Systems, sensors, and methods for detecting CEA based on electrophoresis and ECL principles.

Abstract

This invention belongs to the field of biosensing and clinical testing technology, specifically relating to a system, sensor, and method for detecting CEA based on electrophoresis and ECL principles. The carcinoembryonic antigen (CEA) detection system comprises an electrochemiluminescence immunoassay sensor and an electrophoresis apparatus. The electrophoresis apparatus is an electrolytic cup with a platinum wire disc at the bottom, connected to the negative terminal of a power supply. The working electrode of the electrochemiluminescence immunoassay sensor is suspended in the electrolytic cup via a fixed stage and connected to the positive terminal of the power supply. The analyte is driven to the electrode surface by an upward electric field for enrichment. A method for detecting CEA using this system is also provided, which can shorten the incubation time of CEA analytes to 5 minutes. This detection method has higher sensitivity and a lower detection limit. The sensor provided by this patent offers another strategy for the rapid detection of CEA and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and clinical testing technology, specifically relating to a system, sensor and method for detecting CEA based on electrophoresis and ECL principles. Background Technology

[0002] Carcinoembryonic antigen (CEA) is a broad-spectrum cancer biomarker and has been identified as one of the essential clinical biomarkers for cancer diagnosis, monitoring, and evaluation of treatment efficacy. Advances in techniques such as enzyme-linked immunosorbent assay (ELISA), fluorescence-based methods, surface-enhanced Raman scattering (SERS), and electrochemical methods have improved the identification and quantification of cancer biomarkers. However, these methods suffer from drawbacks such as long processing times, low sensitivity, and high cost.

[0003] Electrochemiluminescence (ECL) immunosensors have garnered significant attention in biological detection and clinical fields due to their excellent stability, sensitivity, and selectivity. Electrochemiluminescence refers to the process by which substances generated on the electrode surface undergo electron transfer reactions with chemical substances in solution or electrode products, forming an excited state, and then emitting light as they return to the ground state. The detection principle of ECL immunosensors involves immobilizing the immune complex resulting from the interaction between a labeled antigen and antibody at the electrode sensing interface. Then, by applying a suitable voltage using cyclic voltammetry, the labeled electrochemiluminescent material generates an ECL signal. Finally, statistical data is collected to determine the relationship between the target analyte concentration and the ECL signal intensity, enabling quantitative detection of the sample. ECL immunosensors offer high controllability and fast sample analysis speed, which can shorten measurement time to some extent. They also provide a rapid, highly sensitive, and highly selective method for detecting target analytes. Furthermore, ECL immunosensors also boast advantages such as low background signal, simple operation, ease of use, low cost, miniaturization, automation, and wide applicability. However, current ECL sensors still face significant challenges in achieving both sensitive analyte detection and extremely short detection times. Traditional ECL sensors typically employ incubation methods involving drop-on incubation at the sensing interface. The binding time for antigen-antibody immune complexes generally exceeds one hour, resulting in excessively long detection times for target antigens in clinical applications. Furthermore, there are significant limitations in the antigen-binding sites of antibodies and the spatial sites on the electrode surface, affecting the sensitivity and selectivity of the sensor.

[0004] Electrophoresis, particularly Western blotting, is an effective method for the rapid enrichment of target proteins and reduction of background interference. Its detection principle involves denaturing polyacrylamide gel electrophoresis to concentrate and separate proteins, transferring them to an NC or PVDF membrane, and then detecting specific signals using antigen-antibody reactions. While this technique has been widely applied in protein expression studies, antibody activity detection, and early disease diagnosis, it suffers from drawbacks such as cumbersome procedures and long detection times, necessitating the use of alternative detection tools. Summary of the Invention

[0005] In view of this, one of the objectives of this invention is to provide a carcinoembryonic antigen (CEA) detection system based on the principles of electrophoresis and electrochemiluminescence. Based on the principle of directional migration of proteins during electrophoresis, this invention designs a novel electrophoresis device for an ECL immunosensor, which efficiently and rapidly enriches and identifies target proteins, significantly shortens the incubation time of biomarkers, and achieves rapid and sensitive detection of target proteins.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A carcinoembryonic antigen (CEA) detection system based on electrophoresis and electrochemiluminescence principles comprises an electrochemiluminescence immunosensor for CEA and an electrophoresis apparatus. The electrophoresis apparatus is an electrolytic cup with a platinum wire disc at the bottom, which is connected to the negative terminal of the power supply of the electrophoresis apparatus. The electrochemiluminescence immunosensor is suspended in the electrolytic cup, and its working electrode is connected to the positive terminal of the power supply. The analyte is driven to the electrode surface by an upward electric field for enrichment.

[0008] In this technical field, dielectrophoresis (DEP) and capillary electrophoresis (CE) are also widely used in biosensing. Dielectrophoresis is based on the interaction between the dipoles of particles and the spatial gradient of the electric field, causing particles to move in a non-uniform electric field. This technique operates on neutral particles; in a non-uniform electric field, the magnitude and direction of the induced dipole moments differ due to variations in dielectric properties, causing the particles to move towards areas of higher or lower electric field strength. Dielectrophoresis is based on the stable trapping field generated by the electric field force. Depending on the positive and negative dielectrophoretic forces, particles move towards directions with different electric field strengths. It is a method well-suited for micron-scale biological particle manipulation and microsystem integration, and has been widely applied in microfluidics. Capillary electrophoresis is a novel liquid-phase separation technique that uses a capillary as the separation channel and a high-voltage DC electric field as the driving force. Capillary electrophoresis actually encompasses electrophoresis, chromatography, and their intersections. It has enabled analytical chemistry to move from the microliter to the nanoliter level, making single-cell and even single-molecule analysis possible, and can also be used for the separation and detection of biomacromolecules such as proteins. However, the electrophoresis principles of these two electrophoresis techniques are fundamentally different from the electrophoresis system used in this invention. Furthermore, the electrophoresis device we constructed is inexpensive, portable, easy to operate, and has minimal interference signals. Combined with an ECL immunosensor, the electric field-enhanced ECL immunosensor can achieve antigen incubation in a very short time, enabling rapid antigen detection. Simultaneously, it allows for more accurate detection in larger electrophoresis systems.

[0009] Furthermore, the electrochemiluminescence immunosensor can be suspended in the electrolytic cup using a fixing device such as an iron stand.

[0010] Furthermore, the electrochemiluminescence immunosensor employs a three-electrode system, including a working electrode, a counter electrode, and a reference electrode; the working electrode is a glassy carbon electrode, the counter electrode is a platinum wire electrode, and the reference electrode is Ag / AgCl.

[0011] Furthermore, the working electrode is parallel to the Pt disk.

[0012] Furthermore, the working electrode is immersed in the buffer solution of the electrophoresis apparatus at a height of 1 mm above the upper surface of the buffer solution.

[0013] Furthermore, the electrochemiluminescent immunosensor uses Au-CNNs as its electrochemiluminescent material; the Au-CNNs are prepared as follows:

[0014] (1) AuNPs were prepared by chemical reduction using HAuCl4·4H2O solution and C6H5Na3O7·2H2O.

[0015] (2) After ultrasonication, CNNs nanosheets are added to AuNPs to obtain Au-CNNs.

[0016] Further, step (1) specifically involves: adding HAuCl4·4H2O solution to ultrapure water, heating it to boiling with magnetic stirring, then quickly adding C6H5Na3O7·2H2O for chemical reduction, continuing to heat and stir for 30 minutes to obtain AuNPs, and storing them sealed and protected from light at 4°C.

[0017] Preferably, the concentration of the HAuCl4·4H2O solution is 0.01M; the concentration of the C6H5Na3O7·2H2O is 0.01M.

[0018] Preferably, the volume ratio of ultrapure water, HAuCl4·4H2O solution, and C6H5Na3O7·2H2O is 99:1:2.5.

[0019] Further, step (2) specifically involves: sonicating the CNNs nanosheets and adding them to the AuNPs solution, stirring at room temperature for 24 hours, centrifuging the mixed materials, washing them thoroughly three times with PBS, and redispersing them in 1 mL of 0.1 M PBS to obtain Au-CNNs.

[0020] Preferably, the mass-to-volume ratio of CNNs nanosheets to AuNPs is 0.8:1 (mg / ml).

[0021] As a preferred preparation method, the Au-CNNs are prepared as follows: 100 μL of HAuCl4·4H2O solution is added to 9.9 mL of ultrapure water, and the mixture is heated to boiling with magnetic stirring. Then, 250 μL of C6H5Na3O7·2H2O is quickly added for chemical reduction. The mixture is heated and stirred for another 30 min to obtain AuNPs, which are then stored at 4°C in a sealed container protected from light. 0.8 mg of CNNs nanosheets are sonicated and added to 1 mL of AuNPs solution. The mixture is stirred at room temperature for 24 h. After centrifugation, the mixture is thoroughly washed three times with PBS and redispersed in 1 mL of 0.1 M PBS to obtain Au-CNNs.

[0022] Furthermore, the working electrode of the electrochemiluminescence immunosensor is prepared by electropolymerizing GCE in an ATA solution, then activating the electropolymerized GCE in a PBS solution containing K2S2O8 and H2O2, and then adding EDC and NHS as coupling agents to activate the carboxyl groups of ATA to obtain the working electrode.

[0023] As a preferred technical solution, the preparation method of the working electrode is as follows: electropolymerization is performed in 10 ml of ATA solution at 50 mV / s, scanning from 0.2 to 1.2 V for 4 cycles, requiring strict protection from light. Activation treatment is then performed in PBS containing K₂S₂O₈ and H₂O₂, scanning from 0 to -1.6 V for 4 cycles. 10 μL of EDC and NHS (100 mg / mL) are added dropwise as coupling agents to activate the carboxyl groups of ATA for 30 min, thus obtaining the working electrode.

[0024] Furthermore, the ATA solution is prepared by dissolving 10 mM ATA in 0.01 M PBS.

[0025] Furthermore, the concentration of K2S2O8 is 0.1M; the concentration of H2O2 is 0.06M.

[0026] Furthermore, prior to electropolymerization, the bare GCE was polished with 0.3 μm alumina powder and ultrasonically cleaned in ultrapure water for 15 seconds. Then, the bare GCE was polished with 0.05 μm alumina powder and ultrasonically cleaned successively in ultrapure water, pure ethanol, and ultrapure water for 15 seconds each. This process was repeated in the presence of 5 mM [Fe(CN)6]. 3- / 4- The response of the bare GCE was recorded by CV scanning in PBS solution. The voltage was cycled from -0.2V to +0.6V at a rate of 100mV / s until a stable cyclic voltammogram was obtained. Finally, the electrode was rinsed with ultrapure water and dried at room temperature.

[0027] The second objective of this invention is to provide an electric field-enhanced ECL immunosensor for detecting carcinoembryonic antigen.

[0028] To achieve the above objectives, the present invention adopts the following technical solution:

[0029] An electric field-enhanced ECL immunosensor for detecting carcinoembryonic antigen (CEA), comprising an electrochemiluminescent immunosensor for CEA and an electrophoresis apparatus.

[0030] Furthermore, the electrochemiluminescence immunosensor employs a three-electrode system, including a working electrode, a counter electrode, and a reference electrode; the working electrode is a glassy carbon electrode, the counter electrode is a platinum wire electrode, and the reference electrode is Ag / AgCl.

[0031] Furthermore, the electrophoresis apparatus is an electrolytic cup with a platinum wire disc at the bottom, the platinum wire disc being connected to the negative terminal of the power supply of the electrophoresis apparatus; the working electrode of the electrochemiluminescence immunosensor is suspended in the electrolytic cup via a fixed stage and connected to the positive terminal of the power supply; the analyte is driven to the electrode surface by an upward electric field force for enrichment.

[0032] The method for constructing the electric field-enhanced ECL immunosensor includes: (1) preparing Au-CNNs; (2) assembling the electrophoresis incubation system: coiling a platinum wire around the bottom of the electrolytic cup of the device and connecting it to the negative terminal of the power supply of the electrophoresis apparatus, placing the working electrode 1 mm away from the upper liquid surface and connecting it to the positive terminal of the power supply, and parallel to the Pt disk; (3) using GCE as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode to construct an electrochemiluminescent immunosensor, and connecting the electrochemiluminescent immunosensor to the electrophoresis apparatus to construct an electric field-enhanced ECL immunosensor.

[0033] The third objective of this invention is to provide a method for detecting carcinoembryonic antigen (CEA) using the aforementioned CEA detection system. This method utilizes an electrophoresis system to directly shorten the incubation time from the traditional 60 minutes to 5 minutes, greatly reducing the CEA detection time.

[0034] To achieve the above objectives, the present invention adopts the following technical solution:

[0035] The method for detecting carcinoembryonic antigen using the aforementioned carcinoembryonic antigen detection system includes the following steps:

[0036] (1) Add Ab2 to Au-CNNs for coupling, incubate overnight at 4°C with stirring; after sealing, obtain the Au-CNNs / Ab2;

[0037] (2) Add Ab1 to the surface of the working electrode for incubation and then seal it. Then add the analyte to the electrophoresis apparatus for incubation, and then add Au-CNNs / Ab2 obtained in step (1) for incubation to obtain GCE / ATA / AB1 / CEA / Au-CNNs / Ab2.

[0038] (3) Perform ECL detection on the GCE / ATA / AB1 / CEA / Au-CNNs / Ab2 obtained in step (2) to obtain the ECL signal intensity.

[0039] Furthermore, the buffer solution in the electrophoresis apparatus is PBS with a concentration of 0.1M and a pH of 8.0.

[0040] Substances with opposite or no charges in the electrophoresis buffer solution migrate in different directions and at different speeds than those in CEA. They are less likely to move to the electrode surface and occupy space sites, further reducing interference from background signals.

[0041] Furthermore, the blocking solution is a 2wt% BSA solution.

[0042] Furthermore, in step (1), after overnight incubation at 4°C, the free secondary antibody needs to be removed by centrifugation.

[0043] Furthermore, in step (1), the closure time is 1 hour.

[0044] As a preferred technical solution, step (1) is as follows: add 100ul 10ug / ml Ab2 to Au-CNNs for conjugation, incubate overnight at 4℃ with stirring, centrifuge to remove free secondary antibody, add 500μL 2wt% BSA solution and incubate for 1h to block non-specific binding sites, centrifuge and wash to a final volume of 1ml to obtain functionalized Au-CNNs / Ab2.

[0045] Furthermore, the incubation of CEA in the electrophoresis apparatus was as follows: After adding CEA to the electrophoresis apparatus, the DC power supply was turned on to provide a voltage of 0.3V to the electrophoresis system. After the system ran for 5 minutes, the DC power supply was turned off, completing the rapid incubation of CEA.

[0046] Furthermore, in step (2), the three incubation times are 1 hour, 5 minutes, and 1 hour in sequence.

[0047] As a preferred technical solution, step (2) specifically involves: adding 10 μL of 10 μg / mL Ab1 to the surface of the working electrode and incubating for 1 h, followed by adding 2.0 wt% BSA solution for 30 min to block non-specific binding. Then, adding 10 μL of CEA to the electrophoresis apparatus for enrichment and incubation for 5 min. Finally, adding the Au-CNNs / Ab2 conjugate and incubating for 1 h forms GCE / ATA / AB1 / CEA / Au-CNNs / Ab2.

[0048] Furthermore, the incubation process was carried out at 37°C. After each modification, the electrode was eluted with 0.1M PBS and allowed to air dry before proceeding to the next step.

[0049] Furthermore, in step (2), the electrophoresis incubation time is 5 min, the external voltage is 0.3 V, and the height of the electrode surface from the liquid surface is 1 mm.

[0050] Furthermore, in step (3), the electrolyte for ECL detection is a PBS solution containing K2S2O8 and H2O2; the concentration of K2S2O8 is 0.1M; the concentration of H2O2 is 0.06M; and the concentration of PBS is 0.1M.

[0051] Furthermore, in step (3), the potential is continuously scanned from 0 to -1.6V at a scan rate of 0.1V / S. The high voltage of the photomultiplier tube (PMT) is set to 600V. When a stable ECL response is obtained, the ECL signal intensity is recorded for immunoassay.

[0052] The beneficial effects of this invention are as follows:

[0053] 1. This invention applies a novel electrophoresis device based on the principle of electrophoresis to the ECL immunosensor. In this electrophoresis incubation system, CEA is driven to the electrode surface by an upward electric field force for enrichment. The electrophoresis system directly shortens the incubation time from the traditional 60 min to 5 min, greatly reducing the detection time.

[0054] 2. This invention uses an electrophoresis system to incubate CEA, allowing CEA in the solution to move more accurately to the primary antibody binding site. Furthermore, by loading AuNPs onto CNNs, the conductivity of the CNNs is increased, thereby amplifying the ECL signal. The resulting electric field-enhanced ECL immunosensor has better sensitivity, with a linear range of 10 fg / mL to 10 ng / mL and a detection limit as low as 2.33 fg / mL.

[0055] 3. In this invention, substances with opposite charges or no charges in the electrophoresis buffer solution migrate in different directions and at different speeds than those in CEA, making them less likely to reach the electrode surface and occupy spatial sites, thus further reducing background signal interference. The electrophoretic immune system provides a novel mechanism for the selective focusing of analytes in large-volume solutions, ensuring accurate, reliable detection and high specificity.

[0056] 4. The electric field-enhanced ECL immunosensor constructed in this invention significantly shortens the detection time for CEA, further improves sensitivity, and lowers the detection limit, making it a potential means for rapid detection and diagnostic monitoring of cancer biomarkers in clinical practice. Furthermore, this application uses a standard spiking method to detect CEA in serum samples diluted in healthy individuals, achieving a standard recovery rate of 97.95%-106.00%, with RSD values ​​all less than 5.00%. In addition, the feasibility of detecting CEA in serum samples from clinical cancer patients was evaluated, and the detection results of our method were essentially consistent with those of the reference method. Both of these detection methods demonstrate that this detection has good application value in clinical samples. Attached Figure Description

[0057] Figure 1 This diagram illustrates the specific construction process of the electrophoresis apparatus and the electric field-enhanced ECL immunosensor of the present invention.

[0058] Figure 2 CV curves of electrodes at different modification stages;

[0059] Figure 3 For Nyquist plot;

[0060] Figure 4 A graph showing the relationship between electrophoretic incubation time and CEA enrichment;

[0061] Figure 5 A linear relationship graph of CEA concentration;

[0062] Figure 6 This is a selective results graph;

[0063] Figure 7 The stability results are shown in the figure.

[0064] Figure 8 This is a model diagram of the carcinoembryonic antigen detection system of this application, in which 1 is an electric field enhanced ECL immunosensor, 2 is an electrolytic cup, 3 is a Pt disk, 4 is an electrophoresis apparatus; the black triangle symbol represents a clamp. Detailed Implementation

[0065] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0066] In this embodiment of the invention, chloroauric acid (HAuCl4·4H2O), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Admas-Beta (Shanghai, China). Graphite-phase carbon nitride nanosheets (CNNs) were purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd. Bovine serum albumin (BSA), sodium citrate, 2-aminoterephthalic acid (ATA), potassium ferrocyanide (K3[Fe(CN)6]), and potassium ferrocyanide (K4[Fe(CN)6·3H2O]) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Potassium dihydrogen phosphate (KH2PO4) was purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd. Anhydrous disodium hydrogen phosphate (Na2HPO4) was provided by Chengdu Kelong Chemical Co., Ltd. Potassium persulfate (K2S2O8) was purchased from Shanghai Ron Chemical Technology Co., Ltd. 30% hydrogen peroxide (H2O2) was purchased from Chengdu Kelon Chemical Co., Ltd. The carcinoembryonic antigen (CEA) DIY kit was purchased from Wuhan Yunclone Co., Ltd.

[0067] In this embodiment of the invention, all reagents are analytical grade reagents, and all solutions are prepared using ultrapure water (18.2 MΩcm).

[0068] In this embodiment of the invention, all electrochemical experiments were performed on a CHI 660E electrochemical workstation (Chenhua Instruments Co., Ltd., Shanghai, China). ECL measurements were performed on an MPI-E multifunctional electrochemiluminescence analyzer (Xi'an Remex Analytical Instruments Co., Ltd.). A three-electrode system was used: a glassy carbon electrode (GCE, Ф=4mm) as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. The UTP1306S series economical DC stable power supply was from Uni-Trend Technology Co., Ltd. (China), with a continuously adjustable voltage range of 0–32V and a maximum output current of 0–6.0A.

[0069] In this embodiment of the invention, the collection and study of human serum samples (Human 1 and Human 2) were conducted with the consent of the relevant students of the Cell and Histology Laboratory of Chongqing Medical University; the clinical serum samples involved in this application have also been approved and authorized by the Ethics Committee of Guizhou Provincial People's Hospital, and the consent of the patients and their families has been obtained.

[0070] Example 1. Construction of an electric field-enhanced ECL immunosensor

[0071] The construction process of the electrophoresis device and the electric field-enhanced ECL immunosensor of the present invention is as follows: Figure 1 As shown; the model diagram of the carcinoembryonic antigen detection system of this application is shown below. Figure 8 As shown, it consists of an electric field enhanced ECL immunosensor 1, an electrolytic cup 2, a Pt disk 3, and an electrophoresis apparatus 4; the Pt disk 3 is connected to the negative electrode of the electrophoresis apparatus 4, and the working electrode of the electric field enhanced ECL immunosensor 1 is connected to the positive electrode of the electrophoresis apparatus 4; the electric field enhanced ECL immunosensor 1 can be fixed and suspended in the electrolytic cup 2 by means of a device such as an iron stand, and the working electrode is about 1 mm away from the upper liquid surface of the buffer solution in the electrophoresis apparatus.

[0072] The construction of an electric field-enhanced ECL immune sensor specifically includes the following steps:

[0073] 1. Preparation of electrochemiluminescent gold nanoparticles-carbon nitride (Au-CNNs): 100 μL of HAuCl4·4H2O (0.01 M) solution was added to 9.9 mL of ultrapure water. After heating to boiling with magnetic stirring, 250 μL of C6H5Na3O7·2H2O (0.01 M) was quickly added for chemical reduction. The mixture was heated and stirred for another 30 min to obtain AuNPs, which were then stored at 4 °C in a sealed container protected from light. 0.8 mg of CNNs nanosheets were sonicated and added to 1 mL of AuNPs solution. The mixture was stirred at room temperature for 24 h. After centrifugation, the mixture was thoroughly washed three times with PBS and redispersed in 1 mL of 0.1 M PBS to obtain Au-CNNs. Add 100 μL of 10 μg / ml Ab2 for conjugation, incubate overnight at 4°C with stirring, centrifuge again to remove free secondary antibody, add 500 μL of 2 wt% BSA solution and incubate for 1 h to block non-specific binding sites, centrifuge and wash to a final volume of 1 ml to obtain functionalized Au-CNNs / Ab2.

[0074] 2. Assembly of the electrophoresis incubation system: A platinum wire was coiled around the bottom of the electrolytic cup and connected to the negative terminal of the electrophoresis apparatus power supply. The working electrode was placed 1 mm above the liquid surface, connected to the positive terminal of the power supply, and parallel to the Pt disk. The buffer solution system in the electrophoresis apparatus consisted of 5 ml of PBS (0.1 M, pH = 8.0) with 10 μL of CEA at different concentrations added. Then, the DC power supply was turned on to provide 0.3 V to the electrophoresis system. After running for 5 minutes, the DC power supply was turned off to complete the rapid incubation of CEA. The electrode was gently eluted with PBS to remove free CEA. Optimization results showed that the optimal electrophoresis incubation time was 5 minutes, the electrode surface height from the liquid surface was 1 mm, the applied external voltage was 0.3 V, and the optimal pH in the electrophoresis system was 8.0.

[0075] 3. Construction of an electric field-enhanced ECL immunosensor: Before electropolymerization, bare GCEs were polished with 0.3 μm alumina powder and ultrasonically cleaned in ultrapure water for 15 seconds. Then, bare GCEs were polished with 0.05 μm alumina powder and ultrasonically cleaned successively in ultrapure water, pure ethanol, and ultrapure water for 15 seconds each. [The text then abruptly shifts to a description of a 5 mM Fe(CN)6 immunosensor.] 3- / 4-The response of the bare GCE was recorded by CV scanning in PBS solution. The voltage was cycled from -0.2V to +0.6V at a rate of 100mV / s until a stable cyclic voltammogram was obtained. Finally, the electrode was rinsed with ultrapure water and dried at room temperature. Electropolymerization was performed in 10 mL of ATA solution (10 mM ATA dissolved in 0.01 M PBS) with CV scanning from 0.2 to 1.2V for 4 cycles at 50 mV / s, under strict light protection. Activation was performed in PBS containing K2S2O8 (0.1 M) and H2O2 (0.06 M) with CV scanning from 0 to -1.6V for 4 cycles. The carboxyl groups of ATA were activated by adding 10 μL of EDC and NHS (100 mg / mL) as coupling agents for 30 min. 10 μL of 10 μg / mL Ab1 was added to the modified GCE surface and incubated for 1 h, followed by blocking with 2.0 wt% BSA solution for 30 min to prevent nonspecific binding. 10 μL of CEA at different concentrations was added to the electrophoresis apparatus for enrichment and incubation for 5 min. Then, Au-CNNs / Ab2 conjugate was added and incubated for 1 h to form GCE / ATA / AB1 / CEA / Au-CNNs / Ab2. All incubation processes were performed at 37 °C. After each modification, the electrode was eluted with 0.1 M PBS and allowed to air dry before proceeding to the next step. The constructed sensor was stored at 4 °C for subsequent use.

[0076] 4. Detection of ECL: The electrolyte for ECL measurement was a 0.1M PBS solution containing K2S2O8 (0.1M) and H2O2 (0.06M). The potential was continuously scanned from 0 to -1.6V at a scan rate of 0.1V / s. The photomultiplier tube (PMT) voltage was set to 600V. When a stable ECL response was obtained, the ECL signal intensity was recorded for immunoassay.

[0077] Example 2. Electrochemical characterization of an electric field-enhanced ECL immunosensor

[0078] To verify the successful construction of the ECL immunosensor, we performed electrochemical characterization using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results are as follows: Figures 2-3 As shown, Figure 2The CV curves of the electrodes at different modification stages are shown. Due to the poor conductivity of ATA, when fixed to the electrode surface, it restricts the transfer of the electrochemical redox probe, resulting in a sharp drop in peak current. After electroactivation with H2O2 and K2S2O8, the current increases significantly, which helps to reduce impedance. After successive fixation with Ab1, BSA, and CEA, the peak current continues to decrease due to the poor conductivity of biological protein molecules. After modification with Au-CNN / Ab2, the current response improves. Electrochemical impedance spectroscopy (EIS) is another electrochemical technique. Each point on the Nyquist plot represents the impedance at a certain frequency. The semicircular part of the curve is positively correlated with the charge transfer resistance (Rct), such as... Figure 3 As shown, after gradual modification, the successful construction of the novel ECL immunosensor was also confirmed based on the electron transfer relationship of Ret on the electrode surface.

[0079] Example 3. Optimization of electrophoresis incubation time

[0080] In the buffer system of the electrophoresis apparatus, CEA is driven by an upward electric field to accumulate on the electrode surface. The time required for CEA to specifically bind to the electrode surface is optimized. As the electrophoresis apparatus runs longer, more CEA accumulates on the electrode surface, and the ECL signal increases accordingly. When the CEA binding amount reaches saturation, the ECL signal remains essentially unchanged. Figure 4 The results showed that the ECL signal reached its maximum at an electrophoresis time of 5 minutes, therefore 5 minutes was chosen as the optimal incubation time.

[0081] Example 4. Linear relationship of CEA concentration

[0082] Figure 5 The study demonstrated that the ECL signal of the electric field-enhanced ECL immunosensor gradually increased with increasing CEA concentration. Within the concentration range of 10 fg / ml to 10 ng / ml, a strong linear correlation was found between the ECL intensity and the logarithm of the CEA concentration, with the linear regression equation being Y = 2406.04lgC. CEA +6702.26, correlation coefficient R 2 The value was 0.9965, and the limit of detection (LOD) was 2.33 fg / ml (S / N = 3).

[0083] Example 5. Selective Experiment

[0084] To test the selectivity of the electric field-enhanced ECL immunosensor prepared in Example 1, the present invention tested the response to some interfering substances. The results are as follows: Figure 6As shown, compared with CEA, BSA, Muc1, ErBb2 and ERa all have very low ECL signal responses. The specific binding of the antigen and antibody themselves gives them high selectivity. Moreover, the pI and molecular weight of different interfering substances are quite different. In the specific electrophoresis system of this work, the migration effect is not as good as CEA. The ECL immunosensor using the electrophoresis device has better selectivity. This electric field principle can also provide a new anti-interference approach.

[0085] Example 6. Stability Test

[0086] Sensors prepared in the same batch were stored at 4°C, and CEA was detected at different time intervals. The results are as follows: Figure 7 As shown, the ECL signal did not change significantly after 11 days and remained at 93.77% of its initial intensity, indicating acceptable long-term stability.

[0087] Example 7. Detection of real samples and clinical serum samples

[0088] 1. Real sample testing

[0089] Human serum samples (Human 1 and Human 2) were obtained from students in the Cell and Histology Laboratory of Chongqing Medical University; venous blood was drawn and centrifuged by students majoring in clinical medicine. Both Human 1 and Human 2 were from healthy individuals. CEA levels in the diluted serum samples were detected using a standard spiking method. Due to individual variability, serum samples from different individuals were used in this application, with standard CEA concentrations of 100 pg / ml and 1 pg / ml, respectively. As shown in Table 1, the standard recoveries ranged from 97.95% to 106.00%, with RSD values ​​less than 5.00%.

[0090] Table 1

[0091]

[0092] 2. Clinical serum sample testing

[0093] This invention also evaluated the feasibility of using an electric field-enhanced ECL immunosensor to detect clinical serum samples. In the method of this invention, clinical serum samples were diluted 1000-fold with PBS (0.1M, pH 8.0). The reference method used a commercially available ECLIA from Elecsys CEA, performed on a hospital electrochemiluminescence analyzer. Ultimately, the results of this method were largely consistent with those of the reference method, indicating that the method of this invention has reliable applicability in clinical sample analysis. Table 2 compares the results of the ECL immunosensor constructed using the method of this invention with those detected by a commercially available ECL immunosensor in a hospital. The clinical serum samples were from different cancer patients at Guizhou Provincial People's Hospital.

[0094] Table 2. Results of immunoassay of CEA in clinical serum samples using the method of the present invention and the reference method.

[0095] 1 21.7 22.21 2.4 2 0.81 0.73 -9.9 3 92.9 94.03 1.2 4 29.2 32.07 9.8 5 14.9 12.85 -13.7 6 1.24 1.34 8.1

[0096] The above results demonstrate that the electric field-enhanced ECL immunosensor of this application can achieve CEA incubation within 5 minutes, greatly shortening the detection time of CEA. It also has good detection sensitivity, low detection limit, and excellent selectivity and stability, which has good application significance for the detection of clinical samples.

Claims

1. A carcinoembryonic antigen detection system based on electrophoresis and electrochemiluminescence principles, characterized in that, The carcinoembryonic antigen (CEA) detection system consists of an electrochemiluminescent immunosensor and an electrophoresis apparatus. The electrophoresis apparatus is an electrolytic cup with a platinum wire disc at the bottom, which is connected to the negative terminal of the power supply of the electrophoresis apparatus. The electrochemiluminescent immunosensor is suspended in the electrolytic cup, and its working electrode is connected to the positive terminal of the power supply. The analyte is driven to the electrode surface by an upward electric field for enrichment. The electrochemiluminescence immunosensor employs a three-electrode system, wherein the working electrode is a glassy carbon electrode, the counter electrode is a platinum wire electrode, and the reference electrode is Ag / AgCl; the electrochemiluminescence material of the electrochemiluminescence immunosensor is Au-CNNs. The working electrode is immersed in the buffer solution of the electrophoresis apparatus, and the height of the electrode from the upper liquid surface of the buffer solution is 1 mm. The method for preparing the Au-CNNs is as follows: (1) AuNPs were prepared by chemical reduction using HAuCl4•4H2O solution and C6H5Na3O7•2H2O; (2) After ultrasonication, CNNs nanosheets are added to AuNPs to obtain Au-CNNs.

2. The carcinoembryonic antigen detection system according to claim 1, characterized in that, The working electrode of the electrochemiluminescence immunosensor is prepared by electropolymerizing GCE in a 2-aminoterephthalic acid (ATA) solution, activating the electropolymerized GCE in a PBS solution containing K2S2O8 and H2O2, and then adding EDC and NHS as coupling agents to activate the carboxyl group of ATA to obtain the working electrode.

3. A method for detecting carcinoembryonic antigen using the carcinoembryonic antigen detection system according to claim 1, characterized in that, Includes the following steps: (1) Add Ab2 to Au-CNNs and incubate overnight at 4°C with stirring; after sealing, Au-CNNs / Ab2 is obtained; (2) Add Ab1 to the surface of the working electrode for incubation and then seal it. Then add the analyte to the electrophoresis apparatus for incubation, and then add Au-CNNs / Ab2 obtained in step (1) for incubation to obtain GCE / ATA / AB1 / CEA / Au-CNNs / Ab2. (3) Perform ECL detection on the GCE / ATA / AB1 / CEA / Au-CNNs / Ab2 obtained in step (2) to obtain the ECL signal intensity.

4. The method according to claim 3, characterized in that, The buffer solution in the electrophoresis apparatus is PBS with a concentration of 0.1M and a pH of 8.

0.

5. The method according to claim 3, characterized in that, In step (2), the electrophoresis incubation time is 5 min and the external voltage is 0.3 V.

6. The method according to claim 3, characterized in that, In step (3), the electrolyte for ECL detection is a PBS solution containing K2S2O8 and H2O2.

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