CN115337968A

CN115337968A - Semi-closed digital microfluidic system based on SLIPS insulating hydrophobic membrane

Info

Publication number: CN115337968A
Application number: CN202210970852.8A
Authority: CN
Inventors: 何小东; 徐劲松
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2022-08-13
Filing date: 2022-08-13
Publication date: 2022-11-15
Anticipated expiration: 2042-08-13
Also published as: CN115337968B

Abstract

The invention belongs to the technical field of microfluidic chips, and particularly relates to a semi-closed digital microfluidic system based on a SLIPS insulating hydrophobic membrane. The system comprises a PCB substrate, an electrode array, an upper electrode plate and a controller for controlling liquid drops; the electrode array is arranged on the surface of the PCB substrate as a lower electrode plate; the shape of the electrode comprises a round part and a square part with a sawtooth-shaped edge; the round electrode is used as a liquid storage tank, and the square electrode is used as a driving electrode; a SLIPS insulating hydrophobic film covers the electrode array; the upper electrode plate is made of ITO conductive glass, and the lower surface of the upper electrode plate is covered with a SLIPS insulating hydrophobic film; the upper electrode plate covers the electrode array; the upper electrode plate can move left and right to form a mode of combining a double-layer structure and a single-layer structure. The invention uses the asymmetric electrowetting acting force on the modified smooth liquid injection film to complete the control of liquid drops between the independent electrodes, can effectively reduce the control cost of the digital microfluidic control, can effectively prevent biological deposition and reduce the pollution of a control plane.

Description

Semi-closed digital microfluidic system based on SLIPS insulating hydrophobic membrane

Technical Field

The invention belongs to the technical field of microfluidic chips, and particularly relates to a semi-closed digital microfluidic system.

Background

With the development of the micro-electro-mechanical system technology, the digital micro-fluidic technology based on the dielectric wetting effect has a wide application prospect in the fields of chemical reaction, drug synthesis, sample analysis, biological detection and the like due to an accurate, efficient and rapid micro-droplet control mode. The digital microfluidics based on electrowetting has the advantages of flexible control mode, low power consumption, small size and the like. However, the digital microfluidic technology based on electrowetting still has the problems of high continuous driving voltage, easy breakdown of high voltage of a dielectric layer, complex electrode structure and control circuit, low parallel quantity and the like. Particularly, the digital microfluidic control method constructed based on the PCB has the problems of complex coating process, easy breakdown of a dielectric layer, high driving voltage and the like.

Microfluidic technologies can be divided into channel-based continuous microfluidic control technologies and single discrete droplet-based digital microfluidic control technologies. The former has complex processing technology and higher cost, and needs to etch a permanent physical structure to limit liquid and guide fluid transmission, so that the chip design is specialized, and the production cost is increased sharply aiming at different application scenes. In the structure, the liquid is limited in the closed pipeline, and the advantages are that the flux is higher, and the reagent is not easy to be contacted with the external environment to cause reagent pollution; however, mixing and reaction between different liquids also cause contamination between liquid reagents, and it is not easy to clean and replace liquid droplets. The digital microfluidic technology can solve the problems, and the diversity of the droplet generation technology, such as a micro valve, a magnetic field, electrowetting and the like; the diversity of electrode designs, such as materials and layouts, and the flexible use of open and closed structures enable the use thereof in multiple scenarios.

The early digital microfluidic technology adopts a micro pump and a micro valve structure to realize the driving of liquid drops and the generation of discrete liquid drops, and has the advantages of quick and reliable liquid drop generation, and the following disadvantages of high cost, complex structure, easy biological or chemical pollution and the like because the liquid drop generation still depends on a channel type structure to carry out fluid control, and is not beneficial to large-scale and multi-scene use. Compared with the prior art, the droplet generation equipment based on the electrowetting technology has the advantages of simple structure, easy operation and lower cost, can realize that the driving electrode and the hydrophobic membrane are used and replaced at once, and reduces the pollution among droplet reagents.

Conductive electrodes (such as ITO glass and precious metals) prepared by utilizing a photoetching technology are common methods for realizing dielectric wetting (EWOD) digital microfluidics, have smooth surfaces and good conductivity, are small in resistance when fluids are controlled, and are easy to control microfluidics. In recent years, due to the development of the electronic manufacturing industry, the production of PCBs has unified and standardized manufacturing facilities and processes, and has low cost and high integration level, and the multilayer structure also solves the problems of difficult wiring caused by the photolithography technology, and thus, the multilayer structure is more and more widely used for manufacturing EWOD devices. The control of the liquid drops on the PCB based on the EWOD technology has strong flexibility, the size of the electrode and the distance between the double-layer plates can determine the size of the generated liquid drops, and the control method has good uniformity. In the closed structure (double-layer structure), four basic operations of liquid drops, namely generation, transportation, combination and splitting, can be realized. The open structure can better perform some experiments (such as fluorescence monitoring), and liquid drops can be added or replaced conveniently.

Electrowetting technology on a smooth injection surface (SLIPS) has attracted great interest due to its numerous advantages, with the advantages of small contact angle hysteresis, high breakdown voltage resistance, self-cleaning, self-healing, freeze protection, and bio-contamination resistance. Especially in digital microfluidics, SLIPS droplet manipulation shows efficient antifouling properties. However, the activation voltage to drive the Deionized (DI) water droplets on its surface is up to 300V, which limits its application in digital microfluidics. After SLIPS is modified by fluorosilane, the driving voltage of the liquid drop is also obviously improved. As the voltage is increased, contact angle differences of up to 55 ° are observed at the modified SLIPS surface, with low voltage electrowetting occurring at positive bias voltages, but not negative bias voltages. Therefore, this electrowetting phenomenon is referred to as polarity-dependent low voltage electrowetting. Under the voltage of 20V to 500V, a thin film with the thickness of 25 mu m is adopted, the continuous driving speed of liquid drops can reach 0.075mm/s to 123mm/s, the driving voltage is reduced to 8V, the continuous liquid drop driving voltage is reduced by at least 15 times compared with an unmodified SLIPS thin film, and the thin film has the breakdown voltage of 1kV and the self-repairing capability, and has good robustness. Droplet manipulation on such flexible two-in-one films with insulating and hydrophobic properties is a promising curved droplet manipulation, lab-on-a-chip biology and chemical reaction technology. The invention utilizes the low-voltage electrowetting technology related to the polarity of the perfluoro silane modified SLIPS surface and combines the PCB electrode preparation technology to realize the high-efficiency and high-precision control of liquid drops on open, closed and semi-closed structures.

Disclosure of Invention

The invention aims to provide a semi-closed digital microfluidic system based on a SLIPS modified insulating hydrophobic membrane, which realizes high-efficiency and high-precision liquid drop control on the surfaces of open, closed and semi-closed perfluorosilane modified SLIPS.

The structure of the semi-closed digital microfluidic system based on the SLIPS modified insulating hydrophobic membrane is shown in figure 1. The PCB substrate, the electrode array and the upper electrode plate are arranged from bottom to top in sequence; and a droplet manipulation controller; wherein:

the PCB substrate is used as a lower electrode plate;

the electrode array is arranged on the surface of the PCB substrate; forming a single electrode by adopting a pad tin spraying mode; the electrode array comprises a square array consisting of M multiplied by N square small electrodes with sawtooth edges and a plurality of circular electrodes uniformly distributed on two sides of the square array; the electrical connection is made through the electrode center via to the control circuit.

Specifically, the size of the M × N electrode array may be determined according to actual needs, such as a 24 × 12 array, a 13 × 8 array, and so on; the number of the circular electrodes can be multiple, and the circular electrodes are uniformly distributed on two sides of the M multiplied by N electrode array, for example, the number of the circular electrodes can be 5, the number of the circular electrodes on the left side is 3, and the number of the circular electrodes on the right side is 2;

the round electrodes are respectively connected with the square array electrodes through 3 square small electrodes, wherein the first two small electrodes are overlapped with the round electrodes to form a liquid drop generating structure; the circular electrode is used for storing a large liquid drop, a small liquid drop is generated on a third small electrode adjacent to the circular electrode, and the small liquid drop is controlled through the electrode array of M multiplied by N;

the circular electrode is used as a liquid storage tank for storing reacted liquid drops or storing a large liquid drop source for generating small liquid drops. The diameter of the general circular electrode is 10-13.5 mm;

the square small electrode with the sawtooth-shaped edge is a driving electrode and is used for controlling generation, transportation, splitting and combination of liquid drops. Compared with square or crescent electrodes, the electrode with the sawtooth edge has better droplet control stability and higher driving speed, and the liquid drop is easier to move between the electrodes because the sawtooth is arranged across different electrodes, so that the liquid drop is subjected to higher tension when being transported across the electrodes (the longer the electrode edge is contacted with the liquid drop), and the like. In addition, the depth of the teeth and the angle of the teeth tips can also affect the droplet transport capacity and efficiency of the electrode, and too small a tooth tip angle, too deep a tooth tip angle, or too large a tooth tip angle, too shallow a tooth tip angle can all reduce the transport capacity of the electrode (compared to square, crescent, etc.). The side length of a common square small electrode is 2-3 mm, the tooth depth is 0.2-0.3 mm, the tooth tip angle is 60-120 degrees, and the distance between the electrodes is 80-100 mu m.

In the electrode array, each electrode is controlled by a single pin, two HV507 high-voltage push-pull output chips are adopted for cascading in the whole electrode array, and 128 paths of pins are output in parallel; the specific selection can be determined according to the size of the electrode array.

A layer of SLIPS insulating hydrophobic membrane covers the electrode array;

the upper electrode plate is made of ITO conductive glass, and the lower surface of the upper electrode plate is also covered with a layer of SLIPS insulating hydrophobic film; a glass gasket is arranged between the edge of the upper electrode plate and the electrode array; for controlling the distance between the two; typically the distance is 50-300. Mu.m.

The upper electrode plate covers the electrode array, and the upper electrode plate and the square electrode array form a structure which is half closed and half open; the upper electrode plate can move left and right, and the edge of the electrode plate at one end of the circular electrode can completely cover the circular electrode and can also cover half of the circular electrode, so that liquid drops can be added or removed conveniently; the edge of the upper electrode plate at one end of the square electrode array is arranged in the middle of the electrode array so as to realize coexistence of an open area and a closed area, namely, a structural mode combining a double-layer structure and a single-layer structure is adopted.

In the invention, in a closed structure mode (namely a double-layer mode), the upper polar plate can realize the control of liquid drops without electrifying; the top plate can also be powered or used as a reference electrode when needed.

In the invention, the SLIPS insulating hydrophobic membrane is obtained by coating silicone oil with the viscosity of 10cst on the surface of a polymer compound PTFE membrane modified by 0.01-0.1wt% (the optimal concentration is 0.031 wt%) of PFOTS-ethanol solution, and is used as a dielectric membrane.

The controller for controlling the liquid drops comprises a single chip microcomputer, a high-voltage push-pull output chip and a booster circuit.

The single chip microcomputer (such as STC 12) is used for downloading programs, is a Micro Control Unit (MCU) of the whole digital microfluidic system, and is adopted due to perfect functions, simple use, small volume and high integration level.

The high-voltage push-pull output chip (such as HV 507) is respectively connected with the singlechip and the electrode array, receives the control signal of the singlechip on one hand, and outputs a voltage signal to corresponding electrodes in parallel on the other hand; and a plurality of high-voltage push-pull output chips can be cascaded to expand the output.

The booster circuit boosts the 5V direct current power supply voltage of the singlechip to 40 to 300V, and inputs the boosted voltage to a corresponding electrode through a high-voltage push-pull output chip to realize control of a power-on time sequence; the output voltage may be regulated by a digital potentiometer (e.g., MCP 41050).

In the invention, the ratio of the closed area and the open area of the electrode array can be changed by moving the upper polar plate so as to adapt to different application scenes.

In the invention, in a scene with less electrode requirements, the electrodes can adopt a 1-to-1 control mode (namely one control pin is connected with 1 electrode); in a use scene of a large-scale electrode array, control pins can be added through cascade connection of high-voltage push-pull chips, or a 1-to-many electrode control mode (namely, one control pin is connected with a plurality of electrodes) is adopted on the premise of not influencing each other.

The invention adopts a structure mode of combining a double-layer structure and a single-layer structure, namely a mode of combining a closed structure and an open structure to realize the control of microfluid. In the two-layer structure mode, the liquid is confined in the space between the parallel plates, the lower plate being a SLIPS insulating hydrophobic film covering the electrode array; the upper flat plate (i.e. the upper electrode plate) is ITO glass covered with a SLIPS insulating hydrophobic film; the ITO electrode of the upper plate is not electrified and is not used as a reference potential, and only plays a role in limiting liquid drops to a set space. In a bilayer structure, therefore, the driving of the droplets is dependent on the electrodes covered by the hydrophobic insulating layer of the lower plate. When the electrodes are powered up, the hydrophobic insulating layer covers the electrodes and an electric field is generated between the electrodes on the opposite plates. This electric field creates a surface tension gradient that causes the shape of the droplet to change and move towards the electrodes in the preset direction. By appropriate arrangement and control of the electrodes, droplets can be transported to any location covered by the electrodes by continuously transporting the droplets between adjacent electrodes. The space around the droplets may be filled with a gas, such as air or nitrogen, or with a liquid that is immiscible with each other, such as silicone oil. The basic operations of generating, transporting, mixing and splitting four kinds of liquid drops can be realized in the double-layer structure. In the single-layer open structure mode, the driving principle of the liquid drop is consistent, the limitation of an upper plate is avoided, the liquid drop driving between the electrodes needs the liquid drop with larger volume compared with the double-layer structure, and meanwhile, in the open space, the liquid drop is difficult to realize the generation and the division operation. The invention better integrates the advantages of the two structures, and leads the application scene to be wider.

The invention also provides a liquid drop control method based on the semi-closed digital microfluidic system, which comprises the steps of liquid drop generation, liquid drop transportation, liquid drop combination and liquid drop splitting, and specifically comprises the following steps:

(1) Generating liquid drops; small droplets are generated from larger droplets in a closed bilayer structure in the following manner: firstly, placing a larger liquid drop (for example, 20 to 25 mu l) on a circular electrode, and keeping a bottom plate electrode in a positive bias voltage state, and not electrifying an upper electrode plate; then, the circular electrode where the larger liquid drop is positioned is under negative bias voltage, so that the larger liquid drop is uniformly distributed on the circular electrode, and the small liquid drop is favorably generated; subsequently, the small electrode (1 st small electrode) completely wrapped by the circular electrode is subjected to negative bias voltage, and the circular electrode is subjected to positive bias voltage at the same time; when the liquid column is conveyed to the 1 st small electrode and completely covers the small electrode, the small electrode (the 2 nd small electrode) which is semi-surrounded by the circular electrode is connected with a negative bias voltage, and the 1 st small electrode is maintained to be the negative bias voltage; when the liquid column completely covers the 2 nd small electrode, the small electrode (the 3 rd small electrode) adjacent to the 2 nd small electrode is also under the negative bias voltage, and meanwhile, the 1 st small electrode and the 2 nd small electrode are maintained to be under the negative bias voltage; after the liquid column covers the 3 rd small electrode, the circular electrode where the large liquid drop is located and the 3 rd small electrode are simultaneously under negative bias voltage, other electrodes are under positive bias voltage, and the small liquid drop can be generated smoothly after the liquid column is maintained for a period of time;

(2) And (3) transporting liquid drops: the liquid drop transportation can be carried out in a single-layer structure, a double-layer structure and a semi-closed structure; the control methods are the same; the specific operation method is that the electrode where the liquid drop is located is connected with a negative bias voltage to enable the electrode to be located at the central position of the electrode where the liquid drop is located, and the adjacent electrodes are all positive bias voltages; subsequently, determining the polarity of the adjacent electrode according to the transport direction of the liquid drop; if the liquid drop needs to move to the adjacent electrode at the left side in the next step, setting the adjacent electrode as a negative bias voltage, and simultaneously connecting the electrode where the liquid drop is located with a positive bias voltage, and when the liquid drop completely moves to the adjacent electrode, carrying out the next step of liquid drop transportation operation; when the target position of the liquid drop is far away from the current position, the path can be flexibly set according to the requirement;

(3) Merging of droplets: the small drops on different electrodes can be transported to the same electrode on a square electrode array by control to be combined (the volume ratio of the two drops before combination is unlimited, namely the small drops can be combined with larger drops or with equal volume drops), and the combined drops are circularly transported according to a certain path to be fully mixed; the operations of merging and mixing of the droplets can be carried out in both closed and open configurations; the operation can be applied to chemical reaction, biological culture, fluorescence monitoring and the like;

(4) Splitting of the droplets: a larger droplet occupying 2 electrodes (in a splitting operation, the electrodes are square electrodes with sawtooth-shaped edges) in the closed structure can be split into two droplets occupying only 1 electrode, specifically, a negative bias voltage is simultaneously applied to the electrodes adjacent to both sides of the larger droplet occupying 2 electrodes (namely, two adjacent electrodes in line with the two electrodes where the droplet is located), and a positive bias voltage is applied to the other electrodes (including the two electrodes where the larger droplet is located); furthermore, the invention can also split a plurality of electrode-sized droplets in the closed structure into droplets of any electrode size; for example, a droplet with a size of 4 electrodes is split into a droplet with a size of 1 electrode and a droplet with a size of 3 electrodes, and the specific operation method is that a negative bias voltage is applied to an electrode where the droplet with the size of 3 electrodes is located, a negative bias voltage is applied to an electrode adjacent to the other side of the electrode where the droplet with the size of 1 electrode is located (the other side of the electrode is in a straight line with the droplet with the size of 4 electrodes), and a positive bias voltage is applied to other electrodes; or the liquid drop is split into two liquid drops with the same volume and occupying the size of 2 electrodes, the specific operation method is that positive bias voltage is applied to the middle two electrodes occupied by the liquid drop, negative bias voltage is applied to the other two electrodes occupied by the liquid drop and the two adjacent electrodes at the two sides which are in a straight line with the other two electrodes, the total four electrodes are applied with negative bias voltage, and the other electrodes are all applied with positive bias voltage; the droplet splitting operation can be applied to extraction and purification of DNA, and the like.

The invention provides a semi-closed digital microfluidic system based on a SLIPS modified insulating hydrophobic membrane, which is a liquid processing and control system based on an electrowetting technology. Droplets of sizes in the microliter range can be manipulated by controlling the voltage polarity at the various electrodes. The control principle of the liquid drop is that the wettability of the liquid drop on the substrate is changed by changing the voltage (including the size and the polarity) between the liquid drop and the insulating substrate, namely, the contact angle is changed, so that the liquid drop has the electrowetting effect of deformation and displacement. The chip can re-plan the manipulation path of the liquid drop during the execution of the preset task, thereby having strong flexibility. The combination of the double-layer structure and the single-layer structure of the electrode plate enables the invention to have stronger practicability compared with the traditional microfluidic system. The present invention is capable of performing several different types of processing and manipulation tasks on individually controllable droplet samples, including movement, detection, irradiation, incubation, reaction, dilution, mixing, and the like. Furthermore, the invention can be used to generate droplets from a continuous flow of liquid (reservoir), the volume of the droplets being controlled by the size of the electrodes and the spacing of the biplates, and the structure having good uniformity (substantially uniform droplet volume) when multiple droplets are generated. In addition, because the electrodes in the invention adopt a 1-to-1 control mode (namely, one control pin is connected with 1 electrode), the invention can well meet the requirements in a scene with less electrode requirements, and in a use scene of a large-scale electrode array, the control pins can be increased through the cascade connection of high-voltage push-pull chips, or on the premise of not influencing each other, a one-to-many electrode control mode (namely, one control pin is connected with a plurality of electrodes) can be adopted, which is favorable for reducing the high-voltage control pins of the electrodes, thereby reducing the number of the control chips and lowering the cost.

The PTFE polymer film is used as a dielectric layer, and has excellent chemical stability, high temperature resistance, corrosion resistance, sealing property, high lubrication non-sticking property, electrical insulation property and good ageing resistance. Because PTFE has a porous surface layer, it needs to be soaked in lubricating fluid to produce an ultra-smooth surface (i.e., SLIPS) on a rough surface. The ultra-smooth surface is smooth and resists or reduces the adhesion of particles or immiscible liquids. These unique features of SLIPS allow various biomaterials to pass smoothly over their surfaces without clumping, adhering, or otherwise contaminating the SLIPS, i.e., with superior anti-biocontamination properties. In general, SLIPS can be manufactured by providing a liquid (e.g., a high density fluid with chemical inertness) on a roughened surface characterized by micro-scale or nano-scale topography. In the invention, the ratio of the fluorosilane-ethanol solution is 0.01-0.1wt% (the optimal concentration is 0.031 wt%), and the fluorosilane-ethanol solution is used as lubricating liquid to be injected into the PTFE porous surface to manufacture SLIPS. PTFE needs to be soaked in fluorosilane-ethanol solution for modification, and can be used after being fully combined.

The modified SLIPS has a smoother surface, and compared to PTFE, the droplet has less resistance to transport on its surface, but still requires a larger driving voltage, and it is considered to coat its surface with a hydrophobic layer to solve this problem. The hydrophobic layer should be chosen to be chemically and biologically inert (i.e., unreactive or immiscible with most chemical or biological agents), and thus silicone oil is a good choice. According to experimental research of the invention, when silicone oil with 10cst viscosity is coated on the SLIPS surface, the liquid drop has smaller driving voltage, and the control efficiency and precision of the liquid drop are obviously improved.

The liquid drop control controller adopts an STC12 single chip microcomputer, the functions are complete, and the programming is relatively simple. The control circuit comprises a booster circuit, a 5V power supply provided by the singlechip is converted into 40-300V large-range voltage output, and a large-scale voltage source is integrated on the PCB, so that the portability of the system is greatly improved. The MAX1771 boost chip and the MCP41050 digital potentiometer are adopted in the boost circuit, the structure is simpler, the output high voltage is convenient to adjust, and the requirements of liquid drop control under different scenes can be met. And an HV507 high-voltage push-pull output chip is used for receiving a time sequence control instruction of the singlechip, so that accurate droplet control is realized.

Compared with a continuous flow system, the system has a simple structure and is more convenient and flexible to control. The manipulation mode and path of the droplets can be set according to specific use scenarios. Specifically, the invention utilizes asymmetric electrowetting force on a modified smooth liquid injection film to complete the control of liquid drops between independent electrodes. And the transport, combination, splitting and mixing operation of the liquid drops on the two-dimensional plate are completed by controlling the polarity of the voltage applied to the electrodes so that the liquid drops move from the independent high-potential electrode to the low-potential electrode. The replaceable droplet operation on the smooth liquid injection surface can effectively reduce the digital microfluidic operation cost, simplify the coating process of the PCB electrode dielectric hydrophobic layer, effectively prevent biological deposition and reduce the pollution of the operation plane. The invention can effectively reduce the complexity and the cost of the liquid drop control chip.

The invention also has the following advantages:

(a) The compact electrode array layout brings a high degree of parallelism handling capability;

(b) The mixing of the droplet samples with different proportions can be realized by controlling the amount of the droplets, because the generated droplets have better uniformity (namely the volumes of the generated droplets are basically consistent), when reagents with different volume ratios are needed to react, the amount of the droplets generated by different reagents is only needed to be controlled (the volumes of the generated droplets can be controlled by designing the size of an electrode and adjusting the distance between an upper polar plate and a lower polar plate, and the volume of the droplets generated in the structure is 1~3 mul), so that the mixing has strong controllability and higher accuracy;

(c) The design of a plurality of liquid storage tanks improves the throughput capacity of liquid drops and enhances the parallelism;

(d) The electrode plate can be replaced at any time, and has low cost and strong reconfigurability;

(e) In the double-layer structure, the distance between the polar plates can be flexibly changed, and the volume of generated liquid drops is further controlled;

(f) The SLIPS polymer film is used as a dielectric layer, so that the flatness of the surface of an electrode can be increased, and the control performance of liquid drops is improved; and the SLIPS film can be replaced at any time to reduce the cross contamination among reagents;

(g) The proportion of the closed area to the open area can be changed by moving the upper polar plate, and the method has strong flexibility for different application scenes;

(h) The programmable control improves the universality of the system, and can provide various liquid drop control schemes under the same scene.

Drawings

Fig. 1 is a top view and partial cross-sectional view of a digital microfluidic system.

Fig. 2 is a structural diagram of a droplet generation unit.

Fig. 3 is a diagram showing a state of transport of a droplet in a closed structure.

Fig. 4 is a diagram showing a state of liquid droplet transportation in the open structure.

Fig. 5 is a diagram showing a state of liquid droplet transport from open to closed.

Fig. 6 is a diagram showing a state of the droplet transport from closed to open.

Fig. 7 is a surface microscale plot of a PTFE membrane.

Fig. 8 is a surface microscale plot of a SLIPS film formed after modification with a fluorosilane-ethanol solution.

Reference numbers in the figures: 1 is a PCB substrate; 2 is an electrode; 3 is SLIPS insulating hydrophobic membrane; 4 is a gasket; 5 is an ITO film; 6 is transparent glass; 7 denotes the generation state of the microfluidic droplets; 8 denotes the transport state of the microfluidic droplets in the closed structure; 9 represents the transport state of the microfluidic droplets in the open structure; 10 denotes the transport state of the microfluidic droplets from the open structure to the closed structure; and 11 represents the transport of the microfluidic droplets from the closed to the open configuration.

Detailed Description

The invention will be further described with reference to the drawings and examples, but the invention is by no means limited to the examples described.

Example 1: preparation of digital microfluidic system

And (5) preparing a PCB electrode plate. The PCB board can be made of FR-4 flame-retardant materials, and can also be made of an aluminum substrate, the whole circuit board is of a double-layer structure, the surface electrode is a welding disc, and the spraying mode during processing can be lead spraying, lead-free spraying, gold immersion and the like. The electric connection is that a through hole is arranged in the central area of the electrode, and the bonding pad (electrode) is connected to the control circuit through the bottom layer routing. The shape of the PCB electrode is designed with a larger circular electrode as a liquid storage tank (used for storing reacted liquid drops or storing large liquid drop sources for generating small liquid drops), and the diameter of the electrode is 10-13.5 mm; the square electrode with the smaller sawtooth-shaped edge is formed by cutting a square electrode with the side length of 2-3 mm, the tooth depth is 0.2-0.3mm, and the tooth tip angle is 90 degrees; the pitch of the electrodes is 100 μm, and the above parameters may be varied within a certain range.

Preparation of SLIPS insulating hydrophobic integral layer. In EWOD devices, the dielectric layer is an important component determining the performance of the device. The dielectric materials used for the EWOD at present are mainly divided into polymer dielectric materials and inorganic dielectric materials, and the high polymer materials are widely used due to the characteristics of high pressure resistance, breakdown resistance and corrosion resistance. Since SLIPS is modified with fluorosilane, asymmetric electrowetting is generated, and thus the preparation method is considered. 1H, 2H-Perfluorooctyltrichlorosilane (PFOTS) modified SLIPS was prepared approximately as follows: firstly, mixing PFOTS into absolute ethyl alcohol, and ultrasonically dissolving for 30 min to obtain a 2% volume fraction PFOTS ethyl alcohol solution. To ensure minimum lag angle and asymmetric electrowetting effect of the film, the solution was diluted from 2wt% to 0.03wt%. Then, a porous PTFE membrane (consisting of a nanofiber network, with a thickness of 25 μm and an average pore diameter of 0.1 μm) was cut out according to the size of the PCB, immersed in a 0.03% wt PFOTS ethanol solution for 1h, and a self-assembled perfluorosilane molecular monolayer was grafted on the PTFE membrane. Secondly, the film is applied on a clean PCB electrode substrate and is moderately stretched to be flat. After the absolute ethyl alcohol is completely evaporated, the film is fixed on the electrode substrate by utilizing capillary force. Finally, H201 methyl silicone oil with a viscosity of 10cst was injected into the modified nanoporous PTFE film by capillary action to form a transparent SLIPS. To remove excess silicone oil, the sample was placed vertically for about 20 minutes and then horizontally for 30 minutes to achieve a uniform thickness lubricant layer.

And preparing an upper electrode plate. The method is characterized in that smooth ITO glass is used as an upper polar plate, a proper size is cut and the upper polar plate is cleaned, the modified SLIPS film is used as a medium layer and is laid on the surface of the glass, and bubbles between the SLIPS film and the glass are removed. The ITO conductive layer is paved on one side of the ITO conductive layer as much as possible for use when the upper polar plate needs to be electrified, and the ITO conductive layer is respectively placed horizontally and vertically for 30 minutes for use after being evenly coated with silicone oil.

The distance control between the upper and lower electrode plates is realized by using a glass slide with a certain thickness, and the thickness of the glass slide is dozens of micrometers to hundreds of micrometers (generally 50-300 micrometers). The glass slide is placed above the electrode plate of the film, and the size of the glass slide is properly selected, so that the normal use of the electrode cannot be influenced. The thickness of the slide is selected taking into account the desired droplet size.

The whole digital microfluidic system adopts a single chip microcomputer to control the high-voltage output time sequence. The singlechip is connected with the booster circuit and an HV507 (64-bit parallel output) or HV513 (8-bit parallel output) high-voltage push-pull output chip to form a complete control circuit, and when the number of controlled electrodes is large, the push-pull output port can be expanded in a cascading mode. The connection mode of the high-voltage output pins and the electrodes can be 1 to 1 or 1 to more, when the connection mode of 1 to more is used, the two controlled electrodes are kept at a certain distance as far as possible, and the control of liquid drops is guaranteed to be not influenced by each other. In addition, the boost circuit can also use an external power source, but it is not favorable for the portability and miniaturization of the whole system.

Example 2 manipulation of droplets

The distance between the upper and lower polar plates in the closed structure is set to be 170 μm, and 20 μ l of large liquid drops with different colors (which is convenient for observing the mixing and splitting states of the liquid drops) are added on each circular electrode at the open side on a plurality of semi-closed circular electrodes. The large droplets respectively generate 2 mul of small droplets with consistent volume on the tail small electrode (the 3 rd small electrode) in a plurality of small droplet generating structures; the droplets are then fed into an M x N rectangular array, and the droplets of different samples (e.g., differentiated by color) are circulated according to a predetermined path in a closed or open configuration for thorough mixing (the droplets are transported back and forth between multiple electrodes as much as possible during mixing), and can be split or otherwise manipulated in the closed configuration after mixing. Furthermore, if a larger number of droplets are required in the closed structure, i.e., a larger flux, the top plate can be moved to an open system or can be chosen appropriately before the experiment to increase the closed area fraction. If the liquid drops need to be removed in the experimental process, the liquid drops in the closed system can be transported to an open area, and the operation is convenient.

For example, it can be used in the extraction process of nucleic acid, first generating a small droplet from each of lysis buffer, biological sample and large droplet of magnetic bead suspension, transporting three droplets to the first mixing region for fusion (pH = 5), moving left and right for thorough mixing, and then moving the droplets and small droplets generated by purification buffer to the second mixing region for thorough mixing (pH = 7) to complete the binding of DNA/RNA and magnetic beads (pH = 5); then applying a magnetic field to enable the magnetic beads combined with the DNA to be precipitated to the bottom of the liquid drop (the magnetic field can be applied by fixing a neodymium magnet on the other side of the PCB), enabling other impurities to be in the supernatant, moving the liquid drop to a waste liquid pool, and keeping the magnetic beads combined with the nucleic acid in a magnetic field area; then moving the small drops generated by the elution buffer to the magnetic field area to complete the separation of the DNA/RNA from the magnetic beads (pH = 8.8), wherein the elution process can be repeated for 2-4 times to fully extract the DNA/RNA bound on the magnetic beads; finally, a large liquid drop containing nucleic acid molecules is obtained; if the extracted sample needs to be subjected to parallel experiments, the liquid drops can be transported to a closed rectangular array electrode area, splitting operation is carried out, and a plurality of small liquid drops with the same volume are generated. Finally, the nucleic acid sample droplets are transported to an amplification reaction tank (a circular liquid storage tank), and are fused with the main mixed solution to perform nucleic acid amplification reaction and analysis. According to the detection requirement, the reaction product can be flexibly transported to an open or closed structure. If the liquid drop needs to be detected by fluorescence or the like (the liquid drop needs to be kept to be aggregated into a sphere), the small liquid drop generated by the reaction product is transported into the open structure for multiple times and then is fused into a large liquid drop.

In the experimental process, a plurality of liquid drops of different buffer solutions can be generated simultaneously, the liquid drops of the same type are combined to form a strip-shaped liquid column, different liquid columns are operated according to the same sequence, and the flux of the system can be improved, so that the reaction efficiency is improved. If the liquid drops which do not react completely need to be removed in the experimental process, the liquid drops can be transported from the closed structure to the open area, and then the liquid drops can be conveniently removed. When the experiment begins at every turn, can change SLIPS insulating hydrophobic membrane again to reduce or eliminate the pollution between different reagents, also prevent simultaneously that the film live time is overlength and lead to hydrophobicity to reduce, insulating nature weakens and cause the easy scheduling problem that punctures of film, and then increase equipment reuse rate, reduce the experiment cost by a wide margin.

Claims

1. A semi-closed digital microfluidic system based on an SLIPS insulating hydrophobic membrane is characterized in that a PCB substrate, an electrode array and an upper electrode plate are sequentially arranged from bottom to top; and a droplet manipulation controller; wherein:

the PCB substrate is used as a lower electrode plate;

the electrode array is arranged on the surface of the PCB substrate; forming a single electrode by adopting a pad tin spraying mode; the electrode array consists of a square array consisting of M multiplied by N square small electrodes with sawtooth edges and a plurality of circular electrodes, and the circular electrodes are uniformly distributed on two sides of the square array; the electrical connection is realized by connecting the electrode center through hole to a control circuit;

the circular electrode is used as a liquid storage tank for storing the reacted liquid drops or storing a large liquid drop source for generating small liquid drops;

the square small electrode with the sawtooth-shaped edge is a driving electrode and is used for controlling the generation, movement, splitting and combination of liquid drops;

a layer of SLIPS insulating hydrophobic membrane covers the electrode array;

the upper electrode plate is made of ITO conductive glass, and the lower surface of the upper electrode plate is also covered with a layer of SLIPS insulating hydrophobic film; a glass gasket is arranged between the edge of the upper electrode plate and the electrode array; for controlling the distance between the two;

the upper electrode plate covers the electrode array, and the upper electrode plate and the square electrode array form a structure which is half closed and half open; the upper electrode plate can move left and right, and the edge of the electrode plate at one end of the circular electrode can completely cover the circular electrode and can also cover half of the circular electrode, so that liquid drops can be added or removed conveniently; the edge of the upper electrode plate at one end of the square electrode array is arranged in the middle of the electrode array to realize coexistence of an open area and a closed area, namely a structural mode combining a double-layer structure and a single-layer structure is adopted;

the controller for controlling the liquid drops comprises a single chip microcomputer, a high-voltage push-pull output chip and a booster circuit;

the single chip microcomputer is used for downloading programs and is a micro control unit of the whole digital micro-fluidic system;

the high-voltage push-pull output chip is respectively connected with the single chip microcomputer and the electrode array, receives a control signal of the single chip microcomputer on one hand, and outputs a voltage signal to a corresponding electrode on the other hand in parallel; the plurality of high-voltage push-pull output chips can be cascaded to expand output;

the booster circuit boosts the 5V direct current power supply voltage of the singlechip to 40 to 300V, and inputs the boosted voltage to a corresponding electrode through a high-voltage push-pull output chip to realize control of a power-on time sequence; the output voltage can be adjusted by a digital potentiometer.

2. The semi-closed digital microfluidic system according to claim 1, wherein said circular electrode has a diameter of 10-13.5 mm; the side length of the square electrode with the sawtooth-shaped edge is 2-3 mm, the tooth depth is 0.2-0.3mm, the tooth tip angle is 60-120 degrees, and the distance between the electrodes is 80-100 mu m.

3. The semi-enclosed digital microfluidic system according to claim 1, wherein the distance between said upper electrode plate and the electrode array covering the SLIPS insulating hydrophobic membrane is 50-300 μm.

4. The semi-enclosed digital microfluidic system of claim 1, wherein in the enclosed structural mode, the upper plate can be operated without being powered up; either the top plate is powered up or serves as a reference electrode when needed.

5. The semi-closed digital microfluidic system according to claim 1, wherein the SLIPS insulating hydrophobic membrane is obtained by coating silicone oil with viscosity of 10cst on the surface of a polymer compound PTFE membrane modified by 0.01-0.1% wt PFOTS-ethanol solution, and is used as a medium hydrophobic integral membrane.

6. The semi-enclosed digital microfluidic system of claim 1, wherein the ratio of the enclosed and open areas of the electrode array is changed by moving the top plate to accommodate different application scenarios.

7. The semi-closed digital microfluidic system according to claim 1, wherein in a scene with less electrode requirements, the electrodes are controlled in a 1-to-1 manner, i.e. one control pin is connected to 1 electrode; in a large-scale electrode array use scene, control pins can be added through cascade connection of high-voltage push-pull chips, or on the premise of not influencing each other, a control mode of 1 pair of more electrodes is adopted, namely one control pin is connected with a plurality of electrodes.

8. The semi-closed digital microfluidic system according to one of claims 1 to 7, wherein the control of microfluidics is achieved by a structural mode combining a double-layer structure and a single-layer structure, i.e. a closed structure and an open structure; in the two-layer structure mode, the liquid is confined in the space between the parallel plates, the lower plate being a SLIPS insulating hydrophobic film covering the electrode array; the upper flat plate, namely the upper electrode plate, is ITO glass covered with a SLIPS insulating hydrophobic film; the upper plate electrode is not connected with electricity and is not used as a reference potential, and only plays a role in limiting the liquid drop to a set space; in a two-layer structure, therefore, the driving of the droplets is dependent on the electrodes covered by the hydrophobic insulating layer of the lower plate; when the upper plate electrode is electrified, the hydrophobic insulating layer covers the electrode, and an electric field is generated between the electrodes on the opposite flat plates; this electric field creates a surface tension gradient that causes the shape of the droplet to change and move towards the electrode in a preset direction; by suitable arrangement and control of the electrodes, by continuously transporting droplets between adjacent electrodes; to any location covered by the electrode; the space around the liquid drop is filled with gas or liquid which is not dissolved mutually; the basic operations of generating, transporting, mixing and splitting four kinds of liquid drops are realized in a double-layer structure.

9. A droplet control method based on the semi-closed digital microfluidic system of claim 8, comprising:

(1) Generating liquid drops;

small droplets are generated from larger droplets in a closed bilayer structure in the following manner: firstly, placing larger liquid drops on a circular electrode, and keeping a bottom plate electrode in a positive bias voltage state, wherein an upper electrode plate is not electrified; then, the circular electrode where the larger liquid drop is positioned is under negative bias voltage, so that the liquid drop is uniformly distributed on the circular electrode; then, the small electrode completely wrapped by the circular electrode, namely the 1 st small electrode is under negative bias voltage, and the circular electrode is under positive bias voltage; when the liquid column is conveyed to the 1 st small electrode and completely covers the small electrode, the small electrode which is semi-surrounded by the circular electrode, namely the 2 nd small electrode, is switched into negative bias voltage, and meanwhile, the 1 st small electrode is maintained to be negative bias voltage; when the liquid column completely covers the 2 nd small electrode, the small electrode adjacent to the 2 nd small electrode, namely the 3 rd small electrode is under the negative bias voltage, and the 1 st small electrode and the 2 nd small electrode are maintained under the negative bias voltage; after the liquid column covers the 3 rd small electrode, the circular electrode where the large liquid drop is located and the 3 rd small electrode are simultaneously under negative bias voltage, other electrodes are under positive bias voltage, and the small liquid drop can be generated smoothly after the liquid column is maintained for a period of time;

(2) And (3) transporting liquid drops: the liquid drop transportation is carried out in a single-layer structure, a double-layer structure and a semi-closed structure; the specific operation method is that the electrode where the liquid drop is located is connected with negative bias voltage to enable the electrode to be located at the central position of the electrode where the liquid drop is located, and the adjacent electrodes are all positive bias voltage; subsequently, determining the polarity of the adjacent electrode according to the transport direction of the liquid drop; if the liquid drop needs to move to the adjacent electrode at the left side in the next step, setting the adjacent electrode as a negative bias voltage, and simultaneously connecting the electrode where the liquid drop is located with a positive bias voltage, and carrying out the next liquid drop transportation operation when the liquid drop completely moves to the adjacent electrode; when the target position of the liquid drop is far away from the current position, the path is flexibly set according to the requirement;

(3) Merging of droplets: through control, small droplets on different electrodes on the square electrode array are conveyed to the same electrode to be combined, and then the combined droplets are circularly conveyed according to a certain path to be fully mixed; the operations of merging and mixing of the droplets can be carried out in both closed and open configurations;

(4) Splitting of the droplets: the method comprises the following steps of splitting a larger liquid drop occupying 2 electrodes in a closed structure into two liquid drops occupying 1 electrode, wherein the specific operation method comprises the steps of simultaneously applying negative bias voltage to two adjacent electrodes on two sides of the larger liquid drop occupying 2 electrodes, and applying positive bias voltage to other electrodes; furthermore, it is also possible to break up droplets of multiple electrode sizes in a closed structure into droplets of arbitrary electrode sizes.