A microfluidic chip for detecting blood biochemical indexes on demand and a preparation method thereof

Abstract

The application provides a microfluidic chip for detecting blood biochemical indexes on demand and a preparation method thereof. The microfluidic chip comprises an interface layer, a separation layer, a transducer layer, a detection layer and a substrate layer. The interface layer comprises an interface layer body, a blood cell sample outlet, a blood sample inlet and a serum sample outlet. The separation layer comprises a separation layer body, a separation flow channel, a sample outlet channel, a transducer pin interface, a plasma channel and a serum channel. The transducer layer comprises a transducer layer body, a sample outlet channel, an interdigital transducer and a serum sample inlet channel. The detection layer comprises a detection layer body, a detection cavity, a sample outlet channel, an electrochemical sensor reference electrode interface, an electrochemical sensor working electrode interface and a sample inlet channel. The substrate layer comprises a substrate layer body and a sensor electrode. The microfluidic chip is used for separating plasma and serum and can be used for rapid, accurate and wide-range detection of whole blood biochemical indexes.

Description

Technical Field

[0001] This invention relates to the field of blood biochemical index detection equipment technology, specifically to a microfluidic blood biochemical index detection chip and its preparation method. Background Technology

[0002] In clinical practice, patients with acute, critical, or severe conditions require multiple blood samples to be collected for blood biochemical tests in order to provide clinicians with a basis for timely and accurate judgment of their condition. Therefore, monitoring and testing of patients' blood biochemical indicators is of great significance for the control of patients' physical condition and diagnosis and treatment.

[0003] Traditionally, hospitals primarily use distal sampling and venous blood testing to detect blood biochemical indicators. However, both methods have limitations in practice. Distal sampling results are affected by peripheral circulation, potentially leading to inaccuracies in patients with shock or hypothermia. Repeated blood draws can also increase patient discomfort and workload for healthcare workers. Venous blood testing allows for direct laboratory testing of various biochemical indicators using analyzers, but it still requires multiple blood draws, is more complex, and produces slower results. There is a time lag between routine blood tests and biomarker detection, limiting its flexibility and on-demand measurement capabilities. Furthermore, the presence of blood cells, especially white blood cells, can significantly affect the results when testing certain biomarkers.

[0004] Intravenous catheterization is an arterial puncture technique that has been applied clinically in recent years. It involves collecting venous blood samples using an indwelling venous needle. This method allows for rapid sample acquisition, saving time in emergency situations, and avoids the local damage and complications caused by repeated punctures, thus preventing disruptions to diagnosis and treatment. Given the need for blood sample collection via indwelling needles, a microfluidic chip for on-demand blood biochemical indicator detection is required. This chip should be able to separate serum and plasma for separate testing according to the actual needs of the physician during treatment, removing interference from blood cells and reducing the adhesion of viscous blood cells to the sensor, layer, and detection chamber, thereby extending the sensor's lifespan. Accurate indicators can be detected using only a small amount of blood, achieving more precise detection with shorter testing time, simple operation, and low cost. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic chip for on-demand detection of blood biochemical indicators, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A microfluidic chip for on-demand blood biochemical index detection includes an interface layer, a separation layer, a transducer layer, a detection layer, and a base layer. The interface layer includes an interface layer body 1, a blood cell sampling port 2, a blood sampling port 3, and a serum sampling port 4. The blood sampling port 3 is connected to a threaded catheter connector and penetrates into the interface layer body 1. The blood cell sampling port 2 penetrates into the interface layer body 1 and is connected to an external receiving manifold via a flexible tube. The serum sampling port 4 penetrates into the interface layer body 1 and is sequentially connected to a first sampling channel 7, a second sampling channel 10, and a third sampling channel 13, and is then connected to a waste liquid cylinder via a flexible tube.

[0008] The separation layer includes a separation layer body 5, a separation channel 6, a first sample outlet channel 7, a transducer pin interface 8, a plasma channel 21, and a serum channel 22. The blood inlet 3 is connected to the inlet of the separation channel 6 through a conduit. The end of the separation channel 6 branches into the plasma channel 21 and the serum channel 22. The separation channel 6, the plasma channel 21, and the serum channel 22 penetrate the separation layer body 5. The end of the plasma channel 21 is connected to the blood cell outlet 2. The first sample outlet channel 7 penetrates the separation layer body 5. The transducer pin interface 8 is located on the side of the separation layer body 5.

[0009] The transducer layer includes a transducer layer body 9, a second sample outlet channel 10, an interdigital transducer 19, and a serum sample inlet channel 20. The serum sample inlet channel 20 penetrates the transducer layer 9, and the second sample outlet channel 10 penetrates the transducer layer 9. The interdigital transducer 19 is externally connected to a power amplifier and a signal generator through a transducer pin interface 8. The interdigital transducer 19 is attached to the surface of the transducer layer body 9 by screen printing. From top to bottom, the layers are a reference electrode layer 11', a double conductive layer 4', and a substrate layer 3'.

[0010] The detection layer includes a detection layer body 11, a detection cavity 12, a third sample outlet channel 13, an electrochemical sensor reference electrode interface 16, an electrochemical sensor working electrode interface 17, and a sample inlet channel 18. The sample inlet channel 18 penetrates the detection layer body 11. The outlet of the serum channel 22, the serum sample inlet channel 20, and the sample inlet channel 18 are sequentially connected and injected into the detection cavity 12. The detection cavity 12 is composed of a rugby ball-shaped main cavity 1' and a rectangular flow channel 2'. The rectangular flow channel 2' is connected to the main cavity 1'. The detection cavity 12 is located at the bottom of the detection layer body 11. The third sample outlet channel 13 is located at the end of the detection cavity 12 and penetrates the detection layer body 11.

[0011] The substrate layer includes a substrate body 14 and a sensor electrode 15. The sensor electrode 15 is attached to the surface of the substrate layer 14 by screen printing. The sensor electrode 15 has a dual-electrode structure or a triple-electrode structure. The electrochemical sensor reference electrode interface 16 is externally connected to the reference electrode of the detector and internally connected to the sensor electrode 15. The electrochemical sensor working electrode interface 17 is externally connected to the working electrode of the matching detector and internally connected to the sensor electrode 15.

[0012] The shape and size of the interface layer, separation layer, transducer layer, detection layer, and substrate layer can be arbitrarily changed to meet the requirements of different application environments. Similarly, the shape and thickness of the sensor electrode 15 can also be changed to match the size of the interface layer, detection layer, and sensor layer. The shape, size, and thickness of the detection cavity 12 can also be changed according to actual usage requirements to better cope with different detection environments. The number and position of the detection cavity 12 and sensor electrode 15, as well as the type of enzyme-chitosan layer 9', can be changed according to different detection needs.

[0013] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the dual-electrode structure includes a working electrode strip wire 5', a working electrode 6', a reference electrode 7', and a reference electrode strip wire 8'. The reference electrode 7' is arranged around the working electrode 6', and the shape of the reference electrode 7' is a ring with a central angle exceeding 120°.

[0014] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the three-electrode structure includes a working electrode wire 5', a working electrode 6', a reference electrode strip wire 8', a reference electrode 7', a counter electrode strip wire 14', and a counter electrode 13'. The working electrode is connected to a working electrode wire, the reference electrode is connected to a reference electrode strip wire, and the counter electrode is connected to a counter electrode strip wire. The reference electrode and the counter electrode are arranged around the working electrode. The reference electrode is a ring with a central angle exceeding 30°, and the counter electrode is a ring with a central angle exceeding 90°.

[0015] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the working electrode 6' is layered and printed with an enzyme-chitosan layer 9', a working electrode layer 10', and a conductive layer 12' from top to bottom; the reference electrode 7' is layered and printed with a reference electrode layer 11' and a conductive layer 12' from top to bottom.

[0016] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the interface layer 1, transducer layer 9 and base layer 14 are made of thin and stable materials such as acrylic or glass, which are low in cost and easy to obtain.

[0017] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the materials of the separation layer 5 and the detection layer 11 are glass, acrylic, polymethyl methacrylate, or a mixture of polydimethylsiloxane and polydimethylsiloxane crosslinking agent that is cured by heating. The amount of materials required for each preparation is small, and the cost is easy to control.

[0018] Preferably, in the above-mentioned microfluidic chip for on-demand blood biochemical index detection, the mass ratio of polydimethylsiloxane to polydimethylsiloxane crosslinking agent is 15-5:1.

[0019] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the reference electrode layer 11' of the interdigital transducer 19 is made of Ag / AgCl; the double conductive layer 4' is made of Cr and Au, wherein the Cr layer is in close contact with the substrate layer 3' and the Au layer is in close contact with the reference electrode layer 11'. The substrate layer 3' is made of lithium niobate and is used to generate surface acoustic waves, providing basic support and piezoelectric properties for the interdigital transducer 19.

[0020] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the working electrode 6' is connected to the external detection device by being inserted into the working electrode interface 17 of the electrochemical sensor through the working electrode strip wire 5', and the reference electrode 7' is connected to the external detection device by being inserted into the working electrode hole 16 of the electrochemical sensor through the reference electrode strip wire 8'.

[0021] Preferably, in the above-mentioned microfluidic chip for on-demand blood biochemical index detection, the working electrode strip wire 5', the reference electrode strip wire 8', and the conductive layer 12' are made of Au, Ag, Cu, or Pt.

[0022] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the working electrode layer 10' is made of carbon and the reference electrode layer 11' is made of Ag / AgCl. This material configuration enables the electrodes to have good conductivity while not reacting with ions in the solution, resulting in good consistency of detection results.

[0023] Preferably, in the aforementioned on-demand blood biochemical index detection microfluidic chip, the enzyme-chitosan layer 9' material is obtained by mixing chitosan, glacial acetic acid, and glycerol, followed by the addition of glucose oxidase, lactate oxidase, or HMG coenzyme A synthase. This material configuration allows the enzyme to have a higher catalytic rate, and the cross-linked network formed between the enzyme and chitosan protects the enzyme from being washed away by liquid. The type and quantity of enzymes can be selected according to actual application requirements.

[0024] Preferably, in the above-mentioned on-demand blood biochemical index detection microfluidic chip, the enzyme-chitosan layer 9' material enzyme-chitosan solution is prepared by the following method: chitosan and deionized water are mixed at a mass ratio of 1:50-200, and then glacial acetic acid and glycerol solution are added at a volume ratio of 1-10:100 after stirring. The volume ratio of the two mixed solutions is 15-5:1. After stirring at 60℃-90℃ for 1h-4h, glucose oxidase solution, lactate oxidase solution or HMG coenzyme A synthase solution with a concentration of 5mg / ml-20mg / ml is added.

[0025] Beneficial effects

[0026] The aforementioned on-demand blood biochemical index detection microfluidic chip allows users to connect the indwelling needle interface to the sample inlet, introducing blood through the inlet. After automatically filling the entire separation chamber, a high-frequency surface acoustic wave generated by an interdigital transducer separates plasma and serum. The plasma is collected by an external receiving tube through the blood cell outlet and can be used for further routine blood tests or discarded as needed. The serum fills the rugby ball-shaped detection chamber, using a small amount of blood to detect the desired blood biochemical indexes, and the separated plasma is used for further blood component analysis. Blood biochemical index detection can be achieved by reading the current data through an external electrochemical detection device. The operation is simple and convenient, and the manufacturing cost of the device is very low. Chitosan, as a natural polysaccharide, is widely used in biosensors due to its good biocompatibility, biodegradability, and abundant amino functional groups. The enzyme-chitosan layer in the electrochemical sensor, which is a mixture of chitosan, glacial acetic acid, and glycerol, exhibits excellent catalytic performance. The cross-linking structure of chitosan helps enhance the mechanical strength and stability of the mixed solution, while glycerol helps enhance the flexibility and mechanical strength of the mixed solution. Glacial acetic acid adjusts the pH and promotes the cross-linking reaction of the membrane, providing a stable catalytic environment for the reaction. This greatly improves the detection sensitivity and accuracy and solves the problem of enzyme shedding in each detection. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the microfluidic chip for on-demand blood biochemical index detection described in this invention;

[0028] Figure 2 This is a schematic diagram of the detection cavity described in this invention;

[0029] Figure 3 This is a schematic diagram of the structure of each layer of the interdigital transducer described in this invention;

[0030] Figure 4 This is a schematic diagram of the structure of the dual-electrode sensor electrode described in this invention;

[0031] Figure 5 This is a schematic diagram of the layer structure of the dual-electrode sensor electrode described in this invention;

[0032] Figure 6 This is a schematic diagram of the structure of the three-electrode sensor electrode described in this invention;

[0033] Figure 7 This is a schematic diagram of the layer structure of the three-electrode sensor electrode described in this invention.

[0034] In the diagram: 1-Interface layer, 2-Blood cell outlet, 3-Blood inlet, 4-Serve outlet, 5-Separation layer, 6-Separation channel, 7-First outlet channel, 8-Transducer pin interface, 9-Transducer layer, 10-Second outlet channel, 11-Detection layer, 12-Detection chamber, 13-Third outlet channel, 14-Base layer, 15-Sensor electrode, 16-Electrochemical sensor reference electrode interface, 17-Electrochemical sensor working electrode interface, 18-Inlet Sample channel, 19-interdigital transducer, 20-serum sample inlet channel, 21-plasma channel, 22-serum channel, 1'-main cavity, 2'-flow channel, 3'-substrate layer, 4'-double conductive layer, 5'-working electrode strip wire, 6'-working electrode, 7'-reference electrode, 8'-reference electrode strip wire, 9'-enzyme-chitosan layer, 10'-working electrode layer, 11'-reference electrode layer, 12'-conductive layer, 13'-counter electrode, 14'-counter electrode strip wire. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0036] Example 1

[0037] like Figure 1-6 As shown, the on-demand blood biochemical index detection microfluidic chip includes an interface layer, a separation layer, a transducer layer, a detection layer, and a base layer. The interface layer includes an interface layer body 1, a blood cell sampling port 2, a blood sampling port 3, and a serum sampling port 4. The blood sampling port 3 is connected to a conduit connector with external threads and penetrates into the interface layer body 1. The blood cell sampling port 2 penetrates into the interface layer body 1 and is connected to an external receiving manifold through a flexible tube. The serum sampling port 4 penetrates into the interface layer body 1 and is sequentially connected to a first sampling channel 7, a second sampling channel 10, and a third sampling channel 13, and is then connected to a waste liquid cylinder through a flexible tube.

[0038] The separation layer includes a separation layer body 5, a separation channel 6, a first sample outlet channel 7, a transducer pin interface 8, a plasma channel 21, and a serum channel 22. The blood inlet 3 is connected to the inlet of the separation channel 6 through a conduit. The end of the separation channel 6 branches into the plasma channel 21 and the serum channel 22. The separation channel 6, the plasma channel 21, and the serum channel 22 penetrate the separation layer body 5. The end of the plasma channel 21 is connected to the blood cell outlet 2. The first sample outlet channel 7 penetrates the separation layer body 5. The transducer pin interface 8 is located on the side of the separation layer body 5.

[0039] The transducer layer includes a transducer layer body 9, a second sample outlet channel 10, an interdigital transducer 19, and a serum sample inlet channel 20. The serum sample inlet channel 20 penetrates the transducer layer 9, and the second sample outlet channel 10 penetrates the transducer layer 9. The interdigital transducer 19 is externally connected to a power amplifier and a signal generator through a transducer pin interface 8. The interdigital transducer 19 is attached to the surface of the transducer layer body 9 by screen printing. From top to bottom, the layers are a reference electrode layer 11', a double conductive layer 4', and a substrate layer 3'.

[0040] The detection layer includes a detection layer body 11, a detection cavity 12, a third sample outlet channel 13, an electrochemical sensor reference electrode interface 16, an electrochemical sensor working electrode interface 17, and a sample inlet channel 18. The sample inlet channel 18 penetrates the detection layer body 11. The outlet of the serum channel 22, the serum sample inlet channel 20, and the sample inlet channel 18 are sequentially connected and injected into the detection cavity 12. The detection cavity 12 is composed of a rugby ball-shaped main cavity 1' and a rectangular flow channel 2'. The rectangular flow channel 2' is connected to the main cavity 1'. The detection cavity 12 is located at the bottom of the detection layer body 11. The third sample outlet channel 13 is located at the end of the detection cavity 12 and penetrates the detection layer body 11.

[0041] The substrate layer includes a substrate body 14 and a sensor electrode 15. The sensor electrode 15 is attached to the surface of the substrate layer 14 by screen printing. The sensor electrode 15 has a dual-electrode structure or a triple-electrode structure. The electrochemical sensor reference electrode interface 16 is externally connected to the reference electrode of the detector and internally connected to the sensor electrode 15. The electrochemical sensor working electrode interface 17 is externally connected to the working electrode of the matching detector and internally connected to the sensor electrode 15. Wherein, as... Figure 4 As shown, the dual-electrode structure includes a working electrode strip wire 5', a working electrode 6', a reference electrode 7', and a reference electrode strip wire 8'. The working electrode 6' is circular in shape, and the reference electrode 7' is arranged around the working electrode 6'. The reference electrode 7' is an annular shape with a central angle exceeding 120°. This shape design occupies little space and is suitable for most detection environments. Figure 6As shown, the three-electrode structure includes a working electrode wire 5', a working electrode 6', a reference electrode strip wire 8', a reference electrode 7', a counter electrode strip wire 14', and a counter electrode 13'. The working electrode is connected to a working electrode wire, the reference electrode is connected to a reference electrode strip wire, and the counter electrode is connected to a counter electrode strip wire. The reference electrode and counter electrode are arranged around the working electrode. The reference electrode is a ring with a central angle exceeding 30°, and the counter electrode is a ring with a central angle exceeding 90°. Figure 5 and Figure 7 As shown, the working electrode 6' is layered and printed with an enzyme-chitosan layer 9', a working electrode layer 10', and a conductive layer 12' from top to bottom; the reference electrode 7' is layered and printed with a reference electrode layer 11' and a conductive layer 12' from top to bottom; the counter electrode 13' is layered and printed with a counter electrode layer 15' and a conductive layer 12' from top to bottom. The material of the counter electrode layer 15' is carbon, and the material of the conductive layer 12' is Au (or Ag, Cu, or Pt). The working electrode 6' is connected to an external detection device via a working electrode strip wire 5' inserted into the working electrode interface 17 of the electrochemical sensor, and the reference electrode 7' is connected to an external detection device via a reference electrode strip wire 8' inserted into the working electrode hole 16 of the electrochemical sensor. The working electrode strip wire 5', the reference electrode strip wire 8', and the conductive layer 12' are made of Au (or Ag, Cu, or Pt), the working electrode layer 10' is made of carbon, and the reference electrode layer 11' is made of Ag / AgCl. This material configuration allows the electrode to have good conductivity while not reacting with ions in the solution, resulting in good consistency of the detection results. The enzyme-chitosan layer 9' material is an enzyme-chitosan solution, prepared by the following method: chitosan and deionized water are mixed at a mass ratio of 1:100 (or any ratio within the range of 1:50-200), and then glacial acetic acid and glycerol solution are added at a volume ratio of 1:1000 (or any ratio within the range of 1:500-1500) after stirring. The volume ratio of the glycerol solution to the aforementioned mixed solution is 1:10 (or any ratio within the range of 1:5-15). After stirring at about 80°C (60-90°C) for 2 hours (or any ratio within the range of 1-4 hours), a glucose oxidase solution (or lactate oxidase solution or HMG coenzyme A synthase solution) with a concentration of 10 mg / ml (or any ratio within the range of 5 mg / ml-20 mg / ml) is added.

[0042] The aforementioned microfluidic chip for on-demand blood biochemical index detection uses acrylic (or other thin and stable materials such as glass) as the material for the interface layer 1, transducer layer 9, and base layer 14. The separation layer 5 and detection layer 11 are made of a mixture of polydimethylsiloxane and a polydimethylsiloxane crosslinking agent, cured by heating. This mixture is poured into a laser-engraved acrylic mold for casting and then cured by heating, thus obtaining the inlet / outlet holes, flow channels, and detection chamber of the detection layer. The polydimethylsiloxane... The mass ratio of oxane and polydimethylsiloxane crosslinking agent is 10:1 (or can be selected in the range of 15-5:1); the material of the reference electrode layer 11' in the interdigital transducer 19 is AgCl (or Ag); the material of the double conductive layer 4' is Cr and Au, wherein the Cr layer is in close contact with the substrate layer 3', the Au layer is in close contact with the reference electrode layer 11', and the material of the substrate layer 3' is lithium niobate, which is used to generate surface acoustic waves and provide basic support and piezoelectric properties for the interdigital transducer 19;

[0043] The manufacturing method of the aforementioned microfluidic chip for on-demand blood biochemical index detection comprises the following steps:

[0044] (1-1) Interface layer fabrication

[0045] The interface layer can be made of materials such as acrylic or glass, and is rectangular in shape with a length of 90mm (selectable from 50mm-100mm) and a width of 60mm (selectable from 30mm-60mm), and a thickness of 1mm (selectable from 1mm-5mm). First, a hole of suitable position and size is laser-cut. Then, a tap is used to tap threads matching the screw into the drilled hole, ensuring a secure connection between the inlet hole and the threaded conduit connector, preventing loosening and leakage.

[0046] (2-1) Preparation of the separation layer

[0047] The separation layer 5 can be obtained by mixing 30g (optional, 10g-50g) of polydimethylsiloxane and a polydimethylsiloxane crosslinking agent, with a mass ratio of 10:1 (optional, 5-15:1). The mixture is poured into an acrylic mold obtained by laser engraving, and after vacuum treatment to remove air bubbles, it is placed in an oven for heating and curing. The thickness of the mold is 3mm (optional, 3mm-6mm). The height of the central vortex-shaped part of the mold must exceed that of the separation chamber 6. The sample outlet channel 7 of the mold must be coaxial with the serum outlet 4 on the interface layer 1. The transducer pin interface 8 of the mold must be aligned with the pins of the interdigital transducer 19. The oven heating temperature is around 70℃ (optional, 60℃-80℃), and the heating time is 2 hours (optional, 1 hour-3 hours). After curing, it is cut to a size that matches the interface layer, detection layer, and substrate layer.

[0048] (2-2) Fabrication of interdigital transducers

[0049] like Figure 3 As shown, the double conductive layer 4', substrate layer 3', and reference electrode layer 11' of the interdigital transducer 19 are screen-printed. Before printing, corresponding screens are prepared according to the structure of each layer shown in the figure, and LiNbO3, AgCl (or Ag) paste, Au paste, and Cr paste are prepared. First, the substrate layer 3' is printed. After applying LiNbO3 paste to the corresponding substrate layer 3' structure screen, the screen is placed down and pressed tightly against the transducer layer. The squeegee is moved from top to bottom to evenly print the paste onto the glass substrate through the pattern. After confirming that the electrode pattern is completely printed, the substrate is removed and placed in a baking pan or oven and heated at 120°C for 10 minutes for sintering. The double conductive layer 4' is made of Au paste and Cr paste, and the reference electrode layer 11' is made of AgCl (or Ag) paste. The printing steps are the same as described above.

[0050] (3-1) Fabrication of interdigitated transducer layer

[0051] The interdigital transducer layer 9 can be made of acrylic material (or glass, etc.), and is rectangular in shape with a length of 90mm (selectable from 50mm-100mm), a width of 60mm (selectable from 30mm-60mm), and a thickness of 1mm (selectable from 1mm-5mm). After completing the electrode preparation process, wipe the surface of the interdigital transducer layer 9 with an alcohol swab without damaging the electrodes, ensuring no watermarks, dust, or fingerprints are left. Place the cut separation layer 5 into an ultrasonic cleaner and clean it at a frequency of 60kHz (selectable from 20kHz-90kHz) for 10 minutes (selectable from 5 minutes to 20 minutes). The cleaning medium can be anhydrous ethanol or deionized water. To bond the interdigital transducer layer 9 to the separation layer 5 using a plasma bonding machine, the electrode parts are first covered with tin foil (or black cardstock, etc.) to prevent oxidation during plasma activation. During bonding, the pressure inside the bonding chamber must reach 1 Pa (selectable from 0.1 Pa to 1 Pa). Oxygen is introduced as the reactive gas, and the plasma is excited using an RF or microwave power supply. The entire process typically lasts 60 seconds (selectable from 30 seconds to 180 seconds). Over-activation or repeated activation may damage the surface. After bonding, a hydrophilic bonding layer will form on the plasma-activated surface. The interdigital transducer layer 9 and the separation layer 5 are aligned and gently pressed together at room temperature, achieving initial bonding through van der Waals forces or hydrogen bonds. The mixture is then allowed to stand for 60 minutes (selectable from 30 minutes to 120 minutes).

[0052] (4-1) Preparation of the detection layer

[0053] The detection layer 6 is obtained by mixing 30g (optional, 10g-60g) of polydimethylsiloxane and polydimethylsiloxane crosslinking agent. The mass ratio of polydimethylsiloxane to polydimethylsiloxane crosslinking agent is 10:1 (optional, 5-15:1). The mixed material is poured into an acrylic mold obtained by laser engraving. After removing air bubbles through vacuum treatment, it is placed in an oven for heating and curing. The thickness of the mold is 1mm (optional, 0.5mm-3mm). The mold is required to mold an oval-shaped detection cavity 10 that can accommodate the electrode structure of the sensor layer. The two ends of the flow channel 2' are required to be coaxially aligned with the sample inlet channel 10 and the serum sample inlet channel 20, respectively. The mold is required to align the reference electrode interface 16 of the electrochemical sensor with the end of the reference electrode strip wire 8'. The mold is required to align the working electrode interface 17 of the electrochemical sensor with the end of the working electrode strip wire 5'. The oven heating temperature is around 70℃ (optional, 60℃-80℃), and the heating time is 2h (optional, 1h-3h). After curing, it is cut to a size that matches the interface layer, release layer, and base layer.

[0054] (4-2) Electrode preparation

[0055] A schematic diagram of the screen-printed electrode structure is shown below. Figure 4 The structure of the working electrode strip wire 5', working electrode 6', reference electrode 7', and reference electrode strip wire 8' is shown in the figure. Before printing, prepare the corresponding stencils according to the structure of each layer shown in the figure, and prepare carbon paste, Ag / AgCl paste, and Ag paste. First, print the conductive layer 12'. After applying Ag paste to the stencil of the corresponding conductive layer 12' structure, lower the stencil and press it firmly against the substrate. Move the squeegee from top to bottom through the pattern to evenly print the paste onto the glass substrate. After confirming that the electrode pattern is completely printed, remove the substrate and place it in a baking pan or oven to heat at 120°C for 10 minutes for sintering. The material of the working electrode layer 10' is carbon paste, and the material of the reference electrode layer 11' is AgCl (or Ag) paste. The printing steps are the same as described above. After electrode preparation, 20 μl (15 μl-30 μl is optional) of enzyme-chitosan solution is dropped onto the working electrode and placed at approximately 4°C (0°C-4°C is optional) for 2 hours (1 hour-5 hours is optional). The enzyme-chitosan solution is prepared by mixing chitosan and deionized water at a mass ratio of 1:100 (1:50-200 is optional), then adding glacial acetic acid and glycerol solution mixed and stirred at a volume ratio of 1:1000 (1:500-1500 is optional). The volume ratio of glycerol solution to the aforementioned mixed solution is 1:10 (1:5-15 is optional). The mixture is stirred at approximately 80°C (60°C-90°C is optional) for 2 hours (1-4 hours is optional), and then a glucose oxidase solution (or lactate oxidase solution or HMG coenzyme A synthase solution) with a concentration of 10 mg / ml (5 mg / ml-20 mg / ml is optional) is added.

[0056] (5-1) Preparation of the base layer

[0057] The substrate 14 can be made of a structurally stable material such as acrylic or glass, and is rectangular in shape with a length of 90mm (selectable from 50mm to 100mm) and a width of 60mm (selectable from 30mm to 60mm), and a thickness of 1mm (selectable from 1mm to 5mm). After completing the electrode preparation process, wipe the surface of the substrate 14 with an alcohol swab without damaging the electrodes, ensuring no watermarks, dust, or fingerprints are left. Waterproof tape or similar materials can be used to cover the working electrode strip wire 5' and the reference electrode strip wire 8' to increase sensor lifespan. Place the cut detection layer 11 into an ultrasonic cleaner and clean it at a frequency of 60kHz (selectable from 20kHz to 90kHz) for 10 minutes (selectable from 5 minutes to 20 minutes). The cleaning medium can be anhydrous ethanol or deionized water. To bond the substrate 14 to the detection layer 11 using a plasma bonding machine, the electrode parts are first covered with tin foil or black cardstock to prevent oxidation during plasma activation. During bonding, the pressure inside the bonding chamber must reach 0.1 Pa (selectable from 0.1 Pa to 1 Pa). Oxygen gas is introduced as the reaction gas, and plasma is excited using an RF or microwave power supply. The entire process typically lasts 60 seconds (selectable from 30 seconds to 180 seconds). Over-activation may damage the surface. After bonding, a hydrophilic bonding layer forms on the plasma-activated surface. The substrate 14 and detection layer 11 are aligned and gently pressed together at room temperature, achieving initial bonding through van der Waals forces or hydrogen bonds. The mixture is then allowed to stand for 60 minutes (selectable from 30 minutes to 120 minutes).

[0058] Example 2

[0059] like Figure 1 As shown, a microfluidic chip for on-demand blood biochemical index detection includes an interface layer, a separation layer, a transducer layer, a detection layer, and a substrate layer. When it is necessary to detect the concentration of multiple small molecules at once, such as glucose, lactic acid, and ketone bodies, the overall chip size can be appropriately modified. Multiple detection chambers 12 and electrochemical sensors 15 can be added to the detection layer 11, and corresponding enzyme solutions can be added to meet different needs in actual situations.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications are considered to be within the scope of protection of the present invention.

Claims

1. A microfluidic chip for on-demand detection of blood biochemical indicators, characterized in that: It includes an interface layer, a separation layer, a transducer layer, a detection layer, and a base layer. The interface layer includes an interface layer body, a blood cell sampling port, a blood sampling port, and a serum sampling port. The blood sampling port is connected to a threaded catheter connector and penetrates the interface layer body. The blood cell sampling port penetrates the interface layer body and is connected to an external receiving manifold via a flexible tube. The serum sampling port penetrates the interface layer body and is sequentially connected to a first sampling channel, a second sampling channel, and a third sampling channel, and is then connected to a waste liquid cylinder via a flexible tube. The separation layer includes a separation layer body, a separation channel, a first sample outlet channel, a transducer pin interface, a plasma channel, and a serum channel. The blood inlet is connected to the inlet of the separation channel via a conduit. The end of the separation channel branches into the plasma channel and the serum channel. The separation channel, plasma channel, and serum channel penetrate the separation layer body. The end of the plasma channel is connected to the blood cell outlet. The first sample outlet channel penetrates the separation layer body. The transducer pin interface is located on the side of the separation layer body. The transducer layer includes a transducer layer body, a second sample outlet channel, an interdigital transducer, and a serum sample inlet channel. The serum sample inlet channel penetrates the transducer layer, and the second sample outlet channel penetrates the transducer layer. The interdigital transducer is externally connected to a power amplifier and a signal generator through a transducer pin interface. The interdigital transducer is attached to the surface of the transducer layer body by screen printing. From top to bottom, the layers are a reference electrode layer, a double conductive layer, and a substrate layer. The detection layer includes a detection layer body, a detection cavity, a third sample outlet channel, an electrochemical sensor reference electrode interface, an electrochemical sensor working electrode interface, and a sample inlet channel. The sample inlet channel penetrates the detection layer body. The serum channel outlet, the serum sample inlet channel, and the sample inlet channel are sequentially connected and injected into the detection cavity. The detection cavity consists of an oval-shaped main cavity and a rectangular flow channel. The rectangular flow channel is connected to the main cavity. The detection cavity is located at the bottom of the detection layer body. The third sample outlet channel is located at the end of the detection cavity body and penetrates the detection layer body. The substrate layer includes a substrate body and a sensor electrode. The sensor electrode is attached to the surface of the substrate layer by screen printing. The sensor electrode has a dual-electrode structure or a triple-electrode structure. The electrochemical sensor reference electrode interface is externally connected to the reference electrode of the detector and internally connected to the sensor electrode. The electrochemical sensor working electrode interface is externally connected to the working electrode of the matching detector and internally connected to the sensor electrode.

2. The on-demand blood biochemical index detection microfluidic chip according to claim 1, characterized in that: The dual-electrode structure includes a working electrode strip wire, a working electrode, a reference electrode, and a reference electrode strip wire. The reference electrode is arranged around the working electrode and is a ring with a central angle exceeding 120°. The three-electrode structure includes a working electrode wire, a working electrode, a reference electrode strip wire, a reference electrode, a counter electrode strip wire, and a counter electrode. The working electrode is connected to a working electrode wire, the reference electrode is connected to a reference electrode strip wire, and the counter electrode is connected to a counter electrode strip wire. The reference electrode and the counter electrode are arranged around the working electrode. The reference electrode is a ring with a central angle exceeding 30°, and the counter electrode is a ring with a central angle exceeding 90°.

3. The on-demand blood biochemical index detection microfluidic chip according to claim 2, characterized in that: The working electrode is printed with an enzyme-chitosan layer, a working electrode layer and a conductive layer in layers stacked from top to bottom; the reference electrode is printed with a reference electrode layer and a conductive layer in layers stacked from top to bottom.

4. The on-demand blood biochemical index detection microfluidic chip according to claim 1, characterized in that: The interface layer, transducer layer, and substrate layer are made of acrylic or glass; the separation layer and detection layer are made of glass, acrylic, polymethyl methacrylate, or a mixture of polydimethylsiloxane and polydimethylsiloxane crosslinking agent that has been cured by heating.

5. The on-demand blood biochemical index detection microfluidic chip according to claim 4, characterized in that: The mass ratio of polydimethylsiloxane to polydimethylsiloxane crosslinking agent is 15-5:

1.

6. The on-demand blood biochemical index detection microfluidic chip according to claim 1, characterized in that: The reference electrode layer in the interdigital transducer is made of Ag / AgCl; the double conductive layer is made of Cr and Au, wherein the Cr layer is in close contact with the substrate layer and the Au layer is in close contact with the reference electrode layer, and the substrate layer is made of lithium niobate.

7. The on-demand blood biochemical index detection microfluidic chip according to claim 2, characterized in that: The working electrode is connected to the external detection device via a working electrode strip wire inserted into the working electrode interface of the electrochemical sensor, and the reference electrode is connected to the external detection device via a reference electrode strip wire inserted into the working electrode hole of the electrochemical sensor.

8. The on-demand blood biochemical index detection microfluidic chip according to claim 3, characterized in that: The working electrode strip wire, the reference electrode strip wire, and the conductive layer are made of Au, Ag, Cu, or Pt; the working electrode layer is made of carbon, and the reference electrode layer is made of Ag / AgCl.

9. The on-demand blood biochemical index detection microfluidic chip according to claim 3, characterized in that: The enzyme-chitosan layer material is obtained by mixing chitosan, glacial acetic acid and glycerol, and then adding glucose oxidase, lactate oxidase or HMG coenzyme A synthase.

10. The on-demand blood biochemical index detection microfluidic chip according to claim 9, characterized in that: The enzyme-chitosan layer material is prepared by the following method: chitosan and deionized water are mixed at a mass ratio of 1:50-200, and then glacial acetic acid and glycerol solution are added at a volume ratio of 1-10:100 after stirring. The volume ratio of the two mixed solutions is 15-5:

1. After stirring at 60℃-90℃ for 1 h-4 h, glucose oxidase solution, lactate oxidase solution or HMG coenzyme A synthase solution with a concentration of 5 mg / ml-20 mg / ml is added.

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