A multi-target cancer detection chip based on dielectric wetting technology

Abstract

The present application relates to the technical field of microfluidic biological detection, and particularly relates to a multi-target cancer detection chip based on dielectric wetting technology, which adopts a double-plate EWOD architecture, forms a sealed chamber through a gasket and fills the chamber with silicone oil, sets up a five-target three-inlet structure (sample, gold nanoparticles, microcapsule inlet), integrates a 532nm LED light source to induce surface plasmon resonance (SPR), and realizes detection in combination with a visualization module. The core innovation lies in that: the five-target parallel processing improves the throughput, the LED-SPR triggers the release of biotin from the microcapsule to enhance the signal, and the lateral flow strip develops color through the biotin-streptavidin effect. The chip solves the problems of droplet evaporation, pollution and low-efficiency mixing in the existing EWOD system, supports high-temperature processes such as DNA denaturation, can precisely control droplets to complete sample preparation, mixing and detection, realizes high-sensitivity and intuitive detection of multi-target cancer markers, and is suitable for integration into a lab-on-a-chip (LOC) system.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic bioassay technology, and in particular to a multi-target cancer detection chip based on dielectric wetting technology. Background Art

[0002] EWOD (electrowetting on dielectric) technology is widely used in biochemical and medical research. Its versatility and reconfigurability address key challenges in the development of lab-on-a-chip (LOC) technology. EWOD-based digital microfluidic (DMF) devices offer precise control of small volumes of liquid without the need for micropumps, microvalves, or complex three-dimensional fluidic channels. This simplifies device design, enables dynamic reconfiguration, and allows integration with external systems such as optical, electronic, and magnetic systems.

[0003] Over the past decade, DMF technology has driven progress in the field of biomolecular analysis, including nucleic acid, protein, and hormone research, as well as cell culture, screening, and single-cell analysis. However, existing EWOD-based microfluidic systems still have problems in practical applications, such as droplet evaporation leading to sample concentration drift, high risk of cross-contamination during multi-target detection, and low droplet mixing efficiency under traditional electrode design. These problems limit their application in high-precision biomarker detection and high-throughput analysis scenarios. Summary of the Invention

[0004] One object of the present invention is to provide a multi-target cancer detection chip based on dielectric wetting technology to solve the problems of droplet evaporation, contamination and inefficient mixing.

[0005] To achieve the above objectives, an embodiment of the present invention provides a multi-target cancer detection chip based on dielectric wetting, which uses a digital microfluidic system based on dielectric wetting technology, including: a lower plate of a dual-plate EWOD architecture, a first upper plate, and a second upper plate, wherein the lower plate and the first and second upper plates are separated by a gasket to form a sealed chamber, and the sealed chamber is filled with a transmission medium;

[0006] The chip settings are:

[0007] Five-target three-inlet structure, processing five independent targets, each target includes a sample inlet, a gold nanoparticle inlet and a microcapsule droplet inlet;

[0008] LED light source integrated module, using a 532nm wavelength LED light source to induce the surface plasmon resonance effect of gold nanoparticles;

[0009] The visualization module receives the processed droplets and visualizes the results through biotin-streptavidin interaction.

[0010] In the above technical solution, a driving electrode is provided on the lower plate, a plurality of connection pads are arranged on both sides of the driving electrode, a first SU-8 photoresist is coated on the driving electrode, a first polytetrafluoroethylene plate is stacked on the first SU-8 photoresist, and a spacer layer is connected to both sides of the upper surface of the first polytetrafluoroethylene plate;

[0011] A second polytetrafluoroethylene plate, a first indium tin oxide plate, a first upper plate and a heater are stacked in sequence on one spacer layer, and a second SU-8 photoresist is wrapped on the heater; a second indium tin oxide plate and a second upper plate are stacked in sequence on another spacer layer, and the second indium tin oxide plate and the corresponding detection layer are connected by conductive glue; a third polytetrafluoroethylene plate is connected to one side of the conductive glue.

[0012] In the above technical solution, the thickness of the gasket is 200-333μm, the area of ​​the connection pad and the driving electrode is 1.905mm², and the driving electrode interval is 35μm.

[0013] In the above technical solution, the lower plate is provided with a first mixing point and a second mixing point, and the LED light source integrated module is fixed directly above the first mixing point and the second mixing point by a bracket. The LED light source with a wavelength of 532nm is used, the distance between the light source and the droplet surface is 3mm, the light source power is 0.052W, and the power density is 2.6W / cm 2 .

[0014] In the above technical solution, the lower plate is provided with a first mixing point and a second mixing point. The LED light source integrated module is fixed directly above the first mixing point and the second mixing point by a bracket. A 532nm wavelength LED light source is used. The distance between the light source and the droplet surface is 4mm. The light source power is 0.052W, and the power density is 1.7W / cm 2 .

[0015] In the above technical solution, the three inlets of each target in the five-target three-inlet structure are controlled by an independent electrode control unit for droplet injection. The three inlets are used to introduce DNA sample droplets, functionalized gold nanoparticle droplets and microcapsule droplets, respectively. The droplets at each inlet are merged in the mixing area under the drive of the electric field.

[0016] In the above technical solution, the visualization module contains five independent lateral flow strips, each of which corresponds to a target. The treated droplets are transported to the lateral flow strip detection area under the drive of the electric field, and a visible signal is generated through the biotin-streptavidin interaction.

[0017] In the above technical solution, the chip integrated heater size is 8.75mm 2 ,The heater works with the temperature control module to heat the droplets to the specified temperature to achieve DNA denaturation and keep the temperature stable.

[0018] In the above technical solution, the transmission medium is dimethyl silicone oil, which is injected through the injection hole of the lower plate and then sealed.

[0019] In the above technical solution, when the LED light source induces the surface plasmon resonance effect, it triggers the microcapsule to release biotin, and the biotin combines with the streptavidin on the lateral flow strip to form a visible signal.

[0020] Compared with the prior art, the present invention has the following significant advantages:

[0021] 1. The five-target, three-inlet independent control structure can process multiple detection channels simultaneously, significantly improving sample processing efficiency and enabling simultaneous analysis of multiple targets. The droplet paths of each target are isolated, effectively reducing the risk of cross-contamination.

[0022] 2. Integrating LED-induced surface plasmon resonance technology enhances signal amplification through the resonance effect of gold nanoparticles, significantly improving the detection sensitivity of target DNA sequences and achieving highly specific detection without complex labeling;

[0023] 3. The dual-plate structure is filled with silicone oil as a transmission medium, effectively reducing droplet evaporation and biomolecule adsorption. Combined with the independent channel design, it significantly reduces the risk of sample concentration drift and cross-contamination, ensuring the stability of test results.

[0024] 4. The asymmetric electrode design improves droplet mixing efficiency and shortens reaction time. The integrated heater supports high-temperature processes such as DNA denaturation, solving the problems of inefficient mixing and insufficient temperature control in existing systems, thereby achieving efficient mixing and high-temperature compatibility.

[0025] 5. The lateral flow strip integrates a biotin-streptavidin colorimetric system, enabling visual interpretation of test results without the need for specialized instruments, simplifying the operating process and reducing testing costs, making it suitable for rapid on-site diagnosis.

[0026] 6. The chip highly integrates droplet manipulation, reaction, signal amplification and detection functions, supports docking with external automation systems, and facilitates the construction of an integrated diagnostic platform to meet the expansion needs of high-throughput clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0028] Figure 1 This is a schematic diagram of the configuration of the first upper plate, the second upper plate and the lower plate layer by layer according to the present invention;

[0029] Figure 2 Schematic diagram of the chip of the present invention;

[0030] in, Figures 1 to 2The corresponding relationship between the reference numerals and component names is as follows:

[0031] 1. Connection pad; 2. Sample inlet; 3. Gold nanoparticle inlet; 4. Microcapsule droplet inlet; 5. Heater; 6. Drive electrode; 7. First mixing point; 8. Second mixing point; 9. Lateral flow strip; 10. First SU-8 photoresist; 11. Second SU-8 photoresist; 12. First polytetrafluoroethylene plate; 13. Second polytetrafluoroethylene plate; 14. Spacer layer; 15. First indium tin oxide plate; 16. Second indium tin oxide plate; 17. First upper plate; 18. Second upper plate; 19. Lower plate; 20. Conductive adhesive; 21. Third polytetrafluoroethylene plate. DETAILED DESCRIPTION

[0032] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the specific embodiments disclosed below.

[0034] like Figure 1 and Figure 2 As shown, an embodiment of the present invention provides a multi-target cancer detection chip based on dielectric wetting technology, which adopts a digital microfluidic system based on dielectric wetting technology, including: a lower plate 19, a first upper plate 17 and a second upper plate 18 of a double-plate EWOD architecture, the lower plate 19 and the first upper plate 17 and the second upper plate 18 are separated by a gasket to form a sealed chamber, and the sealed chamber is filled with a transmission medium.

[0035] In one embodiment, the chip is provided with:

[0036] The five-target three-inlet structure processes five independent targets. Each target includes a sample inlet 2, a gold nanoparticle inlet 3, and a microcapsule droplet inlet 4. Each target can independently introduce samples, introduce gold nanoparticles and microcapsules, and carry out subsequent reaction processes.

[0037] In one embodiment, independent control of each target allows for simultaneous analysis of multiple different samples or different targets in the same sample without mutual interference.

[0038] In one embodiment, during the DNA detection process, the sample, GNPs, and microcapsules are mixed in a specific area, and the droplets are manipulated by an electric field to allow them to fully contact and react to achieve the detection of the target DNA sequence.

[0039] The LED light source integrated module uses a 532nm wavelength LED light source to induce the surface plasmon resonance effect of gold nanoparticles. This resonance phenomenon can change the local environment around the gold nanoparticles. The energy changes or physical field changes generated by the resonance affect the structure of the microcapsules, triggering the microcapsules to release biotin to enhance signal amplification.

[0040] The visualization module receives the processed droplets and visualizes the results through biotin-streptavidin interaction.

[0041] In one embodiment, a driving electrode 6 is provided on the lower plate 19, and a plurality of connection pads 1 are arranged on both sides of the driving electrode 6. The driving electrode 6 is wrapped with a first SU-8 photoresist 10, and a first polytetrafluoroethylene plate 12 is stacked on the first SU-8 photoresist 10. Spacer layers 14 are connected to both sides of the upper surface of the first polytetrafluoroethylene plate 12; a second polytetrafluoroethylene plate 13, a first indium tin oxide plate 15, a first upper plate 17 and a heater 5 are stacked in sequence on one spacer layer 14, and the heater 5 is wrapped with a second SU-8 photoresist 11; a second indium tin oxide plate 16 and a second upper plate 18 are stacked in sequence on the other spacer layer 14, and the second indium tin oxide plate 16 and the corresponding detection layer are connected by a conductive glue 20; and a third polytetrafluoroethylene plate 21 is connected to one side of the conductive glue 20.

[0042] Specifically, the lower plate 19 is separated from the first upper plate 17 and the second upper plate 18 by a gasket to form a sealed chamber and filled with silicone oil. This stacking method can stably and accurately control the droplets. The sealed chamber formed by the gasket is filled with silicone oil. Silicone oil can reduce the surface tension of the droplets, so that the droplets can move and react stably under the action of the electric field, while reducing the contact area between the droplets and the outside world, reducing the evaporation rate, avoiding sample concentration drift, and maintaining detection accuracy. At the same time, the dual-plate structure and the sealed chamber isolate the droplet paths of each target. The three inlets of each target in the five-target three-inlet structure are controlled by an independent electrode control unit for droplet injection, which do not interfere with each other and reduce the risk of cross-contamination during multi-target detection.

[0043] In one embodiment, wetting points are provided on the lower plate 19, which cooperate with the driving electrodes to realize the manipulation and movement of DNA sample droplets, functionalized gold nanoparticle droplets and microcapsule droplets. At the same time, the wetting points help reduce the evaporation and contamination of the above droplets during the movement and reaction process.

[0044] The wetting point is the area where the droplet contacts the electrode under the action of the electric field. Through the wetting point, the accuracy and efficiency of the above-mentioned droplet manipulation can be significantly improved, ensuring that they can accurately mix and react at the specified location.

[0045] Specifically, a gasket with a thickness of 200-333μm is used to press the first upper plate 17, the second upper plate 18 and the lower plate 19 together to form a sealed chamber. The electrode layer of the lower plate 19 is produced by a photolithography process. The area of ​​the connecting pad 1 and the driving electrode 6 are both 1.905mm², with a spacing of 35μm. Dimethyl silicone oil is filled in the chamber to reduce the surface tension by using silicone oil, thereby achieving stable control of the droplets while reducing evaporation.

[0046] In one embodiment, a 333 μm thick gasket is selected to increase the gap between the first upper plate 17, the second upper plate 18 and the lower plate 19, thereby reducing the electric field strength, making it more suitable for low-voltage driving scenarios and effectively reducing droplet evaporation.

[0047] In one embodiment, each target site is equipped with an independent sample inlet 2, gold nanoparticle (GNP) inlet, and microcapsule droplet inlet 4. The droplet injection is driven by an electrode control unit. For example, the sample inlet 2 introduces DNA sample droplets, the GNP inlet introduces functionalized GNP droplets, and the microcapsule droplet inlet 4 introduces biotin-containing microcapsule droplets. Driven by the electric field, the droplets merge in the mixing zone.

[0048] In one embodiment, an LED is positioned to illuminate the mixing point where the sample GNPs and microcapsules combine. When a mixed droplet (containing the sample, GNPs, and microcapsules) is at this mixing point, the LED emits 532nm wavelength light to illuminate the mixed droplet. This light induces surface plasmon resonance of the gold nanoparticles in the mixed droplet, triggering the release of biotin from the microcapsules to enhance signal amplification.

[0049] In one embodiment, a first mixing point 7 and a second mixing point 8 are provided on the lower plate 19, and a 532nm wavelength LED is integrated directly above the first mixing point 7 and the second mixing point 8, 3mm or 4mm away from the droplet surface, with a power of 0.052W, to induce the SPR effect of gold nanoparticles.

[0050] The first mixing point 7 and the second mixing point 8 are both arranged on the lower plate 19, so as to achieve precise control, mixing and reaction of the droplets, and work in coordination with other structures on the lower plate 19 (such as the driving electrode 6, the wetting point, etc.).

[0051] In one embodiment, the first mixing point 7 is a mixing point between DNA and GNPs, and the second mixing point 8 is a mixing point between the preceding mixture and the microcapsules.

[0052] Specifically, the distance between the light source and the droplet surface is 3 mm, the light source power is 0.052 W, and the power density is 2.6 W / cm 2 The distance between the light source and the droplet surface is 4 mm, the light source power is 0.052 W, and the power density is 1.7 W / cm 2 .

[0053] In one embodiment, five independent lateral flow strips 9 correspond to five target spots, and the droplets are transported to the detection area of ​​the lateral flow strip 9 under the drive of the electric field, and are colored through the biotin-streptavidin reaction.

[0054] In one embodiment, when five targets were processed in parallel, each target was sequentially introduced into a droplet through three inlets: sample inlet 2 was injected with a DNA buffer, the GNP inlet was injected with a functionalized GNP solution, and the microcapsule droplet inlet 4 was injected with a PBS solution containing biotin microcapsules. Electrodes then moved the droplets to a mixing zone, achieving simultaneous multi-target reactions.

[0055] In one embodiment, when a single target is processed independently, the other four target electrodes are turned off and only the single target is operated to reduce electric field interference, which is suitable for trace sample detection.

[0056] In one embodiment, the droplet moves to the top of heater 5 (area 8.75 mm², length 130 mm, voltage 5 V), is heated to 95°C and maintained for a period of time to achieve complete denaturation of the DNA.

[0057] In one embodiment, an LED illuminates the mixed droplets, inducing a surface plasmon resonance (SPR) effect, triggering the release of biotin from the microcapsules and enhancing the detection signal.

[0058] In one embodiment, each target site has a corresponding lateral flow strip 9. The treated droplets enter the corresponding lateral flow strip 9 under the drive of the electric field. Biotin binds to streptavidin to generate a visible signal, and the result is interpreted by observation or image recognition.

[0059] In one embodiment, silicone oil is injected through the injection hole of the lower plate 19. After the injection hole is sealed, the silicone oil covers the surface of the droplet, reducing evaporation and contamination.

[0060] In the combined detection of multiple cancer markers, the parallel processing capability of five targets is utilized to simultaneously detect multiple gene mutations, significantly shortening the entire detection process time.

[0061] In single-cell DNA analysis, a single-target mode is used to process single-cell DNA, combined with LED-SPR signal amplification to provide targeted guidance for subsequent sequencing.

[0062] Compared to existing technologies, the five-target design increases throughput, while independent control of each target enhances accuracy. The application of LED-SPR technology enhances signal amplification, and through the specific biotin-streptavidin interaction, the test results are clearly displayed on the lateral flow strip 9. This design enables the entire system to achieve highly sensitive, specific, rapid, and intuitive multi-target cancer detection, which is difficult to achieve with existing technologies.

[0063] In the description of the application, it should be understood that the terms "inner", "outer", and the like indicate the positional or location relationship based on the positional or location relationship shown in the drawings, and are only for the convenience of describing the application and simplifying the description, and do not indicate or imply that the device or unit referred to must have a specific direction, be constructed and operated in a specific position, therefore, it cannot be understood as a limitation on the application.

[0064] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "a specific embodiment" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In the present application, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0065] The above is only the preferred embodiment of the present application, and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A multi-target cancer detection chip based on dielectric wetting technology, characterized in that: A digital microfluidic system using dielectric wetting technology comprises: a lower plate (19) of a double-plate EWOD structure, a first upper plate (17) and a second upper plate (18), wherein the lower plate (19) is separated from the first upper plate (17) and the second upper plate (18) by a gasket to form a sealed chamber, wherein the sealed chamber is filled with a transmission medium; The chip is provided with: Five-target three-inlet structure, processing five independent targets, each target includes a sample inlet (2), a gold nanoparticle inlet (3) and a microcapsule droplet inlet (4); The LED light source integrated module uses a 532nm wavelength LED light source to induce the surface plasmon resonance effect of gold nanoparticles and trigger the release of biotin from the microcapsules. Biotin binds to the streptavidin on the lateral flow strip (9) to form a visible signal; A visualization module receives the processed droplets and realizes visualization of the results through biotin-streptavidin interaction, wherein the visualization module comprises five independent lateral flow strips (9), each of which corresponds to a target site, and the processed droplets are transported to the detection area of ​​the lateral flow strips (9) under the driving of the electric field; The lower plate (19) is provided with a driving electrode (6), and the lower plate (19) is provided with a first mixing point (7) and a second mixing point (8). The LED light source integrated module is fixed directly above the first mixing point (7) and the second mixing point (8) through a bracket. The first mixing point (7) is a mixing point of the sample and the gold nanoparticles, and the second mixing point (8) is a mixing point of the pre-sequence mixture and the microcapsules.

2. The detection chip according to claim 1, characterized in that A plurality of connection pads (1) are respectively arranged on both sides of the driving electrode (6); a first SU-8 photoresist (10) is wrapped on the driving electrode (6); a first polytetrafluoroethylene plate (12) is stacked on the first SU-8 photoresist (10); and a spacer layer (14) is respectively connected to both sides of the upper surface of the first polytetrafluoroethylene plate (12); A second polytetrafluoroethylene plate (13), a first indium tin oxide plate (15), a first upper plate (17) and a heater (5) are sequentially stacked on a spacer layer (14), and a second SU-8 photoresist (11) is wrapped on the heater (5); a second indium tin oxide plate (16) and a second upper plate (18) are sequentially stacked on another spacer layer (14), and the second indium tin oxide plate (16) and the corresponding detection layer are connected via a conductive adhesive (20); and a third polytetrafluoroethylene plate (21) is connected to one side of the conductive adhesive (20).

3. The detection chip according to claim 2, characterized in that: The thickness of the gasket is 200-333 μm, the area of ​​the connection pad (1) and the driving electrode (6) are both 1.905 mm², and the driving electrodes (6) are spaced 35 μm apart.

4. The detection chip according to claim 1, characterized in that The distance between the light source and the droplet surface is 3 mm, the light source power is 0.052 W, and the power density is 2.6 W / cm 2 .

5. The detection chip according to claim 1, characterized in that: The distance between the light source and the droplet surface is 4 mm, the light source power is 0.052 W, and the power density is 1.7 W / cm 2 .

6. The detection chip according to claim 1, characterized in that: The three inlets of each target in the five-target three-inlet structure are controlled by an independent electrode control unit for droplet injection. The three inlets are used to introduce DNA sample droplets, functionalized gold nanoparticle droplets and microcapsule droplets, respectively. The droplets at each inlet are merged in the mixing area under the drive of the electric field.

7. The detection chip according to claim 2, characterized in that: The size of the heater (5) is 8.75mm 2 The heater (5) cooperates with the temperature control module to heat the droplet to a specified temperature to achieve DNA denaturation and maintain a stable temperature.

8. The detection chip according to claim 1, characterized in that: The transmission medium is dimethyl silicone oil, which is injected through the injection hole of the lower plate (19) and then sealed.

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