A DMF chip, a rapid PCR system and a PCR method

Abstract

The application discloses a DMF chip, a rapid PCR system and a PCR method, and relates to the technical field of polymerase chain reaction. The DMF chip comprises a bottom plate, electrodes, a dielectric layer and a hydrophobic layer. The dielectric layer is arranged on the bottom plate with the electrodes fixed on one side of the bottom plate. The hydrophobic layer covers the surface of the dielectric layer. The dielectric layer is made of raw material polydimethylsiloxane. The hydrophobic layer is made of raw material expanded PTFE film. The DMF chip and the rapid PCR system are developed on the digital microfluidic platform based on dielectric wetting. The chip or the system improves the robustness of droplet movement at high temperature by adopting PDMS as the dielectric and expanded PTFE film as the hydrophobic layer. The PDMS can protect the electrodes in the high-temperature area above 90 DEG C from being broken down and will not hinder the shuttle movement of the droplets between the high-temperature area and the low-temperature area. The PCR reaction on the chip can be completed in 4 minutes through the above measures.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of polymerase chain reaction technology, in particular to a DMF chip, a rapid PCR system and a PCR method. BACKGROUND

[0002] Polymerase chain reaction (PCR) is a core technology in modern molecular biology and diagnostics. This temperature-dependent technique exponentially amplifies DNA through repeated cycles of heating and cooling. Traditional thermal cyclers are bulky, and a typical PCR usually takes 1-2 hours. In clinical point-of-care diagnostics, especially for infectious diseases, reaction time, reaction specificity, and instrument portability are crucial for rapid clinical decision-making at the point-of-care. With the miniaturization feature of microfluidic technology or capillary reactors, many rapid PCR methods have been developed and applied to point-of-care diagnostics. However, these technologies still require large and complex accessories to support the thermal cycling process. Other rapid PCR setups using laser or plasmonic technology require expensive illumination instruments as light sources.

[0003] Digital microfluidics (DMF) is a branch technology in the field of microfluidics, which can control single microliter to nanoliter size droplets. Digital microfluidics technology based on electrowetting-on-dielectric (EWOD) can handle single droplets on an electrode array through electrowetting force. Its electrically driven function and small footprint make it a promising technology for point-of-care diagnostics.

[0004] The DMF technology contains four basic elements: substrate, electrodes, dielectric layer and hydrophobic layer. The substrate is usually made of glass and silicon wafer, but the cost is high, and more and more DMF chips use printed circuit board (PCB) substrate, which can realize mass production at low cost. The electrode material is usually metal (such as chromium, aluminum, gold, copper, platinum, etc.) or non-metallic conductive material (such as indium tin oxide ITO, doped polysilicon, etc.). An insulating dielectric layer needs to be covered on the electrode to form an electric field effect for droplet movement. The commonly used dielectric layer is Parylene, Ta2O5, amorphous fluorine-containing polymer, Si3N4, PDMS or SU-8. A hydrophobic layer needs to be covered on the dielectric layer to reduce the surface energy required to drive the droplet, which is usually Teflon AF, and some studies also use SiOC, Cytop and expanded PTFE super-hydrophobic film. The DMF chip structure is divided into single board type (open type) and double board type (closed type). In the single board structure, the driving electrode and the ground electrode are located on the same substrate; in the double board structure, the droplet is sandwiched between the top plate and the bottom plate, the driving electrode is usually on the bottom plate, and the ground electrode is on the top plate, which is a continuous transparent conductive layer (usually ITO), and the surface of the ground electrode of the top plate only needs to be hydrophobic and does not need a dielectric layer. Both single board and double board structures can use air or oil (such as silicone oil, dodecane, hexadecane, etc.) as medium. The medium oil can reduce the droplet driving voltage and prevent the droplet from evaporating. The double board structure can realize a wider range of droplet operations, such as dispensing, moving, splitting and merging, while the single board structure cannot realize droplet separation and splitting.

[0005] The PCR method on the digital microfluidic system is divided into two categories: one is the time-domain stationary PCR, and the other is the space-domain shuttling PCR. In the experimental setup of stationary PCR, the PCR reaction droplet stays on an electrode for in-situ PCR reaction, and the thermal cycle is realized by controlling the heating time. The heater can be a large external heater that heats and cools the entire chip, or a chip-on-heater with a smaller heating volume.

[0006] Representative studies using static PCR on DMF include: (1) The first DMF chip-based PCR used a glass substrate with titanium-platinum-gold electrodes, 150-300 nm Si3N4 as the dielectric layer, 50 nm Teflon as the hydrophobic layer, the PCR reaction electrode was treated with O2 plasma to make it hydrophilic, the cover plate was Teflon-coated ITO glass as the ground electrode, the dielectric oil was 1 cSt silicone oil. The heater was a platinum resistance heating electrode and a temperature sensing electrode on the chip. The PCR reaction droplet volume was 15 μL, and the total time for 25 cycles was 55 minutes.

[0007] (2) A hybrid chip of capillary and DMF for automated DNA next-generation sequencing library construction. The chip used ITO glass etched electrodes, 4 μm Parylene-C as the dielectric layer, 50 nm Teflon-AF as the hydrophobic layer, and a Teflon-coated ITO glass as the top plate. The capillary was inserted between the cover plate and the bottom plate as the sample inlet and outlet. The PCR reaction solution was prepared on the chip and then sucked into the PCR capillary, and the heat cycle was performed by the aluminum block and the closed-loop temperature controller below. The total time for 10 cycles of PCR was more than 40 minutes.

[0008] (3) PCR on a 4 mm x 4 mm COMS integrated circuit DMF chip. The dielectric layer was 2 μm thick Parylene-C, the hydrophobic layer was 100 nm Teflon, the top plate was ITO-coated polyethylene naphthalate (PEN), and the dielectric oil was dodecane. This study used micrometer-scale on-chip heating wires and temperature sensors for temperature control, and a DNA thermometer was used to calibrate the sensors. Three heating wires nested in the chip together controlled the temperature of the entire surface, achieving overall heating. The PCR reaction system was 1.2 nL, the heat cycle time was more than 32 s / cycle, and the total time for 35 cycles was more than 18 minutes.

[0009] (4) DMF chip for single cell isolation, mRNA purification and multiplex PCR. DMF chip is coated with 200 nm TiAlCu electrode on a silicon substrate, 300 nm Si3N4 as dielectric layer, 1 μm and 250 nm SiOC as hydrophobic layer on the silicon substrate and ITO glass cover, respectively, and 5 cSt silicone oil as medium oil. The chip is placed on a copper heat sink and heated on a Peltier heating plate, and a temperature sensor is placed in the copper heat sink. The temperature sensor placed in the copper heat sink is corrected by a micro-thermocouple inserted into the chip. The PCR reaction system is 64-128 nL, the thermal cycle time is 60 s / cycle, the cycle number is 40-50, and the total time is 40-50 minutes. Static PCR avoids electrode breakdown and bubble generation caused by movement of PCR droplets at high temperature. The disadvantage is that slow heat conduction leads to longer reaction time and the accuracy of temperature sensing during rapid temperature rise and fall.

[0010] In the experimental setup of shuttle PCR, PCR reaction droplets move back and forth between different temperature zones. Some studies use three temperature zones (denaturation, annealing, and extension) to implement three-step PCR. Some use two temperature zones (denaturation, annealing + extension) to implement two-step PCR. PCR droplets in shuttle PCR move between temperature zones with stable temperature control, which can achieve faster reaction time. Representative studies using shuttle PCR on DMF include:

[0011] (1) DMF chip with PCB as the bottom plate and ITO glass as the top plate, and hexadecane as the medium oil. This study uses two external aluminum heating strips and a thermistor temperature sensor to control the temperature, and the PCR reaction system is 0.6 μL, 95°C for 5 s, 60°C for 7-13 s, and 40 cycles with a total time of less than 12 minutes.

[0012] (2) DMF chip with glass bottom plate, Cr / Au electrode, SiO2 dielectric layer, and Teflon hydrophobic layer. A 0.1 mm thick layer of 1 cSt silicone oil covers the bottom of the chip, and the cover plate is Teflon-coated ITO glass. The top of the cover plate is coated with a curved Cr / Au as a heating electrode and a thermistor temperature sensor, and the temperature correction uses fine-element (FEM) simulation. The volume of the PCR droplet is 0.7 μL, and the total reaction time for 35 cycles is 2.4 h.

[0013] (3) The DMF chip with air medium adopts 6-layer PCB board, uses 15 μm resistive layer as dielectric layer, and 50 nm Teflon as hydrophobic layer. The 1.0 x 7.0 mm cavity is used as temperature insulation layer to insulate the temperature of the three temperature zones of PCR. The heating module and the resistance temperature sensor are attached to the reverse gold-plated copper plate of the PCB board by using heat-conducting glue, and are connected to the electrode on the front of the PCB by the via filled with heat-conducting glue. The top plate is ITO glass coated with Teflon as the grounding electrode. A very thin thermocouple inserted into the liquid drop is used as the RTD temperature sensor on the back of the PCB board. The PCR reaction liquid is 1.5 μL, and the liquid drop is shuttled among the three temperature zones of 95℃, 50℃ and 68℃. The total time of 33 thermal cycles is about 45 minutes.

[0014] In the prior art, the fastest PCR completion time on the DMF is not less than 10 minutes.

[0015] Therefore, the present application is proposed. SUMMARY

[0016] The present application aims to provide a DMF chip, a fast PCR system and a PCR method to solve the above technical problems.

[0017] The inventor found that the following four factors affect the stable and fast PCR reaction of the shuttle PCR: (1) Because of the large temperature difference between the high temperature zone (90-95℃) and the low temperature zone (60-65℃) of PCR, the moving distance is quite long; (2) The breakdown voltage of the electrode decreases at high temperature. The movement of the liquid drop in the high temperature zone is easy to cause electrode breakdown, thereby causing electrode power failure, electrolysis of the liquid drop, etc.; (3) The temperature of the high temperature zone causes the dielectric layer to expand, thereby hindering the movement of the liquid drop. (4) When moving at a high temperature higher than 80℃, bubbles are easily generated, causing inaccurate temperature of the temperature zone and affecting the PCR reaction efficiency.

[0018] Therefore, the inventor proposes a DMF chip that meets the liquid drop shuttling, uses polydimethylsiloxane (PDMS) as the dielectric layer, and uses expanded PTFE film as the hydrophobic layer, so that the liquid drop can move smoothly between the high and low temperature zones. The PDMS can protect the electrode in the high temperature zone above 90℃ from being broken down, and will not hinder the shuttle movement of the liquid drop between the high and low temperature zones. Through the above measures, the PCR reaction on the chip can be completed in 4 minutes, and the reaction cycle time is reduced to 5.5 seconds / cycle.

[0019] The present application is implemented as follows:

[0020] The present application provides a DMF chip, comprising: a bottom plate with an electrode layer, a top plate with an electrode layer, a dielectric layer and a hydrophobic layer, the dielectric layer is arranged on the bottom plate with the electrode layer fixed on one side, the hydrophobic layer covers the surface of the dielectric layer, the dielectric layer is made of raw material polydimethylsiloxane, and the hydrophobic layer is made of raw material expanded PTFE film.

[0021] PTFE is polytetrafluoroethylene, and the expanded PTFE film is made of polytetrafluoroethylene resin through special processing methods such as stretching, and is elastic and flexible.

[0022] The material of the bottom plate substrate includes but is not limited to: glass, PCB board, silicon. The electrode layer includes but is not limited to ITO, chromium, copper, gold, silver, platinum and other metal layers.

[0023] In an alternative embodiment, the top plate is a transparent substrate with a transparent electrode, and the top plate substrate includes but is not limited to glass, polyethylene terephthalate (PET) and the like. The transparent conductive layer includes but is not limited to indium tin oxide ITO, aluminum-doped zinc oxide AZO, fluorine-doped tin oxide FTO, antimony-doped tin oxide ATO and the like.

[0024] The selection of the dielectric layer and the hydrophobic layer is crucial for the shuttle PCR on the DMF chip. When the temperature exceeds 90℃, many materials cannot maintain the smooth movement of the droplets. The driving electrode is easy to be broken down at high temperature, because the breakdown voltage will decrease when the temperature rises. The substance with high stiffness will expand when heated and hinder the droplets from entering the temperature zone. Most importantly, the bubbles generated at high temperature will cause the temperature of the reaction area to be uneven and hinder the movement of the droplets.

[0025] The inventors have tried various combinations of dielectric materials and hydrophobic materials, including SU-8 and Teflon, Parylene C and Teflon, NOA61 / 68 and Teflon, 50-μm ultra-thin glass (bonded with NOA61) and Teflon. These materials are suitable for droplet transmission at ambient temperature and 50℃. However, when the temperature rises above 80℃, they will experience electrode breakdown, movement obstacles or, most commonly, bubble formation.

[0026] We observed that the breakdown of the dielectric layer is related to three factors: (a) the thicker the dielectric layer, the less breakdown; (b) the larger the electrode gap, the less breakdown; (c) the softer the dielectric, the less breakdown. And the softer the dielectric substance, the less the thermal expansion of the dielectric layer hinders the movement of the droplets.

[0027] We also observed that bubbles often form on Teflon surfaces when the temperature exceeds 80 °C. It is well known that perfluorinated surfaces (e.g., Teflon and Cytop coatings) are water (hydrophobic) and oil (oleophobic) repellent. The low wettability of oil on perfluorinated surfaces causes air to be easily trapped in micro- and nano- geometrical structures or surface cracks. At high temperatures, the air trapped in the cracks can form bubble nuclei. When a water droplet contacts the bubble nuclei at high temperatures, the bubble grows rapidly with the evaporation of water.

[0028] To bond the expanded PTFE film to the glass substrate, we chose polydimethylsiloxane (PDMS) as the bonding material and dielectric layer. PDMS is an organic silicon-based elastomer. Its elastic properties can protect the electrodes from being severely damaged at high temperatures. And the surface expansion at high temperatures will not hinder the movement of the droplets.

[0029] In a preferred embodiment of the application, the raw material of the dielectric layer further comprises a diluent and a curing agent, and the mass ratio of the polydimethylsiloxane to the curing agent is (2-19): 1; the mass ratio of the sum of the polydimethylsiloxane and the curing agent to the diluent is 1:(0-5). The hydrophobic layer contains a lubricating liquid.

[0030] In an alternative embodiment, the mass ratio of the polydimethylsiloxane to the curing agent in the raw material is (2-10): 1; the mass ratio of the sum of the polydimethylsiloxane and the curing agent to the diluent is 1:(1-5).

[0031] To find a durable dielectric and hydrophobic layer for on-chip rapid PCR, we tested a slippery liquid infused porous surface (SLIPS) of expanded PTFE film (ePTFE) as the hydrophobic layer. This material has both oleophilic and hydrophobic properties.

[0032] The porous expanded PTFE film without lubricating liquid is hydrophobic by itself. After absorbing lubricating liquids such as hexadecane oil, the film becomes translucent and forms a thin liquid film on the surface. The liquid-liquid interaction reduces the contact angle of the droplet from 130.3° to 105.7°. Studies have shown that SLIPS surfaces with low contact angle hysteresis can be used as a hydrophobic layer on DMF chips, and they have the effect of resisting the adhesion of biomolecules. Lubricating liquids can easily expel all air from the nanoscale pores of the film. Therefore, it greatly alleviates the problem of bubble formation at high temperatures by limiting the formation of bubble nuclei.

[0033] In an alternative embodiment, the diluent is n-hexane, cyclohexane, tetrahydrofuran, chloroform, toluene, xylene, n-heptane. The lubricating liquid is hexadecane. In other embodiments, the lubricating liquid can also be selected from other mineral oils, synthetic oils, animal and vegetable oils, and water-based liquids.

[0034] The hydrophobic layer has a reticular or porous structure with micrometer-scale pores, and the thickness of the hydrophobic layer is 5-100 μm;

[0035] Preferably, the contact angle of the hydrophobic layer with oil droplets is 0-30° (with lipophilic properties), and the contact angle of the hydrophobic layer with water droplets is 100-150° (with hydrophobic properties); the micrometer-scale pore size is 50-500 μm. The inventors have found that a film with such characteristics has the function of avoiding bubble generation at high temperatures in a digital microfluidic chip.

[0036] The PDMS-bonded expanded PTFE surface can support smooth droplet transport at high temperatures. However, it still experiences dielectric breakdown and causes surface bulging in a time-dependent manner. We further address these issues by enlarging the electrode gap and diluting the PDMS to make the dielectric softer and more elastic. The inventors have found that the enlargement of the electrode gap in the high-temperature zone effectively eliminates electrode breakdown. Diluting the PDMS with n-hexane can reduce surface expansion. All these measures result in a durable hydrophobic surface that can withstand at least 240 PCR shuttle PCR thermal cycles.

[0037] In a preferred embodiment of the application, the electrodes described above comprise driving electrodes, the path of the driving electrodes is composed of two rows of rectangular electrodes, and the two rows of rectangular electrodes are arranged in parallel adjacent to each other on the bottom plate, and the rectangular electrodes of the two adjacent rows are distributed in staggered positions, forming an interlocking electrode pattern, and the plurality of rectangular electrodes can realize high-voltage driving and grounding functions at different times, and when any one of the electrodes is connected to a high-voltage electrical signal, the surrounding rectangular electrodes are in a grounded state.

[0038] If a one-piece long ground electrode is used, only half of the droplet can be driven. In order to increase flexibility, the one-piece long ground electrode is divided into a plurality of rectangular electrodes, however, non-staggered electrodes can only support droplet movement at room temperature, but cannot support droplet movement at high temperatures. The movement obstacle is caused by the failure of the droplet to contact the next electrode. The top of the droplet is not provided with a contact ground electrode in the present application, and the electrode arrangement of the present application adopts a novel interlocking structure, so that the parallel ground driving electrodes can support the smooth movement of PCR droplets at high temperatures. Through these measures, the PCR reaction on the chip can be completed in 4 minutes.

[0039] In an alternative embodiment, the plurality of rectangular electrodes are arranged in two rows of staggered form on the bottom plate.

[0040] In a preferred embodiment of the application, the DMF chip further comprises a top plate and a fence (also as a height-limiting gasket) arranged between the top plate and the bottom plate, the top plate is provided with a plurality of heat insulation grooves on the side away from the bottom plate, and a micro-heater is arranged between any two heat insulation grooves.

[0041] The heat insulation grooves are cut around the high-temperature zone to expand the temperature difference, thereby shortening the shuttle distance of the PCR droplet on the chip. The inventors found that the arrangement of the heat insulation grooves greatly reduces the temperature difference of the low-temperature zone, and has no effect on the temperature difference of the high-temperature zone.

[0042] The temperature regulation consists of two parts: a bottom heater that heats the entire chip and a micro-heater on the top plate that generates the DNA denaturation temperature. The double-heater can significantly reduce the vertical temperature difference in the droplet. The temperature sensor uses a chemical sensor in the droplet to ensure temperature calibration and accurate temperature reading of the PCR reaction.

[0043] In an alternative embodiment, the depth of the heat insulation groove is 0-0.9mm (0%-82% of the total thickness of the top plate), the width of the heat insulation groove is 0.25-0.45mm, and the distance between the heat insulation groove and the micro-heater is 0.15-0.30mm. In an alternative embodiment, the total thickness of the top plate is 1.1mm.

[0044] The inventors found that the width and distance of the different heat insulation grooves have no difference in temperature distribution.

[0045] The micro-heater is a transparent electrode in a meander shape and left-right symmetry, which is distributed in the heat insulation groove of the top plate; and the transparent electrode is wide in the middle and narrow on both sides, so that the temperature distribution is uniform when direct current passes through the electrode; the bottom plate has a high-temperature zone and a low-temperature zone, and the heat insulation groove is arranged in the high-temperature zone.

[0046] The application also provides a rapid PCR system, which comprises the DMF chip.

[0047] In a preferred embodiment of the application, the rapid PCR system further comprises an electronic control device for droplet manipulation and temperature regulation, a computer control terminal with control software, and a fluorescence microscope for detecting DNA amplification products; the electronic control device comprises a bottom heater arranged at the bottom of the DMF chip, an FPGA unit, a high-voltage generation module, and an electrode switch array; the FPGA unit is in communication connection with the computer control terminal.

[0048] In an alternative embodiment, the rapid PCR system further comprises a chemical temperature indicating reagent in the droplet.

[0049] Chemical temperature indicating reagent has significant advantages compared with traditional sensors. First, traditional sensors are usually far away from the droplet, some embedded under the dielectric layer, and some even outside the device. Physical temperature sensors inserted into the droplet can cause damage to the hydrophobic coating and are not suitable for use in DMF systems. In this case, the sensor temperature lags behind the droplet temperature. Temperature calibration and compensation inside the droplet are needed to correct the temperature. From this point of view, chemical temperature sensors can directly and instantaneously detect the droplet temperature, especially in the case of rapid temperature transition. Second, traditional temperature sensors can only sense a single point or the average temperature of the entire droplet. In contrast, chemical indicating reagents can achieve analysis of the horizontal temperature distribution of the droplet.

[0050] We chose a temperature-sensitive inert dye, sulfonyl rhodamine B (SRB). It has been proven to be compatible with PCR and does not inhibit PCR reactions, and can be used as a temperature indicating dye in PCR reactions. It is reported that no photobleaching and thermal degradation of SRB dye is observed at 50℃. The thermal degradation rate at 80℃ is 2.2% / h, and at 94℃ it is 5.4% / h.

[0051] In an alternative embodiment, the above-mentioned chemical temperature indicating reagent is selected from rhodamine dyes and derivatives thereof;

[0052] In an alternative embodiment, the chemical temperature indicating reagent includes but is not limited to red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC) and the like or analogs thereof;

[0053] In an alternative embodiment, the chemical temperature indicating reagent is selected from sulfonyl rhodamine B (SRB). Sulfonyl rhodamine B (SRB) is a temperature-sensitive dye added to the aqueous calibration droplet. The fluorescence it emits changes with temperature. SRB has been used in PCR solutions to monitor reaction liquid temperature and has been proven to be compatible with PCR reactions.

[0054] The present application also provides a method for performing PCR using the above-mentioned rapid PCR system, comprising:

[0055] The chemical temperature sensing droplet containing a chemical temperature indicating reagent is introduced into the DMF chip by the method of droplet shuttling, and then the chemical temperature sensing droplet is moved to the high temperature zone. The droplet moves back and forth between the high temperature zone and the low temperature zone according to the command script, and the fluorescence intensity under the first fluorescence channel is recorded. The direct current input to the heating electrode located on the top plate is controlled. When the temperature of the chemical temperature sensing droplet in the high temperature zone of the DMF chip reaches 95℃, the voltage value of the direct current is recorded. The chemical temperature sensor droplet is taken out. The PCR reagent droplet is added to the high temperature zone on the DMF chip. The heating electrode is heated to 95℃ using the same direct current voltage value. After 0-10s preheating at 95℃, the PCR reagent droplet shuttles between the high temperature zone and the low temperature zone according to the command script, and the fluorescence intensity under the second fluorescence channel when the droplet is in the low temperature zone is recorded.

[0056] The PCR method provided by the application has high PCR amplification efficiency, sensitivity and specificity on the rapid chip.

[0057] In an alternative embodiment, the first fluorescence channel is a Cy3 channel, the second fluorescence channel is a GFP channel, and the chemical temperature sensor is selected from SRB.

[0058] In an alternative embodiment, the above-mentioned fluorescence channels include but are not limited to the fluorescence channels of fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), FAM, TET, etc. or their analogues), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), TAMRA, TRITC, etc. or their analogues), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogues), Alexa series dyes and their derivatives (for example, including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogues), and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0059] In a preferred embodiment of the application, a reversible hot start reagent is added to the above-mentioned PCR reagent droplet;

[0060] In an alternative embodiment, the reversible hot start reagent is ThermaStop TM .

[0061] Reversible hot start reagents can improve the specificity of amplification and greatly reduce the generation of non-specific products.

[0062] In an alternative embodiment, the concentration of polymerase in the PCR reagent droplet is 0.1-0.5 U / μL, and the concentration of primer in the PCR reagent droplet is 0.2-20 μM.

[0063] The concentration of primer and the concentration of polymerase are particularly crucial for rapid PCR, and at the above concentrations, PCR performance is good, while too high a primer concentration can lead to the formation of non-specific products.

[0064] In a preferred embodiment of the application, the denaturation dwell time in the above method is 0.1-10 s. Longer denaturation time can result in lower yield, possibly due to the fact that the longer the droplet stays in the high-temperature zone, the greater the vertical temperature difference.

[0065] In a preferred embodiment of the application, the annealing and extension time in the above method is 0.5-60 s.

[0066] In an alternative embodiment, the annealing and extension time in the method is 1 s. In terms of total reaction time, the total amplification time of PCR with 2-second annealing / extension time is the longest. In order to obtain faster PCR reaction time, 1 s of annealing / extension time is more optimal.

[0067] The present application has the following beneficial effects:

[0068] The present application develops a DMF chip and a rapid PCR system based on a digital microfluidic platform of dielectric wetting, which improves the robustness of droplet movement at high temperature (more than 90℃) by using PDMS as the dielectric and ePTFE film as the hydrophobic layer. PDMS can protect the electrodes in the high-temperature zone above 90℃ from being punctured and will not hinder the shuttle movement of droplets between high and low temperature zones. Through the above measures, the PCR reaction on the chip can be completed in 4 minutes, and the reaction cycle time is reduced to 5.5 seconds / cycle. BRIEF DESCRIPTION OF DRAWINGS

[0069] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0070] Figure 1 Schematic diagram of rapid PCR on a digital microfluidic chip;

[0071] Figure 2Figure 1. Fabrication and assembly of DMF chip;

[0072] Figure 3 Figure 2. Schematic of DMF chip and electronic control system;

[0073] Figure 4 Figure 3. Wettability of water and hexadecane on Teflon surface and SLIPS surface;

[0074] Figure 5 Figure 4. Morphology of expanded PTFE and Teflon surface under scanning electron microscope;

[0075] Figure 6 Figure 5. Morphology of expanded PTFE surface under atomic force microscope and scanning electron microscope;

[0076] Figure 7 Figure 6. Surface defects under high temperature and high driving voltage;

[0077] Figure 8 Figure 7. Schematic illustration of driving electrode;

[0078] Figure 9 Figure 8. Shuttle-like thermal cycling movement trajectory of PCR droplet on chip;

[0079] Figure 10 Figure 9. Calibration of chemical temperature sensor;

[0080] Figure 11 Figure 10. FEM simulation results of horizontal temperature with and without insulation groove;

[0081] Figure 12 Figure 11. FEM simulation results of temperature distribution inside droplet;

[0082] Figure 13 Figure 12. Temperature distribution under different insulation groove parameters;

[0083] Figure 14 (a) Rapid PCR amplification curve on DMF chip and agarose gel results. (b) Rapid PCR amplification curve on chip under different base chip temperature. (c) Non-chip PCR amplification curve under different polymerase concentration. (d) Rapid PCR amplification curve on chip under different polymerase concentration;

[0084] Figure 15 (a) Rapid chip PCR amplification curve under different DNA denaturation residence time. (b) Rapid chip PCR amplification curve under different annealing and extension time;

[0085] Figure 16 (a) Rapid chip PCR amplification curve of serially diluted DNA template. (b) C qStandard curve for linear regression analysis of values and Log DNA copy number. DETAILED DESCRIPTION

[0086] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, conventional conditions or conditions suggested by manufacturers are adopted. If manufacturers of reagents or instruments are not indicated, all are conventional products that can be obtained by market purchase.

[0087] The features and performances of the present application are further described in detail below in combination with embodiments.

[0088] Embodiment 1

[0089] This embodiment provides a digital microfluidic chip design and manufacturing method.

[0090] Both the bottom plate and the top plate are 1.1 mm single-sided ITO glass. As shown in FIG. 1, the electrode array, wiring and contact electrode on the bottom plate, ITO heater, thermal isolation groove, inlet hole and integral fence (0.31 mm) on the top plate are designed and drawn by AutoCAD and patterned by laser cutting. Figure 2

[0091] The polydimethylsiloxane (PDMS) elastomer is spin-coated on the ITO bottom plate with the moving electrode engraved as a dielectric layer after being diluted with n-hexane. The mass ratio of PDMS: curing agent: n-hexane is 10:1:11 (w / w / w). Then the bottom plate is heated at 74°C for 3-5 minutes to semi-cure the PDMS, and a expanded PTFE membrane (30 μm, pore size 250-450 μm) is covered on the semi-cured PDMS. Then the bottom plate is heated at 80°C for 10 minutes to fully cure the PDMS. The expanded PTFE membrane should be partially adhered to the PDMS, but not fully infiltrated by the PDMS to cause translucency.

[0092] The integral glass fence gasket (0.31 mm thick) with separated reaction chambers is adhered to the other side of the ITO heater on the top plate with Norland optical adhesive 61 (NOA61). The NOA61 is cured by ultraviolet light. Subsequently, a layer of Teflon AF1601 hydrophobic layer is spin-coated on the surface of the fence-adhered top plate. TM AF1601 hydrophobic layer.

[0093] When assembled, the expanded PTFE membrane on the bottom plate is fully infiltrated with hexadecane or other oily lubricating liquid (such as silicone oil). The bottom plate (expanded PTFE facing up) and the top plate (ITO facing up, fence gasket facing down) are tightly assembled together. Then the PCR separated reaction chambers are filled with hexadecane. A 0.2 mm diameter ultrafine thermocouple is inserted into the oil to monitor the temperature of the reaction chamber. ​

[0094] Example 2

[0095] This example provides a digital microfluidics system for rapid PCR. As shown in the system overview in FIG. 1, the system includes the following elements: an electronic control device for droplet manipulation and temperature regulation; a computer control terminal with control software; a DMF chip customized for rapid PCR; and a fluorescence microscope for detecting DNA amplification products. Rapid PCR on the digital microfluidics chip uses a droplet shuttle scheme. Figure 1 and Figure 3 The electronic control system is automatically controlled by computer software, which can achieve droplet manipulation and heating of the DMF chip. The FPGA unit in the system is used to communicate with the internally customized software on the computer through Bluetooth. The high-voltage (HV) generation module generates a high-voltage alternating current signal with adjustable waveform, frequency and amplitude for droplet driving. The electrode switch array can transmit the high-voltage signal to the designated driving electrode.

[0096] The heating system is composed of a bottom heater and a thin film micro-heater on the chip. The bottom heater is placed below the metal bracket of the DMF chip, and the heating area is the entire chip. The bottom closed-loop temperature regulator includes a positive temperature coefficient (PTC) resistor heater and a thermocouple temperature sensor inserted into the oil chamber. The ceramic heater and thermocouple sensor form a closed-loop feedback control.

[0097] The bottom heater provides the basic temperature (50-60°C) for the entire DMF chip. In addition, the ITO thin film micro-heater on the top plate generates a small area of high temperature (90-95°C) at the site of the reaction high temperature zone. The material of the micro-heater is ITO, which is a light-transmitting transparent non-metallic conductive material. By connecting a direct current to the ITO micro-heater to generate resistance heating. In order to obtain more accurate temperature indication, this study uses a chemical temperature sensor. The chemical temperature sensor - sulfonated rhodamine B (SRB) is a temperature-sensitive dye added to the aqueous calibration droplet. Its emitted fluorescence changes with temperature. SRB has been used in PCR solutions before to monitor the temperature of the reaction solution and has been proven to be compatible with PCR reactions.

[0098] The bottom heater provides the basic temperature (50-60°C) for the entire DMF chip. In addition, the ITO thin film micro-heater on the top plate generates a small area of high temperature (90-95°C) at the site of the reaction high temperature zone. The material of the micro-heater is ITO, which is a light-transmitting transparent non-metallic conductive material. By connecting a direct current to the ITO micro-heater to generate resistance heating. In order to obtain more accurate temperature indication, this study uses a chemical temperature sensor. The chemical temperature sensor - sulfonated rhodamine B (SRB) is a temperature-sensitive dye added to the aqueous calibration droplet. Its emitted fluorescence changes with temperature. SRB has been used in PCR solutions before to monitor the temperature of the reaction solution and has been proven to be compatible with PCR reactions.

[0099] Example 3

[0100] This example provides calibration of the chemical temperature sensor.

[0101] The SRB temperature calibration solution includes 1 x KAPA2G PCR Buffer A, 3 mM MgCl2, 200 nM dNTP, 500 pg^L BSA, 600 nM SRB, 0.6 x EvaGreen, 1.3% glycerol, and 0.1 U^L KAPA2G polymerase. 0.6 pL of SRB temperature calibration droplet was introduced into the DMF chip. A 200 pm diameter ultrafine thermocouple was inserted from the inlet hole on the top plate and contacted with the SRB droplet. For low temperature zone temperature calibration, the droplet was moved to the low temperature zone. The whole chip temperature was then ramped to 80 °C at 5 °C intervals. The fluorescence intensity of the SRB calibration droplet was monitored and recorded using a fluorescence microscope. For high temperature zone temperature calibration, the droplet was moved to the high temperature zone. The chip temperature was set to 50 °C. The ITO heater was turned on and the fluorescence intensity was recorded at 5 °C temperature increments. The calibration curve was derived from a non-linear curve fit to the fluorescence intensity.

[0102] Example 4

[0103] This example provides PCR reagents.

[0104] The off-chip and on-chip PCR reaction solutions were prepared using KAPA2G Fast PCR Kit following the optimized protocol for fast on-chip PCR. For off-chip PCR, the total reaction volume was 10 pL and was run on a real-time PCR detection system. The primer sequences used throughout the process are listed below:

[0105] HIV-1 gag forward primer: 5'-GGAGCCACCCCACAAGAT-3'

[0106] HIV-1 gag backward primer: 5'-CATTGCTGCCTGGTGTCC-3'

[0107] Amplification product length: 63 bp.

[0108] The final optimized reaction mixture contains 1 x KAPA2G PCR Buffer A, 3 mM MgCl2, 200 nM dNTP, 500 nM of each primer, 500 pg^L BSA, 0.6 x EvaGreen, 1.3% glycerol, 0.1 U^L KAPA2G / pL 0.1 ThermaStop TM The positive sample contains 0.4 pL of DNA template, and the no template control (NTC) contains 0.4 pL of ddH2O, and the final reaction volume was adjusted to 10 pL with ddH2O. The DNA template for HIV-1 was constructed into plasmid pUC57. The DNA copy number was calculated as follows:

[0109]

[0110] where X = mass of amplicon (ng); N = length of double stranded DNA amplicon (approximately 3.2 billion bp per haploid genome for human genomic DNA; approximately 2973 bp for HIV-1 -gag-pUC57 circular double stranded DNA plasmid); and 660 g / mole = average mass of 1 bp of dsDNA.

[0111] Each off-chip reaction was set up in a thermal cycler to run at 95 °C for 1 second and 58 °C for 1 second (fluorescence was recorded for each cycle). A total of 40 cycles.

[0112] Example 5

[0113] This example provides a PCR method on a chip.

[0114] The SRB droplet was first introduced into the DMF chip through inlet hole 1. The droplet was then moved to the high temperature zone. The ITO heater was turned on and the droplet started to shuttle between the high and low temperature zones according to the command script. The fluorescence intensity under the Cy3 channel was recorded in time-lapse fashion at 50 ms intervals. The DC voltage of the ITO heater was increased to bring the droplet temperature in the high temperature zone to 95 °C. The droplet was removed from inlet hole 1. The PCR droplet was subsequently introduced into the DMF chip through inlet hole 2. The droplet was directed to the high temperature zone. The ITO heater was then supplied with the voltage value previously adjusted to bring the droplet to 95 °C. After 10 seconds of pre-heating, the droplet started to shuttle between the high and low temperature zones using the same command script as for the SRB droplet to achieve consistent temperatures with the calibration droplet. The fluorescence intensity under the GFP channel was recorded in time-lapse fashion at 100 ms intervals. After the reaction was completed, the PCR droplet was recovered through inlet hole 2 for agarose gel electrophoresis check. The PCR amplification curve was obtained by collecting the fluorescence intensity of the last frame where the droplet was in the low temperature zone. The minimum fluorescence of the amplification curve was normalized to 1.

[0115] Example 6

[0116] This example provides an agarose gel electrophoresis experimental method.

[0117] An agarose gel with a concentration of 2.4% (w / v) was prepared using agarose and 1 x TAE buffer. The reaction product was mixed with a premixed buffer containing DNA fluorescent reagents and then loaded onto the agarose gel. Low molecular weight DNA markers with a size range of 25 bp to 766 bp were run with the test sample. After the electrophoresis was completed, the gel was exposed to ultraviolet light and recorded by a gel imaging system.

[0118] Example 7

[0119] This example provides a C qValue and PCR efficiency calculation method. Plasmid DNA templates of HIV-1 were used to obtain a 5-log DNA concentration gradient by serial dilution. Each dilution gradient of DNA template was subjected to duplicate PCR analysis. Quantification cycle (C q ) was determined from PCR amplification curve analysis.

[0120] C q refers to the cycle number at which fluorescence reaches a defined threshold. In this study, the threshold was defined according to the following points: (a) the threshold line needs to be sufficiently above the background fluorescence baseline; (b) the threshold line is in the log phase of the amplification curve; (c) the threshold line is at a position where all log amplification curves are parallel. From the amplification curves of serially diluted DNA templates, the average C q values of each concentration were plotted against log 10 . A standard curve was generated by linear regression analysis. The slope of the standard curve was used to calculate the PCR amplification efficiency:

[0121] E = (10 (-1 / slope) -1) x 100,

[0122] where E is the efficiency of PCR; the slope was determined from linear regression of C q values of each DNA concentration gradient.

[0123] Experimental Example 1

[0124] Investigation of dielectric layer and ePTFE hydrophobic layer properties.

[0125] The selection of dielectric layer and hydrophobic layer is critical for shuttle PCR on DMF chips. Many materials cannot maintain smooth droplet movement when the temperature exceeds 90 °C. The driving electrode is prone to breakdown at high temperatures because the breakdown voltage decreases as the temperature increases. Substances with high stiffness will expand when heated and hinder the droplet from entering the warm zone. Most importantly, bubbles generated at high temperatures will cause uneven temperature in the reaction area and hinder droplet movement.

[0126] Parylene, SU-8, and Si3N4 are commonly used as dielectrics in DMF systems. and are standard choices for hydrophobic layers. We tried various combinations of dielectric and hydrophobic materials, including SU-8 with Teflon, Parylene C with Teflon, NOA61 / 68 with Teflon, and 50-μm ultra-thin glass (bonded with NOA61) with Teflon. These materials are suitable for droplet transport at ambient temperature and 50 °C. However, when the temperature rises above 80 °C, they experience electrode breakdown, movement obstruction, or most commonly—bubble formation.

[0127] We observed that dielectric layer breakdown is related to three factors: (a) the thicker the dielectric layer, the less breakdown; (b) the larger the electrode gap, the less breakdown; (c) the softer the dielectric, the less breakdown. Also, the softer the dielectric substance, the less the thermal expansion of the dielectric layer hinders the movement of the droplet.

[0128] We also observed that bubbles often form on Teflon surfaces when the temperature exceeds 80 °C. It is well known that perfluorinated surfaces (e.g., Teflon and Cytop coatings) are water (hydrophobic) and oil (oleophobic) repellent. As shown in FIG. 1, hexadecane on a Teflon surface exhibits a contact angle of 68.7°. The low wettability of oil on perfluorinated surfaces leads to easy entrapment of air in the micro- and nano-geometries or surface cracks. At high temperatures, the air trapped in the cracks can form bubble nuclei. When a water droplet contacts the bubble nuclei at high temperatures, the bubble grows rapidly with the evaporation of water. Figure 4

[0129] To find a durable dielectric and hydrophobic layer for on-chip rapid PCR, we tested a slippery liquid infused porous surface (SLIPS) of expanded PTFE membrane (ePTFE) infused with lubricating liquid as a hydrophobic layer. The material is oleophilic and hydrophobic, as shown in FIG. 2b. Figure 5

[0130] The porous expanded PTFE membrane without lubricating liquid is hydrophobic by itself. After absorbing lubricating liquid such as hexadecane oil, the membrane becomes translucent with a thin liquid film on the surface. The liquid-liquid interaction reduces the contact angle of the droplet from 130.3° to 105.7°. There are studies that use SLIPS surfaces with low contact angle hysteresis as a hydrophobic layer on DMF chips and find that they have anti-biomolecule adhesion. The lubricating liquid easily expels all the air from the nanoscale pores of the membrane. Therefore, it greatly alleviates the problem of bubble generation at high temperatures by limiting the formation of bubble nuclei.

[0131] Figure 5 The surface morphology of a 30 pm thick expanded PTFE membrane without oil is shown under a scanning electron microscope (SEM). Compared to other traditional dielectric surface coatings (such as SU-8 and Parylene C), the expanded PTFE membrane has much fewer surface cracks. The large pores in the membrane can be infused with lubricating liquid through capillary action, and the air will be completely expelled. Therefore, fewer bubbles are generated at high temperatures.

[0132] ​​To adhere the expanded PTFE film to the glass substrate, we chose polydimethylsiloxane (PDMS) as the adhesive material and dielectric layer. PDMS is an organic silicon-based elastomer. Its elastic properties can protect the electrodes from being severely damaged at high temperatures. And the surface expansion at high temperatures will not hinder the movement of the droplet.

[0133] However, uncured PDMS can easily penetrate the 30-μm expanded PTFE film completely, as shown in Figure 6 The penetrated expanded PTFE film presents partial translucency Figure 6 a). The SEM image shows that the PDMS in the translucent area fills in the nanopores of the expanded PTFE film and exposes to the surface. In this case, the droplet cannot move smoothly at high temperatures. Therefore, we seek an optimized PDMS curing method. PDMS is first semi-cured at a lower temperature, then covered with a 30-μm expanded PTFE film. Subsequently, final curing is performed. In this way, we obtain a non-penetrated expanded PTFE surface that is tightly bound to the dielectric layer.

[0134] The PDMS-adhered expanded PTFE surface can support smooth droplet transport at high temperatures. However, it still experiences dielectric breakdown and causes surface bulging in a time-dependent manner. We further address these issues by enlarging the electrode gap and diluting the PDMS to make the dielectric more flexible and elastic. Figure 7 a-c show that the enlargement of the electrode gap driven in the high-temperature zone effectively eliminates electrode breakdown. Diluting the PDMS with n-hexane can reduce surface expansion. All these measures result in a durable hydrophobic surface that can withstand at least 240 PCR shuttle PCR thermal cycles.

[0135] Experimental Example 2

[0136] This experimental example carries out a distribution exploration experiment of the driving motor.

[0137] The traditional electrode with a grounding electrode on the top is usually designed as a square, and some electrodes are provided with cross-embedded between them. In our system setting, the droplet top does not contact the grounding electrode. Therefore, a transverse grounding electrode is needed. We first designed an integrated long grounding electrode, as shown in Figure 8 a. Since the electric field between the gap of the grounding electrode and the driving electrode is the strongest, the droplet moves along the gap between the grounding electrode and the driving electrode. In this case, only half of the droplet can be driven. In order to increase flexibility, the integrated long grounding electrode is divided into multiple rectangular electrodes, as shown in Figure 8b. According to the setting of the electronic control system of this study, the electrodes are in the ground state if they are not connected to high voltage. This design can support droplet movement at room temperature, but cannot support droplet movement at high temperature. The movement obstacle is caused by the failure of the droplet to contact the next electrode. To solve this problem, we designed a new interlocking electrode pattern by moving a row of electrodes by half an electrode position. As shown in Fig. c, the droplet is initially located on electrode 1. When electrode 2 is driven, the droplet will move to electrode 2. On the way to electrode 2, the droplet is easy to contact with electrode 3. Therefore, continuous droplet movement can be achieved. In this electrode interlocking design, as long as the diameter of the droplet is greater than half of the electrode, the droplet can always contact at least three electrodes, thereby achieving bidirectional movement. The PCR droplet movement path on the chip is shown in Fig. Figure 8 Figure 9 . In the complete shuttle cycle of on-chip PCR, the PCR droplet moves from the high-temperature denaturation zone to the low-temperature zone for annealing and extension. Then the droplet moves back to the high-temperature zone for denaturation and starts the next round of thermal cycling.

[0138] The driving voltage frequency is 2.5 kHz. The effective voltage range is 153 V rms to 210 V rms . The higher the driving voltage, the shorter the droplet movement time. Figure 9 The total cycle time shown in Fig. is 5.5 s / cycle, which includes droplet movement time, 0.1 s high-temperature denaturation zone residence time, and 1 s low-temperature annealing / extension zone residence time.

[0139] Experimental Example 3

[0140] This experimental example conducts an exploratory experiment on the temperature system.

[0141] The temperature distribution map is drawn depending on the temperature sensor. The chemical temperature sensor used in this study has significant advantages compared to traditional sensors. First, traditional sensors are usually far away from the droplet, some are embedded under the dielectric layer, and some are even outside the device. While physical temperature sensors inserted into the droplet can cause damage to the hydrophobic coating and are not suitable for use in DMF systems. In this case, the sensor temperature lags behind the droplet temperature. Temperature calibration and compensation inside the droplet are needed to correct the temperature. From this point of view, the chemical temperature sensor can directly and instantly detect the droplet temperature, especially in the case of rapid temperature transition. Second, traditional temperature sensors can only sense a single point or the average temperature of the entire droplet. On the contrary, chemical sensors can achieve analysis of the horizontal temperature distribution of the droplet.

[0142] ​We selected a temperature-sensitive inert dye—sulfonylrhodamine B (SRB). It has been shown to be compatible with PCR, does not inhibit the PCR reaction, and can be used as a temperature indicator dye in PCR reactions. No photobleaching or thermal degradation of the SRB dye has been observed at 50°C. The thermal degradation rate is 2.2% / h at 80°C and 5.4% / h at 94°C.

[0143] To correlate temperature with SRB fluorescence intensity, we plotted temperature calibration curves. To investigate the differences in temperature calibration curves during heating and cooling, and when the droplet is located in the low-temperature and high-temperature regions, we plotted... Figure 10 The SRB temperature calibration curve is shown. Figure 10 Figure a shows two almost overlapping curves, indicating that the correlation between temperature and fluorescence intensity is the same whether the temperature increases or decreases. Furthermore, although the heating methods in the low-temperature region (bottom heater) and the high-temperature region (ITO microheater) are different, Figure 10 b shows that the fluorescence curves in the low-temperature region are similar to those in the high-temperature region.

[0144] Ri of the fluorescence intensity index fitting curve at temperature point 2 The value is 0.99954. This curve is used to determine the temperature distribution of the droplet in subsequent temperature distribution plotting.

[0145] After temperature calibration, SRB temperature calibration droplets were used to depict the horizontal temperature distribution along the shuttle PCR path. Finite element analysis (FEM) was used to simulate the horizontal and vertical temperature distributions. Figure 11 The temperature distribution in the high-temperature and low-temperature zones with and without insulation grooves is shown. Without insulation grooves, the temperature in the high-temperature zone is slightly lower than that in the zone with insulation grooves. Furthermore, the temperature with insulation grooves drops more at the same distance outside the high-temperature zone than the temperature without insulation grooves. This is because the air in the insulation grooves is a poor conductor of heat.

[0146] We further employed finite element analysis (FEM) to investigate the droplet temperature distribution in both the high-temperature and low-temperature regions. For example... Figure 12 As shown, when the chip temperature is 50℃, the temperature difference between the upper and lower surfaces of the droplets in the hot zone is approximately 20℃ in both the area with and without the heat insulation bath. When the chip temperature is set to 25℃ (Table 1), the temperature difference in the high-temperature zone reaches approximately 35℃ regardless of whether the heat insulation bath is present. Therefore, the temperature difference in the high-temperature zone is essentially the same regardless of the presence or absence of the heat insulation bath. However, the heat insulation bath significantly reduces the temperature difference in the low-temperature zone. When the chip temperature is 50℃, the temperature difference is 13℃ with the heat insulation bath, while it increases to 20℃ without it. This difference widens when the chip temperature is 25℃. FEM analysis was performed under static conditions. However, in real-world scenarios, PCR droplets constantly shuttle between the high-temperature and low-temperature zones. Therefore, the mixing effect during droplet shuttle movement can compensate for the larger temperature difference.

[0147] Table 1. Temperature distribution of droplets in high and low temperature zones from FEM analysis.

[0148]

[0149]

[0150] Next we experimentally investigated how the geometry of the thermal isolation trench affects the temperature. As shown in Figure a, we investigated the depth, width, and distance to the ITO heater of the thermal isolation trench. SRB temperature calibration droplets were used to map the temperature distribution. For the depth, we compared a 0.9 mm deep thermal isolation trench to no trench. The high temperature zone of the no trench was much cooler than the 0.9 mm deep trench when the heating voltage was kept the same due to heat conduction through the glass. Once the heating voltage was increased, the high temperature zone of the no trench reached over 90 °C, and the temperature difference between the low temperature zone and the high temperature zone became large. This result matched the FEM analysis in Figure b. On the other hand, there was no significant difference in the temperature distribution for different trench widths (0.25 mm and 0.45 mm) and distances to the ITO heater (0.15 mm and 0.30 mm), as shown in Figure c. Figure 13 Figure 11 Figure 13

[0151] Figure 13 d shows the constant temperature distribution of droplets in the high and low temperature zones for 1 s and 2 s, indicating that the droplet temperature reached a steady state after 1 s. The temperature distribution at different chip temperatures is shown in Figure e. The experiment showed that the higher the chip temperature, the smaller the horizontal temperature difference inside the droplet. Figure 13

[0152] Experimental Example 4

[0153] This experimental example performed the optimization of PCR conditions.

[0154] From Experimental Example 3, it can be concluded that the temperature distribution of droplets in the high and low temperature zones is not highly uniform. However, PCR reactions can tolerate a certain temperature non-uniformity. For the denaturation temperature, 80-95 °C is sufficient to melt double-stranded DNA. For the annealing and extension temperatures, it depends on the melting temperature of the primers. A wider temperature range can support the annealing and extension process as long as it does not produce non-specific products.

[0155] In the reagents for rapid PCR, it is usually necessary to increase the concentration of primers and polymerase. Increasing the concentration of primers and polymerase can cause non-specific amplification. In order to inhibit non-specific amplification, we added a special PCR additive, ThermaStop TM ​​​​It is a reversible PCR hot-start reagent that effectively enhances PCR specificity. Unlike chemically modified hot-start reagents that bind to the polymerase and become ineffective after a one-time start, ThermaStop... TM This will take effect in every loop. ThermaStop will activate when the temperature falls below a threshold. TM It will inhibit the work of polymerase. When the temperature exceeds the threshold, the polymerase can resynthesize new DNA strands. Figure 14 The middle 'a' proves that ThermaStop was not added. TM The NTC produced nonspecific products, while the addition of ThermaStop... TM The NTC reaction showed good specificity. To validate rapid PCR on the chip, we ran shuttle PCR at a chip temperature of 55°C with a cycle time of 6.5 seconds per cycle (1 second stop in the high-temperature zone for denaturation, and 1 second stop in the low-temperature zone for annealing and extension). The amplification curve reached its peak and plateaued after approximately 30 cycles, with a total reaction time of 195 seconds (3.25 minutes). Agarose gel electrophoresis verified the specificity of the PCR products.

[0156] Figure 14 Figure b shows the PCR amplification curves at different chip temperatures. It demonstrates that dual-heater DMF shuttle PCR can operate at a baseline chip temperature of 50-60°C. The C values ​​at different temperatures... q The values ​​are almost identical. However, PCR at lower temperatures may yield higher yields.

[0157] Next, we optimized the conditions for on-chip rapid PCR. Previous studies have shown that primer and polymerase concentrations are crucial for rapid PCR. In limit PCR (less than 15 seconds), primer and polymerase concentrations were increased 40-100 times compared to conventional PCR reagents. For the KAPA2G rapid PCR kit we used, standard rapid PCR technical documentation recommends using 500 nM primers and 0.02 U / μL KAPA2G polymerase. (Out-of-chip PCR results...) Figure 14 Figure c) shows that PCR performance using the above-mentioned standard primer and polymerase concentrations is optimal. However, running rapid PCR on a chip with the same standard primer and polymerase concentrations did not result in DNA amplification, as... Figure 14 As shown in Figure d. However, increasing the polymerase concentration to 5-fold (0.1 U / μL) and 25-fold (0.5 U / μL) allowed for the amplification of DNA products in rapid on-chip PCR. Unlike findings in limit PCR, a 25-fold increase in polymerase showed amplification inhibition, particularly in conventional off-chip PCR. In rapid on-chip PCR, PCR with 25-fold polymerase showed C... qThis resulted in lag and reduced yield. Therefore, we used a five-fold polymerase concentration, i.e., 0.1 U / μL, in the following experiments. The conventional 500 nM primer concentration is suitable for rapid on-chip PCR. Given that higher primer concentrations may lead to the formation of nonspecific products, we still used 500 nM as the primer concentration for subsequent rapid on-chip PCR.

[0158] Research reports indicate that DNA template denaturation occurs the moment the temperature rises above 90°C. Furthermore, annealing and extension times are crucial for the PCR reaction. To optimize reaction conditions, we investigated the effects of different denaturation and annealing / extension times on microarray PCR. Figure 15 Figure a shows that PCR with denaturation residence times of 0.1s, 0.5s, and 1.0s has similar C values. q Furthermore, the longer denaturation time leads to a lower yield. This is likely due to the longer the droplets remain in the high-temperature region, the greater the vertical temperature difference. Figure 15 In step b, the longer the annealing / stretching time, the better the C. q The smaller the value, the longer the amplification time. However, in terms of total reaction time, a PCR with an annealing / extension time of 2 seconds has the longest total amplification time. To obtain a faster PCR reaction time, an annealing / extension time of 1 second is sufficient.

[0159] Experimental Example 5

[0160] This experimental example explores PCR sensitivity and amplification efficiency.

[0161] To determine the detection limit and obtain PCR efficiency using a standard curve, we performed rapid on-chip PCR at five orders of magnitude serial dilutions. The serial dilution PCR reactions were run at a baseline chip temperature of 50°C with a cycle time of 6.5 seconds per cycle (1 second for denaturation in the high-temperature zone and 1 second for annealing and extension in the low-temperature zone). Figure 16 As shown in Figure a, the limit of detection was 10 copies / reaction, which is consistent with the results of out-of-chip PCR run in a commercial thermal cycler. Repeated sample reactions showed good reproducibility. Figure 16 The standard curve in b is plotted by C on the Y-axis. q The value is generated by dividing the initial DNA copy number on the X-axis by the logarithm. Each plotted point represents the average C of two replicates. q The calculated PCR amplification efficiency was 100.31%, which is within the good PCR amplification efficiency range (90-110%). These results indicate that rapid chip-based PCR has high PCR amplification efficiency, sensitivity, and specificity.

[0162] In summary, we developed a rapid PCR system on a digital microfluidics platform based on dielectrophoresis. By using PDMS as the dielectric and an ePTFE membrane as the hydrophobic layer, we improved the robustness of droplet movement at high temperature (over 90℃). A chemical temperature sensor (SRB) was used to directly and accurately indicate the rapid temperature change of droplets during shuttling between different temperature zones. The SRB calibration solution can be stored at 4℃ for months while the fluorescence remains stable. To facilitate rapid PCR on the chip, the bottom heater and the top ITO micro-heater work together to reduce the temperature difference within the droplet and the heating volume at high temperature. In addition, the heat insulation groove cut around the ITO heater shortens the shuttling distance of the PCR droplet. We also optimized the PCR reagents for rapid PCR on the chip.

[0163] Reversible hot start and cold stop reagent for Taq polymerase-ThermaStop TM The addition of ThermaStop eliminated the non-specific amplification in the rapid PCR process. With these measures, we achieved rapid PCR on a chip with a cycle time as low as 5.5 seconds / cycle. Therefore, 0.6 μL of 35-cycle PCR requires 3.2 minutes. The results of rapid PCR on a chip with serially diluted DNA templates showed that the sensitivity of rapid PCR can be as low as 10 copies / reaction, and the PCR efficiency is 100.3%.

[0164] The preferred embodiments of the present application have been described above with the preferred embodiments; however, all modifications and changes thereto falling within the scope of the present application should be considered as within the purview and scope of the present application.

Claims

1. A DMF chip, characterized by, It comprises a bottom plate with an electrode layer, a top plate with an electrode layer, a dielectric layer and a hydrophobic layer, the dielectric layer is arranged on the bottom plate with the electrode layer on one side, the hydrophobic layer covers the surface of the dielectric layer, the dielectric layer is made of raw material polydimethylsiloxane, and the hydrophobic layer is made of raw material expanded PTFE film; the hydrophobic layer contains a lubricating liquid; the contact angle of the hydrophobic layer with oil droplets is 0-30°, and the contact angle of the hydrophobic layer with water droplets is 100-150°; the hydrophobic layer has a reticular or porous structure with micron-sized pore size; the lubricating liquid is mineral oil, synthetic oil, animal and plant oil and water-based liquid; The raw material of the dielectric layer also includes a diluent and a curing agent; the mass ratio of polydimethylsiloxane to the curing agent in the raw material is (2-19):1; the mass ratio of the total mass of polydimethylsiloxane and the curing agent to the mass of the diluent is 1:(0-5); The preparation method of the dielectric layer and the hydrophobic layer on the bottom plate comprises the following steps: coating a mixture of the raw material polydimethylsiloxane, the diluent and the curing agent on the bottom plate with the electrode layer, then semi-curing the bottom plate coated with polydimethylsiloxane, and then covering the expanded PTFE film on the surface of the semi-cured polydimethylsiloxane and curing; The electrode includes a driving electrode, and the path of the driving electrode is composed of two rows of rectangular electrodes arranged in parallel and adjacent to each other on the bottom plate, and the rectangular electrodes of the two adjacent rows are distributed in a staggered manner to form an interlocking electrode pattern. The DMF chip further comprises a top plate and a fence arranged between the top plate and the bottom plate, the upward-facing surface of the top plate is provided with a heat-insulating groove with an upward-facing opening, and a direct-current electric heating micro-heater is arranged in the heat-insulating groove.

2. The DMF chip of claim 1, wherein, The diluent is n-hexane, cyclohexane, tetrahydrofuran, chloroform, toluene, xylene, n-heptane, and the mineral oil is selected from hexadecane; The thickness of the hydrophobic layer is 5-100µm; and the micron-sized pore size is 50-500µm.

3. The DMF chip of claim 1, wherein, The rectangular electrodes can realize high-voltage driving and grounding functions at different times, and when any one of the electrodes is connected to a high-voltage electric signal, the rectangular electrodes around it are in a grounded state. The rectangular electrodes are arranged in two rows of parallel staggered form on the bottom plate.

4. The DMF chip of claim 3, wherein, The DMF chip is located on the bottom plate heater to form an upper and lower plate double-heating system. The depth of the heat-insulating groove is 0%-90% of the total thickness of the top plate; the width of the heat-insulating groove is 0.25-0.45mm, and the distance between the heat-insulating groove and the micro-heater is 0.15-0.30mm; The depth of the heat-insulating groove is 0-0.9mm.

5. The DMF chip of claim 4, wherein, The micro-heater is a transparent electrode in a meander shape with left-right symmetry, which is distributed in the heat-insulating groove of the top plate; and the transparent electrode is wide in the middle and narrow on both sides, so that the temperature distribution is uniform when direct current passes through the electrode.

6. The DMF chip of claim 1, wherein, The bottom plate has a high-temperature zone and a low-temperature zone, the heat-insulating groove is arranged corresponding to the high-temperature zone, and the bottom plate heater is arranged corresponding to the low-temperature zone; The bottom plate heater makes the temperature in the chip reach 50-65℃.

7. A rapid PCR system characterized by, It comprises: The DMF chip of any one of claims 1-6.

8. The rapid PCR system of claim 7, wherein, The rapid PCR system further comprises an electronic control device for droplet manipulation and temperature regulation, a computer control terminal with control software, and a fluorescence microscope for detecting DNA amplification products; the electronic control device comprises a bottom heater arranged at the bottom of the DMF chip, an FPGA unit, a high-voltage generation module, and an electrode switch array; the FPGA unit is in communication connection with the computer control terminal.

9. The rapid PCR system of claim 8, wherein, The rapid PCR system further comprises a chemical temperature indicating reagent in the droplet. The chemical temperature indicating reagent is selected from rhodamine dyes and derivatives thereof.

10. The rapid PCR system of claim 9, wherein, The chemical temperature indicating reagent is selected from red rhodamine, tetramethyl rhodamine, and rhodamine B.

11. The rapid PCR system of claim 10, wherein, The chemical temperature indicating reagent is selected from sulfonyl rhodamine B (SRB).

12. A method of performing PCR using the rapid PCR system according to any one of claims 7 to 11, characterized by, It comprises: introducing a chemical temperature sensing droplet containing a chemical temperature indicating reagent into a DMF chip by using a droplet shuttle method, then moving the chemical temperature sensing droplet to a high-temperature zone, and moving the droplet back and forth between the high-temperature zone and the low-temperature zone according to a command script, and recording the fluorescence intensity under the first fluorescence channel; controlling the direct current input to the heating electrode on the top plate; when the temperature of the chemical temperature sensing droplet in the high-temperature zone of the DMF chip stabilizes at 95℃, recording the voltage value of the direct current, taking out the chemical temperature sensor droplet, adding a PCR reagent droplet to the high-temperature zone on the DMF chip, using the same direct current voltage value to heat the heating electrode to 95℃, after preheating at 95℃ for 0-10s, the PCR reagent droplet shuttles between the high-temperature zone and the low-temperature zone according to the command script, and records the fluorescence intensity under the second fluorescence channel when the droplet is in the low-temperature zone.

13. The method of claim 12, wherein, The first fluorescence channel is a Cy3 channel, the second fluorescence channel is a GFP channel, and the chemical temperature sensor is selected from SRB.

14. The method of claim 12, wherein, The PCR reagent droplet is added with a reversible hot start reagent.

15. The method of claim 14, wherein, The reversible hot start reagent is ThermaStopTM.

16. The method of claim 14, wherein, The concentration of the polymerase in the PCR reagent droplet is 0.1-0.5 U / μL, and the concentration of the primer in the PCR reagent droplet is 0.2-20µM.

17. The method of claim 12, wherein, The denaturation residence time in the method is 0.1-10s.

18. The method of claim 12, wherein, The annealing and extension time in the method is 0.5-60s.

19. The method of claim 18, wherein, The annealing and extension time in the method is 1s.

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