Bipolar electrochemical luminescence micro-fluidic chip and application thereof

Abstract

The embodiment of the invention discloses a bipolar electrochemical luminescence micro-fluidic chip and application thereof. The bipolar electrochemical luminescence micro-fluidic chip comprises an ITO (Indium Tin Oxide) electrode 1, a PDMS (Polydimethylsiloxane) chip main body and an ITO electrode 2 from top to bottom. According to the invention, horseradish peroxidase (HRP) is used as a signal label to be coupled with a labeled antibody (Ab2), magnetic beads are coupled with a coated antibody (Ab1), during detection, a coupled reagent and a sample to be detected are injected into the chip through an injection pump at the same time, the antibody and a detection substance AFP are mixed in the chip through a spiral microchannel to form an immune sandwich compound, and the immune sandwich compound is used for detecting the AFP in the chip. The electrochemical luminescence sensor is used for detecting the AFP, then is captured on the surface of the ITO electrode in the detection cavity under the action of the electromagnet, and finally outputs an electrochemical luminescence signal through electrical stimulation to realize detection, so that the AFP can be rapidly and simply detected at low cost, and the electrochemical luminescence sensor can be widely applied to the screening and diagnosis process of HCC.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of microfluidics, and in particular to a bipolar electrochemiluminescent microfluidic chip and applications thereof. Background Art

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

[0003] Electrochemiluminescence (ECL), also known as electrochemiluminescence, is based on the principle that the luminescent substrate undergoes an oxidation-reduction reaction under electrical stimulation to generate excited substances, which then release energy back to the ground state in the form of light radiation. ECL has the advantages of rapid, sensitive, in-situ, and real-time detection. Existing ECL research is mostly focused on fixing antibodies or aptamers on the surface of electrodes such as GCE electrodes, or improving and innovating ECL donors to achieve lower detection limits and higher sensitivity.

[0004] Microfluidic chip (microfluidic chip) is a biochip, also known as micro-total analytical system (μTASs) or lab on chip (LOC). It is a science and technology that uses microchannels to accurately control and manipulate small or trace amounts of fluids. It can integrate basic operations of biochemical experiments such as sample reaction, preparation, separation and detection on a chip. It has many advantages such as miniaturization, rapid separation, high efficiency, automation, high sensitivity and high throughput.

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

[0006] To this end, an embodiment of the present invention provides a bipolar electrochemiluminescent microfluidic chip and its application. The bipolar electrochemiluminescent microfluidic chip does not need to replace electrodes after detection is completed, and can achieve continuous detection. At the same time, when used to detect AFP, it has the advantages of high sensitivity, good selectivity, speed, easy operation and low cost.

[0007] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0008] According to a first aspect of an embodiment of the present invention, the present invention provides a bipolar electrochemiluminescent 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 is provided with a hollow cavity 1-2 and channels 1-3, each of which is provided with at least one inlet, and each of which is communicated with the hollow cavity 1 and the channel 3 is communicated with the hollow cavity 2.

[0010] The surface of the ITO electrode 1 attached to the PDMS chip body is provided with a connected ITO area 1, the surface of the ITO electrode 2 attached to the PDMS chip body is provided with discontinuous ITO areas 2-3, the two ends of the hollow cavity 1 are sealed by ITO area 1 and ITO area 2 to form a detection chamber, and the two ends of the hollow cavity 2 are sealed by ITO area 1 and ITO area 3 to form a signal chamber.

[0011] Further, the channel 1 is provided with four entrances, the channel 2 is provided with one entrance, and the channel 3 is provided with one entrance;

[0012] The detection chamber and the signal chamber are respectively provided with an outlet.

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

[0014] According to a second aspect of an embodiment of the present invention, the present invention provides use of the bipolar electrochemiluminescent microfluidic chip as described in any one of the above items in biological molecule analysis and detection, preferably, the biological molecule is alpha-fetoprotein.

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

[0016] (1) An electromagnet is arranged at a position directly above the detection chamber of the ITO electrode 1;

[0017] (2) MB-Ab1, sample, and PS@HRP-Ab2 were injected into channel 1 simultaneously, and then ultrapure water was injected into channel 1;

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

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

[0020] (5) Exciting the reaction system to emit light through electrical stimulation, and collecting and recording the light intensity signal;

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

[0022] Furthermore, in step (2), the preparation method of MB-Ab1 comprises: 10 mg of carboxylated magnetic beads are pretreated and activated, and then incubated with 200 μg of Ab1 in the presence of 20 mM PBS buffer, pH 7.0-7.5, 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;

[0023] The preparation method of PS@HRP-Ab2 comprises: 2.5 mg horseradish peroxidase and 0.5 mg carboxyl polystyrene microspheres are stirred at room temperature overnight in the presence of 10 mM PBS buffer at pH 7.2-7.4 to obtain PS microspheres PS@HRP adsorbed with HRP; EDC / NHS solution is used for carboxyl activation; 27.6 μg Ab2 is incubated at 37° C. for 2 h in the presence of PBS buffer, and then blocked, resuspended in 1 mL of storage solution, and stored 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] Furthermore, in step (4), in the mixed solution, the concentration of terpyridine ruthenium is 5 mM, and the concentration of tripropylamine is 250 mM.

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

[0028] Furthermore, in step (6), the regression equation is Y=3345.79lgC+558.97, wherein 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.

[0029] The detection principle of the bipolar electrochemiluminescent microfluidic chip of the present invention is:

[0030] Due to the presence of the luminescent reagent [Ru(bpy)3] in the anode signal cavity 2+ When the immune sandwich complex is successfully captured in the detection cavity, HRP will further promote the decomposition of H2O2. Due to the charge balance principle of the two-terminal reaction of closed bipolar electrochemiluminescence (c-BPE) (the oxidation rate of the luminescent agent and the co-reactant at the anode is positively correlated with the reduction rate of H2O2 at the cathode), the electron transfer in the BPE system is accelerated, resulting in [Ru(bpy)3] 2+ The ECL signal is increased, achieving sensitive detection of AFP.

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

[0032] TPrA-e - →TPrA +· (1)

[0033] TPV +· →TPrA · +H + (2)

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

[0035] TPV · +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-mentioned cycle process, the measurement signal is continuously amplified, thereby greatly improving the detection sensitivity, so its measurement has the characteristics of high sensitivity.

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

[0039] The bipolar electrochemiluminescent microfluidic chip provided by the present invention has capture antibodies modified on immunomagnetic beads, which is different from the previous modification on electrodes. The immunomagnetic beads and antigens are in a spiral microchannel to achieve a quasi-homogeneous reaction; the ITO electrode surface is not modified with material, and there is no need to replace the electrode after the detection is completed, so continuous detection can be achieved; the detection chamber and the signal chamber do not interfere with each other, reducing the influence of various factors in a complex environment on the signal.

[0040] The bipolar electrochemiluminescent microfluidic chip of the present invention has the advantages of high sensitivity, good selectivity, good stability, simple operation and low cost in AFP detection and analysis, and is very friendly to low-resource areas, providing a new idea for the rapid and low-cost detection of AFP, which is of great significance for the diagnosis of HCC. More importantly, by changing the antibody that captures the target detection object, the sensor chip has sufficient potential to be applied to the analysis and detection of other biomarkers or biomolecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

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

[0043] Figure 2 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 The present invention provides a bipolar electrochemiluminescent microfluidic chip detection principle;

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

[0047] Figure 6 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 A graph showing the relationship between the ECL signal intensity and the AFP concentration on the bipolar electrochemiluminescence microfluidic chip provided by the present invention;

[0049] Figure 8 The test results of the selectivity of the alpha-fetoprotein detection method provided by the present invention;

[0050] Fig. 9 The test results of the stability of the alpha-fetoprotein detection method provided by the present invention;

[0051] Fig.10 This is the test result of the repeatability of the alpha-fetoprotein detection method provided by the present invention. DETAILED DESCRIPTION

[0052] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0054] See also Figure 1-3 The present invention provides a bipolar electrochemiluminescent microfluidic chip, specifically, a bipolar electrochemiluminescent microfluidic chip for AFP detection, which includes, 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 the glass plate. The glass plate of ITO electrode 1 is provided with a connected ITO region 1 (located on the lower surface of ITO electrode 1) on one side, and the glass plate of ITO electrode 2 is provided with two discontinuous ITO regions (located on the upper surface of ITO electrode 2), namely, ITO region 2 and ITO region 3, on one side.

[0056] A hollow cavity 1-2 and channels 1-3 are provided in the main body of the PDMS chip. Channels 1-2 are communicated with the hollow cavity 1, and channel 3 is communicated with the hollow cavity 2.

[0057] ITO region 1 of ITO electrode 1 and ITO region 1 of ITO electrode 2 are respectively located at two ends of hollow cavity 1 and sealed to form a detection chamber, and ITO region 1 of ITO electrode 1 and ITO region 2 of ITO electrode 2 are respectively located at two ends of hollow cavity 2 and sealed to form a signal chamber.

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

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

[0060] The preparation method of the bipolar electrochemiluminescent microfluidic chip of the present invention:

[0061] (1) An ITO electrode 1 and an ITO electrode 2 with surfaces coated with ITO are prepared according to a conventional method 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 the ITO electrode 1 is stacked with the ITO electrode 2, wherein the ITO region 2 of the ITO electrode 2 is located at the bottom of the hollow cavity 1, and the ITO region 3 of the ITO electrode 2 is located at the bottom of the hollow cavity 2, and then placed in a plasma cleaning machine to activate the PDMS for bonding (the instrument power is set to 50W, the time is 2min), and taken out after the bonding is completed. Then, the microfluidic channel side of the chip is bonded to the electrode 2, and pressed. After the PDMS chip is bonded to the electrode 2, it is placed in a 60°C oven overnight to completely complete the bonding.

[0064] (4) Assembly: The inlet and outlet are both made of peek (polyetheretherketone) connector and PTFE (polytetrafluoroethylene) tube to form the sample inlet and outlet channels; the peek connector is connected to the chip with silicone glue to ensure its airtightness, and finally a bipolar electrochemiluminescence microfluidic chip is obtained.

[0065] Example 1

[0066] 1. Reagents and instruments

[0067] 1.1 Reagents

[0068] Phosphate buffer (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 Biosharp. 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-morpholineethanesulfonic acid (MES), and Tris(2'2-bipyridine) dichlororhodium (II) hexahydrate ([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. 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 of 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-visible spectrophotometer (Shimadzu, Kyoto, Japan). Fourier transform infrared spectrometer (FTIR) (Nicolet iS20 Thermo, USA). IFFM-E flow injection chemiluminometer (Xi'an Mindray Analytical Instrument Co., Ltd., China). Constant temperature mixer (Titan, SD2-100, Shanghai Titan Scientific Instrument Co., Ltd., China). Precision syringe pump (Longerpump, TS-1B, Baoding Longer Precision Pump Co., Ltd., China).

[0071] 2. Preparation of MB-Ab1

[0072] Take 400μL (25mg / mL) of carboxylated magnetic beads, magnetically separate and discard the supernatant, add 2mL PBS buffer (10mM, pH7.2-7.4) and vortex mix for 10s, magnetically separate and discard the supernatant, and repeat the operation twice. Add 900μL PBS (10mM, pH 7.2-7.4), 50μL EDC (60mg / mL) and 50μL NHS (60mg / mL) and vortex mix for 10s, then place at 37℃ for activation for 0.5h. After activation, magnetically separate and discard the supernatant, add 2mL PBS (20mM, pH 7.0-7.5), vortex mix, and then magnetically separate and discard the supernatant, and repeat the operation twice. Add 6.7mg / mL Ab1 (30μL, 200μg) and 1970μL PBS (20mM, pH 7.0-7.5), and incubate at 37℃ for 2h. After the coupling is completed, the supernatant is discarded by magnetic separation, and 2 mL of 1% BSA is added to block at 37°C for 0.5 h. After the blocking is completed, the supernatant is discarded by magnetic separation, and 2 mL of preservation solution (pH 7.4 buffer prepared by Tris 6.6 mg / mL, BSA 0.5 mg / mL and Tween-20 0.5 mL / L) is added. Mix well and discard the supernatant by magnetic separation. Repeat the operation three times. Finally, MB-Ab1 is resuspended in 1 mL of preservation solution and kept at 4°C for use.

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

[0074] 2.5 mg horseradish peroxidase (HRP) was dissolved in 990 μL PBS buffer (10 mM, pH 7.2-7.4), and 10 μL carboxyl polystyrene microspheres (PS-COOH, 200 nm, 50 mg / mL) were added thereto, and stirred overnight (8 h) in a mixer at room temperature. PS@HRP-Ab2 was prepared by amide coupling method, and the specific process was as follows: the PS microspheres (PS@HRP) adsorbed with HRP stirred overnight were centrifuged at 17000 rpm for 5 min, and the supernatant was discarded, and the precipitate (PS@HRP) was redissolved in 1 mL MES buffer (0.5 M, pH 6.0), and 20 μL of EDC / NHS solution (10 mg / mL) were added, and the solution was stirred and incubated at 37°C for 30 min to activate the surface carboxyl groups of the PS microspheres. After removal, centrifuge (17000rpm, 5min), redissolve the precipitate in 800μL PBS buffer (10mM, pH 7.2-7.4), add 3μLAb2 (9.2mg / mL, 27.6μg), and incubate at 37℃ for 2h. After incubation, add 200μL BSA (5mg / mL) and incubate at 37℃ for 30min to block nonspecific binding sites. After incubation, centrifuge and wash three times (17000rpm, 5min), redissolve in 1mL PBS buffer (10mM, pH7.2-7.4), and store at 4℃ for later use.

[0075] 4. Operation process

[0076] A precision syringe pump was used to inject 50μL MB-Ab1, 20μL sample and 100μL PS@HRP-Ab2 into channel 1 through inlets 1, 2 and 3 respectively. The injection time was 30min. After the three solutions were fully mixed and incubated in the spiral mixing area, an immune sandwich complex was formed and captured in the detection chamber by the magnetic field of the external electromagnet (located above the detection chamber and above the ITO electrode 1), covering the surface of the ITO region 1 of the ITO electrode 1. At the same time, 100μL of a mixed solution of terpyridine ruthenium and tripropylamine was injected into the signal chamber through inlet 6, [Ru(bpy)3] 2+The concentrations of TPA are 5mM and 250mM respectively. At this time, since there are still three remaining solutions in the spiral microchannel, 1-2mL of ultrapure water is injected into the chip at a flow rate of 1mL / min through inlet 4 to push the immune sandwich complex in the microchannel into the detection chamber. Finally, 200μL of H2O2 solution (2mol / L, 10s) is injected into the chip through inlet 5 to fill the detection chamber with H2O2 solution. Finally, the reaction system is stimulated to emit light and the light intensity signal is collected and recorded by electrical stimulation (i.e., the green alligator clip of the electrochemical workstation is directly connected to the exposed ITO part of the signal chamber end of the ITO electrode 2, i.e., the positive electrode, and the red and white alligator clips are directly connected to the exposed ITO part of the detection chamber end of the ITO electrode 2, i.e., the negative electrode). The scanning voltage range is 2.5-4v, and the photomultiplier tube voltage is set to 300v. After the detection is completed, the detection chamber loses its magnetic field by controlling the electromagnet, and 2 mL of ultrapure water is injected into the chip through inlet 4 at a flow rate of 1 mL / min to flush the inside of the microfluidic chip, and all the immune sandwich complexes in the detection chamber are pushed out of the microfluidic chip.

[0077] 5. Results

[0078] 5.1 Sensitivity

[0079] The AFP detection concentration was diluted from 100 ng / mL to 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 ng / mL.

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

[0081] 5.2 Selectivity

[0082] In the process of verifying the selectivity of the sensor, prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125) and bovine serum albumin (BSA) were selected. In addition to the above five interfering substances, mixed groups were also set up. Mixed group 1 only mixed the above five interfering substances, and mixed group 2 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 (about 5k) significantly higher than those of the interfering substance and blank groups, indicating that the sensor has good selectivity.

[0083] 5.3 Stability

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

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

[0086] 5.4 Repeatability

[0087] The repeatability experiment of the sensor was tested using 5 groups of AFP antigens with the same concentration. The results showed that the light intensity signals of each group were slightly different, but the overall value was close to 5k. The final RSD value of the five groups of data was 2.64%, indicating that the sensor has good repeatability ( Fig.10 ).

[0088] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A bipolar electrochemiluminescent microfluidic chip, characterized in that: From top to bottom, it includes: ITO electrode 1, PDMS chip body and ITO electrode 2; The PDMS chip body is provided with a hollow cavity 1-2 and channels 1-3, each of which is provided with at least one inlet, and each of which is communicated with the hollow cavity 1 and the channel 3 is communicated with the hollow cavity 2. The surface of the ITO electrode 1 attached to the PDMS chip body is provided with a connected ITO area 1, the surface of the ITO electrode 2 attached to the PDMS chip body is provided with discontinuous ITO areas 2-3, the two ends of the hollow cavity 1 are sealed by ITO area 1 and ITO area 2 to form a detection chamber, and the two ends of the hollow cavity 2 are sealed by ITO area 1 and ITO area 3 to form a signal chamber.

2. The bipolar electrochemiluminescent microfluidic chip according to claim 1, characterized in that: The channel 1 is provided with four entrances, the channel 2 is provided with one entrance, and the channel 3 is provided with one entrance; The detection chamber and the signal chamber are respectively provided with an outlet.

3. The bipolar electrochemiluminescent microfluidic chip according to claim 1, characterized in that: The detection chamber and the signal chamber are both cylindrical, with a diameter of 5 mm and a height of 1 mm.

4. Use of the bipolar electrochemiluminescent microfluidic chip according to any one of claims 1 to 3 in biomolecule analysis and detection, preferably, the biomolecule is alpha-fetoprotein.

5. A method for detecting alpha-fetoprotein using the bipolar electrochemiluminescent microfluidic chip according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (1) An electromagnet is arranged at a position directly above the detection chamber of the ITO electrode 1; (2) MB-Ab1, sample, and PS@HRP-Ab2 were injected into channel 1 simultaneously, and then ultrapure water was injected into channel 1; (3) Inject H2O2 solution into channel 2 to fill the detection chamber; (4) injecting a mixed solution containing terpyridine ruthenium and tripropylamine into channel 3 to fill the signal chamber; (5) Exciting the reaction system to emit light through electrical stimulation, and collecting and recording the light intensity signal; (6) According to the regression equation, the alpha-fetoprotein content in the sample to be tested is obtained.

6. The method for detecting alpha-fetoprotein according to claim 5, characterized in that: In step (2), The preparation method of MB-Ab1 comprises: 10 mg of carboxylated magnetic beads are pretreated and activated, and then incubated with 200 μg of Ab1 in the presence of PBS buffer at 37° C. for 2 h, followed by blocking treatment, resuspending to 1 mL of storage solution, and storing at 4° C. for future use; The preparation method of PS@HRP-Ab2 comprises: 2.5 mg horseradish peroxidase and 0.5 mg carboxyl polystyrene microspheres are stirred overnight at room temperature in the presence of PBS buffer, and centrifuged to obtain PS microspheres PS@HRP adsorbed with HRP; after carboxyl activation using EDC / NHS solution, 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.

7. The method for detecting alpha-fetoprotein according to claim 5, characterized in that: In step (3), the concentration of the H2O2 solution is 2 mol / L.

8. The method for detecting alpha-fetoprotein according to claim 5, characterized in that: In step (4), in the mixed solution, the concentration of terpyridine ruthenium is 5 mM, and the concentration of tripropylamine is 250 mM.

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

10. The method for detecting alpha-fetoprotein according to claim 5, characterized in that: In step (6), the regression equation is Y=3345.79lgC+558.97, wherein 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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