Helical separation function integrated H-type electrolytic tank detection device and method

Abstract

The invention discloses a spiral separation function integrated H-type electrolytic tank detection device and a spiral separation function integrated H-type electrolytic tank detection method, which are used for rapid separation and detection of whole blood samples. According to the core design, a spiral microfluidic separation module is in butt joint with a communicating opening in the top of a first cavity of an H-type electrolytic tank, whole blood is separated through a spiral channel, then plasma directly enters a detection area, blood cells are synchronously guided out, and the whole process integration of separation, incubation, cleaning and detection is achieved. The device comprises an H-type electrolytic tank main body (a porous membrane for fixing a biological probe and a first / second chamber separated by the porous membrane) and a spiral microfluidic separation module (comprising a spiral channel, a sample inlet, a to-be-detected object outlet and a waste liquid outlet). The invention aims to solve the problems of tedious pretreatment, long time consumption and the like of the blood sample, realizes the integration of separation and detection of the blood sample and combined detection with the porous membrane, has the advantages of simplicity and convenience in operation, high detection speed, high sensitivity, effective prevention of cross contamination and the like, and is suitable for instant detection and primary medical treatment.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics and electrochemical biological detection technology, and specifically discloses an H-type electrolytic cell detection device and detection method that integrates helical separation function. Background Technology

[0002] With scientific advancements and medical development, blood tests have become an indispensable part of modern medical diagnosis and a crucial means of disease diagnosis. Currently, for clinical laboratory tests, except for some items such as blood lead, glycated hemoglobin, cyclosporine, and tacrolimus that use whole blood samples, most clinical chemistry tests use serum or plasma. Traditional blood separation methods (such as centrifugation) are complex, time-consuming, and rely on specialized equipment, making it difficult to meet the needs of point-of-care testing (POCT) and primary healthcare.

[0003] Microfluidic chips, as a cutting-edge technology platform, have shown broad application prospects in disease diagnosis, drug screening, and environmental monitoring. Based on the principle of inertial microfluidics, the helical microfluidic separation chip, through optimized microchannel structure design, utilizes the differences in inertial lift force and Dean drag force experienced by blood cells and plasma components of different sizes in a helical flow field to achieve efficient, label-free, and continuous separation of whole blood. This provides technical support for early disease diagnosis.

[0004] Electrochemical biosensors possess rapid detection capabilities, converting biological signals into electrical signals. Porous membranes, with their regular micropores and tunable surfaces, provide more stable active sites and are often combined with H-type electrolytic cells for detection. However, in traditional detection processes, blood separation and electrochemical detection are separate operations, requiring manual transfer of pretreated plasma samples followed by incubation, washing, and liquid addition. This is not only cumbersome but also introduces the risk of contamination, reducing detection efficiency. Furthermore, direct injection of whole blood carries the risk of clogging the porous membrane due to larger diameter blood cells, affecting the continuity and accuracy of detection. Although microfluidic helical separation chips can achieve efficient whole blood separation, they have not yet been integrated with H-type electrolytic cell detection, resulting in a still separate "separation-detection" operation and an unsimplified process. This paper proposes integrating the helical microfluidic structure with the H-type electrolytic cell to achieve integrated separation and detection. This technology can reduce operational steps, shorten detection time, reduce the risk of contamination, improve detection efficiency, and, when combined with porous membrane detection, effectively enhance sensitivity. Summary of the Invention

[0005] The purpose of this invention is to provide an integrated H-type electrolytic cell detection device and method with integrated helical separation function, belonging to the field of electrochemical biological detection technology. It aims to solve the problems of cumbersome and time-consuming blood sample pretreatment, achieving integrated blood sample separation and detection, suitable for point-of-care testing and primary healthcare.

[0006] This invention provides an integrated H-type electrolytic cell detection device with a spiral separation function. The detection device includes: an H-type electrolytic cell body, inside which is a porous membrane dividing the H-type electrolytic cell body into a first chamber and a second chamber; a spiral microfluidic separation module connected to the top of the first chamber, which separates whole blood into blood cells and plasma through the synergistic effect of Dean's resistance and inertial lift within the spiral channel; the module has a sample inlet and a Y-shaped or tri-branched extended outlet fluidly connected to the sample inlet, including a analyte outlet and one or more waste liquid outlets; the analyte outlet is fluidly connected to the first chamber and is used to directly introduce the separated plasma into the H-type electrolytic cell; the waste liquid outlet is used to export the separated blood cells.

[0007] Furthermore, the bottom of the second chamber of the H-type electrolytic cell is provided with a liquid outlet.

[0008] Furthermore, the porous membrane is made of polyethylene terephthalate (PET) or polycarbonate (PC), and the pore size on the surface of the porous membrane is 0.01~10 μm, with a pore density of 1×10⁻⁶. 3 ~1×10 15 / cm 2 .

[0009] Furthermore, the porous membrane channels are modified with biological probes for capturing specific disease biomarkers, which may be antibodies or aptamers bound by covalent bonds.

[0010] Furthermore, the spiral microfluidic separation module includes at least one spiral structure or multiple parallel connected spiral structures. The sample inlets of the parallel channels are combined into a single main inlet. The analyte outlets are combined and connected to the first chamber. The waste liquid outlets are combined and discharged uniformly. The analyte outlet of the spiral microfluidic separation module is located on the outside of the spiral channel, and the waste liquid outlet is located on the inside of the spiral channel. The cross-sectional shape of the spiral channel is trapezoidal or rectangular. The width of the spiral channel is 100~1000 μm, the height is 20~400 μm, the number of spiral turns is 2~20, the radius of curvature of the center line of the first turn of the spiral channel is 1~10 mm, and the channel width ratio of the analyte outlet to the waste liquid outlet is 1:5~5:1.

[0011] This invention also provides an integrated H-type electrolytic cell detection method with integrated spiral separation function, comprising the following steps:

[0012] S1. Blood separation: Blood sample is injected into the sample inlet of the spiral microfluidic separation module through the device (a bioprobe modified on a porous membrane for capturing specific disease biomarkers). The sample is separated into plasma and blood cells through the spiral channel. Blood cells are introduced into the waste liquid outlet, and plasma is introduced into the electrolytic cell through the analyte outlet for incubation. After incubation, the liquid outlet at the bottom of the second chamber is opened to discharge the waste liquid.

[0013] S2. Cleaning: Close the waste liquid outlet, inject cleaning solution into the sample inlet of the spiral microfluidic separation module, and directly introduce the cleaning solution into the electrolytic cell, and then discharge it from the liquid outlet.

[0014] S3. Electrochemical detection: Close the liquid outlet and inject electrolyte into the sample inlet of the spiral microfluidic separation module. The electrolyte is directly introduced into the H-type electrolytic cell. The electrode contacts the electrolyte through the electrode hole. The electrochemical response signal of the porous membrane is collected using an electrochemical detection device.

[0015] Furthermore, the flow rate of the injected blood sample is 0.01 mL / min to 3.0 mL / min, the flow rate of the injected washing solution is 0.01 mL / min to 3.0 mL / min, and the flow rate of the injected electrolyte is 0.01 mL / min to 3.0 mL / min.

[0016] The technical principle of this invention is based on the synergistic effect of helical microfluidic separation, H-type electrolytic cell electrochemical detection, and porous membrane biomolecule-specific recognition. The core mechanism of helical microfluidic separation is that when fluid flows through the helical channel, secondary flow is generated due to the channel curvature, forming Dean's resistance (F). D At the same time, the inertia of the fluid generates net inertial lift (F). L The synergistic effect of the two forces exhibits a significant size dependence, with inertial lift being proportional to the fourth power of the particle diameter (F). L ∝a p 4 Dean's drag is proportional to the first power of the particle diameter (F). D ∝a pThis characteristic causes larger blood cells to migrate and aggregate towards the inner side of the channel under the influence of force balance, while smaller plasma components (containing target disease biomarkers) are distributed towards the outer side of the channel. Blood cells are directly discharged through the waste outlet, while plasma is introduced into the H-type electrolytic cell through the analyte outlet. The dynamic inflow of plasma reduces the loss of dead volume and shortens the incubation time of the bioprobe. The porous membrane in the H-type electrolytic cell has both regular micropores and a tunable surface, allowing small molecule analytes in the plasma to diffuse freely. Specific bioprobes (such as antibodies and aptamers) modified on the pore surface capture target disease biomarkers through molecular recognition, forming a stable "membrane-probe-biomarker" complex. This complex blocks the pores of the porous membrane, thereby changing the effective permeability of the membrane and the ion / electron transport capacity across the membrane. After washing, electrolyte is injected. Because the pores are blocked by the complex, the migration of ions or electrons in the membrane is hindered, leading to an increase in membrane resistance or impedance, a decrease in current response, and a change in electrical signal, thus enabling accurate and sensitive detection of the target analyte. Furthermore, the helical microfluidic module of this invention supports a multi-channel parallel design, which can increase the total processing throughput or further shorten the separation time by increasing the number of parallel helical channels. This technology eliminates the cumbersome steps of sample transfer and multiple processing in traditional testing, compressing the whole blood testing cycle to within 30 minutes, ultimately achieving a high degree of integration between blood sample separation and testing, meeting the needs of point-of-care testing and primary healthcare.

[0017] The beneficial effects of this invention are at least as follows:

[0018] The method provided by this invention integrates helical microfluidic separation technology with H-type electrolytic cell electrochemical detection technology into a single device. It achieves rapid separation of whole blood through Dean's resistance and inertial lift generated by the helical channel. The separated plasma is directly introduced into the H-type electrolytic cell. Combined with a dual-chamber drainage design, it realizes integrated operation from whole blood sample separation input to signal output, avoiding the cumbersome and time-consuming blood sample pretreatment problems of traditional methods, significantly simplifying the detection process. The separation process takes only a few minutes and requires no external centrifugation equipment, making it simple to operate and particularly suitable for point-of-care testing (POCT) scenarios.

[0019] The fluid focusing effect of the spiral channel provided by this invention increases the concentration of the target substance in the plasma (compared to the same volume of whole blood). The efficient removal of blood cells reduces non-specific adsorption and porous membrane contamination. At the same time, the dynamic flow of microfluidic samples accelerates the mass transfer process between the target substance and the porous membrane surface, which can reduce the loss of dead volume and shorten the incubation time of biological probes, thereby improving detection efficiency.

[0020] The present invention provides a porous membrane with a modified specific biological probe that can selectively capture target disease biomarkers. The complex formed by the target and the probe can block the porous membrane pores, altering the effective permeability and ion / electron transport capacity of the porous membrane, thus changing the electrochemical signal. No additional labeling step is required, simplifying the detection process. Helical microfluidic separation of blood cells reduces interference and improves capture efficiency. Combined with the specific recognition characteristics of the porous membrane, detection sensitivity can be effectively improved, thereby achieving highly sensitive and specific detection of the analyte. By changing the porous membrane-modified biological probe, it can be adapted to detect different disease biomarkers (such as proteins, nucleic acids, and small molecules), demonstrating strong scalability. Attached Figure Description

[0021] Figure 1 : This is a three-dimensional structural diagram of the detection device.

[0022] Figure 2 : This is a top view of the detection device.

[0023] Figure 3 : This is a detailed diagram of the spiral microfluidic separation module of the detection device.

[0024] Figure 4 Comparison of operation times for different separation methods.

[0025] Reference numerals: 1. H-type electrolytic cell body; 2. Liquid outlet; 3. Porous membrane; 4. Spiral microfluidic separation module; 5. Electrode orifice; 101. First chamber; 102. Second chamber; 401. Analyte outlet; 402. Waste liquid outlet; 403. Sample inlet; 404. Y-type extended bifurcation outlet; 405. Trapezoidal channel cross-section. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any changes made to the technical solutions of the present invention by those skilled in the art should fall within the scope of protection of the present invention. Example 1

[0027] This embodiment specifically provides an H-type electrolytic cell detection device integrating helical separation function for detecting troponin, as shown in the schematic diagram below. Figure 1 , Figure 2 As shown.

[0028] S1: The preparation methods of the H-type electrolytic cell body 1 and the porous membrane 3 in the detection device are as follows: The electrolytic cell is shaped like an "H" and is divided into a first chamber 101 and a second chamber 102 by a vertically placed porous membrane 3. The porous membrane 3 is located at the connection point between the two chambers, allowing ions / electrons to pass through while maintaining chamber separation. A liquid outlet 2 is located at the bottom of the second chamber 102 for discharging waste liquid or cleaning solution. An electrode hole 5 is located above both the first chamber 101 and the second chamber 102 for connecting the electrodes to the workstation.

[0029] The pore size is 5 μm and the pore density is 1×10⁻⁶. 7 / cm 2 PET porous membrane (pore size can be selected from any value in 0.01~10μm, pore density can be selected from 1×10). 3 ~1×10 15 / cm 2 The porous membrane (which can be made of either PET or PC) was placed in a solution of 15 mg / mL EDC and 4.0 mg / mL NHS for 60 min to activate it. Then, the membrane was placed in a PLL@VPAu solution for 60 min, where PLL@VPAu adsorbed electrostatically into the pores. After multiple rinses and drying, the membrane was incubated in a 15 μmol / L aptamer solution (the aptamer sequence is: 5'-SH-C6-CGTGCAGTACGCCAACCTTTCTCATGCGCTGCCCCTCTTA-3') for 60 min. The aptamer covalently bound to the pores via Au-S bonds. Site blocking with 1% bovine serum albumin (BSA) was used to prevent nonspecific adsorption. After rinsing three times with PBS and vacuum drying at 45°C, the membrane was assembled with an H-type electrolytic cell to prepare an electrochemical biosensor based on a PLL@VPAu-modified porous membrane.

[0030] S2: Fabrication of the spiral microfluidic separation module 4: The spiral microfluidic separation module integrated on one side of the H-type electrolytic cell body 1 is made of polydimethylsiloxane (PDMS), and its specific structure is as follows: Figure 3As shown (this spiral structure can be used in parallel with one or more), it includes a four-turn spiral main channel (the number of turns of the spiral channel can be selected from 2 to 20 turns), the radius of curvature of the center line of the first turn is 6.2 mm (this radius can be any value from 1 to 10 mm), the sample inlet 403 is connected to the sample injection device through a medical-grade silicone tube, and the injection method includes, but is not limited to, manual injection and injection pump injection, the channel width ratio of the analyte outlet 401 and the waste liquid outlet 402 of the Y-shaped extended bifurcation outlet 404 is 2:1 (the channel width ratio can also be selected from any ratio from 1:5 to 5:1), the analyte outlet 401 is connected to the first chamber 101, and the waste liquid outlet 402 is connected to an external waste liquid collection system through a silicone tube. The cross-sectional shape of the spiral channel is trapezoidal (the cross-sectional shape can be either trapezoidal or rectangular). The trapezoidal channel section 405 has a horizontal width of 400 μm (any value in the range of 100~1000 μm), an inner vertical height of 40 μm, and an outer vertical height of 70 μm (any value in the range of 20~400 μm). Example 2

[0031] This embodiment specifically provides an H-type electrolytic cell detection method integrating helical separation function for detecting troponin. The specific operation steps are as follows:

[0032] S1: Preparation of whole blood simulant sample: Take 30 mL of phosphate buffer solution (PBS, pH 7.4), add 1.5 g bovine serum albumin (BSA) and 7.5 μL Tween 20, and sonicate until homogeneous to obtain plasma simulant; then add 8 μm polystyrene microspheres (simulating blood cells) and sonicate until uniformly dispersed to finally obtain whole blood simulant for detection;

[0033] S2: Sample detection: The operator manually injects a whole blood simulant sample containing a specific concentration of troponin (e.g., concentrations of 0, 0.01, 0.1, 1, 100 ng / mL, etc.) directly into the sample inlet 403 of the spiral microfluidic separation module 4 at a flow rate of approximately 1.5 mL / min (the flow rate can be any value from 0.01 mL / min to 3.0 mL / min). The sample flows through the spiral channel and is separated into plasma simulant and simulated blood cells by the influence of Dean resistance and inertial lift. The simulated blood cells are introduced into the waste liquid outlet 402 and flow into the waste liquid bottle through the silicone tube. The plasma simulant is introduced into the H-type electrolytic cell through the analyte outlet 401 and incubated at room temperature for 10 min. After incubation, the liquid outlet 2 at the bottom of the second chamber 102 is opened to discharge the waste liquid.

[0034] S3: Close the waste liquid outlet 402, inject PBS (pH 7.4) cleaning solution into the sample inlet 403 of the spiral microfluidic separation module 4, and the cleaning solution directly enters the first chamber 101 to rinse unbound non-specific adsorbents. Continue cleaning for 3 minutes to ensure that the cleaning solution is completely discharged from the liquid outlet 2 of the second chamber 102.

[0035] S4: Close liquid outlet 2, and inject 0.1 mol / L PBS electrolyte into the sample inlet 403 of the spiral microfluidic separation module 4 at a flow rate of 1.0 mL / min, directly introducing it into the electrolytic cell. Insert the two Ag / AgCl electrodes into the electrode holes 5 respectively, and connect them to the electrochemical workstation through wires to form a closed loop, thereby constructing a complete electrochemical detection system. Cyclic voltammetry (CV) is used to detect the system current. The workstation parameters are set to a scan rate of 100 mV / s and a voltage window of -1.0 V to 1.0 V to complete the acquisition of electrochemical signals. Example 3

[0036] To objectively verify the significant advantages of the integrated helical separation function H-type electrolytic cell detection device of the present invention in terms of detection efficiency and ease of operation, this embodiment compares it with conventional blood detection methods (centrifugation method) in an experiment (e.g.) Figure 4 (As shown). The control group used the traditional method, centrifuging 2 mL of whole blood simulant sample at 3000 rpm for 10 minutes using a benchtop centrifuge. The supernatant plasma was then carefully aspirated using a pipette and transferred to a new container. This pretreatment process took approximately 14 minutes. Subsequent steps included 20 minutes of incubation, 6 minutes of manual cleaning of the electrolytic cell, and electrolyte injection, for a total operation time of approximately 40 minutes. The experimental group strictly followed the method of this invention, directly injecting an equal volume of whole blood simulant sample into the spiral microfluidic separation module. The Dean's resistance and inertial lift generated by the spiral channel were used to separate plasma from blood cells. The separated plasma was directly introduced into the H-type electrolytic cell. Separation and incubation took approximately 10 minutes, followed by 5 minutes of automatic cleaning and electrolyte injection, for a total operation time of approximately 15 minutes. Compared with the traditional method, this invention reduces the time by more than 50%, significantly improving detection efficiency.

[0037] The above description is only a preferred embodiment of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A detection device for an H-type electrolytic cell integrating spiral separation function, characterized in that, include: An H-shaped electrolytic cell body is provided, with a porous membrane inside dividing the H-shaped electrolytic cell body into a first chamber and a second chamber; a spiral microfluidic separation module is connected to the top of the first chamber, which separates whole blood into blood cells and plasma through the synergistic effect of Dean's resistance and inertial lift in the spiral channel; the module is provided with a sample inlet and a Y-shaped or tri-branched extended outlet fluidly connected to the sample inlet, including a analyte outlet and one or more waste liquid outlets; the analyte outlet is fluidly connected to the first chamber and is used to directly introduce the separated plasma into the H-shaped electrolytic cell; the waste liquid outlet is used to export the separated blood cells.

2. The H-type electrolytic cell detection device integrating spiral separation function according to claim 1, characterized in that, The bottom of the second chamber of the H-type electrolytic cell is provided with a liquid outlet.

3. The H-type electrolytic cell detection device integrating spiral separation function according to claim 1, characterized in that, The porous membrane is made of polyethylene terephthalate (PET) or polycarbonate (PC), and the pore size on the surface of the porous membrane is 0.01~10 μm, with a pore density of 1×10⁻⁶. 3 ~1×10 15 / cm 2 .

4. The H-type electrolytic cell detection device integrating spiral separation function according to claim 1, characterized in that, The porous membrane channels are modified with biological probes for capturing specific disease biomarkers. These biological probes can be antibodies or aptamers, which are bound together by covalent bonds.

5. The H-type electrolytic cell detection device integrating spiral separation function according to claim 1, characterized in that, The spiral microfluidic separation module includes at least one spiral structure or multiple parallel spiral structures. The analyte outlet of the spiral microfluidic separation module is located on the outside of the spiral channel, and each waste liquid outlet is located on the inside of the spiral channel. The cross-sectional shape of the spiral channel is trapezoidal or rectangular. The width of the spiral channel is 100~1000 μm, the height is 20~400 μm, the number of spiral turns is 2~20, the radius of curvature of the center line of the first turn of the spiral channel is 1~10 mm, and the channel width ratio of the analyte outlet to the waste liquid outlet is 1:5~5:

1.

6. A detection method for an H-type electrolytic cell integrating spiral separation function according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Blood separation: Using the integrated H-type electrolytic cell detection device with integrated spiral separation function (a bioprobe modified on a porous membrane for capturing specific disease biomarkers), a blood sample is injected into the sample inlet of the spiral microfluidic separation module. The sample is separated into plasma and blood cells through the spiral channel. Blood cells are introduced into the waste liquid outlet, and plasma is introduced into the electrolytic cell through the analyte outlet for incubation. After incubation, the liquid outlet at the bottom of the second chamber is opened to discharge the waste liquid. S2. Cleaning: Close the waste liquid outlet, inject cleaning solution into the sample inlet of the spiral microfluidic separation module, and directly introduce the cleaning solution into the electrolytic cell, and then discharge it from the liquid outlet. S3. Electrochemical detection: Close the liquid outlet and inject electrolyte into the sample inlet of the spiral microfluidic separation module. The electrolyte is directly introduced into the H-type electrolytic cell. The electrode contacts the electrolyte through the electrode hole. The electrochemical response signal of the porous membrane is collected using an electrochemical detection device.

7. The integrated H-type electrolytic cell detection method with integrated spiral separation function according to claim 6, characterized in that, The flow rate of the injected blood sample is 0.01 mL / min to 3.0 mL / min, the flow rate of the injected washing solution is 0.01 mL / min to 3.0 mL / min, and the flow rate of the injected electrolyte is 0.01 mL / min to 3.0 mL / min.

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