Sensing method for detecting double tumor markers CEA (carcino-embryonic antigen) and AFP (alpha-fetoprotein) on surfaces of living cells through ultra-sensitive in-situ electrochemiluminescence
By using a closed bipolar electrode array chip and electrochemiluminescence technology, combined with covalent organic framework materials and nanoprobes, ultrasensitive detection of carcinoembryonic antigen and alpha-fetoprotein was achieved, solving the false negative and false positive problems in the detection of multiple biomarkers in early cancer and realizing high-throughput, rapid and cost-effective simultaneous detection of dual tumor biomarkers.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LIAOCHENG UNIV
- Filing Date
- 2026-01-08
- Publication Date
- 2026-04-28
AI Technical Summary
In the early stages of cancer, tumor marker concentrations are low and detection signals are weak, which can easily lead to false negative or false positive results. Single marker detection is not accurate enough, and existing technologies cannot achieve high-throughput, rapid and cost-effective simultaneous detection of multiple markers.
A closed bipolar electrode array chip combined with electrochemiluminescence technology was used. Covalent organic framework (COF) material was used as a carrier, gold nanoparticles (Au) were used for signal amplification, methylene blue (MB) was used as an electroactive substance, and aptamer DNA was used for targeted recognition. A signal amplification nanoprobe COF@Au@MB-Apt was constructed. Combined with antibody-modified electrodes, electrochemiluminescence signal amplification was achieved under potential drive to quantitatively detect carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP).
It achieves ultrasensitive detection of CEA and AFP on the surface of live cells, with detection limits of 6.12 pg mL⁻¹ and 0.25 pg mL⁻¹, respectively. It has high sensitivity, stability and selectivity, supports single-cell quantitative analysis, and reduces sample and cost requirements.
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Figure CN121933729A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biosensing technology, specifically relating to a method for ultrasensitive in-situ electrochemiluminescence detection of two tumor markers on the surface of living cells, carcinoembryonic antigen (CEA) and α-carcinoembryonic antigen (α-carcinoembryonic antigen), based on a closed bipolar electrode array (c-BPE). Sensing methods for fetal protein (AFP). Background Technology
[0002] Tumor markers are a class of bioactive molecules present on the surface of tumor cells or secreted into peripheral blood, tissues, and body fluids. Their quantitative detection and early discovery provide crucial evidence for cancer diagnosis and treatment. However, in the early stages of cancer, tumor marker concentrations are low and detection signals are weak, easily leading to false negatives or false positives, posing a challenge to the accuracy of single-marker detection. This is mainly because most cancers involve more than one tumor marker, and most markers are not tumor-type specific. Therefore, simultaneous analysis of two or more tumor markers in a single-chip assay can yield more accurate results, higher throughput, faster analysis speed, and significantly reduced sample and cost requirements. Cell surface proteins, as one of the tumor markers, provide crucial clinical information for assessing disease progression, evaluating the effectiveness of cancer interventions, and predicting prognosis. Developing analytical techniques capable of simultaneously and ultrasensitively detecting two tumor-related proteins on the surface of living cell membranes will demonstrate greater value and application prospects in the biomedical field.
[0003] To date, various analytical techniques, including colorimetry, fluorescence assays, enzyme-linked immunosorbent assay (ELISA), surface-enhanced Raman scattering (SERS), electrochemical methods, photoelectrochemical methods, chemiluminescence (CL), and electrochemiluminescence (ECL), have been widely applied to the multi-path detection of tumor markers. Among them, ECL technology combines the advantages of both electrochemistry and chemiluminescence, thus becoming a detection method renowned for its high signal-to-noise ratio, excellent controllability, and outstanding sensitivity. Multi-labeling strategies and multi-electrode platforms are two core methodologies in multi-path bioanalysis: the former relies on differentiated signal labels to achieve target identification, while the latter utilizes independent sensor interface arrays to achieve parallel identification and detection.
[0004] The combination of closed bipolar electrode arrays and electrochemiluminescence technology has been successfully applied to the simultaneous detection of various biomarkers. Bipolar electrodes are conductors or semiconductors immersed in an electrolyte, not connected by direct wires. Under an applied electric field, complementary electrochemical reactions occur at both ends of the bipolar electrode due to field-induced polarization. The closed bipolar electrode effectively prevents signal crosstalk between the two half-cells by physically isolating the cathode and anode chambers with insulating materials, thus making it widely applicable in multi-channel biosensing. Based on its large-scale electrode array design, the system requires only a single power supply for overall control, and the states of each electrode can be read in parallel, facilitating high-throughput screening.
[0005] This invention addresses the key technical bottlenecks and the problem of "insufficient accuracy of single marker detection" in the early stages of cancer, where low concentrations of tumor markers and weak detection signals easily lead to false negative or false positive results. It proposes to simultaneously analyze two or more tumor markers in a single chip detection process, which can obtain more accurate results, higher throughput, faster analysis speed, and significantly reduce sample and cost requirements. Summary of the Invention
[0006] This invention addresses the key technical bottlenecks and the problem of "insufficient accuracy of single marker detection" in the early stages of cancer, where low concentrations of tumor markers and weak detection signals easily lead to false negative or false positive results. It proposes to simultaneously analyze two or more tumor markers in a single chip detection process, which can obtain more accurate results, higher throughput, faster analysis speed, and significantly reduce sample and cost requirements.
[0007] This invention addresses the problems of existing technologies by providing an ultrasensitive in-situ detection method for dual tumor markers on the surface of living cells, comprising a closed bipolar electrode array chip, a signal-amplified nanoprobe, and an electrochemiluminescence detection technique; the signal-amplified nanoprobe comprises a functional covalent organic framework (COF) material, gold nanoparticles (Au NPs), signal molecules, and aptamer DNA targeting tumor markers on the cell surface.
[0008] Furthermore, the dual tumor markers on the cell surface are any two of carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), mucin MUC1, and prostate antigen (PSA), with carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) being preferred.
[0009] Furthermore, the signal amplification nanoprobe is COF@Au@MB-Apt.
[0010] Preferably, the preparation process of the signal amplification nanoprobe is as follows: Based on COF material, gold nanoparticles are deposited on the COF surface by in-situ reduction of chloroauric acid, and then the electrochemical signaling molecule methylene blue (MB) is modified on the COF material. The aptamer DNA of cell surface tumor markers is linked through Au-SH bonds to form COF@Au@MB-Apt, which has both signal amplification and targeted recognition functions.
[0011] More preferably, the cathode of the closed bipolar electrode is modified with an antibody against a tumor cell-specific antigen, the tumor cells are cultured in situ, and then modified with a signal-amplifying nanoprobe COF@Au@MB-Apt; [Ru(bpy)3] is added to the anode of the closed bipolar electrode. 2+ / TPA tripropylamine.
[0012] Furthermore, the concentration of the antibody is 1-40 μg / mL. -1 Preferred, 20 μg mL -1 The concentration of COF@Au@MB-Apt is 25-70 µg / mL. - ¹, The preferred concentration is 50 µg / mL - ¹, The tumor cells mentioned are MCF-7 breast cancer cells.
[0013] Furthermore, the [Ru(bpy)3]² + The concentration of added substance is 0.5-2 mM, preferably 1 mM; the concentration of added tripropylamine TPA is 20-70 mM, preferably 50 mM.
[0014] Preferably, the aptamer DNA strand sequence of the tumor marker carcinoembryonic antigen (CEA) is as follows: 5'-SH-(CH2)6-ATACCAGCTTTATTCAATT-3'; The aptamer DNA strand sequence of the tumor marker alpha-fetoprotein (AFP) is as follows: 5'-SH-(CH2)6-GGCAGGAAGACAAACAAGCTTGGCGGCGGGAAGGTGTTTAAATTCCCGGGTCTGCGTGGTCTGTGGTGCTGT-3'.
[0015] More preferably, the detection limits for CEA and AFP are 6.12 pg mL, respectively. - ¹ and 0.25 pg mL - ¹.
[0016] Preferably, the closed bipolar electrode array chip includes a lower indium tin oxide (ITO) electrode array and an upper polydimethylsiloxane (PDMS) chip; the PDMS chip is fabricated by soft photolithography, and the PDMS has a microfluidic channel structure that matches the lower ITO electrode.
[0017] Furthermore, a sensing method for ultrasensitive in-situ detection of dual tumor markers on the surface of live cells is provided. The specific detection method involves modifying the cathode of a closed bipolar electrode with an antibody containing specific antigens on tumor cells, at a concentration of 20 μg / mL. -1 Based on this, different concentrations of MCF-7 breast cancer cells were cultured in situ, and then 50 µg / mL of the modified [cell] was used. - ¹Signal amplification nanoprobe COF@Au@MB-Apt. 1 mM [Ru(bpy)3]² was added to the anode of the closed bipolar electrode. + And 50 mM tripropylamine (TPA). With a potential of 5 V applied to the driving electrode, MB undergoes electrochemical reduction at the cathode surface, a process triggered by a charge balance mechanism within the anolyte chamber [Ru(bpy)3]². + The tripropylamine (TPA) system undergoes a Faradaic oxidation reaction, generating a strong ECL signal. The intensity of the anodic ECL signal is positively correlated with the amount of signal molecules loaded on the cell surface at the cathode, and thus correlated with the expression levels of tumor markers on the cell surface. Consequently, the expression levels of tumor cell surface antigens can be quantified by measuring the intensity of the anodic ECL signal.
[0018] The following is an explanation and description of the terminology used in this invention: The abbreviation for Carcinoembryonic Antigen is CEA; AFP stands for Alpha-Fetoprotein, hence its standard abbreviation is AFP. Similar to CEA, AFP is also a tumor marker.
[0019] c-BPE-ECL refers to an advanced electrochemiluminescence sensing platform based on a closed bipolar electrode. Through a spatially separated design, it perfectly combines the biochemical reaction sensing end with the optical detection signal reporting end, representing a key technology for achieving high sensitivity, high selectivity, and applicability to complex real-world sample detection. It is currently a cutting-edge research direction in the fields of biosensing and microfluidic analysis.
[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.
[0021] Beneficial technical effects of the present invention: 1. This invention leverages the advantages of a closed bipolar electrode (c-BPE) array and electrochemiluminescence (ECL) technology to establish a c-BPE-ECL platform for the simultaneous detection of two tumor-associated proteins on the cell surface: carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Based on the inherent properties of covalent organic frameworks (COFs), such as high specific surface area, excellent stability, porous structure, and simple synthesis, a bifunctional signal probe, COF@Au@MB-Apt, was designed and successfully synthesized. This probe is constructed through the strategic assembly of four functional components: a covalent organic framework (COF) as a carrier, gold nanoparticles (Au) for signal amplification, methylene blue (MB) as an electroactive substance, and an aptamer (Apt) targeting a specific target for molecular recognition. In the BPE system, using MCF-7 cells as a biological model, the recognition event between the aptamer and the protein facilitated the selective anchoring of the COF@Au@MB-Apt signal probe to the cell membrane within the cathode chamber. After applying an optimized potential, MB molecules anchored to the cell surface undergo electrochemical reduction at the BPE cathode. Due to the principle of charge neutrality, this cathodic process induces a corresponding Faraday reaction at the anolyte, ultimately leading to [Ru(bpy)3]. 2+ The electrochemiluminescence signal is generated in the / TPA co-reaction system. Therefore, the expression of tumor cell surface protein markers can be quantified based on the intensity of the ECL signal. This c-BPE-ECL platform achieves ultrasensitive detection of CEA and AFP. Moreover, this ECL biosensor possesses high sensitivity, stability, selectivity, and reproducibility, and can effectively analyze the expression of CEA and AFP on the surface of various cell lines.
[0022] 2. During the testing process, through antigens Antibody-specific binding to MCF Seven cells were immobilized on the surface of the BPE cathode, and nanoprobes were used to target and bind to cell surface markers via a sandwich-immunoassay. Upon application of an appropriate potential, MB on the cathode surface underwent electrochemical reduction, a process triggered by a charge balance mechanism within the anolyte chamber [Ru(bpy)3]². + The tripropylamine (TPA) system undergoes a Faradaic oxidation reaction, resulting in a strong ECL signal. The constructed c BPE The ECL sensing platform achieves detection limits of 6.12 pg·mL for CEA and AFP, respectively. - ¹ and 0.25 pg·mL - ¹, and can achieve single-cell quantitative analysis (single MCF) The expression levels of CEA and AFP on the cell surface were 37 fg and 0.25 fg, respectively. This platform also possesses excellent stability, high selectivity, and good intra-batch / inter-batch reproducibility, providing a sensitive, reliable, and promising technique for in situ analysis of multiple targets on the surface of live cells. Attached Figure Description
[0023] Figure 1 COF@Au@MB of the present invention Apt signal probe preparation and c-based A schematic diagram of the BPE array chip simultaneously detecting CEA and AFP; Figure 2 The TEM and elemental analysis diagrams of the nanoprobe COF@Au@MB-Apt of this invention are shown below. Figure 3 This is a schematic diagram illustrating the principle verification of the dual tumor marker sensor for CEA and AFP of the present invention. Figure 4 ECL spectra and standard curves for the detection of CEA and AFP by the dual tumor marker sensor of the present invention; Figure 5 This is a graph showing the MCF-7 cell viability assay in the c-BPE microfluidic sensing channel of the present invention. Figure 6 ECL spectra and standard curves for detecting CEA and AFP on the surface of MCF-7 cells at different concentrations using the dual tumor marker sensor of this invention. Detailed Implementation
[0024] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.
[0025] Example 1: Preparation process of the nanoprobe COF@Au@MB-Apt like Figure 1 As shown, 1,3,5-tris(4-aminophenyl)benzene (TAPB, 17.6 mg, 0.05 mmol) and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP, 21.4 mg, 0.11 mmol) were added to 40 mL of acetonitrile. The mixture was sonicated for 3 min to ensure complete dispersion. Then, 1 mL of 12 M acetic acid was added with stirring, and the reaction was continued at room temperature for 4 h. After the reaction was complete, the yellow precipitate was collected by centrifugation, washed three times each with acetonitrile and anhydrous ethanol, and finally dried under vacuum at 60 °C to obtain COF nanomaterials.
[0026] To synthesize COF@Au, 15 mg of COF was first ultrasonically dispersed in 10 mL of methanol for 5 min. After stirring for 5 h, 80 µL of 1% HAuCl4 solution was slowly added at 0 °C. Subsequently, 0.5 mL of 0.2 M NaBH4 methanol solution was added dropwise, and stirring was continued at 0 °C for 3 h. The precipitate was collected by centrifugation at 10,000 rpm for 10 min, washed three times with methanol, and dried under vacuum at 60 °C to obtain brown COF@Au.
[0027] Disperse 5 mg COF@Au in 25 mL of deionized water, sonicate, and then add 2 mL of MB (1 mg·mL⁻¹). - ¹), stir for 12 h. Centrifuge (10,000 rpm, 10 min) to obtain a grass-green precipitate, wash with water until the supernatant is colorless, and then disperse in 2 mL of 10 mM Tris-HCl solution to obtain COF@Au@MB.
[0028] To assemble the COF@Au@MB-Apt signal probe, AFP or CEA aptamer sequences (200 μL, 5 μM) were incubated with tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP, 1 mM) under shaking conditions for 1 h to reduce disulfide bonds to the corresponding thiol groups. At 4 °C, 2 mL of COF@Au@MB nanocomposite material at a concentration of 1 mg / mL was added, and the reaction was allowed to proceed with shaking for 10 h, allowing the AFP or CEA aptamer to bind to the COF@Au@MB surface via Au-S bonds. The precipitate was collected by centrifugation, washed three times with 0.01 M PBS, and finally redispersed in 2 mL of 0.01 M PBS buffer for later use.
[0029] Example 2: Transmission electron microscopy characterization of COF@Au@MB-Apt like Figure 2 As shown, the results indicate that COF@Au@MB-Apt exhibits good dispersion with a size of approximately 300 nm, and the Au NPs are uniformly dispersed. To verify the functionalization of aptamer DNA on the surface of the COF@Au@MB composite material, HAADF-STEM imaging and EDX elemental distribution analysis were performed on COF@Au@MB-Apt. The EDX elemental distribution results confirmed that C, N, O, Au, S, and P elements were uniformly distributed throughout the probe. Notably, S elements in MB and Au nanoparticles achieved uniform deposition on the COF substrate surface, and the presence of P elements demonstrated successful grafting of DNA aptamers onto the COF@Au@MB surface.
[0030] Example 3: Principle Verification Experiment like Figure 3 As shown, only when the cathode is modified with a specific nanoprobe and the anode chamber contains Ru(bpy)3²+ Significant ECL signals can only be observed in the anodic detection zone when TPA is used. Conversely, if an unmodified cathode (i.e., without fixed antibodies, antigens, or nanoprobes) is used, or if only PBS buffer is added to the cathode chamber, the ECL signal is negligible due to the lack of necessary electroactive materials. Furthermore, when Ru(bpy)3² is present in the anodic chamber... + After the TPA system was replaced with PBS, no obvious ECL signal was detected in the voltage range of 3.5-5.0 V. The fundamental reason is that the system lacks the ECL active substance necessary to trigger the core redox reaction, thus verifying the feasibility of detecting CEA and AFP.
[0031] Example 4: Experiment on the determination of CEA and AFP standard curves Based on bipolar electrode (BPE) technology, this invention constructs a multiplex ECL sensing platform on a single chip for the simultaneous detection of two tumor markers, CEA and AFP. A significant concentration-dependent relationship is observed between ECL intensity and CEA concentration; as CEA concentration increases, ECL intensity gradually increases. Figure 4 A). Within 0.01–50 ng·mL - ¹Within the concentration range, there is a good linear relationship between the ECL signal and the logarithm of the CEA concentration. Figure 4 B), the fitted calibration curve equation is I = 1308.9log C CEA (ng·mL) - ¹) + 3084.5 (R² = 0.994), based on a signal-to-noise ratio of 3:1, the limit of detection (LOD) is calculated to be 6.12 pg·mL. - ¹.
[0032] Meanwhile, AFP in 10 - ³~100 ng·mL - ¹The concentration-dependent ECL signal enhancement trend is also observed within the concentration range. Figure 4 (C), the signal intensity showed a significant linear relationship with the logarithm of AFP concentration, and the fitting equation was I = 1091.5 logC. AFP (ng·mL) - ¹) + 4523.8 (R² = 0.998). The detection limit of this c-BPE-ECL platform for AFP is 0.25 pg·mL. - ¹(S / N = 3). A comparison with methods in the literature shows that the platform constructed in this invention has higher detection sensitivity.
[0033] Example 5: Determination of MCF-7 cell viability The activity of MCF-7 cells in the sensing channel was measured by applying a 5 V driving voltage to the bipolar electrode array chip of Example 1. In this invention, calcein-AM and propidium iodide (PI) were used to co-stain MCF-7 cells in the microfluidic sensing channel. These two fluorescent dyes can respectively label live cells (green fluorescence) and dead cells (red fluorescence). Figure 5 As shown, the widespread distribution of green fluorescence at 5.0 V indicates that cell viability is well maintained. This result demonstrates that the voltage applied by the driving electrode does not have a significant adverse effect on cell viability.
[0034] Example 6: Determination of Standard Curves for CEA and AFP on Cell Surface A standard curve determination experiment was conducted to measure the cell surface CEA and AFP standard curves of a sensing method for the ultrasensitive in situ detection of dual tumor markers on the surface of living cells, as described in Example 1. The ECL signal intensity of the BPE anode was directly proportional to the concentration of the nanoprobe captured by the cathode, and thus positively correlated with the expression levels of CEA and AFP on the cell surface. Figure 6 As shown, this invention investigated the ECL response intensity corresponding to different concentrations of MCF-7 cells. In CEA assays, the ECL peak intensity increased with increasing MCF-7 cell concentration. Figure 6 A), and it shows a significant linear relationship with the logarithm of cell concentration. Figure 6 B). The linear regression equation is I = 2957log C(cells·mL). - ¹) – 6370 (R² = 0.998), and the detection limit was calculated to be 165 cells·mL based on a signal-to-noise ratio of 3:1. - ¹. Quantitative analysis determined the expression level of CEA on the surface of individual MCF-7 cells to be 37 fg per cell.
[0035] The ECL signal intensity gradually increases with increasing AFP concentration, which is attributed to the interaction between AFP molecules and COF@Au@MB-Apt. AFP The binding amount at the aptamer recognition sites on the nanoprobe surface increased. The ECL intensity was linearly correlated with the logarithm of MCF-7 cell concentration, and its calibration curve can be expressed as: I = 4473 log C (cells·mL). - ¹) – 13251 (R² = 0.993). Based on a signal-to-noise ratio of 3:1, the detection limit was calculated to be 1000 cells / mL. - ¹ Based on this, the average expression level of AFP on the surface of a single MCF-7 cell was found to be approximately 0.25 fg.
[0036] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A sensing method for ultrasensitive in-situ detection of dual tumor markers on the surface of living cells, characterized in that, The invention includes a closed bipolar electrode array chip, signal-amplified nanoprobes, and electrochemiluminescence detection technology; the signal-amplified nanoprobes include functional covalent organic framework (COF) materials, gold nanoparticles (Au NPs), signaling molecules, and aptamer DNA targeting tumor markers on the cell surface.
2. The sensing method according to claim 1, characterized in that: The cell surface dual tumor markers are any two of carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), mucin MUC1, and prostate antigen (PSA), with carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) being preferred.
3. The sensing method according to claim 1, characterized in that: The signal amplification nanoprobe is COF@Au@MB-Apt.
4. The sensing method according to any one of claims 1-2, characterized in that: The signal amplification nanoprobe preparation process is as follows: Based on COF material, gold nanoparticles are deposited on the COF surface by in-situ reduction of chloroauric acid. Then, the electrochemical signal molecule methylene blue (MB) is modified on the COF material and linked to the aptamer DNA of cell surface tumor markers through Au-SH bonds to form COF@Au@MB-Apt, which has both signal amplification and targeted recognition functions.
5. The sensing method according to any one of claims 1-2, characterized in that: Antibodies against tumor cell-specific antigens were modified at the cathode of a closed bipolar electrode, and the tumor cells were cultured in situ. Then, a signal amplification nanoprobe, COF@Au@MB-Apt, was modified on the cathode. [Ru(bpy)3] was added to the anode of the closed bipolar electrode. 2+ / TPA tripropylamine.
6. The sensing method according to claim 1, characterized in that: The concentration of the antibody is 1-40 μg mL⁻¹, preferably 20 μg mL⁻¹; the concentration of COF@Au@MB-Apt is 25-70 µg mL⁻¹. - ¹, The preferred concentration is 50 µg / mL - ¹, The tumor cells mentioned are MCF-7 breast cancer cells.
7. The sensing method according to claim 6, characterized in that: The [Ru(bpy)3]² + The concentration of added substance is 0.5-2 mM, preferably 1 mM; the concentration of added tripropylamine TPA is 20-70 mM, preferably 50 mM.
8. The sensing method according to claim 2, characterized in that: The aptamer DNA strand sequence of the tumor marker carcinoembryonic antigen (CEA) is as follows: 5'-SH-(CH2)6-ATACCAGCTTTATTCAATT-3'; The aptamer DNA strand sequence of the tumor marker alpha-fetoprotein (AFP) is as follows: 5'-SH-(CH2)6-GGCAGGAAGACAAACAAGCTTGGCGGCGGGAAGGTGTTTAAATTCCCGGGTCTGCGTGGTCTGTGGTGCTGT-3'.
9. The sensing method according to claim 8, characterized in that: The detection limits for CEA and AFP were 6.12 pg / mL, respectively. - ¹ and 0.25 pg mL - ¹.
10. The sensing method according to claim 1, characterized in that, The closed bipolar electrode array chip includes a lower indium tin oxide (ITO) electrode array and an upper polydimethylsiloxane (PDMS) chip; the PDMS chip is fabricated by soft photolithography, and the PDMS has a microfluidic channel structure that matches the lower ITO electrode.