10.00 10.16 1.9 101.6
[0057] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for preparing a sandwich ZnNi-MOF electrochemical immunosensor, characterized in that: The biotin Ab1 solution was dropped onto the CeO2-Pt / streptavidin-modified GCE, incubated, and then sequentially soaked in BSA and incubated with CA15-3. Finally, the obtained electrode was incubated with the ZnNi-MOF / PEI-L012@Ab2 bioconjugate to obtain the sandwich ZnNi-MOF electrochemical immunosensor.
2. The method for preparing a sandwich ZnNi-MOF electrochemical immunosensor according to claim 1, characterized in that: The specific steps of CeO2-Pt / streptavidin modification of GCE are as follows: CeO2-Pt composite material is dispersed in ultrapure water, streptavidin is added, mixed evenly, then dripped on GCE, and dried.
3. The method for preparing a sandwich ZnNi-MOF electrochemical immunosensor according to claim 2, characterized in that: The preparation method of CeO2-Pt composite material is as follows: add CeO2 powder to ethylene glycol, perform ultrasonic treatment, add NaOH ethylene glycol solution and H2PtCl6·6H2O ethylene glycol solution in sequence under magnetic stirring, react at 180°C for 2h, collect the obtained product by centrifugation after cooling, and dry to obtain CeO2-Pt composite material.
4. The method for preparing a sandwich ZnNi-MOF electrochemical immunosensor according to claim 3, characterized in that: Preparation method of CeO2 powder: dissolve Ce(NO3)3·6H2O in water, add NaOH solution dropwise under magnetic stirring, react at 100°C for 24h, cool and collect the product by centrifugation to obtain CeO2 powder.
5. The method for preparing a sandwich ZnNi-MOF electrochemical immunosensor according to claim 1, characterized in that: The preparation method of ZnNi-MOF / PEI-L012@Ab2 bioconjugate is as follows: activating ZnNi-MOF / PEI-L012 in GA solution, centrifugally washing to obtain activated ZnNi-MOF / PEI-L012, covalently coupling the activated ZnNi-MOF / PEI-L012 solution with Ab2, adding BSA, centrifugally washing, and obtaining ZnNi-MOF / PEI-L012@Ab2 bioconjugate.
6. The method for preparing a sandwich ZnNi-MOF electrochemical immunosensor according to claim 5, characterized in that: The preparation method of ZnNi-MOF / PEI-L012 is as follows: ZnNi-MOF powder and PEI solution are mixed, ultrasonically treated, and centrifuged to obtain ZnNi-MOF coated with PEI; GA solution and L012 solution are added to the ZnNi-MOF coated with PEI, ultrasonically treated, stirred, and centrifuged to obtain ZnNi-MOF / PEI-L012.
7. The method for preparing a sandwich ZnNi-MOF electrochemical immunosensor according to claim 6, characterized in that: The preparation method of ZnNi-MOF powder is as follows: dissolve Zn(NO3)2·6H2O, Ni(acac)2, PVP and NH2-BDC in a solvent mixture consisting of ethanol, DMF and DMAC, react at 150°C for 4 hours, cool and collect the obtained product by centrifugation to obtain ZnNi-MOF.
8. A sandwich ZnNi-MOF electrochemical immunosensor, characterized in that: The sandwich ZnNi-MOF electrochemical immunosensor is prepared by the preparation method described in any one of claims 1 to 7.
9. Use of the sandwich ZnNi-MOF electrochemical immunosensor as claimed in claim 8 in the analysis and detection of breast cancer biomarkers.
10. The use of the sandwich ZnNi-MOF electrochemical immunosensor according to claim 9 in the analysis and detection of breast cancer biomarkers, characterized in that: The breast cancer biomarker is CA15-3.