A microfluidic protein sensor based on bias-regulated electrophoretic shift and electrical parameter detection and a detection method thereof

Abstract

The application provides a microfluidic protein sensor based on bias regulation and electrophoretic shift and electrical parameter detection and a detection method thereof, and relates to the field of protein detection. The sensor comprises a bias modulation module and an electrical parameter detection module. The bias modulation module comprises a first substrate and a first top plate arranged oppositely, a first microchannel is arranged between the first substrate and the first top plate, parallel microchannels are arranged between the first microchannels, and the first substrate is provided with a bias regulation electrode located in the parallel microchannels. The electrical parameter detection module comprises a second substrate and a second top plate arranged oppositely, a second microchannel is arranged between the second substrate and the second top plate, and the second substrate is provided with parallel electrodes located in the second microchannel and electrically connected with an external sensing circuit. The outlet of the bias modulation module is in sealed communication with the inlet of the electrical parameter detection module through a pipeline. The application improves the problem that the existing protein detection technology is difficult to consider low cost, simple operation and precise detection of multiple proteins simultaneously.

Description

Technical Field

[0001] This invention relates to the field of protein detection, and in particular to a microfluidic protein sensor and its detection method based on bias-controlled electrophoretic shift and electrical parameter detection. Background Technology

[0002] Blood proteins are key biomarkers reflecting the physiological and pathological states of the human body, and their types, concentrations, and structural changes are closely related to a variety of diseases. Abnormal albumin levels indicate liver and kidney dysfunction, elevated C-reactive protein is a sensitive indicator of inflammation and infection, cardiac troponin is the gold standard for diagnosing acute myocardial infarction, and the combined detection of multiple tumor marker proteins is of great value for early cancer screening. Therefore, accurate detection of blood proteins plays an irreplaceable role in clinical diagnosis, disease monitoring, prognostic assessment, and personalized treatment.

[0003] Currently, commonly used clinical blood protein detection technologies mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, mass spectrometry, electrochemical sensors, and optical protein sensors. ELISA, chemiluminescence immunoassay, and mass spectrometry require large and expensive equipment, involve numerous and time-consuming steps, are difficult to perform in parallel detection of multiple proteins, have low detection efficiency, and rely on manual comparison analysis. Electrochemical sensors and optical protein sensors have the advantages of easy integration and miniaturization, but the fabrication cost of optical protein sensors is higher than that of electrochemical sensors. Therefore, there is an urgent need to develop a solution to address these problems. Summary of the Invention

[0004] The purpose of this invention is to provide a microfluidic protein sensor and its detection method based on bias-controlled electrophoretic shift and electrical parameter detection, which improves the problem that existing protein detection technologies are difficult to achieve in terms of low cost, ease of operation and accurate detection of multiple proteins in parallel.

[0005] The present invention provides a microfluidic protein sensor and its detection method based on bias-controlled electrophoretic shift and electrical parameter detection, which adopts the following technical solution:

[0006] Firstly, a microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection, specifically comprising:

[0007] It consists of a bias modulation module and an electrical parameter detection module;

[0008] The bias modulation module includes a first substrate and a second top plate disposed opposite to each other. The first top plate is provided with a first test solution inlet, a first test solution outlet, a buffer solution inlet, and a buffer solution outlet. A first microchannel is provided between the first substrate and the first top plate. The first test solution inlet and the first test solution outlet, and the buffer solution inlet and the buffer solution outlet are respectively connected through the first microchannel. A parallel microchannel is provided between the two first microchannels. The first substrate is provided with a bias control electrode, which is located in the parallel microchannel.

[0009] The electrical parameter detection module includes a second substrate and a second top plate arranged opposite to each other. The second top plate is provided with a second inlet and a second outlet for the solution to be tested. A second microchannel is provided between the second substrate and the second top plate. The second substrate is provided with a parallel electrode, which is located in the second microchannel and is electrically connected to an external sensing circuit.

[0010] The outlet of the first test solution and the inlet of the second test solution are connected in a sealed manner through a pipeline.

[0011] Optionally, the first substrate includes a rigid carrier or a flexible film, wherein the rigid carrier includes glass or silicon wafer, and the flexible film includes a polymer film made of polyimide, polytetrafluoroethylene or polyethylene terephthalate.

[0012] Optionally, the first top plate includes a rigid carrier sheet or a flexible film, wherein the rigid carrier sheet includes glass or quartz, and the flexible film includes a polymer film made of polydimethylsiloxane, polymethyl methacrylate, or a cyclic olefin copolymer.

[0013] Optionally, the bias control electrode may be made of gold, platinum, or graphite.

[0014] Optionally, the second substrate includes a rigid carrier or a flexible film, wherein the rigid carrier includes glass or silicon wafer, and the flexible film includes a polymer film made of polyimide, polytetrafluoroethylene or polyethylene terephthalate.

[0015] Optionally, the second top plate includes a rigid carrier sheet or a flexible film, wherein the rigid carrier sheet includes glass or quartz, and the flexible film includes a polymer film made of polydimethylsiloxane, polymethyl methacrylate, or a cyclic olefin copolymer.

[0016] Optionally, the parallel electrode includes a metal electrode, a metal oxide electrode, a silver chloride electrode, or a metal electrode coated with a protein aptamer.

[0017] Optionally, the material of the pipe includes polytetrafluoroethylene, polymethyl methacrylate, polydimethylsiloxane, glass, quartz or stainless steel.

[0018] Optionally, the bias control electrode is two electrodes symmetrically disposed on the first substrate, with a gap between the two bias control electrodes; the parallel electrode is two electrodes symmetrically disposed on the second substrate, with a gap between the two parallel electrodes.

[0019] The beneficial effects of the microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection provided by this invention are as follows: By setting parallel microchannels and bias-controlled electrodes in the bias modulation module, multiple proteins can be separated by electrophoresis on the same chip. The parallel electrodes in the electrical parameter detection module enable in-situ acquisition of electrical signals of the separated proteins. The two modules are integrated in series on the microfluidic chip, which is compact, easy to miniaturize and portable, and does not require expensive optical detection equipment and complex labeling steps, thus reducing detection costs and operational difficulty. It improves the problem that existing protein detection technologies cannot simultaneously achieve low cost, easy operation and accurate parallel detection of multiple proteins.

[0020] Secondly, a detection method for a microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection, employing the microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection described in the first aspect, specifically includes:

[0021] A buffer solution and a test protein solution are injected into the buffer solution inlet and the first test solution inlet of the bias modulation module, respectively; the test protein solution flows into the second test solution inlet of the electrical parameter detection module through the first test solution outlet and flows out through the second test solution outlet, while the buffer solution flows out through the buffer solution outlet; a voltage is applied to the bias control electrode; and the electrical parameters of the test protein solution are measured through the parallel electrode.

[0022] The beneficial effect of the microfluidic protein sensor detection method based on bias voltage-controlled electrophoretic migration and electrical parameter detection provided by this invention is that the bias voltage control module applies a bias voltage to the protein solution to be tested, causing different proteins to undergo electrophoretic migration in parallel microchannels to achieve spatial separation. Then, the electrical parameters of the separated proteins are measured by parallel electrodes in the electrical parameter detection module. This enables parallel, rapid, and highly sensitive detection of multiple proteins in a trace sample. Moreover, it is simple to operate and has low equipment cost, which improves the problem that existing protein detection technologies cannot simultaneously achieve low cost, simple operation, and accurate parallel detection of multiple proteins. Attached Figure Description

[0023] Figure 1A schematic diagram of a microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection is provided in an embodiment of the present invention.

[0024] Figure 2 This is a schematic diagram of the bias modulation module in a microfluidic protein sensor based on bias-controlled electrophoretic migration and electrical parameter detection, provided in an embodiment of the present invention.

[0025] Figure 3 The top view (A), longitudinal cross-sectional view (B), and planar structural schematic diagram (C) of the bias modulation module in a microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection are provided for embodiments of the present invention.

[0026] Figure 4 This is a schematic diagram of the electrical parameter detection module in a microfluidic protein sensor based on bias-controlled electrophoretic migration and electrical parameter detection, provided in an embodiment of the present invention.

[0027] Figure 5 The diagram shows a top view (D), a longitudinal cross-sectional view (E), and a schematic diagram (F) of the planar structure of the electrical parameter detection module in a microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection, provided for embodiments of the present invention.

[0028] Figure 6 The diagram illustrates the impedance measurement results of a microfluidic protein sensor based on bias-controlled electrophoretic migration and electrical parameter detection, used in this invention to measure bovine hemoglobin at different concentrations in different pH buffer solutions under +1.2V and -1.2V bias voltages. Figure 6 a to Figure 6 e is a graph showing the relationship between the impedance ratio and pH. Figure 6 f is a graph showing the relationship between the absolute value of the sensor impedance and the concentration of bovine hemoglobin.

[0029] Figure 7 The present invention provides a microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection, which measures the impedance ratio changes of bovine hemoglobin, bovine serum albumin, and bovine γ-globulin at a concentration of 40 g / L under different pH buffer solutions when +1.2 V and -1.2 V bias voltages are applied.

[0030] Figure 8 This invention provides a microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection, which measures the impedance ratio of blood from four animals (cattle, horses, pigs, and sheep) under different pH buffer solutions when +1.2V and -1.2V bias voltages are applied.

[0031] Explanation of reference numerals in the attached figures:

[0032] 1. First substrate; 2. First top plate; 3. Bias control electrode; 4. Second substrate; 5. Second top plate; 6. Parallel electrode; 7. Channel. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0034] See Figure 1 The microfluidic protein sensor based on bias-controlled electrophoretic shift and electrical parameter detection provided in this embodiment of the invention consists of a bias modulation module and an electrical parameter detection module. The test solution outlet of the bias modulation module and the test solution inlet of the electrical parameter detection module are sealed and connected through a pipe 7. The test solution flows out of the bias modulation module and enters the electrical parameter detection module through the pipe 7.

[0035] See Figure 2 and Figure 3 The bias modulation module includes a first substrate 1 and a first top plate 2 that are disposed opposite to each other. For example... Figure 3 As shown in the longitudinal cross-sectional view of B, the first substrate 1, the bias control electrode 3, and the first top plate 2 are stacked sequentially to form a three-dimensional microchannel structure; combined with Figure 3 As shown in the schematic diagram of planar layout C, the first top plate 2 has an inlet and an outlet for the solution to be tested, as well as an inlet and an outlet for the buffer solution. A first microchannel is arranged between the first substrate 1 and the first top plate 2. The inlet and outlet for the solution to be tested, and the inlet and outlet for the buffer solution, are connected through the first microchannel, and several parallel microchannels are arranged between the two first microchannels. Two symmetrical bias control electrodes 3 are arranged on the first substrate 1, located in the area covered by the parallel microchannels. A gap exists between the two bias control electrodes 3, ensuring electrical isolation between them. When the solution to be tested and the buffer solution flow through this gap, a conductive path is formed. After the first top plate 2 is bonded to the first substrate 1, the boundary of the first top plate 2 intersects with the bias control electrode 3, allowing the bias control electrode 3 to be connected to an external circuit to achieve voltage biasing.

[0036] See Figure 4 and Figure 5The electrical parameter detection module includes a second substrate 4 and a second top plate 5 that are positioned opposite each other. For example... Figure 5 As shown in the longitudinal cross-sectional view of E, the second substrate 4, the parallel electrode 6, and the second top plate 5 are stacked sequentially; combined with Figure 5 As shown in the planar layout diagram of F, the second top plate 5 has an inlet and an outlet for the solution to be tested. A second microchannel is arranged between the second substrate 4 and the second top plate 5, connecting the inlet and outlet of the solution to be tested. Two symmetrical parallel electrodes 6 are disposed on the second substrate 4, located within the area covered by the second microchannel, and electrically connected to an external sensing circuit. A gap exists between the two parallel electrodes 6, providing electrical isolation. When the solution to be tested flows through this gap, a conductive path is formed. After the second top plate 5 is bonded to the second substrate 4, the boundary of the second top plate 5 intersects with the parallel electrodes 6, allowing the parallel electrodes 6 to be connected to an external sensing circuit for electrical parameter measurement.

[0037] In some embodiments, regarding material selection, the first substrate 1 and the second substrate 4 are independently selected from rigid substrates or flexible films. The rigid substrate can be glass or silicon, and the flexible film can be a polymer film made of polyimide, polytetrafluoroethylene, or polyethylene terephthalate. The first top plate 2 and the second top plate 5 are independently selected from rigid substrates or flexible films. The rigid substrate can be glass or quartz, and the flexible film can be a polymer film made of polydimethylsiloxane, polymethyl methacrylate, or cyclic olefin copolymers. The bias control electrode 3 is made of conductive materials such as gold, platinum, or graphite. The parallel electrode 6 is made of metal electrodes, metal oxide electrodes, silver chloride electrodes, or metal electrodes coated with protein aptamers. The conduit 7 is made of one of polytetrafluoroethylene, polymethyl methacrylate, polydimethylsiloxane, glass, quartz, or stainless steel.

[0038] In some embodiments, the spacing between the first substrate 1 and the first top plate 2, and the spacing between the second substrate 4 and the second top plate 5, are designed to allow for solution flow. The width of the first microchannel, the second microchannel, and the parallel microchannel is 50 μm, and the height is 20 μm. The gap width between the bias control electrodes 3 is 5 mm, and the gap width between the parallel electrodes 6 is 5 mm. The top plate and the substrate can be fixed by adhesive bonding, specifically by chemically modifying the silicon oxide wafer and the PDMS surface to achieve a tight bond. Both ends of the microchannels in the first top plate 2 and the second top plate 5 are provided with through holes, and the inflow and outflow rates of the solution can be controlled by inserting sealed conduits into the through holes. Furthermore, the shapes of the substrate and the top plate can be designed using template graphics to construct microchannel structures of different shapes to meet the needs of different detection scenarios.

[0039] In some preferred embodiments, the fabrication method of the microfluidic protein sensor of the present invention is as follows:

[0040] First, substrates for the bias modulation module and the electrical parameter detection module are fabricated separately. A silicon oxide wafer is used as the first substrate 1, and a 100 nm thick gold electrode is deposited on the surface of the first substrate 1 by electron beam evaporation to form two mutually symmetrical bias modulation electrodes 3. Similarly, a silicon oxide wafer is used as the second substrate 4, and a 100 nm thick gold electrode is deposited on the surface of the second substrate 4 by electron beam evaporation to form parallel electrodes 6.

[0041] Next, the top plates for the bias modulation module and the electrical parameter detection module were fabricated. Using a silicon wafer as a substrate, microchannel photolithography templates for the bias modulation module and the electrical parameter detection module were fabricated using photolithography. In a glove box, trimethylchlorosilane was evaporated onto the photolithography template to form a silane film. Polydimethylsiloxane and a catalyst were mixed uniformly at a volume ratio of 10:1 and poured onto the evaporated photolithography template. The mixture was dried and cured at 70°C for 2 hours, and then peeled off to obtain the first top plate 2 and the second top plate 5. 0.5 mm diameter circular holes were punched into the PDMS top plates using a puncher to serve as the inlets and outlets.

[0042] Next, the prepared substrate and PDMS top plate were simultaneously placed in a reactive ion etching machine for oxygen plasma treatment for 120 seconds at a power of 100W. After treatment, the PDMS top plate was immediately kept in contact with the silicon oxide substrate and heated on an 80°C heating stage for 50 minutes, while pressure was applied to bond the two together, resulting in the bias modulation module and the electrical parameter detection module, respectively.

[0043] Finally, the solution outlet of the bias modulation module and the solution inlet of the electrical parameter detection module are connected in series and sealed through pipe 7, thus completing the construction of the microfluidic protein sensor.

[0044] In some embodiments, when using the aforementioned microfluidic protein sensor for detection, a buffer solution and a test protein solution are injected into the buffer solution inlet and test solution inlet of the bias modulation module, respectively. The buffer solution flows into the first microchannel through the buffer solution inlet and then flows out from the buffer solution outlet; the test protein solution flows into the first microchannel through the test solution inlet and then flows through a parallel microchannel. During this process, a bias voltage is applied through the bias modulation electrode 3, causing the test protein to undergo electrophoretic shift within the parallel microchannel, achieving preliminary separation of different proteins. The test solution, after being processed by the bias modulation module, flows out from the test solution outlet, flows through pipe 7 into the test solution inlet of the electrical parameter detection module, flows along the second microchannel through the parallel electrode 6, and DC and AC voltages are applied through the parallel electrode 6 to measure the electrical parameter signals of the test solution. Finally, it flows out from the test solution outlet. Based on the electrical parameters measured by the parallel electrode 6, qualitative and quantitative detection of the test protein can be achieved.

[0045] Experimental Example 1: Impedance Detection of Different Concentrations of Bovine Hemoglobin

[0046] Using the microfluidic protein sensor provided by this invention, the impedance ratios of bovine hemoglobin at different concentrations (40 g / L, 80 g / L, 120 g / L, 160 g / L, 200 g / L) in buffer solutions at different pH values ​​(6.0, 6.5, 7.0, 7.5, 8.0) were measured under +1.2V and -1.2V bias voltages relative to the unbiased condition. During detection, a bias voltage was applied to the bovine hemoglobin in the solution through the bias-controlled electrode 3 to induce electrophoretic migration, and an excitation signal was applied through the parallel electrode 6 to collect the impedance response. The results are as follows: Figure 6 As shown, where Figure 6 a- Figure 6 e indicates that, at different concentrations, the impedance ratio of bovine hemoglobin at a +1.2V bias voltage decreases with increasing pH, while it increases with a -1.2V bias voltage. The two curves intersect at pH=7.0 at 1.0, which corresponds to the isoelectric point of bovine hemoglobin. Figure 6 f indicates that the absolute impedance value of the sensor decreases significantly with the increase of bovine hemoglobin concentration (40 g / L to 200 g / L), indicating that the sensor provided by the present invention can achieve sensitive quantitative detection of bovine hemoglobin at different concentrations.

[0047] Experiment Example 2: Differentiation and Detection of Different Types of Proteins

[0048] Using the microfluidic protein sensor provided by this invention, the impedance ratios of three different proteins—bovine hemoglobin, bovine serum albumin, and bovine gamma globulin—at a concentration of 40 g / L were measured under different pH values ​​(6.0, 6.5, 7.0, 7.5, and 8.0) with applied bias voltages of +1.2V and -1.2V relative to the unbiased condition. The results are as follows: Figure 7 As shown, the three proteins exhibit distinctly different impedance ratio shift trends with pH changes, indicating that the sensor provided by this invention can effectively distinguish different types of proteins by detecting the differences in impedance response of different proteins under a specific bias voltage.

[0049] Experiment Example 3: Differentiation Detection of Blood from Different Animals

[0050] The microfluidic protein sensor provided in this invention was used to test the blood of four animals: cattle, horses, pigs, and sheep. Fresh animal blood was collected, allowed to stand for separation, and then centrifuged at 3000 r / min for 3 minutes to remove blood cells. The supernatant plasma was used as the test sample. The impedance ratio under different pH values ​​(6.0, 6.5, 7.0, 7.5, 8.0) and with +1.2V and -1.2V bias voltages relative to the unbiased condition was measured. The results are as follows: Figure 8 As shown, under different pH and bias conditions, the +1.2V bias impedance ratio of the blood from four animals showed a decreasing trend with increasing pH, while the -1.2V bias impedance ratio showed an increasing trend with increasing pH, and all converged around pH=7.0. However, under the same pH conditions, there were significant differences in the impedance ratios of the blood from different animals. This difference can be used to effectively distinguish the blood from different species, indicating that the sensor provided by this invention has universality and can effectively distinguish the blood from different species.

[0051] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A microfluidic protein sensor based on bias-controlled electrophoretic migration and electrical parameter detection, characterized in that, include: It consists of a bias modulation module and an electrical parameter detection module; The bias modulation module includes a first substrate and a second top plate disposed opposite to each other. The first top plate is provided with a first test solution inlet, a first test solution outlet, a buffer solution inlet, and a buffer solution outlet. A first microchannel is provided between the first substrate and the first top plate. The first test solution inlet and the first test solution outlet, and the buffer solution inlet and the buffer solution outlet are respectively connected through the first microchannel. A parallel microchannel is provided between the two first microchannels. The first substrate is provided with a bias control electrode, which is located in the parallel microchannel. The electrical parameter detection module includes a second substrate and a second top plate arranged opposite to each other. The second top plate is provided with a second inlet and a second outlet for the solution to be tested. A second microchannel is provided between the second substrate and the second top plate. The second substrate is provided with a parallel electrode, which is located in the second microchannel and is electrically connected to an external sensing circuit. The outlet of the first test solution and the inlet of the second test solution are connected in a sealed manner through a pipeline.

2. The microfluidic protein sensor as described in claim 1, characterized in that, The first substrate includes a rigid carrier or a flexible film. The rigid carrier includes glass or silicon wafer, and the flexible film includes a polymer film made of polyimide, polytetrafluoroethylene, or polyethylene terephthalate.

3. The microfluidic protein sensor as described in claim 1, characterized in that, The first top plate includes a rigid carrier sheet or a flexible film, wherein the rigid carrier sheet includes glass or quartz, and the flexible film includes a polymer film made of polydimethylsiloxane, polymethyl methacrylate, or a cyclic olefin copolymer.

4. The microfluidic protein sensor as described in claim 1, characterized in that, The bias control electrode is made of gold, platinum, or graphite.

5. The microfluidic protein sensor as described in claim 1, characterized in that, The second substrate includes a rigid carrier or a flexible film. The rigid carrier includes glass or silicon wafer, and the flexible film includes a polymer film made of polyimide, polytetrafluoroethylene, or polyethylene terephthalate.

6. The microfluidic protein sensor as described in claim 1, characterized in that, The second top plate includes a rigid carrier sheet or a flexible film, wherein the rigid carrier sheet includes glass or quartz, and the flexible film includes a polymer film made of polydimethylsiloxane, polymethyl methacrylate, or a cyclic olefin copolymer.

7. The microfluidic protein sensor as described in claim 1, characterized in that, The parallel electrodes include metal electrodes, metal oxide electrodes, silver chloride electrodes, or metal electrodes coated with protein aptamers.

8. The microfluidic protein sensor as described in claim 1, characterized in that, The materials used for the pipes include polytetrafluoroethylene, polymethyl methacrylate, polydimethylsiloxane, glass, quartz, or stainless steel.

9. The microfluidic protein sensor as described in claim 1, characterized in that, The bias control electrode is two electrodes symmetrically arranged on the first substrate, with a gap between the two bias control electrodes; the parallel electrode is two electrodes symmetrically arranged on the second substrate, with a gap between the two parallel electrodes.

10. A detection method of a microfluidic protein sensor as described in any one of claims 1 to 9, characterized in that, include: The buffer solution and the protein solution to be tested are respectively injected into the buffer solution inlet and the first protein solution to be tested inlet of the bias modulation module; The protein solution to be tested flows into the second solution inlet of the electrical parameter detection module through the first solution outlet and flows out through the second solution outlet, while the buffer solution flows out through the buffer solution outlet. A voltage is applied to the bias control electrode; the electrical parameters of the protein solution to be tested are measured through the parallel electrode.

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