Bipolar electrochemiluminescence microfluidic chip and application thereof

Abstract

The application discloses a kind of bipolar electrochemiluminescence microfluidic chip and its application.The bipolar electrochemiluminescence microfluidic chip of the application includes from top to bottom: ITO electrode 1, PDMS chip main body and ITO electrode 2.The application couples horseradish peroxidase (HRP) as signal label with labeled antibody (Ab2), and couples magnetic beads with coated antibody (Ab1).When detecting, only need to inject the reagent coupled with sample to be detected into chip by injection pump at the same time, antibody and detection material AFP are mixed to form immune sandwich complex in chip through spiral microchannel, then are captured on the surface of ITO electrode in detection cavity under the action of electromagnet, and finally electrochemiluminescence signal is output by electric stimulation to realize detection, which can realize fast, simple operation and low-cost detection of AFP, and can be widely applied in screening and diagnosis of HCC.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, specifically to a bipolar electrochemiluminescence microfluidic chip and its applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate. It is one of the most common malignant tumors worldwide and is often the leading cause of death in patients with cirrhosis. The serum tumor marker alpha-fetoprotein (AFP) is a biomarker that is crucial for identifying early-stage tumors, reducing disease-related mortality, and improving cost-effectiveness, especially in adult men, non-pregnant women, and children over one year of age, particularly high-risk individuals without clinical symptoms.

[0003] Electrochemical luminescence (ECL), also known as electrochemiluminescence, works by stimulating an luminescent substrate to undergo a redox reaction, generating excited-state substances. These excited-state substances then release energy through light radiation, returning to their ground state. ECL offers advantages such as rapid, sensitive, in-situ, and real-time detection. Current ECL research primarily focuses on immobilizing antibodies or aptamers on electrode surfaces such as GCE electrodes, or on improving and innovating ECL donors to achieve lower detection limits and higher sensitivity.

[0004] Microfluidic chips, also known as biochips, micro-total analytical systems (μTASs) or lab-on-chip (LOC), are a scientific and technological application that uses microchannels to precisely control and manipulate small or trace amounts of fluids. They integrate basic biochemical experimental operations such as sample reaction, preparation, separation, and detection onto a single chip, offering numerous advantages including miniaturization, rapid separation, high efficiency, automation, high sensitivity, and high throughput.

[0005] Currently, there are many methods for detecting AFP, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorescence assay (IFA), electrochemical luminescence (ECL), surface enhanced Raman spectroscopy (SERS), and electrochemical detection (ECD), etc. Most of them have disadvantages such as complex operation and high cost. Summary of the Invention

[0006] Therefore, this invention provides a bipolar electrochemiluminescence microfluidic chip and its application. This bipolar electrochemiluminescence microfluidic chip does not require electrode replacement after detection, enabling continuous detection. It also has advantages such as high sensitivity, good selectivity, speed, ease of operation, and low cost when used for AFP detection.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] According to a first aspect of the present invention, the present invention provides a bipolar electrochemiluminescence microfluidic chip, comprising, from top to bottom: an ITO electrode 1, a PDMS chip body, and an ITO electrode 2;

[0009] The PDMS chip body has a hollow cavity 1-2 and a channel 1-3. Each channel 1-3 has at least one inlet. Channel 1-2 is connected to the hollow cavity 1, and channel 3 is connected to the hollow cavity 2.

[0010] The surface of the ITO electrode 1 that is attached to the PDMS chip body has a connected ITO region 1, and the surface of the ITO electrode 2 that is attached to the PDMS chip body has intermittent ITO regions 2-3. The two ends of the hollow cavity 1 are sealed with ITO region 1 and ITO region 2 to form a detection chamber, and the two ends of the hollow cavity 2 are sealed with ITO region 1 and ITO region 3 to form a signal chamber.

[0011] Furthermore, channel 1 has four entrances, channel 2 has one entrance, and channel 3 has one entrance;

[0012] The detection chamber and the signal chamber each have an exit.

[0013] The detection chamber and signal chamber are cylindrical, with a diameter of 5 mm and a height of 1 mm.

[0014] According to a second aspect of the present invention, the present invention provides the application of the bipolar electrochemiluminescence microfluidic chip as described in any of the preceding claims in the analysis and detection of biomolecules, preferably, the biomolecule being alpha-fetoprotein.

[0015] According to a third aspect of the present invention, the present invention provides a method for detecting alpha-fetoprotein using a bipolar electrochemiluminescence microfluidic chip as described in any of the preceding claims, the method comprising the following steps:

[0016] (1) An electromagnet is installed at the position directly above the detection chamber for ITO electrode 1;

[0017] (2) Simultaneously inject MB-Ab1, sample and PS@HRP-Ab2 into channel 1, and then inject ultrapure water into channel 1 after the process is complete;

[0018] (3) Inject H2O2 solution into channel 2 to fill the detection chamber;

[0019] (4) Inject a mixed solution containing ruthenium tripyridine and tripropylamine into channel 3 to fill the signal chamber;

[0020] (5) The system was excited to emit light by electrical stimulation, and the light intensity signal was collected and recorded.

[0021] (6) Based on the regression equation, the alpha-fetoprotein content in the sample to be tested is obtained.

[0022] Further, in step (2), the preparation method of MB-Ab1 includes: 10 mg of carboxylated magnetic beads are pretreated and activated, and then incubated with 200 μg Ab1 at 37°C for 2 h in the presence of 20 mM PBS buffer at pH 7.0-7.5. After that, the mixture is blocked, resuspended in 1 mL of storage solution, and stored at 4°C for later use.

[0023] The preparation method of PS@HRP-Ab2 includes: 2.5 mg horseradish peroxidase and 0.5 mg carboxylated polystyrene microspheres are stirred overnight at room temperature in the presence of 10 mM PBS buffer (pH 7.2-7.4) to obtain PS microspheres PS@HRP adsorbed with HRP; carboxyl activation is performed using EDC / NHS solution; and 27.6 μg Ab2 is incubated with PBS buffer at 37 °C for 2 h, followed by blocking treatment, resuspending in 1 mL of storage solution, and storing at 4 °C for later use.

[0024] The amount of MB-Ab1 used was 50 μL, the amount of sample used was 20 μL, and the amount of PS@HRP-Ab2 used was 100 μL.

[0025] Furthermore, in step (3), the concentration of the H2O2 solution is 2 mol / L.

[0026] Further, in step (4), the concentration of ruthenium tripyridine in the mixed solution is 5 mM and the concentration of tripropylamine is 250 mM.

[0027] Further, in step (5), the conditions for electrical stimulation are: scanning voltage range of 2.5-4V and photomultiplier tube voltage of 300V.

[0028] Further, in step (6), the regression equation is Y = 3345.79lgC + 558.97, where C is the alpha-fetoprotein concentration in ng / mL, Y is the light signal intensity, the correlation coefficient R2 = 0.9938, the linear range is 1.5625 to 100 ng / mL, and the detection limit is 0.15 ng / mL.

[0029] The detection principle of the bipolar electrochemiluminescence microfluidic chip of this invention:

[0030] Because the luminescent reagent [Ru(bpy)3] is present in the anode signal cavity. 2+ Once the immune sandwich complex is successfully captured within the detection chamber, HRP further promotes the decomposition of H2O2. Due to the charge balance principle of the reactions at both ends of the closed bipolar electrochemiluminescence (c-BPE) reaction (the oxidation rate of the luminescent agent and co-reactant at the anode is positively correlated with the reduction rate of H2O2 at the cathode), electron transfer in the BPE system is accelerated, leading to [Ru(bpy)3]. 2+ The increased ECL signal enables sensitive detection of AFP.

[0031] Among them, ruthenium tripyridine (Ru(bpy)3) 2+ The ECL system uses tripropylamine (TPrA) as the luminescent agent and tripropylamine (TPrA) as the co-reactant. The luminescence principle is as follows:

[0032] TPrA-e - →TPrA +· (1)

[0033] TPrA +· →TPrA · +H + (2)

[0034] Ru(bpy)3 2+ -e - →Ru(bpy)3 3+ (3)

[0035] TPrA · +Ru(bpy)3 3+ →[Ru(bpy)3 2+ ] * +P1 (4)

[0036] [Ru(bpy)3 2+ ] * →Ru(bpy)3 2+ +hv (5)

[0037] Through the above-described cyclic process, the measured signal is continuously amplified, thereby greatly improving the detection sensitivity, thus giving it the characteristic of high sensitivity.

[0038] The embodiments of the present invention have the following advantages:

[0039] The bipolar electrochemiluminescence microfluidic chip provided by this invention captures antibodies modified on immunomagnetic beads. Unlike previous methods where the antibodies were modified on electrodes, the immunomagnetic beads and antigens react in a quasi-homogeneous manner within a spiral microchannel. The ITO electrode surface is unmodified, so there is no need to replace the electrode after detection, enabling continuous detection. The detection chamber and signal chamber do not interfere with each other, reducing the influence of various factors in a complex environment on the signal.

[0040] The bipolar electrochemiluminescence microfluidic chip of this invention offers advantages such as high sensitivity, good selectivity, good stability, simple operation, and low cost in AFP detection and analysis. It is particularly suitable for resource-poor regions, providing a new approach for rapid and low-cost AFP detection and holding significant importance for the diagnosis of HCC. More importantly, by modifying the antibody that captures the target analyte, this sensor chip has sufficient potential for application in the analysis and detection of other biomarkers or biomolecules. Attached Figure Description

[0041] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0042] Figure 1 A schematic diagram of the structure of the bipolar electrochemiluminescence microfluidic chip provided by the present invention;

[0043] Figure 2 This is a schematic diagram of the structure of the PDMS chip body provided by the present invention;

[0044] Figure 3 A dimensional diagram of the ITO electrode 2 provided by the present invention;

[0045] Figure 4 This invention provides the detection principle of a bipolar electrochemiluminescence microfluidic chip;

[0046] Figure 5A graph showing the relationship between ECL signal intensity and the logarithm of AFP concentration on a bipolar electrochemiluminescence microfluidic chip provided by this invention;

[0047] Figure 6 This is a graph showing the relationship between ECL peak shape and AFP concentration on the bipolar electrochemiluminescence microfluidic chip provided by the present invention.

[0048] Figure 7 The graph showing the relationship between ECL signal intensity and AFP concentration on the bipolar electrochemiluminescence microfluidic chip provided by this invention;

[0049] Figure 8 The test results are selective for the alpha-fetoprotein detection method provided by this invention;

[0050] Figure 9 The results show the stability test results of the alpha-fetoprotein detection method provided by this invention.

[0051] Figure 10 The results show the repeatability of the alpha-fetoprotein detection method provided by this invention. Detailed Implementation

[0052] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0054] See Figure 1-3 The present invention provides a bipolar electrochemiluminescence microfluidic chip, specifically, a bipolar electrochemiluminescence microfluidic chip for AFP detection, comprising, from top to bottom: an ITO electrode 1, a PDMS chip body and an ITO electrode 2.

[0055] The ITO electrode is an electrode with indium tin oxide modified on one side of a glass plate. The glass plate of ITO electrode 1 has a continuous ITO region 1 on one side (located on the lower surface of ITO electrode 1), and the glass plate of ITO electrode 2 has two discontinuous ITO regions on one side (located on the upper surface of ITO electrode 2), namely ITO region 2 and ITO region 3.

[0056] The PDMS chip body has a hollow cavity 1-2 and a channel 1-3. Channel 1-2 is connected to hollow cavity 1, and channel 3 is connected to hollow cavity 2.

[0057] The ITO region 1 of ITO electrode 1 and the ITO region 1 of ITO electrode 2 are located at both ends of the hollow cavity 1, forming a detection chamber. The ITO region 1 of ITO electrode 1 and the ITO region 2 of ITO electrode 2 are located at both ends of the hollow cavity 2, forming a signal chamber.

[0058] In some specific embodiments, channel 1 consists of two spiral microchannels with four inlets, channel 2 has one inlet, and channel 3 has one inlet. The detection chamber and signal chamber each have one outlet. All inlets and outlets use PEEK connectors and PTFE (tetrafluoroethylene) tubing to form the sample inlet and outlet channels.

[0059] In some specific embodiments, channels 1-3 are 1mm high and 1mm wide; both the detection chamber and the signal chamber are cylindrical with a diameter of 5mm and a height of 1mm. ITO electrode 1 is a 30mm*6mm glass plate, which is fully covered with ITO on one side. ITO electrode 2 is a 70mm*55mm glass plate, which is covered with two ITO regions. The ITO region 2 at the bottom of the detection chamber is 25.18mm*6mm, and the ITO region 3 at the bottom of the signal chamber is 13mm*6mm.

[0060] The fabrication method of the bipolar electrochemiluminescence microfluidic chip of the present invention:

[0061] (1) ITO electrode 1 and ITO electrode 2, with ITO coated on their surfaces, are prepared according to conventional methods in the art.

[0062] (2) When preparing the PDMS chip body, the ITO electrode 1 is placed in the mold in advance. After the PDMS is cured, the ITO area of ​​the ITO electrode 1 is encapsulated in the PDMS chip, and holes are punched to set microchannels.

[0063] (3) The PDMS chip body encapsulated with ITO electrode 1 is stacked together with ITO electrode 2, wherein the ITO region 2 of ITO electrode 2 is located at the bottom of hollow cavity 1, and the ITO region 3 of ITO electrode 2 is located at the bottom of hollow cavity 2. Then, it is placed in a plasma cleaner to activate PDMS for bonding (instrument power set to 50W, time 2min). After bonding is completed, it is removed. Then, the microfluidic channel side of the chip is attached to electrode 2 and pressed. After the PDMS chip and electrode 2 are bonded, it is placed in a 60℃ oven overnight to complete the bonding.

[0064] (4) Assembly: The inlet and outlet are both composed of PEEK (polyether ether ketone) connectors and PTFE (polytetrafluoroethylene) tubes to form sample inlet and outlet channels; silicone glue is used to connect the PEEK connectors to the chip to ensure its airtightness, and finally the bipolar electrochemiluminescence microfluidic chip is obtained.

[0065] Example 1

[0066] 1. Reagents and Instruments

[0067] 1.1 Reagents

[0068] Phosphate-buffered saline (PBS, 10 mM, pH 7.2–7.4) and Tris-HCl (1 M, pH 7.0) were purchased from Solarbio. Bovine serum albumin (BSA) was purchased from Bioharp. Magnetic microbeads (MB 3 μm) were purchased from Vdobiotech. N,N-Dipropyl-1-propylamine (TPA) was purchased from Energy Chemical. 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-morpholine ethanesulfonic acid (MES), and Tris(2',2-bipyridine)rhodium(II) dichlorohexahydrate ([Ru(bpy)3]2+) were purchased from Adamas-beta. Sodium hydroxide (NaOH) was purchased from Tianjin Xinbote Chemical Co., Ltd. Sodium chloride (NaCl) was purchased from Tianjin Shengao Chemical Reagent Co., Ltd. Tween 20 was purchased from Fuchen (Tianjin) Chemical Reagent Co., Ltd. The human αFP (alpha-fetoprotein) ELISA kit was purchased from Elabscience Biotechnology Co., Ltd. AFP antibody and AFP antigen were purchased from Zhengzhou Cell to Antibody & Antigen Biotechnology Co., Ltd. Ultrapure water was used throughout the experiment. All reagents were analytical grade and could be used directly without further purification.

[0069] 1.2 Instruments

[0070] Electrochemical workstation (Chenhua CHI660E, Shanghai Chenhua Instrument Co., Ltd., China). UV-2600 UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). Fourier transform infrared spectrometer (FTIR) (Nicolet iS20 Thermo, USA). IFFM-E flow injection chemiluminescence analyzer (Xi'an Mindray Analytical Instrument Co., Ltd., China). Thermostatic mixer (Titan, SD2-100, Shanghai Titan Scientific Instrument Co., Ltd., China). Precision syringe pump (Longerpump, TS-1B, Baoding Longer Precision Pump Industry Co., Ltd., China).

[0071] 2. Preparation of MB-Ab1

[0072] Take 400 μL (25 mg / mL) of carboxylated magnetic beads, magnetically separate and discard the supernatant. Add 2 mL of PBS buffer (10 mM, pH 7.2-7.4), vortex and mix for 10 s, magnetically separate and discard the supernatant. Repeat this process twice. Add 900 μL of PBS (10 mM, pH 7.2-7.4), 50 μL of EDC (60 mg / mL), and 50 μL of NHS (60 mg / mL), vortex and mix for 10 s, then activate at 37 °C for 0.5 h. After activation, magnetically separate and discard the supernatant. Add 2 mL of PBS (20 mM, pH 7.0-7.5), vortex and magnetically separate and discard the supernatant. Repeat this process twice. Add 6.7 mg / mL Ab1 (30 μL, 200 μg) and 1970 μL of PBS (20 mM, pH 7.0-7.5), and incubate at 37 °C for 2 h. After coupling, magnetic separation was performed, and the supernatant was discarded. 2 mL of 1% BSA was added, and the mixture was blocked at 37°C for 0.5 h. After blocking, magnetic separation was performed, and the supernatant was discarded. 2 mL of preservation solution (prepared as a pH 7.4 buffer with Tris 6.6 mg / mL, BSA 0.5 mg / mL, and Tween-20 0.5 mL / L) was added, mixed well, and magnetic separation was performed again, discarding the supernatant. This process was repeated three times. Finally, MB-Ab1 was resuspended in 1 mL of preservation solution and stored at 4°C for later use.

[0073] 3. Preparation of PS@HRP-Ab2

[0074] Dissolve 2.5 mg of horseradish peroxidase (HRP) in 990 μL of PBS buffer (10 mM, pH 7.2-7.4), and add 10 μL of carboxylated polystyrene microspheres (PS-COOH, 200 nm, 50 mg / mL). Stir overnight (8 h) at room temperature. Prepare PS@HRP-Ab2 using the amide coupling method, as follows: Centrifuge the overnight-stirred HRP-adsorbed PS microspheres (PS@HRP) at 17000 rpm for 5 min, discard the supernatant, and redissolve the precipitate (PS@HRP) in 1 mL of MES buffer (0.5 M, pH 6.0). Add 20 μL each of EDC / NHS solution (10 mg / mL). Incubate the solution at 37 °C for 30 min to activate the carboxyl groups on the PS microsphere surface. After removal, centrifuge (17000 rpm, 5 min), reconstitute the precipitate in 800 μL PBS buffer (10 mM, pH 7.2-7.4), add 3 μL Ab2 (9.2 mg / mL, 27.6 μg), and incubate at 37°C with stirring for 2 h. After incubation, add 200 μL BSA (5 mg / mL) and incubate at 37°C with stirring for 30 min to block non-specific binding sites. After incubation, centrifuge and wash three times (17000 rpm, 5 min), reconstitute in 1 mL PBS buffer (10 mM, pH 7.2-7.4), and store at 4°C for later use.

[0075] 4. Operation process

[0076] Using a precision syringe pump, 50 μL of LMB-Ab1, 20 μL of sample, and 100 μL of PS@HRP-Ab2 were injected into channel 1 through inlets 1, 2, and 3, respectively. The injection time was 30 min. After thorough mixing and incubation in the spiral mixing zone, the three solutions formed an immune sandwich complex, which was then captured within the detection chamber by the magnetic field of an external electromagnet (located above the ITO electrode 1), covering the surface of ITO region 1 of the ITO electrode 1. Simultaneously, 100 μL of a mixed solution of ruthenium terpyridine and tripropylamine was injected into the signal chamber through inlet 6. [Ru(bpy)3] 2+The concentrations of TPA were 5 mM and 250 mM, respectively. Since the three solutions remained in the spiral microchannel, 1-2 mL of ultrapure water was injected into the chip through inlet 4 at a flow rate of 1 mL / min to push the immune sandwich complex into the detection chamber. Finally, 200 μL of H2O2 solution (2 mol / L, 10 s) was injected into the chip through inlet 5 to fill the detection chamber with H2O2 solution. Finally, electrical stimulation (i.e., the green alligator clip of the electrochemical workstation was directly connected to the exposed ITO portion of the ITO electrode 2 signal chamber (positive electrode), and the red and white alligator clips were directly connected to the exposed ITO portion of the ITO electrode 2 detection chamber (negative electrode)) excited the reaction system to emit light, and the light intensity signal was collected and recorded. The scanning voltage range was 2.5-4 V, and the photomultiplier tube voltage was set to 300 V. After the test is completed, the detection chamber is demagnetized by controlling the electromagnet, and 2 mL of ultrapure water is injected into the chip through the inlet 4 at a flow rate of 1 mL / min to flush the inside of the microfluidic chip, thus expelling all the immune sandwich complex in the detection chamber from the microfluidic chip.

[0077] 5. Results

[0078] 5.1 Sensitivity

[0079] AFP detection concentrations were serially diluted from 100 ng / mL, namely 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 ng / mL.

[0080] The regression equation is: Y = 3345.79lgC + 558.97, with a correlation coefficient R² = 0.9938. The detection limit (LOD) of this sensor is calculated to be 0.15 ng / mL using the formula LOD = 3σ / k. Therefore, the linear range of this sensor is 1.5625–100 ng / mL, and the LOD is 0.15 ng / mL.

[0081] 5.2 Selectivity

[0082] In verifying the sensor selectivity, prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), and bovine serum albumin (BSA) were selected. In addition to these five interfering substances, mixed groups were also set up. Mixed group one contained only the above five interfering substances, while mixed group two added alpha-fetoprotein (AFP) to the above five interfering substances. The final experimental results are as follows. Figure 8 The two groups containing the target substance AFP produced light intensity signals (approximately 5 kJ) significantly higher than those of the interfering substance and the blank group, indicating that the sensor has good selectivity.

[0083] 5.3 Stability

[0084] Another experiment used to measure sensor performance is stability. The following figure shows the results of the stability experiment:

[0085] After 10 consecutive voltage cycles, the peak RSD of the optical signal was 2.23%. Figure 9 ).

[0086] 5.4 Repeatability

[0087] The repeatability experiment of the sensor was conducted using five groups of AFP antigens with the same concentration. The results showed slight differences in light intensity signals among the groups, but the overall intensity was close to 5 kJ. The final RSD value for the five groups was 2.64%, indicating that the sensor has good repeatability. Figure 10 ).

[0088] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting alpha-fetoprotein using a bipolar electrochemiluminescence microfluidic chip, characterized in that, The method includes the following steps: (1) An electromagnet is installed at the position of ITO electrode 1 directly above the detection chamber; (2) Simultaneously inject MB-Ab1, sample and PS@HRP-Ab2 into channel 1, and then inject ultrapure water into channel 1 after the process is complete; (3) Inject H2O2 solution into channel 2 to fill the detection chamber; (4) Inject a mixed solution containing ruthenium tripyridine and tripropylamine into channel 3 to fill the signal chamber; (5) The system was excited to emit light by electrical stimulation, and the light intensity signal was collected and recorded; (6) Obtain the alpha-fetoprotein content in the sample to be tested based on the regression equation; The bipolar electrochemiluminescence microfluidic chip comprises, from top to bottom: ITO electrode 1, PDMS chip body and ITO electrode 2; The PDMS chip body has a hollow cavity 1-2 and a channel 1-3. Each channel 1-3 has at least one inlet. Channel 1-2 is connected to the hollow cavity 1, and channel 3 is connected to the hollow cavity 2. The surface of the ITO electrode 1 that is attached to the PDMS chip body has a connected ITO region 1, and the surface of the ITO electrode 2 that is attached to the PDMS chip body has discontinuous ITO regions 2-3. The two ends of the hollow cavity 1 are sealed through ITO region 1 and ITO region 2 to form a detection chamber, and the two ends of the hollow cavity 2 are sealed through ITO region 1 and ITO region 3 to form a signal chamber. Both the detection chamber and the signal chamber are cylindrical, with a diameter of 5 mm and a height of 1 mm; In step (2), The preparation method of MB-Ab1 includes: 10 mg of carboxylated magnetic beads are pretreated and activated, and then incubated with 200 μg Ab1 in PBS buffer at 37°C for 2 h. After that, the mixture is blocked, resuspended in 1 mL of storage solution, and stored at 4°C for later use. The preparation method of PS@HRP-Ab2 includes: 2.5 mg horseradish peroxidase and 0.5 mg carboxylated polystyrene microspheres are stirred at room temperature for 8 h in the presence of PBS buffer, centrifuged to obtain PS microspheres PS@HRP adsorbed with HRP; after carboxyl activation using EDC / NHS solution, it is incubated with 27.6 μg Ab2 in the presence of PBS buffer at 37 °C for 2 h, then blocked, resuspended in 1 mL of storage solution, and stored at 4 °C for later use; The amount of MB-Ab1 used was 50 μL, the amount of sample used was 20 μL, and the amount of PS@HRP-Ab2 used was 100 μL; In step (3), the concentration of the H2O2 solution is 2 mol / L; In step (4), the concentration of ruthenium tripyridine in the mixed solution is 5 mM and the concentration of tripropylamine is 250 mM. In steps (2)-(4), the injection time is 30 min.

2. The method for detecting alpha-fetoprotein according to claim 1, characterized in that, Channel 1 has four entrances, channel 2 has one entrance, and channel 3 has one entrance. The detection chamber and the signal chamber each have an exit.

3. The method for detecting alpha-fetoprotein according to claim 1, characterized in that, In step (5), the conditions for electrical stimulation are: scanning voltage range of 2.5-4 V and photomultiplier tube voltage of 300 V.

4. The method for detecting alpha-fetoprotein according to claim 1, characterized in that, In step (6), the regression equation is Y=3345.79 lgC+558.97, where C is the alpha-fetoprotein concentration in ng / mL, Y is the light signal intensity, the correlation coefficient R2=0.9938, the linear range is 1.5625~100 ng / mL, and the detection limit is 0.15 ng / mL.

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