Two-channel microfluidic electrochemical sensor, preparation method thereof and detection system

Abstract

The invention discloses a dual-channel microfluidic electrochemical sensor which is formed by sequentially stacking a bottom layer, an electrode layer and a channel layer, and the bottom layer is a PDMS base; the electrode layer is symmetrically provided with two groups of three-electrode systems, the two groups of three-electrode systems are respectively embedded in the two electrode grooves, and the channel layer comprises a PDMS micro-channel. The invention also discloses a preparation method of the two-channel microfluidic electrochemical sensor. Under the driving of capillary force and air holes, a to-be-detected solution and an electrolyte in a microchannel of the sensor flow into a three-electrode system detection area, and miR-125b and miR-208b are simultaneously detected by an electrochemical method. The method does not need injection pump outer edge equipment, and has the advantages of simplicity in operation, high sensitivity in detection of miR-125b and miR-208b, good selectivity and wide linear range.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a dual-channel microfluidic electrochemical sensor, its preparation method, and detection system. Background Technology

[0002] Acute myocardial infarction (AMI), also known as myocardial infarction, is myocardial necrosis caused by acute and persistent ischemia and hypoxia of the coronary arteries.

[0003] As a cardiovascular disease, myocardial infarction sometimes presents with atypical symptoms, or even no symptoms at all. However, myocardial infarction is characterized by rapid onset and high mortality, making early diagnosis crucial. Cardiac markers are biological indicators released into the bloodstream after myocardial cells are damaged or die. The European / American Heart Association uses the elevation and decline of cardiac markers as two indicators for diagnosing myocardial infarction. Their specificity, sensitivity, and immediacy are of great value for the early diagnosis of myocardial infarction and represent an important research area in the field of biodetection technology.

[0004] In recent years, an increasing number of myocardial biomarkers have been discovered, but none possess perfect specificity. Clinical statistics show that combined detection of multiple myocardial biomarkers plays a crucial role in diagnosing myocardial injury and predicting infarct size. MicroRNAs (miRNAs), as novel biomarkers, have demonstrated great potential in the early diagnosis of acute myocardial infarction (AMI). Studies have shown that miRNAs not only participate in key pathological processes of AMI such as cardiomyocyte apoptosis, inflammatory responses, and fibrosis, but also exhibit significant changes in peripheral blood within 1 hour of onset.

[0005] miR-125b, an important miRNA molecule, is widely present in human tissues and cells, participating in the regulation of various physiological and pathological processes. In various heart diseases, including myocardial infarction, 25 miRNAs, including miR-125b, are upregulated, while 10 miRNAs are downregulated, making miR-125b an important biomarker for cardiovascular diseases.

[0006] miR-208b, a heart-specific miRNA, can be detected with significant elevations in the blood within 1–2 hours after myocardial injury, offering a significant time advantage over the traditional marker troponin (4–8 hour detection window). Both miR-125b and miR-208b are important indicators for the early diagnosis of myocardial infarction, and their sensitivity, specificity, trace amounts, low cost, and simultaneous detection of two myocardial markers are of significant value.

[0007] Microfluidic chips offer advantages such as small sample volume, low detection cost, and short detection time. Combined with electrochemical sensors, they can form highly sensitive, repeatable, and portable detection systems. However, the flow of sample into the detection zone of microfluidic electrochemical sensors often relies on an epitaxial device with a syringe pump, which limits their application scenarios to some extent. Currently, there are few reports on methods for simultaneously detecting miR-125b and miR-208b using a dual-channel microfluidic electrochemical sensor with self-driven sample introduction. Summary of the Invention

[0008] To address the aforementioned technical problems, the first objective of this invention is to disclose a dual-channel microfluidic electrochemical sensor.

[0009] The second objective of this invention is to disclose a method for fabricating a dual-channel microfluidic electrochemical sensor.

[0010] The three objectives of this invention are to disclose a detection system based on the aforementioned dual-channel microfluidic electrochemical sensor, which enables the simultaneous detection of two myocardial markers, miR-125b and miR-208b, without a pump.

[0011] This invention fully utilizes the carboxyl groups and BN bonds on the surface of the h-BNNS / cMWCNTs / MnFe2O4 magnetic nanocomposite material to covalently bond cRNA to the Au working electrode surface via amide bonds, obtaining an h-BNNS / cMWCNTs / MnFe2O4+cRNA electrochemical sensor, which significantly improves the sensitivity and stability of the electrochemical process. Furthermore, the addition of the detectants miR-125b and miR-208b significantly reduces the DPV peak current values ​​in the two detection regions of the dual-channel microfluidic electrochemical sensor, enabling simultaneous detection of miR-125b and miR-208b. Moreover, the addition of other interfering agents reveals that this sensor exhibits good specificity.

[0012] This invention successfully prepared a hexagonal boron nitride nanoparticle (h-BNNS / cMWCNTs / MnFe2O4) magnetic nanocomposite material with a multi-level "layer-nanotube-particle" structure. The material surface is rich in carboxyl groups and BN bonds, providing abundant active sites for cRNA probe immobilization. h-BNNS / cMWCNTs enhance the effective electrode area and conductivity, while MnFe2O4 improves the stability of cRNA probe immobilization. These components are covalently bound to the working electrode surface, constructing a dual-channel microfluidic electrochemical sensor. Based on this electrochemical sensor, miR-125b and miR-208b can be simultaneously detected. Driven by capillary force and pores, the test solution and electrolyte in the sensor microchannel flow into the detection area of ​​the three-electrode system, simultaneously detecting miR-125b and miR-208b using an electrochemical method. The linear detection range for miR-125b in this invention is 1.0 fM to 10 nM, with a detection limit of up to 0.109 fM; the linear detection range for miR-208b is 1.0 fM to 10 nM, with a detection limit of 0.129 fM. This method does not require an external syringe pump and features simple operation, high sensitivity and selectivity for detecting miR-125b and miR-208b, and a wide linear range.

[0013] Technical solution: A dual-channel microfluidic electrochemical sensor, comprising a bottom layer, an electrode layer, and a channel layer stacked sequentially, wherein:

[0014] The bottom layer is a PDMS base, and two electrode slots are symmetrically arranged on the PDMS base.

[0015] The electrode layer is symmetrically provided with two sets of three-electrode systems. The two sets of three-electrode systems are respectively embedded in the two electrode slots. One set of three-electrode systems consists of a first electrode disk and a first pin facing outward, and the first electrode disk is connected to the first pin. The other set of three-electrode systems consists of a second electrode disk and a second pin facing outward, and the second electrode disk is connected to the second pin.

[0016] The channel layer includes PDMS microchannels.

[0017] Furthermore, the PDMS microchannel is composed of the following components:

[0018] Reagent delivery fluid inlet;

[0019] The front-end main channel has one inlet and two outlets, and the reagent delivery liquid inlet is connected to the inlet of the front-end main channel;

[0020] Two sample channels are provided, each with an inlet and two outlets. The inlets of the two sample channels are connected to the outlets of the main channel at the front end, thus forming a primary branch. One outlet of the sample channel is connected to the inlet of the vortex mixing unit through a first pipe, which has a sample dispensing port. The other outlet of the sample channel is connected to the inlet of the conical microcavity flow control unit through a second pipe, which has a complementary chain dispensing port.

[0021] The two electrode regions consist of an electrode pin interface and an electrode reaction region. The outlet of the vortex mixing unit is connected to the inlet of the electrode reaction region through a third pipe, and the outlet of the conical microcavity fluid control unit is connected to the inlet of the electrode reaction region through a complementary chain channel. The electrode pin interfaces are connected to the first pin and the second pin, respectively, and the positions of the two electrode reaction regions correspond to the positions of the first electrode disk and the second electrode disk, respectively.

[0022] The liquid outlets of the two electrode reaction zones are connected to the liquid inlets of the electrode zones via the sixth pipe, and the liquid outlets of the electrode zones are connected to the liquid outlets via the fifth pipe.

[0023] The specific steps of the above-mentioned dual-channel microfluidic electrochemical sensor fabrication method are as follows:

[0024] (1) The circular gold electrode, the semi-circular arc gold counter electrode, and the semi-circular arc reference electrode are all made by screen printing.

[0025] (2) Circular gold electrode surface modified with L-Cys / cMWCNTs+cRNA nanomaterials;

[0026] (3) The bottom layer and channel layer of the dual-channel microfluidic electrochemical sensor are fabricated by photolithography;

[0027] (4) A dual-channel microfluidic electrochemical sensor was fabricated using the room temperature bond method.

[0028] Furthermore, the specific steps of step (1) are as follows:

[0029] A PET film of a certain shape is ultrasonically treated in an ethanol solution for at least 2 hours and dried at 120°C for at least 15 minutes. The PET film is then laid flat on a substrate and fixed. Silver paste is printed on a screen printer using an oil-based polyurethane squeegee at a speed of 5 cm / s to obtain conductive lines of a three-electrode system. The film is then heat-cured at 120°C for at least 10 minutes. A circular gold working electrode and a semi-circular gold counter electrode are then printed sequentially using conductive gold paste. The film is then heat-cured at 60°C for at least 30 minutes. A semi-circular reference electrode is then printed using Ag / AgCl ink. The film is then heat-cured at 110°C for at least 5 minutes. Finally, an insulating layer is formed by printing with insulating ink, exposing the three electrodes and the ends of the wires. The film is then UV-cured at a speed of 30 W / cm.

[0030] Furthermore, the specific steps of step (2) are as follows:

[0031] (21) The PDMS film covers the circular gold electrode, the semi-circular gold counter electrode and the semi-circular reference electrode obtained in step (1), exposing the surface of the circular gold electrode. The h-BNNS / cMWCNTs / MnFe2O4 composite material is dispersed in deionized water to prepare a uniform suspension with a concentration of 1 mg / mL. The suspension is dropped onto the surface of the circular gold electrode and the surface of the circular gold electrode is dried.

[0032] (22) A circular gold electrode, a semi-circular gold counter electrode, and a semi-circular reference electrode were covered with a PDMS film, exposing the surface of the circular gold electrode covalently bonded with h-BNNS / cMWCNTs / MnFe2O4 nanomaterials. 6 μL of 10 μM 5′ amino-modified cRNA solution was added to the modified circular gold electrode surface and incubated in a 37°C constant temperature incubator for at least 60 min to allow the cRNA to covalently bind to the carboxyl groups on the surface of cMWCNTs through amide bonds. After rinsing with PBS buffer with a pH of 7.4 to remove unbound cRNA, the modified circular gold electrode was immersed in 1% BSA solution and incubated at 37°C for 25 min to block non-specific binding sites.

[0033] (23) The modified circular gold electrode was thoroughly rinsed with PBS buffer at pH 7.4, the PDMS membrane was removed, and the surfaces of the circular gold electrode, the semi-circular gold counter electrode and the semi-circular reference electrode were rinsed with SSC and ultrapure water in sequence. The electrode surfaces were dried and covered with a new PDMS membrane. The cRNA was covalently bound to the carboxyl group on the surface of cMWCNTs through amide bonds to obtain an electrochemical sensor modified with h-BNNS / cMWCNTs / MnFe2O4+cRNA and blocked with BSA.

[0034] Furthermore, the specific steps of step (4) are as follows:

[0035] Two circular gold electrodes made of L-Cys / cMWCNTs+cRNA nanomaterials were covalently bound with miR-125b and miR-208b cRNA, respectively. The two three-electrode systems were embedded into the two detection zone electrode grooves of the bottom layer of the dual-channel microfluidic electrochemical sensor. The bottom layer, electrode layer and channel layer were aligned and placed in a bonding machine. The bonding was carried out at room temperature and 0.2 MPa pressure for 180 s to obtain the dual-channel microfluidic electrochemical sensor.

[0036] A dual-channel microfluidic electrochemical sensor is prepared by any one of the methods described above.

[0037] A detection system comprising the dual-channel microfluidic electrochemical sensor described in any one of the preceding claims.

[0038] Furthermore, the detection system also includes an electrochemical workstation connected to the dual-channel microfluidic electrochemical sensor.

[0039] Beneficial Effects: The dual-channel microfluidic electrochemical sensor, its preparation method, and detection system disclosed in this invention have the following beneficial effects:

[0040] 1. Pump-free sample introduction of dual-channel microfluidic electrochemical sensor - The "pump-free" nature of capillary self-driven pump-free microfluidic chip is to replace the traditional mechanical pump to achieve liquid transport through the synergistic effect of surface tension and microstructure at the microscale. Its core lies in the material wettability design and microchannel geometry optimization. It is suitable for portable and low-cost microfluidic systems, but its flow rate stability and functional complexity need to be balanced.

[0041] 2. Simultaneous detection of two cardiac biomarkers, miR-125b and miR-208b, was achieved by preparing a hexagonal boron nitride nanoparticle (h-BNNS / cMWCNTs / MnFe2O4 magnetic nanocomposite material) with a multi-level structure of "sheet-nanotube-particle". The material surface is rich in carboxyl groups and BN bonds, providing abundant active sites for cRNA probe immobilization. h-BNNS / cMWCNTs enhance the effective electrode area and conductivity, while MnFe2O4 improves the stability of cRNA probe immobilization. Both materials are covalently bound to the working electrode surface to construct a dual-channel microfluidic electrochemical sensor. Based on this electrochemical sensor, miR-125b and miR-208b can be detected simultaneously. Driven by capillary force and pores, the test solution and electrolyte in the sensor microchannel flow into the detection area of ​​the three-electrode system, allowing for the simultaneous detection of miR-125b and miR-208b using an electrochemical method.

[0042] 3. The h-BNNS / cMWCNTs / MnFe2O4 modified electrochemical sensor exhibits a linear detection range of 1 fM to 10 nM for miR-125b, with a LOD of 0.109 fM, outperforming similar electrochemical sensors. This dual-channel microfluidic electrochemical sensor effectively distinguishes miR-125b from other myocardial infarction-related miRNAs. After 14 days of storage, the signal decay was only 5.9%, the RSD for three repeated assays was 3.5%, and the serum recovery rate was 95.2%–104.0%, demonstrating excellent specificity and reliable stability, meeting clinical testing needs. Regarding miR-208b detection performance, a wide linear response range of 1 fM to 10 nM was achieved using DPV (Recovery Time Limit). 2 The detection limit was as low as 0.129 fM (0.9936), and the signal attenuation was only 8.2% after 14 days of storage at 4°C. It exhibits high specificity, anti-interference ability, and good stability and repeatability. The serum spiked recovery rate was 95.2%–96.1%, and the RSD was 1.47%–3.16%, verifying its reliability in complex matrices. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the layered structure of a dual-channel microfluidic electrochemical sensor.

[0044] Figure 2 This is a schematic diagram of the channel layer structure of a dual-channel microfluidic electrochemical sensor.

[0045] Figure 3 This is a three-dimensional view of the channel layer of a dual-channel microfluidic electrochemical sensor.

[0046] Figure 4 This is a three-dimensional view of the electrode layer of a dual-channel microfluidic electrochemical sensor.

[0047] Figure 5 This is a three-dimensional view of the bottom layer of a dual-channel microfluidic electrochemical sensor.

[0048] Figure 6 This is a simulation diagram of the intrachannel flow dynamics of a dual-channel microfluidic electrochemical sensor.

[0049] Figure 7 The DPV curve of miR-125b detected by a dual-channel microfluidic electrochemical sensor.

[0050] Figure 8 This is a schematic diagram illustrating the linear range of miR-125b detection using a dual-channel microfluidic electrochemical sensor.

[0051] Figure 9 A schematic diagram of the selectivity detection of miR-125b using a dual-channel microfluidic electrochemical sensor.

[0052] Figure 10 A schematic diagram of a dual-channel microfluidic electrochemical sensor for detecting the stability of miR-125b.

[0053] Figure 11 This is a schematic diagram illustrating the reproducibility of miR-125b detection using a dual-channel microfluidic electrochemical sensor.

[0054] Figure 12 A schematic diagram of the DPV curve for detecting miR-208b using a dual-channel microfluidic electrochemical biosensor.

[0055] Figure 13 A schematic diagram illustrating the linear range of miR-208b detected by a dual-channel microfluidic electrochemical sensor.

[0056] Figure 14 A schematic diagram of the selectivity detection of miR-208b using a dual-channel microfluidic electrochemical sensor.

[0057] Figure 15 A schematic diagram of the stability detection of miR-208b using a dual-channel microfluidic electrochemical sensor.

[0058] Figure 16 This is a schematic diagram illustrating the reproducibility of miR-208b detection using a dual-channel microfluidic electrochemical sensor. Wherein:

[0059] 1-Channel Layer 2-Electrode layer 3-PDMS base 4-PDMS microchannel 5-Three-electrode system 6-First Electrode Disk 7-First pin 8-Second Electrode Disk 9-Second pin 10-Electrode slot 11-Reagent delivery fluid inlet 12-Front-end Main Channel 13-First-level branch 14 - Sample Channel 15-Sample dispensing port 16-Complementary Chain Channel 17-Complementary chain sample addition port 18-Vortex Hybrid Unit 19-Conical Microcavity Fluid Control Unit 20-Electrode Reaction Zone 21-Electrode Region 22-Electrode Pin Interface 23-Liquid Outlet Detailed Implementation

[0060] The specific embodiments of the present invention are described in detail below.

[0061] The "range" disclosed in this invention is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 10–50 is listed for a specific parameter, it is also expected that ranges of 10–40 and 20–50 are also included. Furthermore, if the minimum range values ​​are 1 and 2, and the maximum range values ​​are 3, 4, and 5, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0–5" means that all real numbers between "0–5" have been listed herein; "0–5" is merely a shortened representation of these numerical combinations.

[0062] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0063] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0064] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0065] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0066] Unless otherwise specified, the reaction will proceed under normal temperature and pressure conditions.

[0067] Unless otherwise specified, all parts or percentages are by weight or by weight percentage.

[0068] In this invention, all the substances used are known substances that can be purchased or synthesized by known methods.

[0069] In this invention, all the devices or equipment used are conventional devices or equipment known in the art and are readily available.

[0070] like Figure 1-5 As shown, a dual-channel microfluidic electrochemical sensor is provided. The dual-channel microfluidic electrochemical sensor is laterally symmetrical and consists of a bottom layer, an electrode layer 2, and a channel layer 1 stacked sequentially.

[0071] The bottom layer is a PDMS base 3, and two electrode slots 10 are symmetrically arranged on the PDMS base 3.

[0072] The electrode layer 2 is symmetrically provided with two sets of three-electrode systems 5. The two sets of three-electrode systems 5 are respectively embedded in the two electrode slots 10. The size of the electrode slots 10 matches the size of the embedded three-electrode systems 5. One set of three-electrode systems 5 consists of a first electrode disk 6 and a first pin 7 facing outward. The first electrode disk 6 is connected to the first pin 7. The other set of three-electrode systems 5 consists of a second electrode disk 8 and a second pin 9 facing outward. The second electrode disk 8 is connected to the second pin 9.

[0073] The channel layer 1 includes PDMS microchannels 4.

[0074] Furthermore, the PDMS microchannel 4 is composed of the following components:

[0075] Reagent delivery fluid inlet 11;

[0076] The front-end main channel 12 has one inlet and two outlets, and the reagent delivery liquid inlet 11 is connected to the inlet of the front-end main channel 12.

[0077] Two sample channels 14 are provided with one inlet and two outlets. The inlets of the two sample channels 14 are connected to the outlets of the main channel 12 at the front end, thus forming a primary branch 13. One outlet of the sample channel 14 is connected to the inlet of the vortex mixing unit 18 through a first pipe. The first pipe is provided with a sample loading port 15. The other outlet of the sample channel 14 is connected to the inlet of the conical microcavity flow control unit 19 through a second pipe. The second pipe is provided with a complementary chain loading port 17.

[0078] Two electrode regions 21 are composed of electrode pin interfaces 22 and electrode reaction regions 20. The outlet of the vortex mixing unit 18 is connected to the inlet of the electrode reaction region 20 through a third pipe. The outlet of the conical microcavity flow control unit 19 is connected to the inlet of the electrode reaction region 20 through a complementary chain channel 16. The electrode pin interfaces 22 are connected to the first pin 7 and the second pin 9 respectively. The positions of the two electrode reaction regions 20 correspond to the positions of the first electrode disk 6 and the second electrode disk 8 respectively.

[0079] Liquid outlet 23, the liquid outlets of the two electrode reaction zones 20 are connected to the liquid inlet of the electrode zone 21 through the sixth pipe, and the liquid outlet of the electrode zone 21 is connected to the liquid outlet 23 through the fifth pipe.

[0080] First-level branch 13 is used to control capillary-driven fluid flow and to simultaneously detect two myocardial markers, miR-125b and miR-208b, enabling three-electrode detection with different fluids entering before and after each step.

[0081] The specific steps of the above-mentioned dual-channel microfluidic electrochemical sensor fabrication method are as follows:

[0082] (1) The circular gold electrode, the semi-circular arc gold counter electrode, and the semi-circular arc reference electrode are all made by screen printing.

[0083] (2) Circular gold electrode surface modified with L-Cys / cMWCNTs+cRNA nanomaterials;

[0084] (3) The bottom layer and channel layer of the dual-channel microfluidic electrochemical sensor are fabricated by photolithography;

[0085] (4) A dual-channel microfluidic electrochemical sensor was fabricated using the room temperature bond method.

[0086] The specific steps of the above-mentioned dual-channel microfluidic electrochemical sensor fabrication method are as follows:

[0087] (1) The three electrodes—a circular gold electrode, a semi-circular gold counter electrode, and a semi-circular reference electrode—are fabricated using screen printing.

[0088] A PET film of a certain shape was ultrasonically treated in an ethanol solution for 2 hours and dried at 120°C for 15 minutes. The PET film was then laid flat on a substrate and fixed. Silver paste was printed on a screen printer using an oil-based polyurethane squeegee at a speed of 5 cm / s to obtain conductive lines of a three-electrode system. The film was then heat-cured at 120°C for 10 minutes. A circular gold working electrode and a semi-circular gold counter electrode were then printed sequentially using conductive gold paste and heat-cured at 60°C for 30 minutes. A semi-circular reference electrode was then printed using Ag / AgCl ink and heat-cured at 110°C for 5 minutes. Finally, an insulating layer was formed by printing with insulating ink, exposing the three electrodes and the ends of the wires. The film was then cured in a UV curing machine at a speed of 30 W / cm.

[0089] (2) The surface of the circular gold electrode was modified with L-Cys / cMWCNTs+cRNA nanomaterials. A PDMS film was then applied to the three electrodes from step (1), exposing the surface of the circular gold electrode. The prepared h-BNNS / cMWCNTs / MnFe2O4 composite material was dispersed in deionized water to prepare a uniform suspension with a concentration of 1 mg / mL. The mixture was then dropped onto the electrode surface, the electrode surface was dried, and the three electrodes were covered with a PDMS film, exposing the surface of the circular gold electrode with covalently bonded h-BNNS / cMWCNTs / MnFe2O4 nanomaterials. Finally, 5% PDMS activated by EDC and NHS was applied to the electrode surface. ′cRNA with amino-terminated miR-125b or miR-208b was incubated, the PDMS membrane was removed, and the electrode surface was rinsed sequentially with SSC and ultrapure water. The electrode surface was dried and covered with a new PDMS membrane. The cRNA covalently binds to the carboxyl groups on the surface of cMWCNTs via amide bonds. After rinsing with PBS buffer (pH 7.4) to remove unbound cRNA, the electrode was immersed in 1% BSA solution and incubated at 37°C for 25 min to block non-specific binding sites. Finally, the electrode was thoroughly washed with PBS buffer to obtain an electrochemical sensor modified with h-BNNS / cMWCNTs / MnFe2O4+ cRNA and blocked with BSA.

[0090] (3) Fabrication of dual-channel microfluidic chip: The channel layer and bottom layer of the dual-channel microfluidic electrochemical sensor are fabricated by photolithography. The photolithography process mainly includes silicon wafer cleaning, spin coating of silicon wafer, pre-baking and post-baking, exposure, development, cleaning, hard molding, PDMS molding, PDMS stripping and drilling.

[0091] (4) The dual-channel microfluidic electrochemical sensor was fabricated using the room-temperature bonding method. The specific steps are as follows:

[0092] Two circular gold electrodes made of L-Cys / cMWCNTs+cRNA nanomaterials are covalently bound to miR-125b and miR-208b cRNA, respectively. The two three-electrode systems are embedded into the two detection area electrode slots of the bottom layer of the dual-channel microfluidic chip in step (3). The bottom layer, electrode layer and channel layer are aligned and placed in a bonding machine. At room temperature and 0.2MPa pressure, bonding is performed for 180s to obtain the dual-channel microfluidic electrochemical sensor.

[0093] The sensor's sealing performance, liquid self-flow within the channel, and flow rate difference entering the detection zone were investigated using trypan blue solution. Trypan blue solution was injected into the inlet until the channel was full, all outlets were sealed, and a positive pressure of 20 kPa was applied. The channel interface, bonding seam, and electrode embedding area were observed under a microscope for 5 minutes. No blue solution seepage and no liquid film diffusion at the edges indicated a satisfactory sealing performance. 10 μL of trypan blue solution was added to the inlet (without external force), the drive device was removed, and the system was allowed to stand. If the solution self-filled the channel and uniformly covered the detection zone (without bubbles / discontinuities), liquid self-flow within the channel was achieved. Trypan blue solution was injected into inlet 15, and colorless buffer solution was injected into inlet 17. Simultaneous dual-channel injection observation revealed that the trypan blue solution and colorless buffer solution entered the detection zone at different flow rates. This also verified the accuracy of the COMSOL software simulation results. The results are as follows: Figure 6 As shown.

[0094] A dual-channel microfluidic electrochemical sensor is prepared by any one of the methods described above.

[0095] A dual-channel microfluidic electrochemical sensor is prepared by any one of the methods described above.

[0096] A detection system comprising the dual-channel microfluidic electrochemical sensor described in any one of the preceding claims.

[0097] Furthermore, the detection system also includes an electrochemical workstation connected to the dual-channel microfluidic electrochemical sensor.

[0098] The sensor electrodes of the dual-channel microfluidic electrochemical sensor are connected to the corresponding interfaces of the electrochemical workstation (such as WE1 / WE2, CE, RE channels) via wires.

[0099] Circular gold electrode: This is the target sensor electrode for the study, connected to the working electrode (WE) interface of the workstation. Semi-circular gold counter electrode: This provides the current loop for the electrochemical reaction. It is connected to the counter electrode (CE) interface of the workstation. Semi-circular reference electrode: This provides a stable potential reference point. It is connected to the reference electrode (RE) interface of the workstation.

[0100] The reagent delivery inlet and liquid outlet of the dual-channel microfluidic electrochemical sensor are connected to the fluid drive unit (such as a syringe pump or peristaltic pump) via hoses to achieve precise sample / reagent delivery.

[0101] The electrochemical workstation is connected to a computer, which is equipped with customized data processing software, including LabVIEW and Python.

[0102] The workflow of the above detection system

[0103] (1) Sample loading: The fluid drive unit injects the sample to be tested into the dual-channel microfluidic electrochemical sensor, and the flow rate is precisely controlled by the pump;

[0104] (2) Electrochemical detection: The electrochemical workstation applies an excitation signal to a designated channel and simultaneously acquires the response signal;

[0105] (3) Data synchronization: The detection signal and fluid status (such as flow rate and injection timestamp) are synchronized with the software to ensure data correlation;

[0106] (4) Real-time analysis: Customized data processing software on the computer visualizes the dual-channel detection results and automatically generates reports or triggers feedback control (such as concentration exceeding the standard alarm).

[0107] A detection method, based on the above-mentioned detection system, comprises the following steps:

[0108] 1. Sample pretreatment

[0109] Serum sample pretreatment: Collect blood samples, dilute 10 times and place them in test tubes. Allow them to coagulate naturally at room temperature for 30-60 minutes. After the blood has coagulated, centrifuge at 3000 rpm / min for 10 minutes, collect the serum from the supernatant, and store it in a -80℃ freezer.

[0110] 2. Two circular gold electrodes made of L-Cys / cMWCNTs+cRNA nanomaterials of the dual-channel microfluidic electrochemical sensor are covalently bound with miR-125b and miR-208b cRNA, respectively. Two pairs of circular gold electrodes, a semi-circular gold electrode pair, and a semi-circular reference electrode are embedded in the two electrode slots of the bottom layer of the dual-channel microfluidic electrochemical sensor. The bottom layer, electrode layer and channel layer are aligned and placed in a bonding machine. Bonding is performed at room temperature and 0.2 MPa pressure for 180 s.

[0111] 3. Construction of a dual-channel microfluidic electrochemical sensor platform:

[0112] A circular gold electrode modified with h-BNNS / cMWCNTs / MnFe2O4+cRNA nanomaterials was used as the working electrode. A three-electrode system was formed by a semi-circular gold counter electrode and a semi-circular reference electrode. The two sets of electrode pins were led out with wires and connected to the electrode lines of two electrochemical workstations respectively to build a dual-channel microfluidic electrochemical sensor platform.

[0113] 4. A dual-channel microfluidic electrochemical sensor simultaneously detects two myocardial biomarkers, miR-125b and miR-208b:

[0114] The sample and TE buffer are added to the sample port and TE buffer port of the microfluidic chip, respectively. Under capillary action, the sample and TE buffer flow freely within the channel. Under the combined control of channel width, functional area, and chip pore switching, the sample arrives at the two detection areas of the microfluidic chip earlier than the TE buffer, with an interval of 20-30 minutes. The electrochemical workstation measures the changes in electrochemical signals of the two three-electrode systems before and after sample addition. Based on the relationship between the electrochemical signal changes and the concentrations of miR-125b and miR-208b, the simultaneous quantitative detection of miR-125b and miR-208b in the sample is achieved.

[0115] 5. Quantitative Detection of miR-125b Using a Microfluidic Electrochemical Biosensor: The diffusion control effect of the aptamer-functionalized electrode surface during miR-125b detection was determined using electrochemical impedance spectroscopy (EIS). Subsequently, differential pulse velocity (DPV) curves of different concentrations of miR-125b were measured. Figure 7 As shown), its peak current (i) p ) and miR-125b concentration (C Mb) relationship and peak current (i p ) and the logarithm of miR-125b concentration (logC) Mb Fit a linear regression equation. The result is as follows: Figure 8 As shown, when the miR-125b concentration is in the range of 1.0 fM to 10 nM, i p and logC Mb A good linear relationship was observed, with a correlation coefficient (r) of 0.9945. Based on the linear equation, the concentration of miR-125b in the sample could be calculated, and its limit of detection (LOD) and lowest detection threshold were 0.109 fM. The results are as follows... Figure 9-11 As shown, the dual-channel microfluidic electrochemical sensor for single-sided detection of miR-125b exhibits good stability, repeatability, and specificity.

[0116] (8) Quantitative detection of miR-208b using a microfluidic electrochemical biosensor, with the same procedure as miR-125b: The diffusion control effect of the aptamer-functionalized electrode surface during miR-208b detection was determined using electrochemical impedance spectroscopy (EIS). Subsequently, differential pulse velocity (DPV) curves of different concentrations of miR-208b were measured. Figure 12 As shown), its peak current (i) p ) and miR-208b concentration (C hsCRP ) relationship and peak current (i p ) and the logarithm of miR-208b concentration (logC) hsCRP Fit a linear regression equation. For example... Figure 13 As shown, when the miR-208b concentration is in the range of 1.0 fM to 10 nM, i p and logC hsCRP A good linear relationship was observed, with a correlation coefficient (r) of 0.9936. Based on the linear equation, the concentration of miR-208b in the sample could be calculated, and its limit of detection (LOD) and lowest detection threshold were 0.129 fM. Figure 14-16 As shown, the dual-channel microfluidic electrochemical sensor miR-208b exhibits good stability, repeatability, and specificity for single-sided detection.

[0117] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A dual-channel microfluidic electrochemical sensor, characterized in that, It is composed of a bottom layer, an electrode layer, and a channel layer stacked sequentially, wherein: The bottom layer is a PDMS base, and two electrode slots are symmetrically arranged on the PDMS base. The electrode layer is symmetrically provided with two sets of three-electrode systems. The two sets of three-electrode systems are respectively embedded in the two electrode slots. One set of three-electrode systems consists of a first electrode disk and a first pin facing outward, and the first electrode disk is connected to the first pin. The other set of three-electrode systems consists of a second electrode disk and a second pin facing outward, and the second electrode disk is connected to the second pin. The channel layer includes PDMS microchannels.

2. The dual-channel microfluidic electrochemical sensor as described in claim 1, characterized in that, The PDMS microchannel consists of the following components: Reagent delivery fluid inlet; The front-end main channel has one inlet and two outlets, and the reagent delivery liquid inlet is connected to the inlet of the front-end main channel; Two sample channels are provided, each with an inlet and two outlets. The inlets of the two sample channels are connected to the outlets of the main channel at the front end, thus forming a primary branch. One outlet of the sample channel is connected to the inlet of the vortex mixing unit through a first pipe, which has a sample dispensing port. The other outlet of the sample channel is connected to the inlet of the conical microcavity flow control unit through a second pipe, which has a complementary chain dispensing port. The two electrode regions consist of an electrode pin interface and an electrode reaction region. The outlet of the vortex mixing unit is connected to the inlet of the electrode reaction region through a third pipe, and the outlet of the conical microcavity fluid control unit is connected to the inlet of the electrode reaction region through a complementary chain channel. The electrode pin interfaces are connected to the first pin and the second pin, respectively, and the positions of the two electrode reaction regions correspond to the positions of the first electrode disk and the second electrode disk, respectively. The liquid outlets of the two electrode reaction zones are connected to the liquid inlets of the electrode zones via the sixth pipe, and the liquid outlets of the electrode zones are connected to the liquid outlets via the fifth pipe.

3. The method for preparing the dual-channel microfluidic electrochemical sensor according to claim 1 or 2, characterized in that, The steps are as follows: (1) The circular gold electrode, the semi-circular arc gold counter electrode, and the semi-circular arc reference electrode are all made by screen printing. (2) Circular gold electrode surface modified with L-Cys / cMWCNTs+cRNA nanomaterials; (3) The bottom layer and channel layer of the dual-channel microfluidic electrochemical sensor are fabricated by photolithography; (4) A dual-channel microfluidic electrochemical sensor was fabricated using the room temperature bond method.

4. The method for fabricating the dual-channel microfluidic electrochemical sensor as described in claim 3, characterized in that, The specific steps of step (1) are as follows: A PET film of a certain shape is ultrasonically treated in an ethanol solution for at least 2 hours and dried at 120°C for at least 15 minutes. The PET film is then laid flat on a substrate and fixed. Silver paste is printed on a screen printer using an oil-based polyurethane squeegee at a speed of 5 cm / s to obtain conductive lines of a three-electrode system. The film is then heat-cured at 120°C for at least 10 minutes. A circular gold working electrode and a semi-circular gold counter electrode are then printed sequentially using conductive gold paste. The film is then heat-cured at 60°C for at least 30 minutes. A semi-circular reference electrode is then printed using Ag / AgCl ink. The film is then heat-cured at 110°C for at least 5 minutes. Finally, an insulating layer is formed by printing with insulating ink, exposing the three electrodes and the ends of the wires. The film is then UV-cured at a speed of 30 W / cm.

5. The method for fabricating the dual-channel microfluidic electrochemical sensor as described in claim 3, characterized in that, The specific steps of step (2) are as follows: (21) The PDMS film covers the circular gold electrode, the semi-circular gold counter electrode and the semi-circular reference electrode obtained in step (1), exposing the surface of the circular gold electrode. The h-BNNS / cMWCNTs / MnFe2O4 composite material is dispersed in deionized water to prepare a uniform suspension with a concentration of 1 mg / mL. The suspension is dropped onto the surface of the circular gold electrode and the surface of the circular gold electrode is dried. (22) A circular gold electrode, a semi-circular gold counter electrode, and a semi-circular reference electrode were covered with a PDMS film, exposing the surface of the circular gold electrode covalently bonded with h-BNNS / cMWCNTs / MnFe2O4 nanomaterials. 6 μL of 10 μM 5′ amino-modified cRNA solution was added to the modified circular gold electrode surface and incubated in a 37°C constant temperature incubator for at least 60 min to allow the cRNA to covalently bind to the carboxyl groups on the surface of cMWCNTs through amide bonds. After rinsing with PBS buffer with a pH of 7.4 to remove unbound cRNA, the modified circular gold electrode was immersed in 1% BSA solution and incubated at 37°C for 25 min to block non-specific binding sites. (23) The modified circular gold electrode was thoroughly rinsed with PBS buffer at pH 7.4, the PDMS membrane was removed, and the surfaces of the circular gold electrode, the semi-circular gold counter electrode and the semi-circular reference electrode were rinsed with SSC and ultrapure water in sequence. The electrode surfaces were dried and covered with a new PDMS membrane. The cRNA was covalently bound to the carboxyl group on the surface of cMWCNTs through amide bonds to obtain an electrochemical sensor modified with h-BNNS / cMWCNTs / MnFe2O4+cRNA and blocked with BSA.

6. The method for fabricating the dual-channel microfluidic electrochemical sensor as described in claim 3, characterized in that, The specific steps of step (4) are as follows: Two circular gold electrodes made of L-Cys / cMWCNTs+cRNA nanomaterials were covalently bound with miR-125b and miR-208b cRNA, respectively. The two three-electrode systems were embedded into the two detection zone electrode grooves of the bottom layer of the dual-channel microfluidic electrochemical sensor. The bottom layer, electrode layer and channel layer were aligned and placed in a bonding machine. The bonding was carried out at room temperature and 0.2 MPa pressure for 180 s to obtain the dual-channel microfluidic electrochemical sensor.

7. A dual-channel microfluidic electrochemical sensor, characterized in that, Prepared by the method described in any one of claims 3-6.

8. A detection system, characterized in that, Includes the dual-channel microfluidic electrochemical sensor described in any one of claims 1-2 and 7.

9. The detection system as described in claim 8, characterized in that, The detection system also includes an electrochemical workstation connected to the dual-channel microfluidic electrochemical sensor.

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