A digital microfluidic device and a method of high-resolution melting curve analysis

Abstract

The application discloses a digital microfluidic device and a high-resolution melting curve analysis method, and relates to the technical field of digital microfluidics.The application provides a functional substrate with hierarchical micro-nano structures, which is used for separating and arraying nL-level thin-layer droplets with high resolution on a digital microfluidic platform, so that precise one-time melting curve analysis is realized.The functional substrate has super-wettability with a contact angle contrast of > 170° through etching treatment.On the super-hydrophilic pattern array with a unit gap of 50-500 mu m, the application realizes passive separation of muL-level DNA samples into high-resolution nL-level thin-layer droplets, and the 50 mu m characterization thickness greatly reduces the temperature difference in the droplets, and the high-resolution melting curve analysis can be used.In addition, compared with traditional digital microfluidic devices, the digital microfluidic device provided by the application can realize high-accuracy and high-resolution melting curve analysis.

Description

Technical Field

[0001] This invention relates to the field of digital microfluidics, and more specifically, to a digital microfluidic device and a method for high-resolution melting curve analysis. Background Technology

[0002] Gene mutation research is crucial for understanding normal and abnormal biological processes, including species evolution, immune system development, connectivity diversity, and cancer. Accurate diagnosis of disease-causing genes is essential for clinical treatment, genetic counseling, and disease prevention strategies. Molecular techniques such as DNA sequencing, probe hybridization, capillary electrophoresis, and melting curve analysis (MCA) can identify deeper genetic information from polymerase chain reaction (PCR) products. Among these, MCA, with its advantages of simplicity, high sensitivity, and good specificity, is often used for single-tube detection of single nucleotide polymorphisms (SNPs). However, using commercial qPCR instruments (such as the Bio-Rad CFX96)... TM MCA studies using real-time quantitative PCR (qPCR) systems can only achieve temperature scanning profiles from 40°C to 90°C with a relatively limited temperature resolution of 1°C. Due to the relatively large sample volume (e.g., 10 μL), a considerable amount of time is required to stabilize the sample temperature and reach melting equilibrium. To enable point-of-care testing and reduce the consumption of expensive reagents, microfluidics have emerged. Microfluidics offer advantages such as high analysis speed, low sample consumption, high miniaturization, and ease of automation. Although current pipeline microfluidics can achieve high-resolution MCA through advanced configurations and sensors, existing pipeline microfluidics require a large amount of support equipment (valves, pumps, and tubing), limiting system flexibility and posing a risk of valve clogging.

[0003] In contrast, digital microfluidics (DMF) can provide high controllability of multiple independent droplets with volumes in the micro / nanoliter range through electrowetting-on-dielectric (EWOD) effects, thus bypassing the dependence on additional pumps and tubing. The melting temperature (T0) of the SNP can be explored by integrating a thermostat beneath the dielectric. m This provides better sensitivity for MCA. However, one of the main obstacles to supporting high-resolution MCA on DMF is the uncontrollable internal temperature difference (ΔT) across large droplets. droplet For a 200 μm thick droplet, the internal temperature difference can reach 7 °C (>10 °C if the droplet diameter exceeds the thermostat boundary). Although rapid heating limits the reliability of the device, a complete MCA can be completed within 7 seconds. Furthermore, it is compatible with the Bio-Rad CFX96. TM Compared to the results, the excessively rapid heating rate and insufficient temperature stabilization time during the melting process resulted in Tm Offset by 5°C and T m The precision is limited to 1.6℃, thus limiting the melting accuracy. This large error can mislead PCR amplification of nonspecific products or unsuitable DNA hybridization. L. Wan et al. (2017) addressed this by extending the temperature stabilization time, allowing T... m The offset decreased to 2°C, but it was still affected by the large ΔT on the thick droplet. droplet Limitations. Although thinner droplets can be expected to have smaller ΔT droplet However, this is not achievable in typical DMF devices where droplet height is usually limited to the spacer thickness. Due to the insufficient wettability contrast (ΔCA < 90°) of existing functional hydrophilic substrate technologies, droplet height limited by spacer thickness still hinders their application in high-precision on-chip MCA. The jetting technique developed by Li et al. (H.Li et al. 2020), based on a sudden change in a strong electric field applied to the electrodes, provides an alternative for distributing tiny droplets (pL to nL). However, uncontrolled droplets with varying volumes can compromise the accuracy and repeatability of on-chip MCA.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a digital microfluidic device and a method for high-resolution melting curve analysis to solve the above-mentioned technical problems.

[0006] This invention provides a functional substrate in which a micro-nano porous structure is formed on a hydrophobic substrate, creating a superhydrophilic pattern. Upon irradiation with ultraviolet or near-ultraviolet light, it achieves a superwetting contrast (ΔCA > 170°) and enables the separation of thin droplets to a thickness of 40 μm without spacer layer thickness constraints, thereby generating a simulated ΔT of <0.4°C between 40°C and 90°C. droplet The dynamic melting process requires precise T-wave analysis using a high-speed microscope. m Measurements are taken on a functional substrate, where a static temperature gradient is used to support one-time exposure to improve MCA throughput.

[0007] This invention is implemented as follows:

[0008] A method for preparing a functional substrate includes: oxidizing and etching the surface of a substrate to be treated to form a surface with a micro-nano porous structure; after annealing, depositing a hydrophobic material onto the substrate surface; using a photomask, irradiating the substrate with the hydrophobic material with ultraviolet or near-ultraviolet light to decompose some of the hydrophobic material and form a superhydrophilic pattern; the superhydrophilic pattern is composed of an array of multiple unit cells; the gap between adjacent unit cells is 50-500 μm; the substrate to be treated is a titanium plate.

[0009] The inventors discovered that when the side length of the unit cell is 150-250 μm, the droplet height on the superhydrophilic pattern can be controlled at 40-70 μm, at which point the internal temperature ΔT of the thin-layer droplet is within this range. droplet Below 0.4 °C, and further, droplets with a height of 40–50 μm exhibit a ΔT of 0.1–0.2 °C. droplet This technology can effectively meet the accuracy requirements of high-resolution MCA on chips where the lower surface temperature of the droplets is below 70°C. Simultaneously, the spacing between these units reduces reagent consumption.

[0010] It should be noted that titanium plates were chosen as the substrate to be processed. On the one hand, this allows for better separation of droplets on the superhydrophilic pattern. On the other hand, the good strength properties of titanium plates can also prevent the accidental peeling off of the micro-nano porous structure after subsequent energization.

[0011] In a preferred embodiment of the present invention, the etching is performed hydrothermally in a strong alkaline solution, wherein the strong alkaline solution is selected from sodium hydroxide, potassium hydroxide or lithium hydroxide; the etching time is 3-7 hours; and the etching temperature is 100-120°C.

[0012] Etching transforms the substrate surface into a superhydrophilic, porous surface with micro / nano structures. When the etching time is controlled between 3-5 hours, the surface film structure is more stable, and the superhydrophilicity is superior. Etching for excessively long periods not only leads to film structure instability and reduced surface rigidity, causing titanium dioxide to easily detach from the substrate, but also reduces surface wettability. A 5-hour hydrothermal treatment results in a fully etched substrate with optimal surface roughness.

[0013] In a preferred embodiment of the present invention, the etching temperature is 110°C.

[0014] In a preferred embodiment of the present invention, the annealing temperature is 500±5℃.

[0015] In a preferred embodiment of the present invention, in the photomask using a photomask, the area through which ultraviolet or near-ultraviolet light can pass is a square pyramid, trapezoid, or series of trapezoids; the shape of the unit is a square pyramid, square, trapezoid, or polygon.

[0016] In a preferred embodiment of the present invention, the gap between adjacent units is 150-250 μm, more preferably 200 μm, and the irradiation time with ultraviolet or near-ultraviolet light is 20-120 minutes. Patterns with a gap of 200 μm enhance sensitivity and droplet uniformity.

[0017] After annealing, the roughened titanium dioxide surface crystal morphology is completely transformed into anatase, forming the structural basis for effective oxidation. Following modification with hydrophobic material deposition, a surface with high hydrophobic properties is formed. Thanks to the high catalytic capacity of titanium dioxide, the hydrophobic material deposited on the surface decomposes under ultraviolet or near-ultraviolet irradiation. A mask can be used to control the catalytic reaction in some of the light-transmitting areas, thus transforming the light-transmitting areas from hydrophobic to superhydrophilic, resulting in a completely wetted surface.

[0018] It should be noted that, unlike existing technologies that rely on insufficient wettability contrast (ΔCA<90°) for μL-level droplet separation, the superwetting contrast (ΔCA≥170°) in this invention can ensure the separation and distribution of nL-level droplets on the prepared superhydrophilic pattern.

[0019] In a preferred embodiment of the present invention, the wavelength of near-ultraviolet light is 365-436 nm.

[0020] In a preferred embodiment of the present invention, the hydrophobic material is a superhydrophobic monolayer; the deposition method is chemical vapor deposition.

[0021] In a preferred embodiment of the present invention, the hydrophobic material is an organosilane; in an optional embodiment, the organosilane is selected from one or a combination of chlorotrimethylsilane, chloropropyldimethylsilane, octyltrimethoxysilane, dodecafluoroheptylpropyltrimethoxysilane, tridecafluorooctyltrimethoxysilane, tridecafluorooctyltriethoxysilane, trichloro(1H,1H,2H,2H-perfluorooctyl)silane, heptadecafluorodecyltrimethoxysilane, and heptadecafluorodecyltriethoxysilane.

[0022] The inventors discovered that when the hydrophobic material is selected from organosilanes and coated on the surface of a titanium plate, superhydrophobicity can be achieved. This configuration reduces the resistance to droplet movement on the surface of the functional substrate, allowing for better separation and distribution of droplets under electro-driven action.

[0023] The deposition conditions were: chemical vapor deposition at 180-200℃ for 25-30 minutes.

[0024] In a preferred embodiment of the present invention, the substrate to be processed is oxidized before etching so that a titanium dioxide film is formed on the surface of the substrate to be processed.

[0025] In a preferred embodiment of the present invention, oxidation is performed as an oxidation pretreatment at a temperature of 400-450°C.

[0026] The present invention also provides a functional substrate prepared by a method for preparing a functional substrate.

[0027] The present invention also provides a digital microfluidic device, which includes the above-mentioned functional substrate, driving plate and spacer layer, wherein the spacer layer is disposed between the driving plate and the functional substrate, and the functional substrate, spacer layer and driving plate form a closed cavity to hold droplets.

[0028] In a preferred embodiment of the present invention, a conductive layer is provided on the side of the drive board near the functional substrate, and a dielectric layer is provided on the side of the conductive layer near the functional substrate. A hydrophobic layer is also provided on the surface of the dielectric layer. The conductive layer, dielectric layer, and hydrophobic layer are all disposed facing the side of the functional substrate with the superhydrophilic pattern.

[0029] In a preferred embodiment of the present invention, the conductive layer is transparent indium tin oxide (ITO), and the electrode spacing of the transparent ITO is 50-60 μm. This electrode spacing allows for better driving of large droplets.

[0030] In a preferred embodiment of the present invention, the dielectric layer is formed by coating with a UV-curable optical adhesive; in a preferred embodiment of the present invention, the UV-curable optical adhesive is selected from NOA68, NOA61, NOA60, NOA63, NOA65, NOA81 or NBA107.

[0031] In a preferred embodiment of the present invention, the hydrophobic layer is Teflon, Cytop, Hyflon, or polysiloxane imide.

[0032] In a preferred embodiment of the present invention, the spacer layers on the left and right sides are made of glass; the height of the spacer layers is 200-300 μm.

[0033] In a preferred embodiment of the present invention, a heat source and a heat sink are further provided at the bottom of the functional substrate, and the heat source and the heat sink are respectively provided on both sides of the functional substrate.

[0034] In a preferred embodiment of the present invention, the functional substrate surface of the digital microfluidic device also has a sample inlet and a sample outlet.

[0035] This invention also provides a method for high-resolution melting curve analysis using digital microfluidic devices, comprising: loading the sample to be tested onto a functional substrate, starting a program, energizing a drive board to automatically drive droplets, thereby passively separating the sample to form high-resolution droplets on a superhydrophilic pattern, applying a static temperature gradient on the functional substrate, and identifying the target gene through high-resolution melting curve analysis. It should be noted that this method is not intended for disease diagnosis.

[0036] In a preferred embodiment of the present invention, the method further includes using a fluorescence detection device to detect the fluorescence of the mixed droplets on the digital microfluidic device;

[0037] The temperature gradient of the functional substrate is set to 25℃-90.2℃, and the applied heat source voltage is set to 13V-14V; the voltage of the driving electrode of the driving board is set to 180-250V. rms .

[0038] Within the aforementioned heat source voltage range, a heat distribution from approximately 39.61°C to 90.20°C is achieved within the chamber while preventing reagent evaporation. Within the aforementioned driving voltage range, the fabricated device exhibits good reliability and can satisfy the requirement of generating a temperature gradient within the chamber. The digital microfluidic device provided by this invention can meet the requirements for the separation and patterning of nL-level droplets and allows the mother droplets to be driven in high-temperature regions (e.g., ≥70°C).

[0039] In a preferred embodiment of the present invention, the temperature gradient of the functional substrate is set to 40℃-90.2℃, and the voltage of the driving electrode is set to 200V. rms .

[0040] The present invention also provides an application of functional substrates or digital microfluidic devices in the preparation of single nucleotide mutation identification chips, digital PCR reaction devices, 3D cell culture devices, or 3D cell separation devices.

[0041] In a preferred embodiment of the present invention, single nucleotide mutation identification is used for KRAS gene mutation type identification.

[0042] Since each superhydrophilic pattern corresponds one-to-one with a specific temperature on the functional substrate, this invention enables on-chip identification of KRAS point mutations, which is superior to off-chip MCA performance.

[0043] The present invention has the following beneficial effects:

[0044] This invention provides a functional substrate with a hierarchical micro / nano structure for high-resolution nL-scale thin-layer droplet separation and arraying on a digital microfluidic platform, thereby achieving accurate one-time melting profile analysis. Etching processes impart superwetting properties (contact angle contrast >170°) to the functional substrate. On a superhydrophilic pattern array with unit cell spacing of 50-500 μm, this invention passively separates μL-scale DNA samples into high-resolution nL-scale thin-layer droplets. The 40-70 μm characterization thickness (i.e., droplet height) significantly reduces the temperature difference within the droplets, enabling high-resolution melting profile analysis. Furthermore, compared to traditional digital microfluidic devices, the digital microfluidic device provided by this invention achieves high accuracy and high-resolution melting profile analysis.

[0045] The digital microfluidic device provided by this invention can achieve precise MCA within 3 seconds with a single exposure. This system can successfully identify KRAS gene targets with single nucleotide mutations, demonstrating its potential for digital PCR, 3D cell culture, and single-cell isolation culture on a DMF platform. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 A simplified diagram and schematic diagram of the DMF device for high-resolution MCA research;

[0048] Figure 2 Figure 1 shows the experimental results of the effects of hydrothermal etching time and photocatalysis time on the fabrication of functional substrates.

[0049] Figure 3 Three-dimensional surface fluorescence images of separated droplets on superhydrophilic patterns and droplet heights on the substrate along the Y-axis for hydrophilic patterns of 150-μm, 200-μm and 250-μm;

[0050] Figure 4 The model of the experimental DMF chip shows the internal temperature distribution of the droplet at different droplet thicknesses.

[0051] Figure 5 Top and side view diagrams for temperature calibration; top view of the DMF device for MCA;

[0052] Figure 6 This is a schematic diagram of the simulation device in COMSOL, a graph showing the relationship between the breakdown voltage of NOA68 and the driving voltage required for droplet distribution, and the COMSOL simulation results.

[0053] Figure 7 This is a schematic diagram of the single-use MCA device on a DMF, an infrared image and a single fluorescence image of the DMF device, and a graph showing the relationship between the mapped temperature and temperature resolution and position of the fluorescence points along the chamber.

[0054] Figure 8 Representative chip melting curves, melting peaks, and corresponding melting temperatures (T) for wild-type and mutant targets. m );

[0055] Figure 9 The image shows the MCA detection results for the wild-type KRAS gene target (K1).

[0056] Figure 10 The image shows the MCA detection results for the mutant KRAS gene target (K2).

[0057] Figure 11 The image shows the MCA detection results for the mutant KRAS gene target (K3).

[0058] Figure 12 The image shows the MCA detection results for the mutant KRAS gene target (K4). Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0060] Chemicals and reagents:

[0061] Isopropanol, acetone, and ethanol were purchased from EMD Millipore, USA. NOA68 UV adhesive was purchased from Norland Products Inc., USA. Teflon-AF 1601S was purchased from DuPont, USA, and its solvent, perfluorosilane, was purchased from 3M, USA. In addition, trichloro(1H,1H,2H,2H-perfluorooctyl)silane, sodium hydroxide, and hexadecane were all purchased from Sigma Aldrich, USA.

[0062] The DNA premix for MCA consists of 1×PCR buffer (Invitrogen, USA), 3 mM MgCl2 (Invitrogen, USA), 10 μM KRAS molecular beacon probe (Sangon, China), and 12 μM synthetic KRAS gene target (Sangon, China), or Tris-EDTA buffer (Sigma, USA) for template-free control (NTC). The sequence listings of wild-type (K1) and mutant (K2-K4) KRAS gene target and molecular beacon probe (K) are as follows:

[0063] KRAS gene target K1:

[0064] 5'-GTAGTTGGAGCTGGTGGCGTAGGCAAGAGT-3'.

[0065] KRAS gene target K2:

[0066] 5'-GTAGTTGGAGCTGATGGCGTAGGCAAGAGT-3'.

[0067] KRAS gene target K3:

[0068] 5'-GTAGTTGGAGCTGCTGGCGTAGGCAAGAGT-3'.

[0069] KRAS gene target K4:

[0070] 5'-GTAGTTGGAGCTGTTGGCGTAGGCAAGAGT-3'.

[0071] KRAS molecular beacon probe K:

[0072] 5'-Cy3-TCTACGCCACCAGCTCA-BHQ2-3'.

[0073] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0074] Example 1

[0075] This embodiment provides a method for fabricating a functional substrate and a DMF device.

[0076] The functional substrate serves as a grounding and superwetting substrate. In this embodiment, the substrate refers to a 2.5-inch titanium plate with a purity of 99.5% and a thickness of 1.1 mm.

[0077] The method for preparing the functional substrate includes: firstly, pretreatment at 400℃ to oxidize the surface and form a titanium dioxide layer; then, hydrothermal etching in 10M NaOH solution to form a porous surface with micro-nano structures; followed by annealing at 500℃, which completely transforms the crystal morphology of the roughened titanium dioxide surface into anatase, forming the structural basis for effective oxidation. Next, a superhydrophobic monolayer of trichloro(1H,1H,2H,2H-perfluorooctyl)silane is deposited at 200℃ for 30 minutes to modify the substrate. Then, using a photomask, the superhydrophobic monolayer of the titanium dioxide is selectively decomposed under collimated near-ultraviolet (NUV, 365-436nm) irradiation for 30 minutes. This transforms the superhydrophobic surface into a superhydrophilic mode.

[0078] The DMF device is assembled from a top driving board, a bottom functional substrate, and a spacer layer. The substrate was continuously cleaned for 15 minutes each by ultrasonic pretreatment in isopropanol, acetone, and deionized water. The treated substrate was then used to fabricate the functional substrate. In this embodiment, 300 μm thick glass was used as the spacer layer between the top and bottom boards.

[0079] A conductive layer, a dielectric layer, and a hydrophobic layer are sequentially arranged outwards from the drive board.

[0080] The conductive layer is transparent indium tin oxide (ITO), and the transparent indium tin oxide electrode size is 2.5 × 2.5 mm. 2 Furthermore, the spacing is 60μm to ensure droplet transport and distribution.

[0081] A 5 μm thick layer of NOA68 adhesive was spin-coated onto the dielectric layer to prevent additional electrochemical reactions. A 100 nm thick hydrophobic Teflon layer was further spin-coated onto the drive board to prevent sample adhesion.

[0082] A 300μm spacer layer ensures that the (super)hydrophobic layers face each other, thus limiting the gap height between the drive board and the functional substrate. NOA68 also acts as an adhesive for assembling the entire DMF device. The prepared DMF device was placed indoors at 60% relative humidity before the experiment.

[0083] The process of analyzing on-chip and off-chip melting profiles using the aforementioned DMF device is as follows:

[0084] Reference Figure 1 As shown, Figure 1 The diagram shows a simplified and schematic representation of the DMF device used in high-resolution MCA research. Figure 1 (a) An overview diagram of the DMF device used for high-resolution MCA research. Figure 1 (b) Surface wettability transformation by UV irradiation. The superhydrophobic substrate (CA: 173°) selectively transforms into superhydrophilic (CA: 2.32°) after 30 minutes of UV irradiation.

[0085] pass Figure 1 (b) The functional substrate with programmable superwetting properties shown was used to establish a thermal gradient from 40°C to 90°C in the MCA chamber by driving a heat source with a 13V DC voltage for at least three minutes. After thermal equilibrium, a 5 μL DNA mixture in hexadecane oil was used as a mother droplet for further processing. Figure 1 (c) nL-level samples were dispensed above the superhydrophilic pattern. The remaining DNA mixture was then effluent from the chamber outlet. A single exposure was then performed to obtain the fluorescence pattern within the MCA chamber on the slide. The spatial distribution and temperature gradient of the on-slide fluorescence followed the superhydrophilic location and directly defined the peak at which dissolution was achieved and the dissolution temperature (T0). m DNA melting curve.

[0086] For off-chip MCA, the analyte solution with the same formulation as the on-chip melting curve analysis was used, with a total volume of 10 μL, and a Bio-Rad CFX96 was used. TM The evaluation results were obtained using a real-time quantitative PCR analysis system. The sample was placed at 95°C for 2 minutes, then at 25°C for 5 minutes, followed by a thermal scan from 40°C to 90°C at intervals of 1°C every 30 seconds. The corresponding fluorescence intensity was acquired at the end of each interval.

[0087] Experimental Example 1

[0088] This experimental example investigated the effects of hydrothermal etching time and photocatalytic time on the fabrication of functional substrates.

[0089] Specifically, we first investigated the effect of hydrothermal etching time on surface wettability. A titanium plate was treated at 400°C to form a titanium dioxide film on its surface. The substrate was then immersed in a 10M sodium hydroxide solution and etched at 110°C for different times, followed by annealing. Finally, trichloro(1H,1H,2H,2H-perfluorooctyl)silane was deposited via chemical vapor deposition. Surface wettability was evaluated using the contact angle (CA) and roll-off angle (SA) of a 10 μL droplet.

[0090] like Figure 2 As shown in (a), the substrate becomes superhydrophilic after 3 hours of etching treatment, and the best superhydrophilicity can be observed when the etching time is 5 hours (CA: 173°±2.2°, SA: 0.53°±0.25°). Figure 2 (b) SEM images of the surface structure of titanium dioxide at different etching times (1 hour, 3 hours, 5 hours, and 7 hours) show that the film structure becomes unstable (cracks appear on the substrate surface) when the etching time is increased to 7 hours. This excessively long etching time not only leads to reduced surface wettability but also to reduced surface rigidity, and the titanium dioxide on the substrate is prone to detachment. In contrast, the 5-hour hydrothermal treatment results in a fully etched substrate with the best surface roughness.

[0091] Thanks to its high level of photocatalytic ability, the monolayer of trichloro(1H,1H,2H,2H-perfluorooctyl)silane on the surface of titanium dioxide decomposes under ultraviolet light. All wettability tests were based on three independent 10 μL water droplet experiments, and all substrates were coated with trichloro(1H,1H,2H,2H-perfluorooctyl)silane before the tests.

[0092] like Figure 2 As shown in (c), the substrate becomes hydrophilic (CA≤90°) after 15 minutes of photocatalytic degradation and becomes completely wetted after more than 30 minutes of UV irradiation, resulting in surface superhydrophilicity (CA: 2.32°±0.33°, SA: 180°±0.0°). Unlike existing techniques that rely on insufficient wettability contrast (ΔCA<90°) for μL-level droplet separation, the superwetting contrast (ΔCA≥170°) in this experimental example ensures the separation and distribution of nL-level droplets on the prepared superhydrophilic pattern.

[0093] Experimental Example 2

[0094] Regarding superwetting properties.

[0095] To obtain thinner droplets and minimize internal temperature differences, we investigated the effect of superhydrophilic patterns with different lateral lengths of 150 μm, 200 μm, and 250 μm on a superhydrophobic substrate. For all experiments, the gap between two adjacent superhydrophilic patterns was set to 200 μm. We drove and transported 5 μL mother droplets containing fluorescently patterned DNA probes through the superhydrophilic regions to separate the thin droplets. Figure 3 The fluorescence image in (a) shows a 3D surface map of the region, indicating that the thin droplet was successfully separated onto the superhydrophilic pattern. Furthermore, no contamination was observed in any of the superhydrophobic regions.

[0096] According to Beer-Lambert's law, the fluorescence intensity measured by the sample can be expressed by equation (1): I = I0Φ f εCΗ,(1);

[0097] Where H is the path length of the fluorescence through the sample (i.e., the droplet height), I0 is the intensity of the incident light, and Φ f ε is the fluorescence quantum yield, ε is the molar absorbance, and C is the sample concentration. Figure 3 (b) illustrates the droplet height along the Y-axis through the substrate, showing that the droplet height is proportional to the size of the superhydrophilic pattern. This is mainly due to the linear relationship with the size of the superhydrophilic contact area. Therefore, individual droplets from 150 μm, 200 μm, and 250 μm superhydrophilic patterns exhibit the lowest to highest droplet heights of 41.71 ± 1.68 μm, 49.82 ± 1.46 μm, and 70.72 ± 1.58 μm, respectively, corresponding to discrete droplet volumes of approximately 0.21 nL, 0.43 nL, and 0.9 nL.

[0098] More based on a larger ΔT droplet This occurs on thicker droplets, which can cause a shift in the dissolution peak, thus affecting ΔT. droplet and T m The accuracy. Figure 4 (a) shows a model of an experimental DMF chip in which hexadecane seals droplets to prevent evaporation, used to study ΔT using COMSOL multiphysics. droplet The heat source / radiator is placed on two wings at the bottom of the device to create a temperature gradient. The model droplet heights (40 μm, 50 μm, and 70 μm) vary with the size of the superhydrophilic sites (150 μm, 200 μm, and 250 μm). Figure 4 (b) The internal temperature distribution of droplets with thicknesses of 40, 50, and 70 μm was investigated, showing the temperature distribution along the X-axis. The observed maximum and minimum temperatures were located at the upper left and lower right corners of the droplets, respectively. This is due to the mismatch in heat dissipation rates between the surrounding hexadecane and the air below the device. Figure 4 (c) Thicker droplets exhibit a larger ΔTdroplet And ΔT droplet It increases at higher temperatures. When the lower surface temperature of a 70 μm thick droplet (T) is... surf ) Reaching 60℃ (i.e., close to T) m When the value is (), the observed ΔT droplet The temperature is 0.4℃, which will significantly affect the effect of MCA on T. m The discrimination accuracy of SNPs with a temperature difference ≤ 0.5℃. Conversely, droplets with a thickness of 40 / 50 μm exhibit a ΔT of 0.1–0.2℃. droplet It can effectively satisfy T surf The accuracy requirements of on-chip high-resolution MCA at <70℃. It has been observed that 150 and 200 μm superhydrophilic patterns provide thin-layer droplets at T... surf Optimal ΔT can be achieved at temperatures below 70℃. droplet To achieve accurate MCA and reduce reagent consumption, a 200 μm pattern was used in all subsequent experiments, considering the enhanced sensitivity and droplet uniformity of the 200 μm pattern.

[0099] Experimental Example 3

[0100] This experimental example demonstrates temperature calibration of the device prepared in Example 1.

[0101] Depend on Figure 1 As shown in (c), the patterned nL-scale droplets have a one-to-one mapping relationship with the temperature gradient established on the substrate. We used an infrared (IR) sensor to spatially resolve the substrate temperature through a single snapshot. However, due to the 300 μm distance between the droplets and the top drive plate, the results may not reflect the actual temperature. Furthermore, although the hierarchical micro / nano structure ensures surface superwetting, the scattering effect of hydrothermal etching on the rough surface weakens the infrared signal. Therefore, we defined a specific region next to the MCA chamber (coated with a 50 μm thick viscous polyimide film for infrared detection). Figure 5 The illustration in (a) shows a top and side view schematic of temperature calibration. The droplet temperature (T) is directly read from a 20 μm film thermocouple on the surface of the intracavity device. droplet The pressure-controlled Peltier directly below acts as a heat source, adjusting the surface temperature from 25°C to 90°C, with the temperature recorded simultaneously by an infrared sensor and a thermocouple. Before calibration, the Peltier voltage is driven for 10 minutes to ensure thermal equilibrium, after which three temperature measurements are performed consecutively with a time resolution of 10 seconds. Figure 5 The calibration curve in (a) matches well with the allometric growth function, derived from T. droplet =1.02916T IR +1.00045 Description.

[0102] Measurement coefficient (R) 2The value is better than 0.9995, indicating that within the range of 25℃ to 90℃, the droplet temperature increases with the surface infrared temperature (T). IR It exhibits a linear change.

[0103] Integrating a viscous polyimide film into the IR sensing region allows for thermal measurement and mapping of the MCA chamber with high spatial resolution. Figure 5 The illustration in (b) shows a top view of the DMF device used for the MCA, where the arrows define an effective 40mm infrared sensing area. The applied Peltier (heat source) voltage varies from 10V to 14V. Figure 5 As shown in (b), the established temperature gradient increases overall with increasing heating voltage. From 10V to 14V, the highest / lowest measured infrared temperatures were 61.88 / 31.66℃, 68.97 / 31.56℃, 78.56 / 34.61℃, 86.67 / 37.52℃, and 92.95 / 38.38℃, respectively. (Reference) Figure 5 The calibration curves in (a) show the calibrated mapped thermal gradients of the MCA chamber as 64.68 / 33.58 °C, 71.98 / 33.48 °C, 81.85 / 36.62 °C, 90.20 / 39.61 °C, and 96.29 / 40.50 °C. In this work, we used a 13 V Peltier voltage to achieve a thermal distribution from approximately 39.61 °C to 90.20 °C within the chamber while preventing reagent evaporation.

[0104] Experiment Example 4

[0105] This experimental example assesses the reliability of the device prepared in Example 1.

[0106] The proposed device can generate a temperature gradient along the MCA chamber. Separation and patterning of nL-scale droplets require the mother droplet to be driven in a high-temperature region (e.g., ≥70°C). This can lead to failure in typical DMF devices with coplanar driving and heating electrodes. To improve the reliability of the device, we place the heating electrode on the bottom plate and the driving electrode on the top plate. Figure 6 As shown in (a), compared to the oil phase, the insulation layer on the top plate can effectively dissipate heat into the air. Figure 6 Simulation results in (b) show that when the bottom base temperature (T) sub When the temperature is 40℃, the temperature of the insulation layer (T) in The temperature dropped by 1.5℃ when T sub At 90℃, T in Further reduce by 6.5℃. According to equation (2), the breakdown voltage V BD With T m Inversely proportional.

[0107] V BD=T0V0 / T in , (2).

[0108] In the formula, V0 is the breakdown voltage at ambient temperature T0. Theoretically, the separation of the driving electrode and the heating electrode allows NOA68 to achieve a breakdown voltage in the range of 40℃ to 90℃. BD It decreased by 0.48% to 1.8%, which is consistent with... Figure 6 (c) Measured V BD The consistency remained. As the substrate temperature increased from 40°C to 90°C, the VA68 (5μm thick) remained consistent. BD From 316V rms Dropped to 242V rms Due to the decrease in surface tension of the liquid at higher temperatures, we can observe a slight decreasing trend in the droplet splitting / separation voltage, from 160V at 40℃. rms 128V dropped to 90℃ rms Therefore, we chose 200V. rms As the driving voltage, droplet separation is achieved over the established temperature gradient.

[0109] Experimental Example 5

[0110] This experimental example demonstrates a one-time high-resolution MCA test on the device prepared in Example 1.

[0111] This experimental example demonstrates that the technique proposed in this invention is applicable to the study of high-resolution MCA to distinguish different KRAS mutation types in codon 12, indicating resistance to cancer drugs such as panitumumab or cetuximab for treating colorectal and lung cancer.

[0112] Specifically, synthetic wild-type targets (GGT) or mutant targets with codon 12 variants (GAT, GCT, and GTT) were mixed with wild-type-specific probes and loaded onto a chip for MCA. Each mutant target had a mismatch with a molecular beacon probe labeled with a Cy3 fluorophore at one end and a BHQ2 quencher at the other. On-chip melting profiles were studied by monitoring fluorescence intensity and device temperature gradients. For comparison, we also performed off-chip DNA experiments using the same DNA samples with a commercial qPCR machine.

[0113] Figure 7 (a) Schematic diagram of a single-use MCA on the DMF. A temperature gradient is established by driving a Peltier heat source at 13V for at least 3 minutes, followed by injection of a 5μL DNA sample containing 10μM molecular beacon probe (K) and 12μM of synthetic wild-type target (K1) or mutant target (K2, K3, and K4) into the channel of the DMF device. The DNA sample is driven by a previously selected driving voltage (200V). rmsThe remaining DNA sample was collected through the chamber outlet via an MCA chamber with 2 rows of 101 patterned superhydrophilic sites. The isolated sample volume was estimated to be approximately 0.2–0.3 μL. Figure 7 (b, c) show infrared and single-pass fluorescence images of the device, with an infrared sensing area in the center. Two chambers contain patterned DNA samples of mutant KRAS target-probe hybrids, located in the upper (K3-GCT) and lower (K4-GTT) chambers, respectively. Figure 7 As shown in (d), the temperature curve fits well to the cubic polynomial function, resulting in a temperature resolution of 0.23℃ to 0.92℃ for the calibration device's temperature gradient from 40.02℃ to 89.49℃. This enables the proposed device to perform better calibrations than the Bio-Rad CFX96. TM A real-time PCR detection system with higher resolution MCA. Especially for K3 / K4 (58℃) and K1 (71℃) T... m The nearby temperature resolutions were 0.49 °C and 0.61 °C, respectively. The fluorescence intensity of the two independent bands in each chamber was averaged to reduce errors in fabricating superhydrophilic patterns and enhance the fluorescence uniformity of the MCA.

[0114] Figure 8 Representative melting curves and their melting peaks for each mutant KRAS gene target (K2-K4) and wild-type KRAS gene target (K1) are shown. We applied an sigmoid fit of the dose-response function to the melting curves on the chip, and the point of maximum slope was determined as the melting temperature (T0). m ), of which 50% of the dsDNA was melted. Since the qPCR instrument does not provide raw fluorescence data, the off-chip melting curve and melting peak can be directly presented without fitting. All off-chip and on-chip measurements of KRAS gene targets were repeated three times. Figure 9-12 (Complete results are provided).

[0115] Unlike ultrafast MCA that tracks the dynamic melting process, this invention completes the entire MCA with a single exposure. Thanks to superwetting properties, two rows of 101 patterned nL-scale samples are uniformly distributed on the substrate. With the pre-construction of thermal gradients on the chip, the proposed technique anticipates a much shorter temperature stabilization time for multiple 1.3 nL droplets. Furthermore, the high temperature resolution from 0.23 °C to 0.92 °C obtained from the patterned superhydrophilic sites also allows for SNP identification. Figure 8 (a) and (b) respectively illustrate the representative chip melting curves, melting peaks, and corresponding melting temperatures (T) for wild-type and mutant targets. m ).

[0116] Figure 8 c, T of wild-type (K1) target-probe hybrid mThe temperature was 69.79 ± 0.22 °C (K1-GGT). With the decrease in the number of hydrogen bonds between the mismatched target-probe hybrids, the mutant exhibited a lower T... m The values ​​were 58.76±0.08℃ (K2-GAT), 57.81±0.06℃ (K3-GCT), and 58.76±0.08℃ (K4-GTT), respectively. For comparison, the off-chip MCA... Figure 8 The parallel processing shown in (d, e, f) displays the reported T. m The values ​​were 71℃ (K1-GGT), 59℃ (K2-GAT), 58℃ (K3-GCT), and 58℃ (K4-GTT). No standard deviation was available for any of the off-chip measurements.

[0117] From the above discussion, we can see that the proposed on-chip single-use MCA has higher resolution than off-chip MCA in SNP site identification. It can not only distinguish between mutations, but also between different types of mutations.

[0118] like Figure 8 As shown in (a, b), this is closely related to the established trends in thermal stability and dissolution temperature of matched and mismatched base pairs (CG>CA>CT>CC). In off-chip MCA, the mutation types of K3-GCT and K4-GTT have the same T... m The two cannot be distinguished. This can be attributed to the temperature stability on and off the slide. Commercial qPCR instruments, limited by their low temperature resolution (1°C), cannot distinguish between K3 and K4 mutants. m The experimental identification accuracy of mutants is less than 1°C. Besides reduced specificity, due to the relatively large volume of reagents involved (e.g., 10 μL), each temperature interval of an off-chip MCA typically requires 30 seconds to stabilize the temperature, making the entire off-chip MCA process take up to 30 minutes. In contrast, the on-chip MCA provided by this invention only requires one exposure within 3 seconds to obtain a fluorescence image.

[0119] It should be noted that, Figure 9 , Figure 10 , Figure 11 and Figure 12 In the figure, (a,c,e) are all off-chip melting curves, (g,i,k) are all on-chip melting curves, and (b,d,f) and (h,j,l) represent off-chip and on-chip melting peaks, respectively.

[0120] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a functional substrate, characterized in that, The preparation method includes: oxidizing and etching the surface of the substrate to be treated to form a surface with a micro-nano porous structure, wherein the etching is performed hydrothermally in a strong alkaline solution; the strong alkaline solution is selected from sodium hydroxide, potassium hydroxide or lithium hydroxide; the etching time is 3-7 hours; and the etching temperature is 100-120℃. After annealing, a hydrophobic material is deposited onto the surface of the substrate. Using a photomask, the substrate with the hydrophobic material is irradiated with ultraviolet or near-ultraviolet light, causing some of the hydrophobic material to decompose and form a superhydrophilic pattern. The superhydrophilic pattern is composed of an array of multiple units. The gap between adjacent units is 150-250 μm. The irradiation time with ultraviolet or near-ultraviolet light is 25-120 minutes. The substrate to be processed is a titanium plate.

2. The method for preparing a functional substrate according to claim 1, characterized in that, The etching time is 3-5 hours and the etching temperature is 110℃.

3. The method for preparing a functional substrate according to claim 2, characterized in that, The annealing temperature is 500±5℃.

4. The method for preparing a functional substrate according to claim 2, characterized in that, A photomask is used, wherein the area through which ultraviolet or near-ultraviolet light can pass is a square pyramid, trapezoid, or series of trapezoids; and the shape of the unit is a square pyramid, square, trapezoid, or polygon.

5. The method for preparing a functional substrate according to claim 4, characterized in that, The gap between adjacent unit cells is 200 μm; The wavelength of the near-ultraviolet light is 365-436nm.

6. The method for preparing a functional substrate according to claim 1, characterized in that, The hydrophobic material is a superhydrophobic monolayer; the deposition method is chemical vapor deposition.

7. The method for preparing a functional substrate according to claim 6, characterized in that, The hydrophobic material is an organosilane.

8. The method for preparing a functional substrate according to claim 7, characterized in that, The organosilane is selected from one or a combination of chlorotrimethylsilane, chloropropyldimethylsilane, octyltrimethoxysilane, dodecafluoroheptylpropyltrimethoxysilane, tridecafluorooctyltrimethoxysilane, tridecafluorooctyltriethoxysilane, trichloro(1H,1H,2H,2H-perfluorooctyl)silane, heptadecafluorodecyltrimethoxysilane, and heptadecafluorodecyltriethoxysilane. The deposition conditions are: chemical vapor deposition at 180-200℃ for 25-30 minutes.

9. The method for preparing a functional substrate according to claim 1, characterized in that, The oxidation of the substrate to be processed before etching is performed at a temperature of 400-450°C.

10. A functional substrate prepared by a method for preparing a functional substrate as described in any one of claims 1-9.

11. A digital microfluidic device, characterized in that, It includes the functional substrate, driving plate and spacer layer as described in claim 10, wherein the spacer layer is disposed between the driving plate and the functional substrate, and the functional substrate, spacer layer and driving plate form a closed cavity to hold droplets.

12. The digital microfluidic device according to claim 11, characterized in that, A conductive layer is provided on the side of the drive board close to the functional substrate, and a dielectric layer is provided on the side of the conductive layer close to the functional substrate. A hydrophobic layer is also provided on the surface of the dielectric layer. The conductive layer, dielectric layer and hydrophobic layer are all provided facing the side of the functional substrate with a superhydrophilic pattern.

13. The digital microfluidic device according to claim 12, characterized in that, The conductive layer is transparent indium tin oxide, and the electrode spacing of the transparent indium tin oxide is 50-60 μm.

14. The digital microfluidic device according to claim 13, characterized in that, The dielectric layer is formed by coating with UV-curable optical adhesive.

15. The digital microfluidic device according to claim 14, characterized in that, The UV-curable optical adhesive is selected from NOA68, NOA61, NOA60, NOA63, NOA65, NOA81 or NBA107.

16. The digital microfluidic device according to claim 12, characterized in that, The hydrophobic layer is Teflon, Cytop, Hyflon, or polysiloxane imide; The spacers on the left and right sides are made of glass; the height of the spacers is 200-300μm.

17. The digital microfluidic device according to claim 12, characterized in that, The bottom of the functional substrate is also provided with a heat source and a heat sink, and the heat source and heat sink are respectively provided on both sides of the functional substrate.

18. The digital microfluidic device according to claim 17, characterized in that, The functional substrate surface of the digital microfluidic device also has a sample inlet and a sample outlet.

19. A method for high-resolution melting curve analysis of a digital microfluidic device as described in any one of claims 11-18, characterized in that, The sample to be tested is loaded onto the functional substrate, the program is started, and the power-driven board automatically drives the droplets, so that the passive separation of the sample to be tested forms a high-resolution thin-layer droplet on the superhydrophilic pattern. A static temperature gradient is applied to the functional substrate, and the target gene is identified by high-resolution melting curve analysis.

20. The method for high-resolution melting curve analysis of a digital microfluidic device according to claim 19, characterized in that, The method also includes using a fluorescence detection device to detect the fluorescence of mixed droplets on a digital microfluidic device; The temperature gradient of the functional substrate is set to 25℃-90.2℃, and the applied heat source voltage is set to 13V-14V; the voltage of the driving electrode of the driving board is set to 180-250V. rms .

21. The method for high-resolution melting curve analysis of a digital microfluidic device according to claim 20, characterized in that, The temperature gradient of the functional substrate is set to 40℃-90.2℃, and the voltage of the driving electrode is set to 200 V. rms .

22. The application of a functional substrate as described in claim 10 or a digital microfluidic device as described in any one of claims 11-18 in the preparation of a single nucleotide mutation identification chip, a digital PCR reaction device, a 3D cell culture device, or a 3D cell separation device.

23. The application according to claim 22, characterized in that, The single nucleotide mutation identification is a KRAS gene mutation type identification.

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