A micro-sensing device based on interdigital electrodes and local hydrophilic dots

Abstract

The application belongs to the technical field of microfluidics, and provides a micro-sensing device based on interdigital electrodes and local hydrophilic points. A fluid chamber for accommodating liquid drops and filler fluid is defined between a first substrate and a second substrate, the first substrate is sequentially covered with an electrode layer and a first liquid-repellent layer, the electrode layer comprises interdigital electrodes and driving electrodes, the interdigital electrodes are located between two adjacent driving electrodes or are arranged adjacent to the driving electrodes; the microfluidic chip further comprises a micro well formed in an upper region of the interdigital electrodes of the first substrate, the micro well is a pit structure of a side of the first substrate facing the fluid chamber; the area of the first substrate other than the micro well maintains the liquid-repellent property of the liquid-repellent layer; the upper surface of the interdigital electrodes forms a convex step-shaped structure at the bottom of the micro well. The above structure realizes passive distribution dominated by the surface tension of the liquid drop and in-situ high-sensitivity electrochemical detection, reduces sample consumption, and can construct a vertical three-electrode system through an auxiliary electrode to improve detection accuracy.

Description

Technical Field

[0001] This invention relates to the field of microfluidics and provides a microsensing device based on interdigital electrodes and local hydrophilic points. Background Technology

[0002] Currently, microfluidic chips are mainly divided into two types: channel-based and droplet-based. Droplet-based microfluidic chips, also known as digital microfluidic chips, are a novel microfluidic manipulation platform developed in recent years that utilizes the electrowetting effect to drive droplet movement. Digital microfluidic chips use discrete droplets as functional carriers for fluid transport and processing (such as dilution, splitting, mixing, extraction, and reaction). These droplets, ranging in volume from picoliters to microliters, can each function as an independent reaction system. Digital microfluidic technology includes droplet generation, transport, merging, and splitting, and utilizes these basic operations to achieve specific purposes. Unlike continuously manipulating microfluidics in closed microchannels, digital microfluidic chips primarily control the on / off state of a series of electrodes to manipulate microdroplets on the chip. The channel exists only in a virtual sense; it can be updated and reset instantly through program updates, offering greater flexibility. In addition to the advantages of channel-based microfluidic chips, digital microfluidics also boasts advantages such as simple structure, high tolerance to manufacturing and drive control errors, and ease of fluid manipulation, making it a promising application in clinical testing, especially in the development of portable testing devices. Prior art US20210322973A1, published on October 21, 2021, provides a microfluidic device comprising: an upper substrate and a lower substrate spaced apart to define a fluid chamber between the upper and lower substrates; an orifice for introducing fluid into the fluid chamber; and a fluid input structure disposed above the upper substrate and having a fluid well for receiving fluid from a fluid applicator inserted into the fluid well. The fluid well communicates with a fluid outlet disposed in the base of the fluid input structure, the fluid outlet being adjacent to the orifice. The fluid well includes a first portion, a second portion, and a third portion, wherein the first portion of the well forms a reservoir for filling fluid; and the second portion of the well is configured to sealably abut against the outer surface of the fluid applicator inserted into the fluid well. The third portion of the well communicates with the fluid outlet, and the diameter of the third portion at the interface between the third portion and the second portion is larger than the diameter of the second portion at the interface between the third portion and the second portion. Summary of the Invention

[0003] This invention employs a micro-well pit structure and interdigitated electrodes to achieve passive distribution of droplets dominated by surface tension and in-situ high-sensitivity electrochemical detection, reducing sample consumption. Furthermore, it can improve detection accuracy by constructing a vertical three-electrode system using auxiliary electrodes.

[0004] A microsensing device based on interdigitated electrodes and local hydrophilic points includes a microfluidic chip substrate. The microfluidic chip substrate includes a first substrate and a second substrate disposed opposite to each other, defining a fluid chamber for containing droplets and filler fluid between the first substrate and the second substrate. An electrode layer, a dielectric layer, and a first hydrophobic layer are sequentially covered on the first substrate. The electrode layer includes interdigitated electrodes and driving electrodes. The interdigitated electrodes are located between or adjacent to two adjacent driving electrodes. The microfluidic chip substrate also includes microwells formed in the region above the interdigitated electrodes of the first substrate. The dielectric layer and the first hydrophobic layer in the region are removed, so that the surface of the interdigitated electrodes is directly exposed to the fluid chamber. The microwell is a pit structure on the side of the first substrate facing the fluid chamber. The region on the first substrate other than the microwell retains the hydrophobicity of the hydrophobic layer. The upper surface of the interdigitated electrodes forms a raised stepped structure at the bottom of the microwell.

[0005] The microfluidic chip substrate employs a dual-substrate design. An electrode layer and a first hydrophobic layer are sequentially formed on the first substrate (the lower substrate). The electrode layer contains two types of electrodes: driving electrodes for electrowetting and interdigitated electrodes for sensing. Both are located on the same plane, with the interdigitated electrodes positioned between or adjacent to two adjacent driving electrodes, facilitating direct droplet access to the interdigitated electrode area under the control of the driving electrodes. The first hydrophobic layer directly above the interdigitated electrodes is partially removed, forming a microwell—a downwardly recessed three-dimensional pit structure. After removing the hydrophobic layer, the surface of the interdigitated electrodes is directly exposed to the fluid chamber, while the area outside the microwell remains hydrophobic. The upper surface of the interdigitated electrodes forms a raised, stepped structure at the bottom of the microwell relative to the surrounding dielectric layer or the surface of the first substrate.

[0006] The microwell of this invention passively confines droplets within the electrode region through a recessed structure, while the raised interdigitated electrodes increase the effective sensing area at the bottom of the microwell. This ensures that even sub-droplets on the picoliter scale can be monitored by the interdigitated electrodes, avoiding detection failures caused by droplets not covering the electrodes. Simultaneously, the recessed microwell and the raised electrodes together enhance the pinning effect at the bottom of the microwell, making it easier for sub-droplets to automatically remain under the combined forces of surface tension and other factors.

[0007] Preferably, the second substrate has a common electrode and a second hydrophobic layer on the side facing the fluid chamber, and the distance between the first and second hydrophobic layers is 100 μm to 200 μm. This height is beneficial for the rapid passage of droplets and passive distribution at the microwell. Compared to a smaller distance, when the droplet resides at the microwell, the upper and lower hydrophobic layers will not excessively compress the droplet, allowing sub-droplets to form naturally based on surface tension. Compared to a larger distance, it avoids excessively high driving voltage, excessively fast droplet movement speed, and deformation instability of the droplet due to gravity during movement.

[0008] Preferably, the depth of the microwell is 100 nm to 5 μm, and the diameter of the microwell is 1 μm to 50 μm. The geometry of the microwell is such that a droplet passing through the microwell can be passively distributed and retained in the microwell by surface tension effect, with the sub-droplet in direct contact with the exposed surface of the interdigitated electrode. When the driving electrode pushes the parent droplet through the microwell, because the exposed surface of the interdigitated electrode inside the microwell is hydrophilic, while the first hydrophobic layer outside the microwell is hydrophobic, under the action of the surface tension gradient, a portion of the liquid remains in the three-dimensional depression of the microwell, forming a sub-droplet.

[0009] Since the depth and diameter of the microwell are much smaller than the distance between the upper and lower substrates and the characteristic size of the mother droplet, when the mother droplet moves above the microwell, the electrowetting driving force and fluid resistance of its main body are almost unaffected by the micro-pit, thus ensuring the stability of global control. This means that the pitted structure of the microwell will not significantly affect the global control of the droplet by the driving electrode, and the driving electrode can still effectively attract the droplet across the microwell region.

[0010] The above setup achieves surface tension-driven passive distribution, enabling precise separation of micro-quantum droplets from the mother droplet without the need for an external electric field or mechanical valve. Compared to traditional digital microfluidics that uses electrowetting for droplet splitting, this invention's passive distribution is entirely driven by surface tension, offering high repeatability and volume consistency. The daughter droplets are significantly smaller than the mother droplet, resulting in a substantial reduction in sample consumption—an advantage particularly pronounced for precious biological samples such as rare cells and trace amounts of serum.

[0011] Preferably, the interdigitated electrodes and the driving electrodes are located in the same plane, and the interdigitated electrodes have a finger spacing of 100 nm to 1 μm.

[0012] Preferably, the surface of the interdigitated electrode has a modification layer or molecular film, which specifically binds to the analyte in the subdroplet. This functionalization upgrades the microsensor of the present invention from general conductivity detection to specific biomolecule detection, greatly expanding its application range. Unmodified electrodes can only measure the bulk properties of the solution, such as the increased ion concentration in the subdroplet after spore germination, while functionalized electrodes can identify specific biomarkers, enabling the monitoring of markers at different stages of spore germination.

[0013] Preferably, the interdigitated electrode can be configured with a first electrical signal or a second electrical signal, wherein the first electrical signal achieves droplet splitting at the microwell through the dielectrophoresis effect; The second electrical signal can perform electrochemical detection of droplets in the microwell.

[0014] Preferably, at least one auxiliary electrode is disposed on the second substrate in the region directly opposite the microwell. The auxiliary electrode is insulated from the common electrode and is exposed to the fluid chamber.

[0015] Preferably, the auxiliary electrode, the interdigitated electrode, and the common electrode constitute a three-electrode electrochemical detection system; the interdigitated electrode serves as the working electrode, the auxiliary electrode serves as the counter electrode or reference electrode, and the common electrode on the second substrate serves as the remaining reference electrode or counter electrode.

[0016] In existing technologies, the common electrode of the second substrate serves only as a reference electrode and cannot participate in local electrochemical measurements. The auxiliary electrode added to the second substrate can act as a counter electrode or reference electrode in electrochemical detection, forming a vertical three-electrode system with the interdigitated electrodes of the lower substrate. Compared to impedance measurement relying solely on the interdigitated electrodes of the first substrate, the introduction of the auxiliary electrode can significantly reduce electrode polarization effects, improving the accuracy and reproducibility of detection. For example, in cyclic voltammetry detection, the auxiliary electrode provides a stable current loop, making the potential scan of the working electrode more precise.

[0017] A method for monitoring spore germination, employing the aforementioned microsensor device based on interdigitated electrodes and local hydrophilic points, includes the following sequential steps: A spore suspension containing ungerminated spores is dripped into the fluid chamber of the microsensor device, and the droplets of the spore suspension are driven to the micro well by electrowetting, so that the spores are deposited into the micro well. The droplets of the spore suspension are driven away from the microwell by electrowetting, leaving the sub-droplets at the microwell; Germination agent is dripped into the fluid chamber of the microsensor and driven to the micro well by electrowetting, where it exchanges substances with the sub-droplets, inducing the spores to transform into a germination state. The interdigitated electrodes are used to monitor the changes in the conductivity of the subdroplets and to monitor the spore germination process.

[0018] Preferably, the droplets of the spore suspension remain at the microwell for 10 to 14 hours, which is sufficient time for the spores to deposit onto the interdigitated electrode surface at the bottom of the microwell.

[0019] This invention has the following outstanding technical effects: the combination of the three-dimensional pit structure of the microwell and the stepped protrusions of the interdigitated electrode allows the mother droplet to automatically retain sub-droplets ranging from picoliters to nanoliters under the action of surface tension, achieving passive distribution without external electric field or mechanical valve. This process requires no external electric field or mechanical valve, has high consistency in distribution volume, and low sample consumption, making it suitable for the analysis of precious samples such as rare cells and trace amounts of serum. The interdigitated electrode is directly exposed to the fluid chamber, forming direct contact with the sub-droplets, which improves sensitivity by several orders of magnitude compared to non-contact capacitive detection covered with a liquefactive layer. An auxiliary electrode is added on the second substrate in the area directly opposite the microwell, forming a vertical three-electrode electrochemical detection system with the interdigitated electrode and common electrode on the first substrate, improving the accuracy and reproducibility of measurements such as cyclic voltammetry. Attached Figure Description

[0020] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of a microsensing device based on interdigital electrodes and local hydrophilic points.

[0022] Figure 2 This is a cross-sectional view of the fluid chamber of the present invention.

[0023] Figure 3 This is a schematic diagram of the global structure of the microfluidic chip according to Embodiment 1 of the present invention.

[0024] Figure 4 For the present invention Figure 3 A magnified view of a portion of the image.

[0025] Figure 5 This is a schematic diagram of the operation of the micro-well-retaining droplet of the present invention.

[0026] Figure 6 This is a schematic diagram illustrating the operation of droplet mass exchange at the microwell of the present invention.

[0027] Figure 7 This is a schematic diagram of the overall structure of the microfluidic chip according to Embodiment 2 of the present invention.

[0028] Figure 8 For the present invention Figure 7 A magnified view of a portion of the image.

[0029] Legend: 1. Driving electrode; 2. Interdigitated electrode; 3. Microwell; 4. First hydrophobic layer; 5. Common electrode; 6. Spore suspension; 7. Spore; 8. Germination agent; 9. Polished glass; 10. Microfluidic chip substrate; 11. First substrate; 12. Second substrate; 13. Fluid chamber; 14. Droplet port. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0031] Example 1 like Figure 1 As shown, a microsensing device based on interdigitated electrodes 2 and local hydrophilic points includes a microfluidic chip substrate 10, wherein the microfluidic chip substrate 10 includes as follows: Figure 2 The first substrate 11 and the second substrate 12 are arranged opposite to each other, and a fluid chamber 13 for containing droplets and filler fluid is defined between the first substrate 11 and the second substrate 12. An electrode layer and a first hydrophobic layer 4 are sequentially covered on the first substrate 11. Figure 3 and Figure 4 As shown, the electrode layer includes interdigitated electrodes 2 and driving electrodes 1, wherein the interdigitated electrodes 2 are located between two adjacent driving electrodes 1; as Figure 5 As shown, the microfluidic chip also includes a microwell 3, which is formed in the region above the interdigital electrode 2 of the first substrate 11. The dielectric layer and the first hydrophobic layer 4 in the region are removed, so that the surface of the interdigital electrode 2 is directly exposed to the fluid chamber 13. The microwell 3 is a pit structure on the side of the first substrate 11 facing the fluid chamber 13. The region on the first substrate 11 other than the microwell 3 retains the hydrophobicity of the hydrophobic layer 4. The upper surface of the interdigital electrode 2 forms a raised step-like structure at the bottom of the microwell 3.

[0032] like Figure 2 and Figure 5The microfluidic chip shown employs a dual-substrate configuration. An electrode layer and a first hydrophobic layer 4 are sequentially formed on the first substrate 11 (the lower substrate). The electrode layer contains two types of electrodes: a driving electrode 1 for electrowetting and an interdigital electrode 2 for sensing. Both are located on the same plane, with the interdigital electrode 2 positioned between or adjacent to two adjacent driving electrodes 1, facilitating direct access of droplets to the area where the interdigital electrode 2 is located under the control of the driving electrodes 1. The first hydrophobic layer 4 directly above the interdigital electrode 2 is partially removed, forming a microwell 3, a downwardly recessed three-dimensional pit structure. After removing the hydrophobic layer, the surface of the interdigital electrode 2 is directly exposed to the fluid chamber 13, while the area outside the microwell 3 remains hydrophobic. The upper surface of the interdigital electrode 2 forms a raised, stepped structure at the bottom of the microwell 3 relative to the surrounding dielectric layer or the surface of the first substrate 11.

[0033] The microwell 3 of this invention passively confines droplets within the electrode region through its recessed structure, while the raised interdigitated electrodes 2 increase the effective sensing area at the bottom of the microwell 3, ensuring that even sub-droplets on the picoliter scale can be monitored by the interdigitated electrodes 2, avoiding detection failures caused by droplets not covering the electrodes. Simultaneously, the recessed structure of the microwell 3 and the raised electrodes together increase the frictional force at the bottom of the microwell 3, making it easier for sub-droplets to automatically remain under the combined forces of surface tension and other factors.

[0034] like Figure 5 As shown, the second substrate 12 has a common electrode 5 and a second hydrophobic layer on the side facing the fluid chamber 13. The distance between the first hydrophobic layer 4 and the second hydrophobic layer is 100 μm to 200 μm. This height is beneficial for the rapid passage of droplets and passive distribution at the microwell 3. Compared with a smaller distance, when the droplet stays at the microwell 3, the upper and lower hydrophobic layers will not excessively compress the droplet, allowing the sub-droplets to form naturally by relying on surface tension. Compared with a larger distance, it avoids excessively high driving voltage, excessively fast droplet movement speed, and deformation instability of the droplet due to gravity during movement.

[0035] The depth of microwell 3 is 100 nm to 5 μm, and the diameter of microwell 3 is 1 μm to 50 μm. The geometry of microwell 3 is such that a droplet passing through microwell 3 can be passively distributed and retained in microwell 3 through surface tension effect, and the sub-droplet is in direct contact with the exposed surface of interdigitated electrode 2. When driving electrode 1 pushes the parent droplet through microwell 3, because the surface of the exposed interdigitated electrode 2 inside microwell 3 is hydrophilic, while the first hydrophobic layer 4 outside microwell 3 is hydrophobic, under the action of surface tension gradient, part of the liquid is retained in the three-dimensional depression of microwell 3, forming a sub-droplet.

[0036] Since the depth and diameter of the microwell are much smaller than the distance between the upper and lower substrates and the characteristic size of the mother droplet, when the mother droplet moves above the microwell, the electrowetting driving force and fluid resistance of its main body are almost unaffected by the micro-pit, thus ensuring the stability of global control. This means that the recessed structure of the microwell 3 will not significantly affect the global control of the droplet by the driving electrode 1, and the driving electrode 1 can still effectively attract the droplet across the region of the microwell 3.

[0037] The above setup achieves passive, passive dispensing, meaning that micro-quantum droplets can be precisely separated from the mother droplet without the need for an external electric field or mechanical valve. Compared to traditional digital microfluidics that uses electrowetting for droplet splitting, the passive dispensing of this invention is entirely driven by surface tension, resulting in high repeatability and volume consistency. The daughter droplets are much smaller than the mother droplets, significantly reducing sample consumption—an advantage that is particularly pronounced for precious biological samples, such as rare cells and trace amounts of serum.

[0038] Combination Figure 3 , Figure 4 and Figure 5 As shown, the interdigitated electrode 2 and the driving electrode 1 are located in the same plane, and the interdigitated electrode 2 has a finger spacing of 100nm to 1μm.

[0039] The surface of the interdigitated electrode 2 has a modification layer or molecular film, which specifically binds to the analyte in the subdroplet. This functionalization upgrades the microsensor of this invention from general conductivity detection to specific biomolecule detection, significantly expanding its application range. Unmodified electrodes can only measure the bulk properties of the solution, such as the increased ion concentration in the subdroplet after spore 7 germination, while functionalized electrodes can identify specific biomarkers, enabling the monitoring of markers at different stages of spore 7 germination.

[0040] The interdigitated electrode 2 can be configured with a first electrical signal or a second electrical signal, wherein the first electrical signal achieves the splitting of the droplet at the microwell 3 through the dielectrophoresis effect; The second electrical signal can perform electrochemical detection of droplets in the microwell 3.

[0041] In this embodiment, the microfluidic chip substrate 10 is fabricated using the following specific steps: A 10-nanometer layer of chromium was sputtered onto a piece of polished glass 9.

[0042] A 70-nanometer layer of gold is sputtered onto the chromium layer.

[0043] Photolithography, in which the electrode shape on the first mask used is as follows Figure 4 As shown.

[0044] Developing, etching, obtaining Figure 3The global electrode structure is shown.

[0045] Photolithography, development, in which a second mask is used to coat only the surface of the annular interdigitated electrode 2 with a layer of photoresist.

[0046] A layer of silicon nitride is deposited on the chip surface using a chemical deposition method to serve as a dielectric layer.

[0047] A layer of Teflon is coated onto the dielectric layer by spin coating to form a liquid-repellent layer 4.

[0048] The photoresist on the annular interdigital electrode 2 is removed using a stripping technique, thereby removing the silicon nitride and Teflon overlying it, exposing the annular interdigital electrode 2. At this point, the surface of the annular interdigital electrode 2 is hydrophilic, while the rest of the chip remains hydrophobic. The first substrate 11 (lower electrode plate) of the digital microfluidic chip is now fabricated.

[0049] A layer of Teflon is coated onto an indium tin oxide glass 9 using a spin coating method as a second hydrophobic layer. The indium oxide and tin oxide on the surface of the glass substrate serve as the common electrode 5, and the upper electrode of the digital microfluidic chip is fabricated.

[0050] The upper and lower plates are arranged as follows Figure 2 and Figure 5 The plates are placed in a 100-200 μm spacing.

[0051] A method for monitoring spore germination, employing the aforementioned microsensor device based on interdigital electrodes 2 and local hydrophilic points, includes the following sequential steps: like Figure 5 As shown, a spore suspension 6 containing ungerminated spores 7 is dripped into the fluid chamber 13 of the microsensor through the drip port 14. The droplets of the spore suspension 6 are driven by electrowetting to the micro well 3, so that the spores 7 are deposited into the micro well 3. The droplets of the spore suspension 6 are driven away from the microwell 3 by electrowetting, and the sub-droplets remain at the microwell 3; like Figure 6 As shown, germination agent 8, which is L-alanine in this embodiment, is dripped into the fluid chamber 13 of the micro-sensor through the drip port 14. The droplets of germination agent 8 are driven by electrowetting to the micro well 3, where they exchange substances with the sub-droplets, inducing the spores 7 to transform into a germination state. The conductivity change of the subdroplet is monitored using the interdigitated electrode 2 to monitor the germination process of spore 7.

[0052] The droplets of spore suspension 6 remain at the microwell 3 for 10 to 14 hours, which is sufficient time for spores 7 to deposit onto the surface of the interdigitated electrode 2 at the bottom of the microwell 3. In some embodiments, a residence time of 12 hours is generally chosen, which ensures that the surface of the interdigitated electrode 2 is covered by a sufficient number of spores 7, so that the amount of electrolyte released during germination can produce a significant change in conductivity.

[0053] Meanwhile, this time window is compatible with experimental schedules on regular workdays—spore suspension 6 can be added before the experiment begins, allowing the chip to settle overnight at room temperature, and germination agent 8 can be added the next day for detection. Furthermore, the microwell 3 maintains a stable humidity environment, avoiding the problem of droplet evaporation and drying caused by prolonged exposure.

[0054] Example 2 like Figure 7 As shown, in this embodiment, the microfluidic chip substrate 10 includes a first substrate 11 and a second substrate 12 disposed opposite to each other. A fluid chamber 13 for containing droplets and filler fluid is defined between the first substrate 11 and the second substrate 12. An electrode layer, a dielectric layer, and a first hydrophobic layer 4 are sequentially covered on the first substrate 11. The difference from Embodiment 1 is that, as shown in the figure... Figure 8 As shown, the electrode layer includes a square interdigitated electrode 2 and a driving electrode 1. The square interdigitated electrode 2 and the driving electrode 1 are arranged adjacent to each other. The electrode is a three-electrode system, namely a working electrode, a counter electrode and a reference electrode.

[0055] The microfluidic chip substrate 10 also includes microwells 3, which are formed in the region above the interdigital electrodes 2 of the first substrate 11. The dielectric layer and the first hydrophobic layer 4 in the region are removed, so that the surface of the interdigital electrodes 2 is directly exposed to the fluid chamber 13. The microwell 3 is a pit structure on the side of the first substrate 11 facing the fluid chamber 13. The region on the first substrate 11 other than the microwell 3 retains the hydrophobicity of the hydrophobic layer 4. The upper surface of the interdigital electrodes 2 forms a raised step-like structure at the bottom of the microwell 3.

[0056] In this embodiment, the microsensor is used to study the potassium iodide solution oxidation electrode. Potassium iodide solution and buffer solution are added through the droplet 14. The potassium iodide-containing droplet is separated from the potassium iodide droplet 14 by electrowetting, and the buffer solution-containing droplet is separated from the buffer solution droplet 14. The two droplets are then driven by electrowetting to mix them. The mixed droplet is then driven to the microwell 3 to start cyclic voltammetry detection.

[0057] Example 3 The difference from Embodiment 1 is that, in this embodiment, at least one auxiliary electrode is provided on the area of ​​the second substrate 12 directly opposite the microwell 3. The auxiliary electrode is insulated from the common electrode 5 and is exposed to the fluid chamber 13.

[0058] The auxiliary electrode is a counter electrode or a reference electrode, which together with the interdigitated electrode 2 on the first substrate 11 forms a three-electrode electrochemical detection system; the interdigitated electrode 2 serves as the working electrode, and the common electrode 5 on the second substrate 12 serves as a reference electrode or a counter electrode corresponding to the auxiliary electrode.

[0059] In existing technologies, the common electrode 5 of the second substrate 12 serves only as a reference electrode and cannot participate in local electrochemical measurements. The auxiliary electrode added to the second substrate 12 can act as a counter electrode or reference electrode in electrochemical detection, forming a vertical three-electrode system with the interdigitated electrode 2 of the lower substrate. Compared to impedance measurement relying solely on the interdigitated electrode 2 of the first substrate 11, the introduction of the auxiliary electrode can significantly reduce electrode polarization effects, improving the accuracy and reproducibility of detection. For example, in cyclic voltammetry detection, the auxiliary electrode provides a stable current loop, making the potential scan of the working electrode more precise.

[0060] The present invention has the following outstanding technical effects: The three-dimensional pit structure of the microwell 3 combined with the stepped protrusions of the interdigitated electrode 2 allows the mother droplet to automatically retain sub-droplets of picoliter to nanoliter size under the action of surface tension, realizing passive distribution without external electric field or mechanical valve. This process does not require an external electric field or mechanical valve, has high consistency in distribution volume, and low sample consumption, making it suitable for the analysis of precious samples such as rare cells and trace amounts of serum. The interdigitated electrode 2 is directly exposed to the fluid chamber 13, forming direct contact with the sub-droplets, which improves the sensitivity by several orders of magnitude compared to non-contact capacitive detection covered with a lyophobic layer. An auxiliary electrode is added on the second substrate 12 in the area opposite to the microwell 3, forming a vertical three-electrode electrochemical detection system with the interdigitated electrode 2 and the common electrode 5 on the first substrate 11, which improves the accuracy and reproducibility of measurements such as cyclic voltammetry.

[0061] The above embodiments and / or implementation methods are merely illustrative of preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art may make some modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but these should still be regarded as the technology or embodiments that are substantially the same as the present invention.

Claims

1. A microsensing device based on interdigitated electrodes and local hydrophilic points, comprising a microfluidic chip substrate (10), the microfluidic chip substrate (10) comprising a first substrate (11) and a second substrate (12) disposed opposite to each other, wherein a fluid chamber (13) for accommodating droplets and filler fluid is defined between the first substrate (11) and the second substrate (12), characterized in that, An electrode layer, a dielectric layer and a first hydrophobic layer (4) are sequentially covered on the first substrate (11). The electrode layer includes interdigitated electrodes (2) and driving electrodes (1). The interdigitated electrodes (2) are located between two adjacent driving electrodes (1) or adjacent to the driving electrodes (1). The microfluidic chip substrate (10) also includes a microwell (3), which is formed in the area above the interdigital electrode (2) of the first substrate (11). The dielectric layer and the first hydrophobic layer (4) in the area are removed, so that the surface of the interdigital electrode (2) is directly exposed to the fluid chamber (13). The microwell (3) is a pit structure on the side of the first substrate (11) facing the fluid chamber (13). The area on the first substrate (11) other than the microwell (3) retains the hydrophobicity of the hydrophobic layer (4); The upper surface of the interdigitated electrode (2) forms a raised, stepped structure at the bottom of the microwell (3).

2. The microsensing device based on interdigitated electrodes and local hydrophilic points according to claim 1, characterized in that, The second substrate (12) has a common electrode (5) and a second hydrophobic layer on the side facing the fluid chamber (13), and the distance between the first hydrophobic layer (4) and the second hydrophobic layer is 100 μm to 200 μm.

3. A microsensing device based on interdigitated electrodes and local hydrophilic points according to claim 2, characterized in that, The depth of the microwell (3) is 100 nm to 5 μm, and the diameter of the microwell (3) is 1 μm to 50 μm. The geometry of the microwell (3) is such that a droplet passing through the microwell (3) can be passively distributed and retained in the microwell (3) through the surface tension effect, and the droplet is in direct contact with the exposed surface of the interdigitated electrode (2).

4. A microsensing device based on interdigitated electrodes and local hydrophilic points according to claim 1, characterized in that, The interdigitated electrode (2) and the driving electrode (1) are located in the same plane, and the interdigitated electrode (2) has a finger spacing of 100 nm to 1 μm.

5. A microsensing device based on interdigitated electrodes and local hydrophilic points according to claim 3, characterized in that, The surface of the interdigitated electrode (2) has a modification layer or molecular film, which specifically binds to the analyte in the subdroplet.

6. A microsensing device based on interdigitated electrodes and local hydrophilic points according to claim 1, characterized in that, The interdigitated electrode (2) can be configured with a first electrical signal or a second electrical signal, wherein the first electrical signal enables the droplet to split at the microwell (3) through the dielectrophoresis effect; The second electrical signal can perform electrochemical detection of droplets in the microwell (3).

7. A microsensing device based on interdigitated electrodes and local hydrophilic points according to claim 1, characterized in that, At least one auxiliary electrode is provided on the second substrate (12) in the area directly opposite the microwell (3). The auxiliary electrode is insulated from the common electrode (5) and is exposed to the fluid chamber (13).

8. A microsensing device based on interdigitated electrodes and local hydrophilic points according to claim 7, characterized in that, The auxiliary electrode, interdigitated electrode (2), and common electrode (5) constitute a three-electrode electrochemical detection system; The interdigitated electrode (2) serves as the working electrode, the auxiliary electrode serves as the counter electrode or reference electrode, and the common electrode (5) on the second substrate (12) serves as the remaining reference electrode or counter electrode.

9. A method for monitoring spore germination, characterized in that, The microsensing device based on interdigitated electrodes and local hydrophilic points as described in any one of claims 1 to 8 comprises the following sequential steps: In the fluid chamber (13) of the microsensor, a spore suspension (6) containing ungerminated spores (7) is dripped into the droplet port (14). The droplet of the spore suspension (6) is driven to the micro well (3) by electrowetting, so that the spores (7) are deposited into the micro well (3). The droplets of the spore suspension (6) are driven away from the microwell (3) by electrowetting, and the sub-droplets remain at the microwell (3); Germination agent (8) is dripped into the fluid chamber (13) of the microsensor through the drip port (14). The droplets of germination agent (8) are driven by electrowetting to the micro well (3) to exchange substances with the sub-droplets, thereby inducing the spores (7) to transform into the germination state. The conductivity of the subdroplets was monitored using the interdigitated electrode (2) to monitor the germination process of the spores.

10. The method for monitoring spore germination according to claim 9, characterized in that, The droplets of the spore suspension (6) remain at the microwell (3) for 10 to 14 hours, which is the time required for the spores (7) to deposit onto the surface of the interdigitated electrode (2) at the bottom of the microwell (3).

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