Dielectric wetting valve micro-fluidic chip for metabolic marker detection and preparation method thereof

Abstract

The invention relates to a dielectric wetting valve micro-fluidic chip for metabolic marker detection and a preparation method of the dielectric wetting valve micro-fluidic chip. A chip middle layer is stacked above a chip lower layer, and an inverted chip upper layer is stacked above the chip middle layer; the chip middle layer comprises a double-sided adhesive layer and a trident micro-channel, the periphery of the trident micro-channel is coated with the double-sided adhesive layer, the trident micro-channel comprises a central area, a first branch channel, a second branch channel and a third branch channel, and the first branch channel, the second branch channel and the third branch channel are uniformly distributed on the periphery of the central area; the head end of the first branch channel, the head end of the second branch channel and the head end of the third branch channel are respectively connected with the central area. Through the trident micro-channel, the detection of various metabolic markers becomes simpler, more convenient and more sensitive, and the detection precision of the metabolic markers is improved. According to the invention, through the trifurcate micro-channel, the sequential analysis of various targeted metabolites can be realized, an external pump is not needed, and the device has the advantages of high portability, accurate fluid control and the like.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and in particular to a dielectric wetting valve microfluidic chip for the detection of metabolic biomarkers and its fabrication method. Background Technology

[0002] The detection of human metabolites, including glucose, lactic acid, pyruvate, and reactive oxygen species (ROS), is crucial for the diagnosis and treatment of various diseases. To monitor the dynamic changes of metabolites in cells or body fluids (such as blood, saliva, and sweat), various detection techniques have emerged, including colorimetry, fluorescence, electrochemistry, chemiluminescence (CL), and electrochemiluminescence (ECL). Among these, chemiluminescence has become a research hotspot due to its unique advantages such as high sensitivity, simple operation, and no need for an external light source.

[0003] Metabolites are typically detected using luminescence analyzers, ELISA readers, or flow injection chemiluminescence systems. However, these platforms often consume large amounts of reagents and samples, involve cumbersome manual operations, and have long detection cycles. To overcome these limitations, microfluidic chemiluminescence detection technologies have emerged. Based on the flow control mechanism, microfluidic chemiluminescence systems can be categorized into flow injection devices with differential pressure flow modes, capillary-driven microfluidic paper analyzers (μPADs), microchip capillary electrophoresis (MCE) devices, and microfluidic chips that utilize electric fields to control droplets.

[0004] The advent of paper-based chips has enhanced the advantages of chemiluminescence technology in terms of fluid control, low cost, and disposableness. Using capillary forces to drive fluids, the sequential transport and reaction of various reagents can be conveniently accomplished on paper-based devices. However, the porous structure of paper-based materials can interfere with the acquisition of light signals, especially in low-concentration samples, thus reducing detection accuracy.

[0005] In contrast, dielectric wetting-based microfluidics allows for precise manipulation of droplets. While electrowetting valve-based microfluidics shows great promise in metabolic analysis, practical applications still face several challenges. First, sequential analysis of multiple targeted metabolites is required within ultra-small sample volumes. Second, sensors must possess low detection limits and wide linear ranges to handle significant variations in metabolite concentrations within samples: glucose and lactate are often in the millimole range, while pyruvate is only in the micromolar range. Furthermore, when detecting cells, the total amount of metabolites produced by the target analyte throughout the culture cycle is limited, requiring sensors to resolve minute changes against high backgrounds (such as high glucose concentrations). Moreover, advancing clinical application requires reducing reliance on large peripheral equipment and complex chip fabrication processes. Currently, most microfluidic systems for metabolic biomarker detection cannot simultaneously meet these requirements, becoming a major bottleneck in clinical translation. Summary of the Invention

[0006] This invention provides a dielectric wetting valve microfluidic chip for the detection of metabolic biomarkers and a method for its fabrication, aiming to solve at least one of the technical problems existing in the prior art.

[0007] The technical solution of this invention is a dielectric wetting valve microfluidic chip for the detection of metabolic biomarkers, comprising: The lower layer of the chip; A middle layer of a chip, which is stacked on top of a lower layer of a chip; The upper layer of the chip, which is placed upside down, is stacked on top of the middle layer of the chip; The middle layer of the chip includes a double-sided adhesive layer and a three-way microchannel. The double-sided adhesive layer covers the perimeter of the three-way microchannel. The three-way microchannel includes a central region, a first branch channel, a second branch channel, and a third branch channel. The first branch channel, the second branch channel, and the third branch channel are evenly distributed around the central region. The beginning ends of the first branch channel, the second branch channel, and the third branch channel are respectively connected to the central region.

[0008] According to some embodiments of the present invention, the upper layer of the chip includes a polydimethylsiloxane layer, a polyethylene terephthalate layer, a first silver electrode, and a second silver electrode. The first silver electrode is stacked on top of the polyethylene terephthalate layer, and the second silver electrode is stacked on top of the polyethylene terephthalate layer. The polydimethylsiloxane layer covers the polyethylene terephthalate layer, the first silver electrode, and the second silver electrode.

[0009] According to some embodiments of the present invention, the first silver electrode is horizontally disposed on the upper part of the polyethylene terephthalate layer, and two second silver electrodes are disposed symmetrically opposite each other and vertically disposed on the lower part of the polyethylene terephthalate layer.

[0010] According to some embodiments of the present invention, the upper layer of the chip includes an inlet hole and an outlet hole. The inlet hole is stacked above the central region, and three outlet holes are provided. The three outlet holes are respectively stacked above the tail end of the first branch channel, the tail end of the second branch channel, and the tail end of the third branch channel.

[0011] According to some embodiments of the present invention, the first branch channel, the second branch channel, and the third branch channel are each provided with an enzyme reaction zone, a dielectric wetting valve, and a detection zone connected in sequence. One end of the enzyme reaction zone is connected to the inlet hole through the central zone, and one end of the detection zone is connected to the outlet hole, so that when the first silver electrode or the second silver electrode is energized, the dielectric wetting valve opens, thereby allowing the solution in the central zone and the enzyme reaction zone to flow to the detection zone through the dielectric wetting valve, triggering a chemiluminescent reaction.

[0012] According to some embodiments of the present invention, the upper layer of the chip is pre-positioned with a pre-embedded enzyme solution and a pre-embedded substrate solution. The pre-embedded enzyme solution is stacked on top of the polydimethylsiloxane layer, and the pre-embedded substrate solution is stacked on top of the polydimethylsiloxane layer. When the middle layer of the chip and the inverted upper layer of the chip are combined, the pre-embedded enzyme solution is placed inside the enzyme reaction zone, and the pre-embedded substrate solution is placed inside the detection zone. The pre-embedded substrate solution is used for chemiluminescent detection of metabolic markers.

[0013] According to some embodiments of the present invention, one end of the first silver electrode is stacked above the dielectric wetting valve, and one end of the second silver electrode is stacked above the dielectric wetting valve.

[0014] According to some embodiments of the present invention, the lower layer of the chip includes an indium tin oxide electrode and a glass layer, wherein the indium tin oxide electrode layer is stacked on top of the glass layer.

[0015] According to some embodiments of the present invention, the dielectric wetting valve is composed of the indium tin oxide electrode and the polydimethylsiloxane layer.

[0016] The technical solution of the present invention also relates to a method for fabricating a dielectric wetting valve microfluidic chip for the detection of metabolic biomarkers, comprising the following steps: S100. Laser etching is performed on the indium tin oxide glass substrate to obtain a glass layer and an indium tin oxide electrode protruding on the surface of the glass layer to obtain the chip lower layer. The chip lower layer is immersed in acetone, rinsed with ethanol and deionized water in sequence to remove residual solvent, and then dried. S200. A three-way microchannel is formed by cutting with double-sided tape to obtain the middle layer of the chip, wherein the three-way microchannel includes a central region, a first branch channel, a second branch channel and a third branch channel; S300: Using conductive silver paste, screen print a first silver electrode and a second silver electrode at different positions on a polyethylene terephthalate layer. Mix a polydimethylsiloxane prepolymer with a substrate and a curing agent at a mass ratio of 10:1 to obtain a polydimethylsiloxane layer. Spin coat the polydimethylsiloxane layer over the polyethylene terephthalate layer, the first silver electrode, and the second silver electrode, and then cure it to obtain the initial upper layer of the chip. S400: Drill holes in the initial upper layer of the chip to form an inlet hole and three outlet holes. The inlet hole is connected to the central region, and the three outlet holes are respectively connected to the tail ends of the first branch channel, the second branch channel, and the third branch channel. A pre-embedded enzyme solution is pre-placed between one end of the first silver electrode and the inlet hole, and the pre-embedded enzyme solution is pre-placed between one end of the second silver electrode and the inlet hole. A pre-embedded substrate solution is pre-placed between one end of the first silver electrode and the outlet hole, and the pre-embedded substrate solution is pre-placed between one end of the second silver electrode and the outlet hole, thus obtaining the upper layer of the chip. The pre-embedded substrate solution is used for the chemiluminescent detection of metabolic markers. S500. Align and attach the inverted upper layer of the chip with the middle layer and the lower layer of the chip in sequence to obtain a microfluidic chip with a dielectric wetting valve. When the first silver electrode or the second silver electrode is energized, the dielectric wetting valve opens, so that the solution in the central region and the enzyme reaction region flows to the detection region through the dielectric wetting valve, triggering a chemiluminescent reaction.

[0017] The present invention also relates to a computer device, including a memory and a processor, wherein the processor executes the method described above when executing a computer program stored in the memory.

[0018] The present invention also relates to a computer-readable storage medium having program instructions stored thereon, which, when executed by a processor, implement the method described above.

[0019] The beneficial effects of the present invention include: the microfluidic chip with dielectric wetting valve includes a lower chip layer, a middle chip layer, and a upper chip layer, with the middle chip layer stacked on top of the lower chip layer and the inverted upper chip layer stacked on top of the middle chip layer; the middle chip layer includes a double-sided adhesive layer and a three-way microchannel, with the double-sided adhesive layer covering the periphery of the three-way microchannel, and the three-way microchannel including a central region, a first branch channel, a second branch channel, and a third branch channel, with the first branch channel, the second branch channel, and the third branch channel evenly distributed around the central region, and the beginning ends of the first branch channel, the second branch channel, and the third branch channel respectively connected to the central region.

[0020] The use of a triangular microchannel simplifies, facilitates, and enhances the sensitivity of detecting multiple metabolic biomarkers, while also improving detection accuracy. This invention, through the triangular microchannel, enables sequential analysis of multiple targeted metabolites without the need for an external pump, offering advantages such as high portability and precise fluid control. The chip exhibits superior sensitivity and a wide linear range in the detection of various metabolic biomarkers, accurately quantifying glucose, lactate, and pyruvate in samples, and shows broad application potential in the automated detection of multiple samples.

[0021] Furthermore, additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a dielectric wetting valve microfluidic chip for detecting metabolic biomarkers in an embodiment of the present invention.

[0023] Figure 2 This is a schematic diagram illustrating the fabrication principle of the microfluidic chip in this embodiment of the invention.

[0024] Figure 3 This is a diagram illustrating the fabrication process of the microfluidic chip in this embodiment of the invention.

[0025] Figure 4 This is a schematic diagram of the sample detection process in an embodiment of the present invention.

[0026] Figure 5 This is a schematic diagram illustrating the situation of fluid flowing through a dielectric wetting valve after a voltage is applied, as described in an embodiment of the present invention.

[0027] Figure 6 This is a schematic diagram of fluid flow control on the dielectric valve chip in an embodiment of the present invention.

[0028] Figure 7 This is a schematic diagram showing the independent detection of the CL intensity of H2O2 in three channels within 30 seconds in an embodiment of the present invention.

[0029] Figure 8 This is an optional flowchart of a method for fabricating a dielectric wetting valve microfluidic chip for detecting metabolic biomarkers in an embodiment of the present invention.

[0030] Figure 9 This is a cross-sectional view of the dielectric wetting valve of the first branch channel in the microfluidic chip of the present invention.

[0031] Figure 10 This is a real-time chemiluminescence curve of glucose detection by the dielectric valve chip in this embodiment of the invention.

[0032] Figure 11 This is a real-time chemiluminescence curve of lactic acid detected by the dielectric valve chip in this embodiment of the invention.

[0033] Figure 12 This is a real-time chemiluminescence curve of pyruvate detected by the dielectric valve chip in this embodiment of the invention.

[0034] Figure 13 This is a detection chart of glucose, lactic acid, and pyruvate in an embodiment of the present invention.

[0035] Figure 14 This is a schematic diagram illustrating the specificity and stability of the chip in the embodiments of the present invention.

[0036] The above figures include the following reference numerals: 100. Lower layer of the chip; 110. Indium tin oxide electrode; 120. Glass layer; 200. Chip middle layer; 210. Double-sided adhesive layer; 220. Tri-channel microchannel; 221. Central area; 222. First branch channel; 223. Second branch channel; 224. Third branch channel; 300, Top layer of chip; 310, Polydimethylsiloxane layer; 320, Polyethylene terephthalate layer; 330, First silver electrode; 340, Second silver electrode; 350, Inlet hole; 360, Outlet hole. Detailed Implementation

[0037] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0038] It should be noted that, unless otherwise specified, when a feature is referred to as "fixed" or "connected" to another feature, it can be directly fixed or connected to the other feature, or indirectly fixed or connected to the other feature. Furthermore, the descriptions of "upper," "lower," "left," "right," "top," and "bottom" used in this invention are only relative to the relative positional relationships of the various components of the invention in the accompanying drawings.

[0039] Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and not for limiting the invention. The term "and / or" as used herein includes any combination of one or more of the associated listed items.

[0040] It should be understood that although the terms first, second, third, etc., may be used to describe various elements in this invention, these elements should not be limited to these terms. These terms are only used to distinguish elements of the same type from each other. For example, a first element may also be referred to as a second element without departing from the scope of this invention, and similarly, a second element may also be referred to as a first element.

[0041] Reference Figures 1 to 14 In some embodiments, the dielectric wetting valve microfluidic chip for metabolic biomarker detection according to the present invention includes: a lower chip layer 100, a middle chip layer 200, and a upper chip layer 300. The middle chip layer 200 is stacked on top of the lower chip layer 100, and the inverted upper chip layer 300 is stacked on top of the middle chip layer 200. The middle chip layer 200 includes a double-sided adhesive layer 210 and a three-way microchannel 220. The double-sided adhesive layer 210 covers the periphery of the three-way microchannel 220. The three-way microchannel 220 includes a central region 221, a first branch channel 222, a second branch channel 223, and a third branch channel 224. The first branch channel 222, the second branch channel 223, and the third branch channel 224 are evenly distributed around the central region 221. The beginning ends of the first branch channel 222, the second branch channel 223, and the third branch channel 224 are respectively connected to the central region 221.

[0042] As can be seen, the triangular microchannel makes the detection of multiple metabolic biomarkers simpler, more convenient, and more sensitive, while also improving the detection accuracy. This invention, through the triangular microchannel, facilitates the sequential analysis of multiple targeted metabolites, eliminating the need for an external pump and offering advantages such as high portability and precise fluid control. This chip exhibits excellent sensitivity and a wide linear range in the detection of multiple metabolic biomarkers, accurately quantifying glucose, lactate, and pyruvate in samples, and has broad application potential in the automated detection of multiple samples.

[0043] exist Figure 1 middle, Figure 1 (a) represents the lower layer 100 of the chip. Figure 1 (b) is the upper layer 300 of the chip. Figure 1 (c) represents layer 200 in the middle of the chip. Figure 1 (d) is a microfluidic chip, which is composed of a lower chip layer 100, a middle chip layer 200 and an upper chip layer 300.

[0044] In some embodiments, the upper chip layer 300 includes a polydimethylsiloxane layer 310, a polyethylene terephthalate layer 320, a first silver electrode 330, and a second silver electrode 340. The first silver electrode 330 is stacked on top of the polyethylene terephthalate layer 320, and the second silver electrode 340 is stacked on top of the polyethylene terephthalate layer 320. The polydimethylsiloxane layer 310 covers the polyethylene terephthalate layer 320, the first silver electrode 330, and the second silver electrode 340.

[0045] Specifically, the other end of the first silver electrode 330 is connected to the positive electrode, the other end of the second silver electrode 340 is connected to the positive electrode, the upper left part of the middle layer 200 of the chip is connected to the negative electrode, and the upper right part of the middle layer 200 of the chip is connected to the negative electrode.

[0046] In some embodiments, the first silver electrode 330 is horizontally disposed on the upper part of the polyethylene terephthalate layer 320, and two second silver electrodes 340 are disposed symmetrically opposite each other and vertically disposed on the lower part of the polyethylene terephthalate layer 320.

[0047] In some embodiments, the upper chip layer 300 includes an inlet hole 350 and an outlet hole 360. The inlet hole 350 is stacked above the central region 221, and there are three outlet holes 360, which are respectively stacked above the tail end of the first branch channel 222, the tail end of the second branch channel 223, and the tail end of the third branch channel 224.

[0048] Specifically, the diameter of the inlet orifice 350 is larger than the diameter of the outlet orifice 360.

[0049] In some embodiments, the first branch channel 222, the second branch channel 223, and the third branch channel 224 are each provided with an enzyme reaction zone, a dielectric wetting valve, and a detection zone connected in sequence. The enzyme reaction zone is connected to the central zone 221, the enzyme reaction zone is connected to the dielectric wetting valve, and the dielectric wetting valve is connected to the detection zone. One end of the enzyme reaction zone is connected to the inlet hole 350 through the central zone 221, and one end of the detection zone is connected to the outlet hole 360. When the first silver electrode 330 or the second silver electrode 340 is energized, the dielectric wetting valve opens, and the solution in the central zone 221 and the enzyme reaction zone flows to the detection zone through the dielectric wetting valve, triggering a chemiluminescent reaction.

[0050] Specifically, dielectric wetting valves enable precise fluid control within capillary channels, thereby triggering enzymatic oxidation and chemiluminescence reactions. Dielectric wetting valves make the detection of a variety of metabolites simpler, more convenient, and more sensitive. See also Figure 9 , Figure 9This is a cross-sectional view of the dielectric wetting valve of the first branch channel in the microfluidic chip. The cross-sectional views of the dielectric wetting valve of the second branch channel and the third branch channel are based on the same principle as the cross-sectional view of the dielectric wetting valve of the first branch channel.

[0051] In some embodiments, the upper chip layer 300 is pre-positioned with a pre-intercalated enzyme solution and a pre-intercalated substrate solution. The pre-intercalated enzyme solution is stacked on top of the polydimethylsiloxane layer 310, and the pre-intercalated substrate solution is stacked on top of the polydimethylsiloxane layer 310. When the middle chip layer 200 and the inverted upper chip layer 300 are combined, the pre-intercalated enzyme solution is placed inside the enzyme reaction region, and the pre-intercalated substrate solution is placed inside the detection region. The pre-intercalated substrate solution is used for the chemiluminescent detection of metabolic markers. Specifically, metabolic markers include glucose, lactate, and pyruvate, etc.

[0052] In some embodiments, one end of the first silver electrode 330 is stacked above the dielectric wetting valve, and one end of the second silver electrode 340 is stacked above the dielectric wetting valve.

[0053] In some embodiments, the lower chip layer 100 includes an indium tin oxide electrode 110 and a glass layer 120, with the indium tin oxide electrode 110 stacked on top of the glass layer 120.

[0054] In some embodiments, the dielectric wetting valve is composed of an indium tin oxide electrode 110 and a polydimethylsiloxane layer 310.

[0055] Specifically, in Figure 4 In this process, the dielectric wetting valve is located between the enzyme reaction zone and the detection zone.

[0056] Reference Figure 8 In some embodiments, the method for preparing the dielectric wetting valve microfluidic chip for metabolic biomarker detection according to the present invention includes at least the following steps: S100: Laser etching is performed on the indium tin oxide glass substrate to obtain a glass layer 120 and an indium tin oxide electrode 110 protruding on the surface of the glass layer 120, so as to obtain a lower chip layer 100. The lower chip layer 100 is immersed in acetone, rinsed with ethanol and deionized water in sequence to remove residual solvent, and then dried. S200. A three-way microchannel 220 is formed by cutting with double-sided tape to obtain the middle layer 200 of the chip. The three-way microchannel 220 includes a central region 221, a first branch channel 222, a second branch channel 223 and a third branch channel 224. S300: Using conductive silver paste, screen print a first silver electrode 330 and a second silver electrode 340 at different positions on the polyethylene terephthalate layer 320. Mix the substrate and curing agent with polydimethylsiloxane prepolymer at a mass ratio of 10:1 to obtain a polydimethylsiloxane layer 310. Spin coat the polydimethylsiloxane layer 310 on top of the polyethylene terephthalate layer 320, the first silver electrode 330 and the second silver electrode 340 and perform a curing treatment to obtain the initial upper layer of the chip. S400: A hole 350 and three outlet holes 360 are formed on the initial upper layer of the chip. The inlet hole 350 is connected to the central region 221, and the three outlet holes 360 are connected to the tail ends of the first branch channel 222, the second branch channel 223, and the third branch channel 224, respectively. A pre-embedded enzyme solution is placed between one end of the first silver electrode 330 and the inlet hole 350, a pre-embedded enzyme solution is placed between one end of the second silver electrode 340 and the inlet hole 350, a pre-embedded substrate solution is placed between one end of the first silver electrode 330 and the outlet hole 360, and a pre-embedded substrate solution is placed between one end of the second silver electrode 340 and the outlet hole 360, thus obtaining the upper layer 300 of the chip. The pre-embedded substrate solution is used for the chemiluminescence detection of metabolic markers. S500, the inverted chip upper layer 300 is aligned and bonded with the chip middle layer 200 and chip lower layer 100 in sequence to obtain a microfluidic chip with a dielectric wetting valve, so that when the first silver electrode 330 or the second silver electrode 340 is energized, the dielectric wetting valve opens, and the solution in the central region 221 and the enzyme reaction region flows to the detection region through the dielectric wetting valve, triggering a chemiluminescent reaction.

[0057] In a specific embodiment, the fabrication method of the dielectric wetting valve microfluidic chip for metabolic biomarker detection includes: Laser etching is performed on the indium tin oxide glass substrate to obtain a glass layer 120 and an indium tin oxide electrode 110 protruding on the surface of the glass layer 120, so as to obtain the lower layer 100 of the chip. The indium tin oxide electrode 110 is used as the ground electrode; The lower layer 100 of the chip is immersed in acetone, then rinsed with ethanol and deionized water in sequence to remove residual solvent, and finally dried. Tri-branch microchannels 220 are formed by cutting double-sided tape to obtain the middle layer 200 of the chip, wherein the tri-branch microchannels 220 include a central region 221, a first branch channel 222, a second branch channel 223 and a third branch channel 224; A first silver electrode 330 and a second silver electrode 340 located at different positions are screen-printed on a polyethylene terephthalate layer 320 using conductive silver paste. The first silver electrode 330 and the second silver electrode 340 are used as excitation electrodes; PDMS prepolymer is mixed with substrate and curing agent at a mass ratio of 10 to 1 to obtain polydimethylsiloxane layer 310. Polydimethylsiloxane layer 310 is spin-coated on top of polyethylene terephthalate layer 320, first silver electrode 330 and second silver electrode 340 and cured to obtain the initial upper layer of chip. One inlet hole 350 and three outlet holes 360 are formed by drilling holes on the initial upper layer of the chip after curing. The inlet hole 350 is connected to the central region 221, and the three outlet holes 360 are connected to the tail ends of the first branch channel 222, the second branch channel 223, and the third branch channel 224, respectively. A pre-embedded enzyme solution is pre-placed between one end of the first silver electrode 330 and the inlet hole 350, a pre-embedded enzyme solution is pre-placed between one end of the second silver electrode 340 and the inlet hole 350, a pre-embedded substrate solution is pre-placed between one end of the first silver electrode 330 and the outlet hole 360, and a pre-embedded substrate solution is pre-placed between one end of the second silver electrode 340 and the outlet hole 360, thus obtaining the upper layer 300 of the chip. The pre-embedded substrate solution is used for the chemiluminescence detection of metabolic markers. The upper layer 300 of the chip is aligned and bonded to the middle layer 200 and the lower layer 100 of the chip in sequence to obtain a microfluidic chip with a dielectric wetting valve. When the first silver electrode 330 or the second silver electrode 340 is energized, the dielectric wetting valve opens, so that the solution in the central region 221 and the enzyme reaction region flows to the detection region through the dielectric wetting valve, triggering a chemiluminescent reaction.

[0058] Specifically, see Figure 2 and Figure 3The fabrication of the microfluidic chip mainly involves three steps: etching the ITO electrode (i.e., the aforementioned indium tin oxide electrode) to serve as the ground electrode, printing a silver electrode as the excitation electrode, and spin-coating a PDMS layer (i.e., the aforementioned polydimethylsiloxane layer) to form a hydrophobic channel surface. First, the ITO glass substrate (ITO layer thickness: 680±50nm) is laser-etched (Femto-IR-50-40) to define the ITO electrode surrounded by a non-conductive region. To clean the ITO electrode surface, the substrate is immersed in acetone for 10 min, rinsed sequentially with ethanol and deionized water to remove residual solvent, and then dried for later use. A three-way microchannel structure is formed using double-sided adhesive tape (total thickness 170µm, including adhesive layers on both sides), including a central node (4mm in diameter) and three branch channels (each 12mm×2mm). Three silver electrodes at different locations were screen-printed on a PET layer (i.e., the aforementioned polyethylene terephthalate layer) (29.7 mm × 21 mm × 0.2 mm) using conductive silver paste. A PDMS prepolymer was mixed with a curing agent at a mass ratio of 10:1 and spin-coated onto the silver electrode layer (located on the PET) at 3500 rpm for 2 min, followed by curing at 60°C for 5 h to form a hydrophobic dielectric layer (20 μm thick film). Holes were drilled in the combined structure of the silver electrode layer and PDMS layer to form one inlet (6 mm) and three outlets (3 mm), and enzymes and probes were pre-placed and dried in designated areas. Starting from the upper left corner, the combined silver electrode layer and PDMS layer was aligned and bonded to the ITO electrode layer containing the three-way channel, thereby sealing the channel and completing the final chip fabrication.

[0059] The silver electrode layer includes a first silver electrode 330 and a second silver electrode 340. Figure 3 (a) represents the lower layer 100 of the chip. Figure 3 (b) is layer 200 in the middle of the chip. Figure 3 (c) represents the combination of the silver electrode layer and the PDMS layer. Figure 3 (d) indicates pre-intercalation of the enzyme. Figure 3 (e) indicates the pre-embedding of the detection substrate. Figure 3 (f) represents the assembly of the microfluidic chip. In Figure 3 In (d), GOX, LOX, and POX represent pre-intercalated enzyme solutions; Figure 3 In (e), the probe represents the pre-embedded substrate solution.

[0060] Specifically, valve-controlled fluid operation was validated using a diluted dye solution at 250V, and the process was recorded by a camera. Subsequently, the sequential chemiluminescence detection performance was evaluated using 1mM H2O2 as the analyte. A 5μL reaction system containing 2.0mM luminol, 1.0mM PIP, and 140U / mL HRP was pre-placed in the detection zone. Chemiluminescence signals were generated by sample introduction and sequential valve triggering, and recorded by the instrument. Figure 7 As shown.

[0061] Specifically, the chip's detection performance for a mixture of glucose, lactate, and pyruvate diluted in PBS was evaluated. 5 μL of enzyme solution (70 U / mL GOX, 20 U / mL LOX, 25 U / mL POX) and 5 μL of substrate solution (containing 2.0 mM luminol, 1.0 mM PIP, and 100 U / mL HRP) were pre-embedded in the enzyme reaction zone before the valves and the detection zone after the valves on the PDMS layer. After assembly, 21 μL of the analyte mixture was added to the chip inlet. The mixture immediately flowed to the area before the valves in the three branch channels. After incubation for 4, 5, and 6 minutes of oxidation reaction, the valves controlling glucose, lactate, and pyruvate were activated at 250 V. Simultaneously, the chemiluminescence (CL) signal was monitored using a photomultiplier tube (PMT, Hamamatsu H7421) at a gate width of 10 ms, and signal changes were recorded within 60 seconds after valve opening.

[0062] See Figure 4 (a) and Figure 4 (b) The dielectric valve chip consists of an ITO electrode and a PDMS dielectric layer forming a hydrophobic valve in the capillary channel. Due to capillary action, the liquid stops flowing at the dielectric valve until a 250V voltage is applied, activating the silver electrode to resume liquid flow. Enzymes and probes are preloaded and dried in designated areas of the Ag-PDMS layer. After the analytes are introduced through the chip inlet, the mixed solution immediately flows to the valve in front of each branch channel. GOX, LOX, and POX oxidize their respective analytes to generate H2O2, which is produced in the corresponding channels. Subsequently, the flow control valves for glucose, lactate, and pyruvate are activated, allowing the H2O2-containing solution to enter the detection area, where the H2O2 oxidizes luminol to produce chemiluminescence.

[0063] Specifically, the dielectric wetting mechanism within the capillary channel was studied through simulation. The process from initial liquid loading to cessation at the hydrophobic region of the dielectric valve, and then the resumption of flow through dielectric wetting control, was simulated. By adjusting the applied voltage of the dielectric wetting valve and the dielectric layer thickness, the contact angle at the hydrophobic valve was reduced, thereby determining the optimal input voltage and dielectric layer thickness.

[0064] exist Figure 5 middle, Figure 5(a) represents the position of the fluid in the pipe at time 0s and voltage 0V; Figure 5 (b) represents the position of the fluid in the pipe at a time of 0.0006s and a voltage of 250V; Figure 5 (c) represents the position of the fluid in the pipe at a time of 0.07s and a voltage of 250V; Figure 5 (d) indicates the position of the fluid in the pipe at a time of 0.15s and a voltage of 250V.

[0065] See Figure 5 In the numerical simulation model, the initial contact angles were set to 120° for the PDMS surface, 80° for the ITO glass surface, and 30° for the non-conductive region. When a voltage of 250V was applied, although the bottom ITO glass surface maintained an 80° contact angle, the gas / liquid interface contact angle of the PDMS layer immediately decreased from 120° to 78.4°. After the voltage was applied, the dielectric wetting effect caused a change in the contact angle of the droplet across the solid-liquid-gas three-phase system, which altered the direction of the capillary force. During flow, the direction of the capillary force shifted from upstream to downstream, effectively changing the fluid behavior and allowing the previously stagnant liquid to continue flowing through the dielectric wetting valve. This process demonstrates the ability of the dielectric wetting effect to regulate liquid flow in microfluidic chips and also showcases the effectiveness and reliability of the dielectric wetting valve.

[0066] Understandably, see Figure 6 and Figure 7 In the control experiment of the dielectric wetting valve, after dye loading, capillary flow spontaneously started from the chip inlet and stopped at the hydrophobic region of the dielectric valve. Subsequently, after sequentially applying a 250V voltage to the silver electrode, the liquid flow resumed and flowed sequentially into the detection regions of glucose, lactic acid, and pyruvate. This experimental process demonstrates the control capability of dielectric wetting technology in microfluidic chips, enabling precise manipulation and sequential flow of liquids, providing an effective solution for the multiplex detection of metabolites. The detection capability of the device was then evaluated using an H2O2 solution (concentration: 1.0 mmol / L) with probe substrates of luminol (2.0 mmol / L), PIP (1.0 mmol / L), and HRP (140 U / mL). The valve controlled the flow as expected, and the CL intensity in the detection region reached its maximum within 0.1 seconds, then decreased sharply.

[0067] Specifically, this dielectric valve capillary microfluidic chip is used to sequentially detect glucose, lactic acid, and pyruvate, and experiments were conducted under optimized detection conditions (luminol -2 mmol / L, PIP -1 mmol / L, and HRP -100 U / mL). The sample flows spontaneously after being added to the chip inlet and stops before the dielectric valve to complete sample incubation. When the sample passes through the dielectric valve, the real-time CL intensity reaches its peak within seconds. The real-time chemiluminescence curve (red line) for glucose detection is shown below. Figure 10 As shown. The real-time chemiluminescence curve (green line) for lactic acid detection is shown below. Figure 11 As shown. The real-time chemiluminescence curve (blue line) for pyruvate detection is shown below. Figure 12 As shown. By Figures 10 to 12 It was found that the chemiluminescence (CL) signal detected in the sample increased rapidly after reagent mixing, reaching its maximum intensity in about 1 second, and then gradually decayed. Furthermore, when the glucose concentration varied from 1.0 to 2000 μmol / L, the CL intensity increased from 59 photons / gate to 121.25 × 10³ photons / gate; when the lactate concentration varied from 0.5 to 2000 μmol / L, the CL intensity increased from 53 photons / gate to 117.45 × 10³ photons / gate; and when the pyruvate concentration varied from 2.0 to 2000 μmol / L, the CL intensity increased from 69 photons / gate to 111.49 × 10³ photons / gate.

[0068] in, Figure 10 (a) represents the change in chemiluminescence intensity over time when the glucose concentration is 0–100 μmol / L; Figure 10 (b) represents the change in chemiluminescence intensity over time when the glucose concentration is 100–5000 μmol / L. Figure 11 (a) represents the change in chemiluminescence intensity over time when the lactic acid concentration is 0–100 μmol / L; Figure 11 (b) represents the change in chemiluminescence intensity over time when the lactic acid concentration is 100–5000 μmol / L. Figure 12 (a) represents the change in chemiluminescence intensity over time when the concentration of pyruvate is 0–100 μmol / L; Figure 12 (b) represents the change in chemiluminescence intensity over time when the pyruvate concentration is 100–5000 μmol / L. Figure 13 (a) represents the chemiluminescence (CL) intensity as a function of analyte concentration (n=3); Figure 13 (b) represents the linear correlation between CL intensity and glucose, lactate and pyruvate concentrations (n=3). Figure 13 (c) represents the correlation between CL intensity and the logarithmic concentrations of glucose, lactate, and pyruvate (n=3). Figure 14 (a) represents the detection specificity of the chip (n=3); Figure 14 (b) represents the storage stability of the chip (n=3).

[0069] See Figure 13 The chemiluminescence intensity (y) showed a linear relationship with the concentrations (Cg, Cl, Cp) or logarithmic concentrations (logCg, logCl, logCp) of glucose, lactic acid, and pyruvate. For glucose, the linear regression models were y = 0.056Cg + 0.03 (R² = 0.974, 1.0–100 μmol / L) and y = 91.39logCg - 176.95 (R² = 0.993, 100–2000 μmol / L). Figure 13 (b) and Figure 13 The red line in (c) shows this. For lactic acid, the linear regression models are y = 0.066Cl + 0.028 (R² = 0.983, 0.5–100 μmol / L) and y = 86.24logCl - 165.46 (R² = 0.996, 100–2000 μmol / L), as shown in the figure. Figure 13 (b) and Figure 13 The green line in (c) shows the results. For pyruvate, the linear regression models are y = 0.019Cp + 0.026 (R² = 0.999, 2.0–100 μmol / L) and y = 81.47logCp - 160.94 (R² = 0.991, 100–2000 μmol / L), as shown in Figure 1. Figure 13 (b) and Figure 13 The blue line in (c) shows the limit of detection (LOD). The LOD is calculated using formula 3STD / S, where S is the slope of the linear regression model and STD is the standard deviation of the blank sample. Therefore, the LOD for glucose is 0.16 μmol / L, for lactic acid is 0.15 μmol / L, and for pyruvate is 0.55 μmol / L.

[0070] Specifically, see Figure 14 The system's specificity tests confirmed the excellent detection selectivity of the dielectric valve microfluidic chip, such as... Figure 14 As shown in (a), the target analytes—glucose, lactate, and pyruvate, and mixtures thereof—exhibited significant chemiluminescent signals, while blank samples and non-target analytes showed almost no chemiluminescent signals. The effects of interfering substances (including ascorbic acid (AA), glutamate, maltose, uric acid (UA), potassium chloride (KCl), and sodium chloride (NaCl)) on metabolite detection were negligible. This indicates that the chemiluminescent biosensor integrated into the dielectric valve chip exhibits high selectivity, primarily attributed to the specific recognition of relevant GOx / LOx / POx enzymatic reactions and the physical isolation function of the dielectric valve reducing cross-reactivity between the sample and the pre-embedded enzyme.

[0071] In addition, the storage stability of the pre-loaded enzyme chip was evaluated, such as... Figure 14 As shown in (b), 21 μL samples (containing 1.0 mmol / L glucose, lactate, and pyruvate) were tested on days 1, 3, 5, 8, and 15. The study found that after 15 days of storage at 4˚C, the CL signal intensity for detecting 1 mmol / L glucose, lactate, and pyruvate remained at 97.4%, 95.3%, and 96.3% of the initial values, respectively. There were no significant changes in the CL signal intensity of any analyte during storage (P>0.05). These results demonstrate that this strategy of integrating biosensors with microfluidic chips not only exhibits good storage stability, making it suitable for mass production, remote transportation, and use, but also maintains high sensitivity to samples, providing crucial technical support for the practical application of POCT devices.

[0072] This invention also provides a computer device including a memory and a processor, wherein the processor performs the above-described method when executing a computer program stored in the memory.

[0073] This invention also provides a computer-readable storage medium storing program instructions thereon, which, when executed by a processor, implement the method described above.

[0074] It should be understood that the method steps in the embodiments of the present invention can be implemented or carried out by computer hardware, a combination of hardware and software, or by computer instructions stored in a non-transitory computer-readable storage medium. The method can use standard programming techniques. Each program can be implemented in a high-level procedural or object-oriented programming language to communicate with the computer system. However, if necessary, the program can be implemented in assembly or machine language. In any case, the language can be a compiled or interpreted language. Furthermore, for this purpose, the program can run on a programmed application-specific integrated circuit (ASIC).

[0075] Furthermore, the procedures described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The procedures described herein (or variations and / or combinations thereof) may be executed under the control of one or more computer systems configured with executable instructions, and may be implemented by hardware or a combination thereof as code (e.g., executable instructions, one or more computer programs, or one or more applications) that commonly executes on one or more processors. The computer program comprises a plurality of instructions executable by one or more processors.

[0076] Furthermore, the method can be implemented in any suitable type of computing platform, including but not limited to personal computers, minicomputers, mainframes, workstations, networked or distributed computing environments, standalone or integrated computer platforms, or in communication with charged particle tools or other imaging devices, etc. Aspects of the invention can be implemented as machine-readable code stored on a non-transitory storage medium or device, whether removable or integrated into a computing platform, such as a hard disk, optical read and / or write storage medium, RAM, ROM, etc., such that it is readable by a programmable computer, and when the storage medium or device is read by the computer, it can be used to configure and operate the computer to perform the processes described herein. Furthermore, the machine-readable code, or portions thereof, can be transmitted via wired or wireless networks. The invention described herein includes these and other different types of non-transitory computer-readable storage media when such media comprises instructions or programs that implement the steps described above in conjunction with a microprocessor or other data processor. When programmed according to the methods and techniques described in the invention, the invention may also include the computer itself.

[0077] A computer program can be applied to input data to perform the functions described herein, thereby transforming the input data to generate output data stored in non-volatile memory. The output information can also be applied to one or more output devices, such as a display. In a preferred embodiment of the invention, the transformed data represents physical and tangible objects, including specific visual depictions of physical and tangible objects generated on the display.

[0078] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention, as long as they achieve the technical effects of the present invention by the same means, should be included within the scope of protection of the present invention. Within the scope of protection of the present invention, the technical solutions and / or implementation methods can have various modifications and variations.

Claims

1. A dielectric wetting valve microfluidic chip for the detection of metabolic biomarkers, characterized in that, include: Lower layer of chip (100); A middle layer (200) is stacked on top of the lower layer (100); The upper chip layer (300) is stacked on top of the middle chip layer (200) in an inverted position; The chip middle layer (200) includes a double-sided adhesive layer (210) and a three-way microchannel (220). The double-sided adhesive layer (210) covers the three-way microchannel (220) around its perimeter. The three-way microchannel (220) includes a central region (221), a first branch channel (222), a second branch channel (223), and a third branch channel (224). The first branch channel (222), the second branch channel (223), and the third branch channel (224) are evenly distributed around the central region (221). The beginning ends of the first branch channel (222), the second branch channel (223), and the third branch channel (224) are respectively connected to the central region (221).

2. The dielectric wetting valve microfluidic chip for metabolic biomarker detection according to claim 1, characterized in that, The upper layer (300) of the chip includes a polydimethylsiloxane layer (310), a polyethylene terephthalate layer (320), a first silver electrode (330), and a second silver electrode (340). The first silver electrode (330) is stacked on top of the polyethylene terephthalate layer (320), and the second silver electrode (340) is stacked on top of the polyethylene terephthalate layer (320). The polydimethylsiloxane layer (310) covers the polyethylene terephthalate layer (320), the first silver electrode (330), and the second silver electrode (340).

3. The dielectric wetting valve microfluidic chip for metabolic biomarker detection according to claim 2, characterized in that, The first silver electrode (330) is horizontally disposed on the upper part of the polyethylene terephthalate layer (320). The number of the second silver electrodes (340) is two, the two second silver electrodes (340) are symmetrical to each other, and the two second silver electrodes (340) are vertically arranged at the lower part of the polyethylene terephthalate layer (320).

4. The dielectric wetting valve microfluidic chip for metabolic biomarker detection according to claim 1, characterized in that, The upper layer (300) of the chip includes an inlet hole (350) and an outlet hole (360), wherein the inlet hole (350) is stacked above the central region (221). The number of outlet holes (360) is three, and the three outlet holes (360) are respectively stacked above the tail end of the first branch channel (222), above the tail end of the second branch channel (223), and above the tail end of the third branch channel (224).

5. The dielectric wetting valve microfluidic chip for metabolic biomarker detection according to claim 2, characterized in that, The first branch channel (222), the second branch channel (223), and the third branch channel (224) are each provided with an enzyme reaction zone, a dielectric wetting valve, and a detection zone connected in sequence. One end of the enzyme reaction zone is connected to the inlet hole (350) through the central area (221), and one end of the detection zone is connected to the outlet hole (360). When the first silver electrode (330) or the second silver electrode (340) is energized, the dielectric wetting valve opens, so that the solution in the central area (221) and the enzyme reaction zone flows to the detection zone through the dielectric wetting valve, triggering a chemiluminescent reaction.

6. The dielectric wetting valve microfluidic chip for metabolic biomarker detection according to claim 5, characterized in that, The upper layer (300) of the chip is pre-positioned with a pre-embedded enzyme solution and a pre-embedded substrate solution. The pre-embedded enzyme solution is stacked on top of the polydimethylsiloxane layer (310), and the pre-embedded substrate solution is stacked on top of the polydimethylsiloxane layer (310). When the middle layer (200) of the chip and the inverted upper layer (300) of the chip are combined, the pre-embedded enzyme solution is placed inside the enzyme reaction area, and the pre-embedded substrate solution is placed inside the detection area. The pre-embedded substrate solution is used for the chemiluminescent detection of metabolic markers.

7. The dielectric wetting valve microfluidic chip for metabolic biomarker detection according to claim 5, characterized in that, One end of the first silver electrode (330) is stacked above the dielectric wetting valve, and one end of the second silver electrode (340) is stacked above the dielectric wetting valve.

8. The dielectric wetting valve microfluidic chip for metabolic biomarker detection according to claim 5, characterized in that, The lower layer (100) of the chip includes an indium tin oxide electrode (110) and a glass layer (120), with the indium tin oxide electrode (110) stacked on top of the glass layer (120).

9. The dielectric wetting valve microfluidic chip for metabolic biomarker detection according to claim 8, characterized in that, The dielectric wetting valve is composed of the indium tin oxide electrode (110) and the polydimethylsiloxane layer (310).

10. A method for fabricating a dielectric wetting valve microfluidic chip for metabolic biomarker detection, applied to the dielectric wetting valve microfluidic chip for metabolic biomarker detection as described in claim 8, characterized in that, Includes the following steps: S100, Laser etching is performed on the indium tin oxide glass substrate to obtain a glass layer (120) and an indium tin oxide electrode (110) protruding on the surface of the glass layer (120) to obtain a chip lower layer (100). The chip lower layer (100) is immersed in acetone, and the chip lower layer (100) is rinsed with ethanol and deionized water in sequence to remove residual solvent. The chip lower layer (100) is dried. S200. A three-way microchannel (220) is formed by cutting with double-sided tape to obtain the middle layer (200) of the chip, wherein the three-way microchannel (220) includes a central region (221), a first branch channel (222), a second branch channel (223) and a third branch channel (224). S300: Using conductive silver paste, screen print a first silver electrode (330) and a second silver electrode (340) located at different positions on a polyethylene terephthalate layer (320). Mix a polydimethylsiloxane prepolymer with a curing agent at a mass ratio of 10:1 to obtain a polydimethylsiloxane layer (310). Spin coat the polydimethylsiloxane layer (310) over the polyethylene terephthalate layer (320), the first silver electrode (330), and the second silver electrode (340) and perform a curing process to obtain the initial upper layer of the chip. S400: A hole (350) and three outlet holes (360) are formed on the initial upper layer of the chip. The inlet hole (350) is connected to the central region (221). The three outlet holes (360) are respectively connected to the tail end of the first branch channel (222), the tail end of the second branch channel (223), and the tail end of the third branch channel (224). A pre-embedded enzyme solution is placed between one end of the first silver electrode (330) and the inlet hole (350). The pre-embedded enzyme solution is placed between one end of the second silver electrode (340) and the inlet hole (350). A pre-embedded substrate solution is placed between one end of the first silver electrode (330) and the outlet hole (360). The pre-embedded substrate solution is placed between one end of the second silver electrode (340) and the outlet hole (360) to obtain the upper layer (300) of the chip. The pre-embedded substrate solution is used for the chemiluminescence detection of metabolic markers. S500, the inverted upper layer (300) of the chip is aligned and bonded to the middle layer (200) and the lower layer (100) of the chip in sequence to obtain a microfluidic chip with a dielectric wetting valve, so that when the first silver electrode (330) or the second silver electrode (340) is energized, the dielectric wetting valve opens, thereby the solution in the central region (221) and the enzyme reaction region flows to the detection region through the dielectric wetting valve, triggering a chemiluminescent reaction.

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