Nucleic acid detection method based on liquid drop control platform

Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a nucleic acid detection method based on a liquid drop control platform. The invention provides a novel nucleic acid detection method based on a liquid drop control platform. The method depends on near-infrared laser-induced photothermal conversion and surface tension gradient-driven Marangei effect: the photothermal conversion capability of PDMS is improved by doping Fe3O4 nanoparticles in PDMS, and heating of reaction liquid drops is realized through a near-infrared laser transmission reaction. Accurate temperature control is achieved through a temperature sensor, and PDMS temperature gradient is formed through photothermal conversion, so that surface tension of liquid drops is changed, and liquid drop driving is triggered. Meanwhile, droplet evaporation is inhibited through oil immersion treatment, a PID algorithm is used for temperature control in the adjusting process, and temperature control is achieved more sensitively and rapidly. The method is expected to solve the limitation problem, and integrated nucleic acid detection which is more convenient and faster, lower in cost and higher in integration degree is realized.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to a nucleic acid detection method based on a droplet manipulation platform. Background Technology

[0002] Nucleic acid detection, as a molecular diagnostic technology for the precise identification of pathogens and genetic material, has become a core tool in modern medicine, public health, and life science research due to its high sensitivity, high specificity, and early diagnostic advantages. Quantitative PCR (qPCR) technology, as the cornerstone of nucleic acid detection, uses fluorescently labeled amplified products and monitors changes in fluorescence intensity in real time during temperature cycling (denaturation, annealing, extension), dynamically tracking the amplification process of DNA / RNA. However, conventional qPCR instruments rely on precise temperature control modules and optical detection systems, resulting in bulky and expensive equipment with high costs per test, limiting their widespread adoption in resource-constrained scenarios. Microfluidics, on the other hand, is a cutting-edge technology for the precise manipulation of fluids in microenvironments. Through microchannel networks, reaction chambers, and functionalized structures, it can integrate fluid mixing, separation, reaction, and detection. Its core advantages lie in the high efficiency, low reagent consumption, and high-throughput parallel processing capabilities brought about by miniaturization. Microfluidics has been widely applied in fields such as biochemical detection, single-cell analysis, point-of-care diagnostics, and organ-on-a-chip. Droplet manipulation technology, as a core branch of microfluidics, achieves directional delivery, mixing, separation, and reaction control of microdroplets through various methods such as electrowetting, pressure, magnetohydrodynamics, and optical actuation. By combining nucleic acid detection with microfluidics, and through miniaturized, integrated, and automated design concepts, it solves the problems of large equipment, high cost, complex operation, and insufficient sensitivity in traditional technologies, thus driving nucleic acid detection towards lower cost, higher sensitivity, faster response, and greater portability and ease of use.

[0003] In existing research, Zou Yaowei's team designed an automated quantitative PCR system based on microfluidic photothermal technology. This system utilizes commercially available black adhesive tape to achieve photothermal conversion under near-infrared laser light, thereby controlling the heating of droplets and providing an efficient and convenient solution for rapid on-site pathogen detection. However, this system still has some limitations: 1) Although the cost of the system has been reduced significantly, it still needs further optimization in large-scale production and application to reduce detection costs and improve accessibility.

[0004] 2) Due to the limited photothermal conversion capability of commercial black adhesives, their temperature sensitivity is low and their heating and cooling rates are slow, making them unsuitable for rapid detection.

[0005] 3) The reaction droplets are fixed and cannot be moved, separated, or mixed, resulting in a low degree of automation.

[0006] 4) It only realizes the single step of droplet reaction and cannot perform subsequent detection operations, resulting in a low degree of integration.

[0007] Furthermore, in microfluidic photothermal PCR systems, reaction droplets are prone to evaporation under laser irradiation, resulting in a reduction in droplet volume and a significant change in the concentration of the reaction system. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a nucleic acid detection method that enables the movement and separation of reaction droplets, achieves precise temperature control, and effectively prevents the reaction droplets from evaporating due to heat.

[0009] To achieve the above-mentioned content of this invention, the technical solution adopted by this invention is as follows: A first aspect of the present invention provides a nucleic acid detection method based on a droplet manipulation platform, comprising the following steps: 1) Add reaction droplets to the droplet manipulation platform; 2) Using lasers to control the movement and temperature of reaction droplets to achieve nucleic acid detection.

[0010] In some embodiments of the present invention, the droplet manipulation platform is prepared from PDMS prepolymer, curing agent and Fe3O4.

[0011] In some embodiments of the present invention, the curing agent comprises a platinum-gold catalyst.

[0012] Crosslinking PDMS prepolymer and curing agent to form polymeric materials is a conventional method in the field. Those skilled in the art can achieve the technical effects of the present invention using commercially available prepolymers and curing agents.

[0013] In some embodiments of the present invention, the mass ratio of the PDMS prepolymer to the curing agent is (5~20):1.

[0014] In some embodiments of the present invention, the Fe3O4 content is 0.8~1.2wt%.

[0015] In some embodiments of the present invention, the PDMS prepolymer, curing agent, and Fe3O4 are mixed and then subjected to a curing reaction.

[0016] In some embodiments of the present invention, the curing reaction temperature is 60~90°C.

[0017] In some embodiments of the present invention, the curing reaction time is 90-120 min.

[0018] In some embodiments of the present invention, vacuum defoaming is performed before the curing reaction.

[0019] In some embodiments of the present invention, the thickness of the droplet manipulation platform is 0.5~1.5 mm.

[0020] In some embodiments of the invention, the oil immersion treatment includes treating the droplet manipulation platform with a SiO2-dimethylsilicone oil mixture.

[0021] In some embodiments of the present invention, the viscosity of the dimethyl silicone oil is 80~120 mPa·s.

[0022] In some embodiments of the present invention, the mass fraction of SiO2 in the mixture is 8-12 wt%.

[0023] In some embodiments of the present invention, the processing time is 12 to 48 hours.

[0024] In some embodiments of the present invention, the wavelength of the laser is 600~1000nm; preferably 808nm.

[0025] In some embodiments of the present invention, the intensity of the laser is 0~2A.

[0026] In some embodiments of the present invention, a PID algorithm is used for temperature control.

[0027] In a second aspect of the present invention, a nucleic acid detection device is provided, comprising: 1) Laser emitting module; 2) Droplet manipulation platform; 3) Temperature control module; 4) Fluorescence detection module.

[0028] In some embodiments of the present invention, the laser emitting module described in 1) is equipped with a laser emitter.

[0029] In some embodiments of the present invention, the droplet manipulation platform described in 2) is prepared from PDMS prepolymer, curing agent and Fe3O4.

[0030] In some embodiments of the present invention, the curing agent comprises a platinum-gold catalyst.

[0031] In some embodiments of the present invention, the mass ratio of the PDMS prepolymer to the curing agent is (5~20):1.

[0032] In some embodiments of the present invention, the Fe3O4 content is 0.8~1.2wt%.

[0033] In some embodiments of the present invention, the PDMS prepolymer, curing agent, and Fe3O4 are mixed and then subjected to a curing reaction.

[0034] In some embodiments of the present invention, the curing reaction temperature is 60~90°C.

[0035] In some embodiments of the present invention, the curing reaction time is 90-120 min.

[0036] In some embodiments of the present invention, vacuum defoaming is performed before the curing reaction.

[0037] In some embodiments of the present invention, the thickness of the droplet manipulation platform is 0.5~1.5 mm.

[0038] In some embodiments of the invention, the oil immersion treatment includes treating the droplet manipulation platform with a SiO2-dimethylsilicone oil mixture.

[0039] In some embodiments of the present invention, the viscosity of the dimethyl silicone oil is 80~120 mPa·s.

[0040] In some embodiments of the present invention, the mass fraction of SiO2 in the mixture is 8-12 wt%.

[0041] In some embodiments of the present invention, the processing time is 12 to 48 hours.

[0042] In some embodiments of the present invention, the temperature control module in 3) includes a temperature sensor, an information processor, and a laser controller.

[0043] In some embodiments of the present invention, the fluorescence detection module in 4) includes a fluorescence detection sensor.

[0044] The beneficial effects of this invention are: This invention proposes a novel nucleic acid detection method based on a droplet manipulation platform. This method relies on near-infrared laser-induced photothermal conversion and the Marangoni effect driven by surface tension gradients: Fe3O4 nanoparticles are doped into PDMS to enhance its photothermal conversion capability; near-infrared laser light is transmitted through the reaction droplets to heat the PDMS, thus heating the droplets. A temperature sensor enables precise temperature control, and the photothermal conversion creates a temperature gradient in the PDMS, leading to a change in the droplet surface tension and triggering droplet manipulation. Simultaneously, oil immersion treatment suppresses droplet evaporation, and a PID algorithm is used for temperature control during the adjustment process, achieving more sensitive and rapid temperature control. A fluorescence detection module is integrated to achieve integrated reaction and detection. This method is expected to overcome the aforementioned limitations, achieving a more convenient, faster, lower-cost, and more integrated nucleic acid detection solution. Attached Figure Description

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram illustrating the principle of the present invention.

[0046] Figure 2 The graph shows the relationship between Fe3O4 doping concentration and time.

[0047] Figure 3 The results show the relationship between the droplet heating rate and the Fe3O4 doping content.

[0048] Figure 4 The final temperature of the laser-heated PDMS substrate after a constant time is shown in Example 2.

[0049] Figure 5 The results show the changes in droplet evaporation rate under different treatment combinations.

[0050] Figure 6 The results show the variation of droplet contact angle under different treatment combinations.

[0051] Figure 7 The result shows the temperature change after manually adjusting the temperature.

[0052] Figure 8 The temperature change results are used to adjust the temperature for the PID algorithm.

[0053] Figure 9 This is the result of the relationship between droplet driving rate and laser current.

[0054] Figure 10 The results show the comparison of fluorescence curves after normalization.

[0055] Figure 11 The results show a comparison of the nucleic acid amplification fluorescence curves of commercial PCR instruments and those obtained using this method. Detailed Implementation

[0056] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0057] The principle of this invention is: For the heating component, a laser emits light above the droplet, which then transmits through the reacting droplet to irradiate the PDMS substrate, heating the PDMS substrate and consequently the droplet above it. A temperature sensor monitors the temperature in real time, and a PID algorithm controls the laser power for rapid and sensitive temperature regulation.

[0058] Regarding the driving mechanism, based on the Marangoni effect, the upper laser can locally heat the droplet, creating a temperature gradient. Since the surface tension of water decreases with increasing temperature, the surface tension in the high-temperature region is lower than that in the low-temperature region, triggering Marangoni flow: the liquid flows from the low surface tension region (high temperature) to the high surface tension region (low temperature). This flow forms a closed loop inside the droplet: the fluid on the droplet surface moves towards the lower temperature, while the fluid at the bottom flows in the opposite direction, thus propelling the entire droplet to slide towards the unheated side.

[0059] The schematic diagram of the present invention is as follows: Figure 1 As shown.

[0060] The material information used in this invention is as follows: PDMS: Dow Corning---01673921, curing agent is platinum catalyst.

[0061] The Fe3O4 nanoparticles (300-500 nm in diameter) were purchased from Macklin (I992749).

[0062] SiO2 nanoparticles (15 nm) were purchased from Macklin (MKL-S861488-500g).

[0063] Dimethyl silicone oil, purchased from Macklin --- D817599-5L.

[0064] Example 1: Effect of Fe3O4 concentration on the properties of PDMS substrate 1. Experimental Methods Raw materials: polydimethylsiloxane (PDMS) prepolymer, curing agent, iron(III) oxide (Fe3O4) nanoparticles (particle size 300-500 nm), SiO2 nanoparticles (particle size 15 nm), dimethyl silicone oil (100 mPa·s).

[0065] Procedure: Mix PDMS prepolymer and curing agent at a mass ratio of 10:1, add Fe3O4 nanoparticles (set 3 gradients, 0.5wt%, 1.0wt%, 2.0wt%), vacuum degas (40min), pour into a petri dish, and cure at 75℃ for 120min. Cut into 1mm thin slices and immerse in dimethyl silicone oil + 10% SiO2 nanoparticles for 24h to form an oil film coating.

[0066] Irradiation with a fixed laser current of 1.8 A was used to test the effect of different Fe3O4 nanoparticle contents on the heating rate of the substrate.

[0067] 2. Experimental Results The heating efficiency of the prepared 0.5wt%, 1.0wt%, and 2.0wt% solutions was tested, and the results are as follows: Figure 2 , 3 As shown.

[0068] The content of doped Fe3O4 nanoparticles affects the heating efficiency of the droplets. In this method, the content of doped Fe3O4 nanoparticles is selected as 1.0 wt%. Lower content will result in low droplet heating efficiency, while excessively high content, limited by the heat resistance of PDMS, will cause the PDMS substrate to heat up too quickly, resulting in white smoke or even burn-through. Using a content of 1.0 wt%, a heating rate of 3.02℃ / s can be achieved.

[0069] Example 2: Effects of thickness and oil immersion on the properties of PDMS substrates 1. Experimental Methods Raw materials: polydimethylsiloxane (PDMS) prepolymer, curing agent, iron(III) oxide (Fe3O4) nanoparticles (particle size 300-500 nm), SiO2 nanoparticles (particle size 15 nm), dimethyl silicone oil (100 mPa·s).

[0070] Steps: Mix PDMS prepolymer and curing agent at a mass ratio of 10:1, add Fe3O4 nanoparticles (1.0wt%), degas under vacuum (40 min), pour into a petri dish, and cure at 75℃ for 120 min. Cut into thin slices of different thicknesses (1 mm and 2 mm groups).

[0071] For the two thickness groups, an oil-immersed group and an oil-free group were designed. The oil-immersed group was immersed in dimethyl silicone oil + 10% SiO2 nanoparticles for 24 h to form an oil film coating.

[0072] Irradiation with a fixed laser current of 1.8 A was used to test the effect of different thicknesses and whether or not the substrate was oil-immersed on the heating rate of the PDMS substrate.

[0073] 2. Experimental Results Experimental results are as follows Figure 4 As shown, by comparing the heating efficiency of reaction droplets on thick and thin PDMS substrates (1 mm and 2 mm) under fixed conditions, it was found that although the heating effect of the thick substrate (2 mm) was better than that of the thin substrate (1 mm) under the condition of no oil immersion, the heating effect of both decreased significantly after oil immersion treatment, and the thick substrate decreased more. In the final result, the difference in heating efficiency between the thick and thin substrates after oil immersion was not significant. Choosing a thickness of 1 mm can further limit the cost.

[0074] Example 3: Effect of dimethyl silicone oil on the evaporation of reaction droplets Dimethyl silicone oil treatment of a PDMS substrate forms an oil film, which covers the added reaction droplets and inhibits their evaporation during heating. However, the oil film can also displace upon heating, exposing the droplets and thus weakening its evaporation-inhibiting effect. Therefore, to maintain the oil film's evaporation-inhibiting effect, it is necessary to ensure complete coverage of the droplets.

[0075] 1. Experimental Methods Substrate preparation: PDMS prepolymer and curing agent were mixed at a mass ratio of 10:1, and Fe3O4 nanoparticles (1.0wt%) were added. After vacuum degassing (40 min), the mixture was poured into a petri dish and cured at 75℃ for 120 min. The substrate was then cut into thin sheets (1 mm).

[0076] Oil immersion treatment: The sheet was immersed in dimethyl silicone oil of different viscosities and SiO2 of different concentrations (20 mPa s, 100 mPa s, 100 mPa s + 0.5% SiO2, 100 mPa s + 5.0% SiO2, 100 mPa s + 10% SiO2) for 24 hours to form an oil film coating.

[0077] Droplets were added to PDMS substrates of different treatment groups, and after irradiation with a fixed laser current of 1.8 A for 100 s, the evaporation amount was measured and the contact angle was characterized.

[0078] 2. Experimental Results Evaporation rate test results are as follows Figure 5 As shown, the higher the viscosity of dimethyl silicone oil, the more obvious the inhibition of droplet evaporation. The addition of SiO2 nanoparticles has a more obvious inhibitory effect on droplet evaporation. Dimethyl silicone oil with 10% SiO2 nanoparticles can inhibit droplet evaporation by about 5.6% (90℃, 100s).

[0079] The contact angle characterization results are shown in Table 1. Figure 6 As shown, Evaporation rate: The higher the viscosity, the more significant the inhibition of droplet evaporation, and the effect of adding SiO2 nanoparticles on inhibiting droplet evaporation is even more significant. Dimethyl silicone oil with 10% SiO2 nanoparticles can inhibit droplet evaporation by about 5.6% (90℃, 100s).

[0080] Contact angle: As viscosity increases, the contact angle of the droplet decreases. The contact angle after adding SiO2 nanoparticles is in the range of 30°-60° (this range is the most suitable for droplet movement). Further addition of SiO2 nanoparticles has little overall impact on the contact angle.

[0081] Table 1

[0082] Example 4: PID algorithm for controlling reaction temperature A laser emits light directly above a droplet, which transmits through the reactive droplet to irradiate the PDMS substrate, heating the substrate and thus the droplet above it. A temperature sensor monitors the temperature in real time, and a PID algorithm controls the laser power for rapid and sensitive temperature regulation.

[0083] The PID algorithm is based on the coordinated action of three components: proportional (P), integral (I), and derivative (D). It adjusts the control output according to the error signal. The proportional component responds to the current error, outputting a control signal proportional to the error. The integral component accumulates historical errors, eliminating steady-state deviations (such as long-term temperature drift). The derivative component predicts temperature change trends and adjusts the output based on the error rate of change, preventing overshoot or oscillation. After data input, the three components work in a closed loop to eventually stabilize the output value to the preset value.

[0084] In terms of temperature regulation, the temperature of the droplet is detected and fed back in real time by a temperature sensor. The feedback information is processed by three components: proportional, differential, and integral, and then output to adjust the laser power of the laser so that the droplet temperature gradually approaches the preset temperature.

[0085] This embodiment tested both manual temperature control and temperature control using a PID algorithm, and the results are as follows: Figure 7 , 8 As shown, the temperature change under the conditions of the PID algorithm is more gradual and stable.

[0086] Example 5 Droplet-driven test Based on the Marangoni effect, the laser from above can locally heat the droplet, creating a temperature gradient. Since the surface tension of water decreases with increasing temperature, the surface tension of the high-temperature region is lower than that of the low-temperature region, triggering Marangoni flow—the liquid flows from the low surface tension region (high temperature) to the high surface tension region (low temperature). This flow forms a closed loop inside the droplet: the fluid on the droplet surface moves towards the low temperature, while the fluid at the bottom flows in the opposite direction, thus propelling the droplet as a whole to slide towards the unheated side.

[0087] A PDMS substrate (1 mm thick) was immersed in 100 mPa s + 10% SiO2 silicone oil, and 20 μL of reaction solution was added dropwise. The droplets were moved by lasers with different currents, and the droplet movement speed was recorded.

[0088] The experimental results are shown in Table 2. Figure 9 As shown.

[0089] Table 2

[0090] With increasing laser current, the droplet driving speed increases, reaching over 1.715 mm / s (1.8 A), enabling rapid movement on a 100×100 mm PDMS substrate. The magnitude of the laser current and the movement rate of the reaction droplets have a good linear relationship under the condition of oil film coverage of a certain thickness, which can be used as a basis for precise control of the droplets.

[0091] Example 6 Nucleic Acid Amplification and Fluorescence Detection 1. Reaction system 1) Reagent composition: The 20 μL system contains 10 μL BeyoFast™ SYBR Green premix (2X), 2 μL cDNA template (5 ng / μL), 2 μL primer (3 μM), and 6 μL deionized water.

[0092] 2) Reaction process: pre-denaturation at 90℃ for 60s, denaturation at 90℃ for 3s, amplification at 60℃ for 5s, 40 cycles.

[0093] 3) Procedure: The complete reaction process was performed using both the method described in this paper and a commercial PCR instrument, and the excitation spectrum of the reaction system was measured. Real-time fluorescence detection was then performed using a self-built fluorescence detection module (detection was performed after each cycle), and the results were compared with the amplification data from the commercial PCR instrument.

[0094] 2. Reaction Results The results are shown in Table 3. Figure 10 , 11 As shown.

[0095] After a complete cycle of reaction, the excitation spectrum of nucleic acid amplification using the method of this invention was highly similar to the normalized excitation spectrum of amplification using a commercial PCR instrument (SYBR Green I was used as a fluorescent probe in this experiment; the fluorescence increased exponentially after nucleic acid amplification, and its excitation spectrum showed a characteristic peak at 520 nm), indicating that they had the same characteristic excitation spectrum and that the target nucleic acid fragment was amplified in large quantities, proving that the method can achieve the purpose of nucleic acid amplification.

[0096] Real-time fluorescence detection of nucleic acid amplification using the method of this invention shows a normalized fluorescence curve with distinct growth and plateau phases, consistent with the characteristics of PCR amplification curves. Compared with commercial PCR instruments, the fluorescence curves of nucleic acid amplification using this method exhibit the same characteristics (exponential growth and plateau phases), but with a larger CT value (cycle number to reach the threshold). As shown in Table 3, compared with commercial PCR instruments, the nucleic acid amplification efficiency of this method is lower (70%), but it has advantages such as wider application range and lower cost.

[0097] Table 3

[0098] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1.A nucleic acid detection method based on a droplet manipulation platform, comprising the following steps: 1) adding reaction droplets on the droplet manipulation platform; 2) moving and heating the reaction droplets by laser to realize nucleic acid detection; The droplet manipulation platform is doped with Fe3O4; The surface of the droplet manipulation platform is treated by oil immersion. 2.The nucleic acid detection method according to claim 1, wherein: The droplet manipulation platform is prepared from PDMS prepolymer, curing agent and Fe3O4. 3.The nucleic acid detection method according to claim 2, wherein: The content of Fe3O4 is 0.8-1.2wt%. 4.The nucleic acid detection method according to claim 2, wherein: The thickness of the droplet manipulation platform is 0.5-1.5mm. 5.The nucleic acid detection method according to claim 1, wherein: The oil immersion treatment includes treating the droplet manipulation platform with a SiO2-dimethyl silicone oil mixture. 6.The nucleic acid detection method according to claim 5, wherein: The viscosity of the dimethyl silicone oil is 80-120 mPa·s. 7.The nucleic acid detection method according to claim 5, wherein: The mass fraction of SiO2 in the mixture is 8-12wt%. 8.The nucleic acid detection method according to claim 5, wherein: The treatment time is 12-48h. 9.The nucleic acid detection method according to claim 1, wherein: PID algorithm is used for temperature control. 10.A nucleic acid detection device, comprising: 1) a laser emission module; 2) a droplet manipulation platform; 3) a temperature control module; 4) a fluorescence detection module.

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