WO2024099079A1 - Amplification structure, and rapid nucleic acid detection chip, device and method - Google Patents

Amplification structure, and rapid nucleic acid detection chip, device and method Download PDF

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Publication number
WO2024099079A1
WO2024099079A1 PCT/CN2023/126623 CN2023126623W WO2024099079A1 WO 2024099079 A1 WO2024099079 A1 WO 2024099079A1 CN 2023126623 W CN2023126623 W CN 2023126623W WO 2024099079 A1 WO2024099079 A1 WO 2024099079A1
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Prior art keywords
droplets
suspended
amplification
film
heating
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PCT/CN2023/126623
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French (fr)
Chinese (zh)
Inventor
程鑫
刘红均
刘荣跃
陈日飞
林国洪
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南方科技大学
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Publication of WO2024099079A1 publication Critical patent/WO2024099079A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6834Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of molecular amplification diagnosis in molecular biology, and specifically relates to an amplification structure, a rapid nucleic acid detection chip, a device and a method.
  • PCR Polymerase chain reaction
  • Nucleic acid replication and amplification have many applications, such as nucleic acid detection.
  • Nucleic acid detection is an important field in biomolecular detection. PCR amplification can achieve accurate and quantitative detection of ultra-trace amounts of nucleic acids (even single nucleic acid molecules).
  • PCR nucleic acid detection has a wide range of applications, including clinical disease diagnosis (such as diagnosis and efficacy evaluation of various infectious pathogenic microorganisms, eugenics detection, tumor marker and tumor gene detection, genetic gene detection, etc.), animal disease detection (such as avian influenza, foot-and-mouth disease, swine fever, parasitic diseases, anthrax Bacillus, etc.), food safety testing (such as foodborne microorganisms, food allergens, genetically modified foods, etc.), scientific research (such as medicine, life sciences, agriculture and animal husbandry and other related molecular biology quantitative research), etc.
  • the application industries of PCR nucleic acid quantitative detection technology include medical institutions, scientific research institutes, universities, disease control centers, inspection and quarantine bureaus, food companies, livestock companies, etc.
  • PCR Polymerase chain reaction
  • PCR amplification technology requires temperature cycling between 95oC-65oC-72oC. Since it takes a certain amount of time to raise and lower the temperature, a single cycle takes 5-30 minutes. Generally, PCR requires 20-40 cycles of expansion, which means that PCR testing takes several hours, and the test must be completed in a qualified testing agency laboratory. PCR testing has poor timeliness and requires professional testing units to operate, which leads to certain limitations in its role in large-scale infectious disease prevention and control.
  • the temperature control system in addition to temperature control of the liquid to be tested, the temperature control system inevitably heats the sample support structure such as the substrate during temperature cycling, resulting in a relatively long time for both heating and cooling.
  • the concentration of the nucleic acid to be tested is relatively low, a certain waiting time is required to allow the reaction to be sufficient.
  • the time required for PCR cycling being about 5-30 minutes. Because multiple PCR cycles (20-40 times) are required, the total time required for detection is relatively long (half an hour to several hours), and the results cannot be obtained in real time (within a few minutes).
  • thermoelectric chips to heat and cool the droplets to achieve temperature cycling.
  • the temperature cycle takes more than 1 minute
  • the droplets and substrates cool naturally.
  • the temperature cycle takes about 5 minutes to 20 minutes
  • the temperature cycle depends on the speed of the droplet drive, and the time can be controlled within a few seconds.
  • this method requires precise manipulation of the droplets, and the device design and difficulty are relatively high. The power consumption of maintaining two temperature zones is high, and bubbles are easily generated when the droplets move at a high temperature of 95oC, which brings great inconvenience to chip design and droplet manipulation.
  • the technical problem to be solved by the present invention is how to achieve rapid, stable and convenient nucleic acid amplification.
  • the present invention provides an amplification structure, including a suspended membrane and a heating device, wherein the heating device is used to heat droplets on the suspended membrane.
  • the liquid droplets on the suspended membrane are coated with an anti-volatile layer.
  • the anti-volatile layer is configured as a non-volatile hydrophobic liquid film and/or a hydrophobic nanoparticle layer.
  • the boiling point of the non-volatile hydrophobic liquid film is higher than the boiling point of the liquid droplets.
  • the non-volatile hydrophobic liquid film is set to be fluorine oil or silicone oil.
  • the anti-volatile layer is formed by self-assembly of a surfactant on the surface of the droplet to form a film.
  • a layer of suspended film is added above the liquid droplets on the suspended film so that the liquid droplets are sealed between two layers of suspended films to form the anti-volatile layer.
  • the heating device is configured as a microwave container, the suspended film and the liquid droplets are placed in the microwave container, and the liquid droplets are heated by the microwave container.
  • the heating device is configured to heat the microneedles, and the droplets on the suspended membrane are heated by inserting the heating microneedles.
  • the heating microneedle is configured as a microwave or ultrasonic probe microneedle.
  • the heating device is configured as a heating plate or a heating wire, and the heating plate or the heating wire is disposed under the suspended membrane for heating.
  • the heating device is provided with a temperature measuring device.
  • the heating plate or heating wire is configured as a heating and temperature measuring micro-resistance wire.
  • the suspended membrane is integrated with the heating wire or heating sheet to form a micro heater.
  • the anti-volatile layer is used to fill the liquid droplets on the suspended membrane and the area below the suspended membrane, so that the anti-volatile layer can completely cover the micro-heater and the liquid droplets on the micro-heater.
  • the microheater is suspended above the substrate, and at least two supporting conductive wires extend from the microheater to be fixed on the substrate and form microheater connection terminals; the supporting conductive wires support the balance of the microheater when it is suspended.
  • a groove is provided on the substrate, the microheater is arranged above the groove, and at least two supporting conductive wires extend from the microheater to the raised edge of the groove to support the microheater to be suspended above the groove; the supporting conductive wires extend to the raised edge of the groove and are fixed to form the microheater connecting terminal.
  • four supporting conductive wires extend from the microheater to the raised edge of the groove to be fixed and form a microheater connection terminal, and the four supporting conductive wires support the balance of the microheater when it is suspended in the air.
  • connection terminals Preferably, different electrical signals are input to the connection terminals, so that the micro-heater can achieve rapid temperature changes so as to conduct heat to the droplets to achieve rapid temperature changes.
  • a hydrophobic or super-hydrophobic coating is provided on the suspended membrane.
  • the suspended membrane is configured to be a thin film selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene (paraxylene polymer) film, metal film, or a composite film formed by the above-mentioned thin films.
  • a heat dissipation device is also included.
  • the heat dissipation device is one or more of a thermoelectric cooling sheet, a planar heat pipe or a microfluidic pipeline fluid arranged on the suspended membrane.
  • the present invention also provides a rapid nucleic acid detection chip including an amplification structure, including the aforementioned amplification structure.
  • a rapid nucleic acid detection chip including an amplification structure, including the aforementioned amplification structure.
  • an enhanced reflection coating is provided on the suspended membrane.
  • the present invention also provides a rapid nucleic acid detection array chip, including the rapid nucleic acid detection chip as described above; in each rapid nucleic acid detection chip, a heating device is used independently or uniformly to heat the droplets to achieve rapid temperature changes.
  • the present invention also provides an amplification method, comprising the following steps:
  • a film is set to be suspended in the air
  • a droplet containing the amplified sample is placed on a suspended membrane
  • an anti-volatile layer is disposed outside the droplet.
  • the anti-volatile layer is formed by one or more of the following methods: using a surfactant to self-assemble into a film on the surface of the droplet to form an anti-volatile layer; covering the surface of the droplet with a layer of hydrophobic nanoparticles to form an anti-volatile layer; adding a layer of suspended film above the droplet to seal the droplet between two layers of suspended film to form an anti-volatile layer.
  • one or more of the following heating methods are used to achieve periodic heating of the droplets: heating with a heating wire or a heating plate under the suspended film; or inserting a microwave or ultrasonic probe into the droplets for heating; placing the entire chip in a microwave oven and using microwaves to heat the droplets and the suspended film without contact.
  • the heating temperature of the droplets is measured.
  • a temperature measuring resistance wire is used to heat and measure the temperature of the droplets on the suspended membrane.
  • the droplets are heated periodically to achieve circulation of the droplets at different temperatures and to achieve amplification, comprising the following steps:
  • the temperature measuring resistance wire is integrated with the suspended film to form a suspended microheater, and the droplets are placed on the upper surface of the suspended film; the anti-volatile layer covers the droplets on the microheater; different electrical signals are applied to the suspended microheater to periodically heat the droplets, so that the droplets circulate at different temperatures to achieve amplification.
  • the rapid nucleic acid detection method further comprises the following steps:
  • the suspended microheater is placed on a substrate with a groove, and at least two supporting conductive wires are extended from the suspended microheater to the raised edge of the substrate groove to support the microheater to be suspended above the groove; the supporting conductive wires are extended to the raised edge of the substrate groove and fixed to form a microheater connection terminal; the droplets and the area below the suspended film are completely filled with an anti-volatile layer so that the microheater and the droplets on the microheater are completely covered;
  • Different electrical signals are applied to the microheater connection terminals of the suspended microheater to periodically heat the droplets, so that the droplets circulate at different temperatures to achieve amplification.
  • the non-volatile hydrophobic liquid film is used as the anti-volatilization layer, and the non-volatile hydrophobic liquid film is set to fluorine oil or silicone oil.
  • the suspended membrane is configured to be a thin film selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene (paraxylene polymer) film, metal film, or a composite film formed by the above-mentioned thin films.
  • an additional heat sink on the suspended membrane is used to dissipate the heat from the droplets.
  • one or more of the following methods including a hot spot cooling sheet, a planar heat pipe or a microfluidic channel fluid, is used on the suspended membrane to dissipate heat from the droplets.
  • the present invention also provides a rapid nucleic acid detection method, which uses the aforementioned amplification method to amplify the sample to be tested.
  • the rapid nucleic acid detection method preferably further comprises the following steps
  • the amplified droplets containing fluorescent markers are placed in a fluorescence detection device to complete the fluorescence detection of nucleic acids;
  • the detection result is determined based on the fluorescence brightness of the droplets after amplification.
  • the rapid nucleic acid detection method is preferably such that the droplets contain a sample to be tested, an amplification primer, an enzyme, dNTP deoxyribonucleoside triphosphates, a template, a fluorescent probe and a buffer.
  • This rapid nucleic acid detection method is preferably performed by coating the suspended film with an enhanced reflection coating to enhance the fluorescent reflection signal.
  • the present invention also provides a rapid large-scale nucleic acid detection method, which divides the same sample or different samples into multiple droplets, and uses the aforementioned rapid nucleic acid detection method to simultaneously detect the multiple droplets.
  • the present invention provides an amplification structure and a corresponding rapid nucleic acid detection chip.
  • the amplification of the sample to be tested in the heated droplets can be quickly realized.
  • the droplets are heated and cooled in situ, and the need for substrate heating is eliminated through the support of the suspended membrane.
  • the fastest droplet heating and cooling can be achieved without driving the droplets, which greatly simplifies the chip design and operation, ensuring ease of use and reliability.
  • the present invention uses micro droplets as reaction containers, with fast mass transfer and heat transfer speeds, and fast nucleic acid amplification reactions; at the same time, the droplets are placed on the suspended membrane to reduce the inevitable substrate heating during the temperature cycle, so that the heating and cooling speed is completed within 0.5 seconds, while the time required for the existing PCR amplification temperature cycle is within 1-5 minutes, and the temperature cycle speed is increased by more than 100 times. Since nucleic acid detection requires PCR amplification for more than 20-30 times, the total time of nucleic acid detection can be shortened from the traditional 30 minutes to several hours to less than 1 minute by using the present invention, greatly shortening the time required for detection and greatly improving the timeliness of nucleic acid detection.
  • the device of the present invention has a simple structure. Each amplification structure and rapid nucleic acid detection chip only detects a single droplet.
  • the single droplet has strong detection capabilities and low detection costs.
  • the use of microdroplets greatly reduces the amount of reagents used, greatly shortening the detection time while controlling the detection cost to an extremely low level. It is suitable for large-scale promotion, such as timely and efficient nucleic acid screening in epidemic prevention and control; rapid detection of pathogenic microorganisms for household use, and other applications.
  • the amplification structure and rapid nucleic acid detection chip provided by the present invention while amplification is performed in the droplets, an anti-volatile layer is set on the droplets.
  • the anti-volatile layer can effectively prevent aerosol pollution during nucleic acid amplification, and also ensure the effect of amplification.
  • the anti-volatile layer here can be formed in a variety of ways, such as using a high-boiling point non-volatile hydrophobic liquid film, such as silicone oil or fluoro oil. At this time, nucleic acid amplification is completed in a closed oil phase, which can effectively prevent the formation of aerosols.
  • the anti-volatile layer can be formed in other ways, such as by amphiphilic surfactants self-assembling into a film on the surface of the droplets to reduce the volatilization of the droplets, or adding a high-boiling point compatible solvent to the droplets (such as adding ethylene glycol or polyethylene glycol to aqueous droplets), or covering the surface of the droplets with a layer of hydrophobic nanoparticles to form liquid marble (droplets wrapped in a solid surface), or adding a layer of suspended film above the droplets to seal the droplets between two layers of suspended films to prevent the droplets from volatilizing during the heating process. Because the chips and reagents are low-cost and disposable, after the amplification test, the positive samples are sealed in an anti-volatile layer such as oil droplets, which can effectively prevent the amplified nucleic acid molecules from contaminating the equipment.
  • a high-boiling point compatible solvent such as adding ethylene glycol or polyethylene glycol to aqueous droplets
  • the present invention also provides a variety of adaptive amplification heating devices for the amplification structure and rapid nucleic acid detection chip, using micro-heating resistor wires prepared by micro-nano processing on a suspended membrane; or using microwave (or ultrasonic) probes to insert into droplets for heating. If all droplets circulate within the same temperature range, the entire chip can also be placed in a microwave oven, and microwaves can be used to heat all droplets simultaneously without contact.
  • the present invention specifically proposes that a cooling device, i.e., an additional heat dissipation device, such as a thermoelectric cooling sheet, or a planar heat pipe, or a microfluidic channel fluid convection heat dissipation can be installed on the suspended membrane.
  • a cooling device i.e., an additional heat dissipation device, such as a thermoelectric cooling sheet, or a planar heat pipe, or a microfluidic channel fluid convection heat dissipation can be installed on the suspended membrane.
  • the present invention also proposes a specific amplification structure and a nucleic acid detection chip structure, wherein a temperature measuring micro-resistance wire is arranged under the suspended membrane, and the temperature measuring micro-resistance wire and the suspended membrane can even be integrated together to form a micro-heater with a surface covered with a thin film, and the micro-heater is suspended and leads out two or more, such as four supporting conductive wires, which not only provide support for the suspended micro-heater, but also extend to become a micro-heater connection terminal, and provide an electrical signal for the suspended micro-heater to amplify.
  • a temperature measuring micro-resistance wire is arranged under the suspended membrane, and the temperature measuring micro-resistance wire and the suspended membrane can even be integrated together to form a micro-heater with a surface covered with a thin film, and the micro-heater is suspended and leads out two or more, such as four supporting conductive wires, which not only provide support for the suspended micro-
  • the specific rapid nucleic acid detection structure provided by the present invention can use MEMS processing technology to control the cost of a single micro-heater to about 1-10 yuan, and use micro-droplets to greatly reduce the amount of reagents, while greatly shortening the detection time, and controlling the detection cost to an extremely low level.
  • the specific nucleic acid detection chip structure proposed by the present invention can effectively and quickly realize nucleic acid amplification, greatly increase the amplification efficiency, and has a low production cost.
  • the nucleic acid control process is well controlled. Since the heater and the suspended membrane are fixed, only different electrical signals need to be provided to achieve rapid amplification, and the operation is very convenient. Compared with the existing amplification methods and devices, there is no need to accurately operate the droplets, the device design is simple, and there is no need to maintain the power consumption of two temperature zones, which has great advantages.
  • the amplification structure and rapid nucleic acid detection chip proposed by the present invention and the required device are compact and ultra-portable: using MEMS processing technology, the size of a single micro-heating chip is between 100 microns * 100 microns and 10 mm * 10 mm, making rapid nucleic acid detection not only suitable for laboratory scenarios, but also for daily scenarios such as homes.
  • the rapid nucleic acid detection chip provided by the present invention also has a hydrophobic or super-hydrophobic coating on the suspended film to prevent the droplets from spreading out.
  • the film can be coated with an enhanced reflection coating such as Au, Pt metal film, or a highly reflective multilayer dielectric film to enhance the fluorescent reflection signal.
  • the present invention also provides a rapid nucleic acid detection device, which integrates multiple rapid nucleic acid detection chips.
  • a rapid nucleic acid detection device which integrates multiple rapid nucleic acid detection chips.
  • hundreds or thousands of microheater arrays can be integrated, and each microheater in the array can quickly perform thermal cycling operations on a droplet.
  • a certain amount of nucleic acid detection is divided into multiple liquids for simultaneous detection to ensure that rare nucleic acid copies are not missed.
  • different samples to be tested can also be thermally cycled on different microheaters to achieve high-throughput sample detection; or different nucleic acid detection reagents can be used for the same sample to achieve simultaneous detection of multiple nucleic acids.
  • the above methods can also be mixed and integrated, that is, multiple nucleic acids can be detected on multiple samples at the same time.
  • the amplification structure proposed in the present invention can be used not only for nucleic acid detection, but also for other scenarios that require amplification. Taking the amplification structure for rapid nucleic acid detection as an example, traditional nucleic acid detection is more carried out in medical, disease control or scientific research PCR laboratories, and the epidemic and future diagnosis and treatment needs require PCR nucleic acid detection to break through the limitations of the laboratory to adapt to more application scenarios, and even go into the home.
  • the present invention can realize nucleic acid on-site detection (POCT), break through the application scenario limitations under the characteristics of low cost, ultra-portability, and low power consumption, and efficiently enable fever clinics, emergency departments, customs, airports, entry and exit checkpoints and other high-traffic gathering places to meet the needs of rapid screening of infectious diseases, and increase the response capabilities and solutions to public health emergencies in multiple scenarios.
  • Rapid and low-cost nucleic acid detection is an important means for the diagnosis of patients with new coronavirus pneumonia (COVID-19), clinical treatment effect evaluation, population screening and epidemiological surveys when an epidemic occurs.
  • Low-cost and rapid nucleic acid detection also plays a role in on-site detection in animal husbandry, agriculture, and food industries. Pathogenic microorganisms and biological warfare pathogens can be detected in the wild or on the battlefield.
  • FIG1 is a schematic diagram of an amplification structure according to a specific embodiment of the present invention.
  • FIG2 is a schematic diagram of the structure of the anti-volatile layer of the hydrophobic liquid film used for the droplets in Example 1 of the present invention.
  • Example 3 is a schematic diagram of the structure of the anti-volatile layer formed by the surface self-organizing active agent in the droplet in Example 1 of the present invention
  • FIG4 is a schematic diagram of the structure of the anti-volatile layer formed by the droplets using hydrophobic nanoparticles in Example 1 of the present invention.
  • Example 5 is a schematic diagram of the structure of preventing the droplets from volatilizing by covering them with suspended films in Example 1 of the present invention
  • FIG6 is a schematic diagram of the structures of Examples 2-1 and 2-2 in Embodiment 2 of the present invention.
  • FIG7 is a schematic diagram of the structure of Example 2-3 in Example 2 of the present invention.
  • FIG8 is a schematic diagram of the structure of an integrated suspended membrane and a heating device integrated micro-heater on the surface of a silicon wafer in Example 3 of the present invention.
  • FIG9 is a schematic structural diagram of the anti-volatile layer in FIG8;
  • FIG10 is a schematic diagram of an actual micro-heater structure in Example 3, wherein the left side is a schematic diagram of a micro-heater structure without droplets, and the right side is a schematic diagram of a micro-heater structure with droplets and an anti-volatile layer;
  • FIG11 is a diagram showing the relationship between the surface temperature of the micro-heater, the heating voltage and the heating time in Example 3, wherein the horizontal axis is the time required for heating, the vertical axis is the heating temperature, and the order is the magnitude of the power-on voltage;
  • Example 12 is a time response curve of the temperature rise and fall cycle of the surface of the micro-heater without droplets in Example 3, wherein the horizontal axis is the time required for heating and the vertical axis is the heating temperature;
  • Example 13 is a temperature change curve diagram when a droplet is placed on the micro-heater in Example 3, wherein the horizontal axis is the time required for heating and the vertical axis is the heating temperature;
  • FIG. 14 is a graph showing the results of a hepatitis B virus inactivated nucleic acid amplification experiment group according to a specific embodiment of the present invention.
  • 15 is a graph showing the results of a control group of hepatitis B inactivated virus nucleic acid amplification performed in a specific embodiment of the present invention.
  • 16 is a graph showing the amplification concentration-cycle number of a control experiment for amplification of inactivated hepatitis B virus nucleic acid in a specific embodiment of the present invention.
  • FIG17 is a graph showing the results of a control group of a novel coronavirus inactivated virus (COVID-19) nucleic acid amplification control experiment performed in a specific embodiment of the present invention
  • FIG18 is a set of result graphs of a control experiment of nucleic acid amplification of the inactivated coronavirus (COVID-19) according to a specific embodiment of the present invention.
  • Figure 19 is a graph showing the results of Experiment 2 after the first experimental group was diluted 100 times in a control experiment of nucleic acid amplification of the inactivated coronavirus (COVID-19) in a specific embodiment of the present invention.
  • Example 1 provides a relatively simple amplification structure in the present invention.
  • a simple rapid nucleic acid detection chip can also be formed, which can only include a suspended membrane 1 and a heating device (not shown in FIG1 ).
  • a thin film is plated on the substrate 2.
  • the thin film 1 is suspended to form a suspended membrane, thus forming a simple amplification structure.
  • a droplet 3 containing an amplified sample is placed on the suspended membrane 1, and periodically heated, such as by energizing a micro-resistance wire 5 or heating the droplet with a microwave, to achieve the circulation of the droplet at different temperatures and achieve PCR amplification.
  • the amplified droplet containing a fluorescent marker is placed in a fluorescence detection device to complete the fluorescence detection of nucleic acid. By recording the fluorescence brightness of the droplet after each temperature cycle (amplification), the melting curve of nucleic acid amplification is completed to determine the detection result.
  • this embodiment uses droplets for amplification to form an amplification container, the droplets are small in size and can achieve the effect of rapid heating and temperature change.
  • the structure of this embodiment can be used alone for amplification to form an amplification structure, and fluorescent markers or probes can be added to achieve rapid nucleic acid detection, becoming a rapid nucleic acid detection chip.
  • the droplet When used for nucleic acid detection to make a rapid nucleic acid detection chip, the droplet contains the sample to be tested, amplification primers, enzymes, dNTPs (deoxyribonucleoside triphosphates), templates, fluorescent probes and buffer. During the amplification process, it needs to be continuously heated and circulated.
  • the anti-volatile layer here can be a hydrophobic liquid film, as shown in Figure 2.
  • the droplets can be wrapped with a high-boiling point non-volatile oil film to form an anti-volatile layer 4.
  • the high boiling point here refers to a boiling point higher than that of the liquid in the droplets to prevent the liquid in the protective layer from volatilizing during heating.
  • the hydrophobic liquid film here can be a surfactant that self-assembles on the surface of the droplets, as shown in Figure 3.
  • an amphiphilic surfactant is used to self-assemble on the surface of the droplets to form an anti-volatile layer 4 to reduce the volatilization of the droplets, or a high-boiling point compatible solvent is added to the droplets (such as adding ethylene glycol or polyethylene glycol to aqueous droplets), as shown in Figure 4, or a layer of hydrophobic nanoparticles is covered on the surface of the droplets to form a liquid marble (liquid droplets coated on a solid surface) to form an anti-volatile layer 4, as shown in FIG5, or a layer of suspended film 1 is added above the liquid droplets to seal the liquid droplets between two layers of suspended films 1 to form an anti-volatile effect, thereby forming an anti-volatile layer.
  • oil sealing can be performed on the liquid droplets at the same time to form an anti-volatile layer 4, or the aforementioned other methods can be used to further prevent volatility.
  • the anti-volatile layer 4 here is not limited to the several methods listed above. In the actual amplification process or rapid nucleic acid detection process, one or a combination of these methods can also be used to achieve a better anti-volatile layer effect.
  • the amplification control experiment of the single amplification structure or rapid nucleic acid detection chip of the present invention is carried out using the inactivated hepatitis B virus.
  • the inactivated hepatitis B virus is subjected to rapid temperature cycling using the scheme of the present invention, 4 seconds at 95oC, 4 seconds at 65oC, a total of 8 seconds per PCR cycle, and the amplification fluorescence brightness changes with the number of cycles as shown in Figure 14.
  • Figure 15 is the result of the blank control group of the scheme of the present invention in which the same amplification primers, enzymes, dNTPs, i.e., deoxyribonucleoside triphosphates, templates, fluorescent probes, and buffers as in the experimental group are added, but there is no amplification virus.
  • Figure 16 is a curve of amplification concentration-cycle number of related control experiments, in which the horizontal axis represents the relative fluorescence intensity and the vertical axis represents the number of thermal cycles. It can be seen that the concentration changes significantly after 30 cycles and can be detected, so it only takes 4 minutes to achieve the amplification of the virus sample and rapid nucleic acid detection.
  • a preliminary experimental control of rapid COVID-19 inactivated virus (COVID-19) detection is performed using microdroplets on a single amplification structure or rapid nucleic acid detection chip provided by the present invention (a specific embodiment can be a microheater), and the same PCR cycle amplification is performed for 4 seconds at 95oC, 4 seconds at 65oC, and once every 8 seconds.
  • Figure 17 is the result of adding the same COVID-19 amplification primers, enzymes, dNTPs, i.e., deoxyribonucleoside triphosphates, templates, fluorescent probes, and buffer as the experimental group, but without the COVID-19 amplification sample, and the blank control group is subjected to the same amplification cycle treatment;
  • Figure 18 is the experimental control result of the COVID-19 amplification sample, i.e., the experimental group 1;
  • Figure 19 is the experimental result of the experimental group 2 after the positive standard at the COVID-19 amplification sample concentration of Figure 18 was diluted 100 times.
  • nucleic acid amplification is effectively and quickly completed in one cycle of 8 seconds, and relatively accurate detection results are obtained. From the experimental results, it can be seen that the concentration changes significantly after 30 cycles and can be detected, and 30 cycles only take 4 minutes.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • Example 2-1 as shown in Figure 6, in this example, an ordinary heating plate 5 is used on the suspended film 1 to perform heating amplification.
  • the heating plate 5 can be an integration of heating wires, or a metal plate containing heating wires.
  • the advantage of this heating method is that since the film 1 is suspended, the suspended film can be directly heated by the heating plate 5. During the heating process, the substrate does not need to be heated in one heating cycle, so there is no need to consider the heating and cooling speed of the substrate, and rapid heating amplification of droplets on the suspended film 1 can be achieved.
  • Example 2-2 relative to Example 2-1, here is a more concise heating amplification method, that is, a heating wire is set on the suspended film, and the heating wire can be directly integrated with the suspended film, also see Figure 6, to form a suspended film with a heating wire, that is, a heating device that is adaptable to rapid heating of droplets.
  • a heating wire that is, a heating device that is adaptable to rapid heating of droplets.
  • the substrate since the suspended film is directly heated by the heating wire, the substrate does not need to be heated during a heating cycle during the heating process, so there is no need to consider the heating and cooling speed of the substrate, and rapid heating amplification of droplets on the suspended film can be achieved.
  • Example 2-3 as shown in FIG7 , we use a heating microneedle 6.
  • a microwave (or ultrasonic) probe 6 is inserted into the droplet for heating.
  • the setting of the suspended membrane makes the heating independent of the substrate, and the droplet can be inserted and heated accurately and conveniently.
  • the temperature barrier of the suspended membrane is removed, and the microneedle heating makes the droplet heat faster.
  • the thermal cycle speed is further increased during amplification.
  • Example 2-4 a simpler heating method is used, which satisfies the requirement of fast heating speed and can be applied to multiple droplets for cyclic amplification within the same temperature range.
  • the entire amplification structure or rapid nucleic acid detection chip is placed in a microwave, such as a microwave oven, and microwaves are used to heat all droplets simultaneously without contact. All droplets circulate within the same temperature range.
  • Example 2-5 in other examples, a temperature measuring device is provided on the heating device, such as in Examples 2-1 to 2-4, so that the heating process can be monitored in real time.
  • a temperature measuring resistance wire can be used and directly integrated into the suspended membrane to form a heater that can both quickly heat and measure temperature in real time, and can quickly perform a heating cycle on the suspended membrane while measuring temperature at the same time.
  • Example 2-6 in the examples of Example 2-1 to Example 2-5, in addition to allowing the heated droplets to cool naturally, a heat sink is provided on the rapid nucleic acid chip to further accelerate the thermal cycle.
  • an additional heat sink can be attached to the suspended membrane, such as a thermoelectric cooler, or a planar heat pipe, or a microfluidic channel fluid convection heat dissipation.
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • a silicon nitride film is prepared by chemical plating (PECVD or LPCVD) on a silicon substrate, and a metal microheater (Ti/Au, or Ti/Pt, or Ti/Pd, etc.) is prepared on the film.
  • a suspended silicon nitride (suspended film 1) is formed on the surface of the silicon wafer, that is, a microheater is integrated.
  • the microheater has a suspended film 1, and a heating device 5 integrated with the suspended film, and a droplet 4 of a sample to be tested is contained.
  • the liquid 4 is heated by a microheater, and the temperature of the droplet is detected in real time by resistance measurement. In this way, a microheater with a suspended film is formed.
  • fluorine oil or silicone oil is used to fill the area below the suspended film, and the droplets to be tested are completely covered to form a droplet anti-evaporation layer 4.
  • a microheater with a suspended film is placed in the middle of a substrate with a groove, and a supporting conductive wire is extended from the heater to the raised edge of the substrate groove so that the supporting conductive wire can support the microheater and extend to the raised edge of the substrate groove to form a connecting terminal of the heater.
  • a droplet with an amplified sample is placed on the suspended silicon nitride microheater platform in the middle and is heated by powering the microheater electrode. The temperature of the microheater changes with the heating power. Because it is suspended, the temperature can change rapidly during heating.
  • the heater with the suspended film can be directly covered on the groove on the substrate, and the raised part of the suspended film near the periphery of the substrate groove can be hollowed out, leaving only the middle microheater part, and heating it by energizing the microheater electrode.
  • the effect can also be achieved.
  • other methods can be used.
  • the microheater or the suspended film is isolated from other parts of the substrate, suspended heating is achieved, which is within the scope of protection of the present invention.
  • the microheater structure in this embodiment can adopt MEMS processing technology.
  • the cost of a single microheater is about 1-10 yuan.
  • the size of a single microheater is between 100 microns * 100 microns and 10 mm * 10 mm.
  • the structure of this embodiment can be used alone for amplification to form an amplification structure, and can also be added with fluorescent markers or probes to achieve rapid nucleic acid detection, thereby becoming a rapid nucleic acid detection chip.
  • micro-heater structure and micro-droplet nucleic acid detection greatly reduces the amount of reagents used, greatly shortens the detection time, and controls the detection cost at an extremely low level, which is suitable for large-scale promotion, such as timely and efficient nucleic acid screening in epidemic prevention and control; rapid detection of pathogenic microorganisms for household use, and other applications.
  • the structure of this embodiment is experimentally verified by experiments as follows. As shown in FIG11 , when the voltage changes, the temperature on the suspended film can quickly reach equilibrium and change between room temperature and 400°C. As shown in FIG12 , when the voltage on the microheater of this structure is added to 0.8 volts, the temperature on the suspended film can reach about 105°C, and the time required to rise from room temperature to 105°C is only 0.02 seconds. When the voltage on the microheater becomes 0, the suspended film cools rapidly, and it only takes 0.02 seconds to go from 105°C to room temperature. Therefore, the suspended microheater can complete a cycle of temperature cycling within 0.04 seconds. PCR nucleic acid testing requires placing droplets on the suspended microheater.
  • the time required for the temperature cycle increases. As shown in FIG13 , this is the temperature change when droplets are placed on the suspended microheater in this embodiment.
  • the droplets are PEG droplets.
  • the voltage of the microheater is 1.5 volts, it takes 0.1 seconds to cool from room temperature to 120°C.
  • the voltage on the microheater is 0 volts, it takes 0.37 seconds to cool from 120°C to room temperature.
  • the time required to complete a temperature cycle is 0.47 seconds.
  • the aqueous droplets are coated with oily liquids, they can withstand temperatures of around 170°C without evaporation or boiling, and exist stably.
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • a portable battery is used to heat the micro heater. Since the micro heater basically only heats the droplets during the process of heating the droplets, without additional power consumption, the energy consumption during the temperature cycle is reduced to a minimum, therefore, it is feasible to use a battery to heat the micro heater in the embodiment of the present invention.
  • the power consumption to complete a 60oC-95oC temperature cycle is only 0.0735 joules, which is particularly suitable for battery-powered portable devices and is very suitable for testing in the field.
  • Embodiment 5 is a diagrammatic representation of Embodiment 5:
  • a rapid nucleic acid detection device or rapid amplification device including several rapid nucleic acid detection chips as described above; in each rapid nucleic acid detection chip, a heating device is used independently or uniformly to heat the droplets on the suspended film to achieve rapid temperature changes.
  • a heating device is used independently or uniformly to heat the droplets on the suspended film to achieve rapid temperature changes.
  • each area is very small, normally around 1 mm, so the microheater can be made and is very suitable for making into large-scale arrays, such as 2*2, 10*10, 100*100 arrays, for high-throughput parallel detection of multiple samples and multiple nucleic acids.
  • the microheater array here can be integrated on the same substrate with a certain area, or it can be set separately on different substrates for unified integration.
  • Each microheater in the array can quickly perform thermal cycling operations on a droplet.
  • each microheater can heat the same droplet to detect a certain volume of droplets.
  • each droplet is 100 nanoliters, and 100 microliters of the liquid to be tested can be divided into 1,000 droplets and placed on 1,000 microheaters for parallel processing to ensure that rare nucleic acid copies are not missed.
  • each rapid nucleic acid detection chip such as a microheater
  • Each rapid nucleic acid detection chip can also be placed with different test samples and nucleic acid detection opportunities. Therefore, in each large-scale microheater array, different samples to be tested can be thermally cycled on different microheaters to achieve high-throughput sample detection; or different nucleic acid detection reagents can be used for the same sample to achieve simultaneous detection of multiple nucleic acids.
  • the above methods can also be mixed and integrated, that is, multiple nucleic acids can be detected simultaneously on multiple samples.
  • Embodiment 6 is a diagrammatic representation of Embodiment 6
  • a specific amplification method including: setting a film suspended in the air;
  • the droplets are heated periodically to achieve circulation of the droplets at different temperatures to achieve amplification.
  • the anti-volatile layer is formed by one or more of the following methods: using a surfactant to self-assemble into a film on the surface of the droplet to form an anti-volatile layer; covering the surface of the droplet with a layer of hydrophobic nanoparticles to form an anti-volatile layer; adding a layer of suspended film above the droplet to seal the droplet between two layers of suspended film to form an anti-volatile layer.
  • one or more of the following heating methods can be used to achieve periodic heating of the droplets: heating with a heating wire or a heating plate under the suspended film; or inserting a microwave or ultrasonic probe into the droplets for heating; placing the entire chip in a microwave oven and using microwaves to heat the droplets and the suspended film without contact.
  • the amplification method further includes the following steps: when the droplets are periodically heated, the heating temperature of the droplets is measured.
  • a temperature measuring resistance wire can be used to heat and measure the temperature of the droplets on the suspended membrane.
  • other methods can also be used to perform heating and temperature measurement during heating amplification.
  • the following steps are included: integrating the temperature measuring resistance wire with the suspended film to form a suspended micro-heater, and placing the droplets on the upper surface of the suspended film; allowing the anti-volatile layer to cover the droplets on the micro-heater; applying different electrical signals to the suspended micro-heater to achieve periodic heating of the droplets, so that the droplets circulate at different temperatures and amplification is achieved.
  • the amplification method in this embodiment can form a rapid nucleic acid detection chip by adding fluorescent markers or fluorescent probes.
  • the amplified droplets containing fluorescent markers are placed in a fluorescence detection device to complete the fluorescence detection of nucleic acids;
  • the detection result is determined based on the fluorescence brightness of the droplets after amplification.
  • the droplet contains a sample to be tested, an amplification primer, an enzyme, dNTP (deoxyribonucleoside triphosphate), a template, a fluorescent probe and a buffer.
  • dNTP deoxyribonucleoside triphosphate
  • An anti-volatile layer is disposed outside the droplet.
  • the following uses a specific rapid nucleic acid detection chip, namely a microheater, to illustrate how to achieve rapid nucleic acid detection:
  • the suspended microheater is placed on a substrate with a groove, and at least two supporting conductive wires of the suspended microheater are extended to the raised edge of the substrate groove to support the microheater to be suspended above the groove; the supporting conductive wires are extended to the raised edge of the substrate groove and fixed to form a microheater connection terminal; an anti-volatile layer is used, and the non-volatile hydrophobic liquid film is used as the anti-volatile layer, and the non-volatile hydrophobic liquid film is set to fluorine oil or silicone oil.
  • the droplets and the area below the suspended film are completely filled so that the microheater and the droplets on the microheater can be completely covered.
  • the fluorescence is detected by using the fluorescent markers in the droplets to obtain the amplification results.
  • the suspended membrane is configured as a thin film selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene (paraxylene polymer) film, metal film, or a composite film formed by the above.
  • an enhanced reflection coating is applied on the suspended membrane to enhance the fluorescence reflection signal.
  • an additional heat sink on the suspended membrane is used to dissipate heat from the droplets.
  • one or more of the following methods including thermoelectric cooling sheets, planar heat pipes, or microfluidic channel fluids, are used on the suspended membrane to dissipate heat from the droplets.
  • the aforementioned rapid nucleic acid detection method and the rapid nucleic acid detection chip provided by the present invention can also be applied on a large scale to realize a rapid nucleic acid large-scale detection method, which divides the same sample or different samples into multiple droplets, and uses the aforementioned rapid nucleic acid detection method to simultaneously detect multiple droplets.
  • the key point of the present invention is to place micro droplets (nanoliter to microliter) on a suspended film, and use a micro heater or microwave heating to achieve a temperature cycle within 0.5 seconds (65oC-95oC). During the cycle, the droplets are covered with oil to prevent the droplets from volatilizing.
  • the present invention uses in-situ heating and cooling of the droplets, and through the support of the suspended film, eliminates the need for substrate heating, and can achieve the fastest droplet heating and cooling without driving the droplets, greatly simplifying chip design and operation, ensuring ease of use and reliability.

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Abstract

Disclosed herein are an amplification structure, a rapid nucleic acid detection chip comprising the amplification structure, a device comprising the rapid nucleic acid detection chip, a corresponding amplification method, a rapid nucleic acid detection method, and a large-scale nucleic acid detection method. The amplification structure comprises a suspended thin film and a heating device, the heating device being configured for heating droplets on the suspended thin film. According to the microdroplet-based rapid nucleic acid detection chip provided by the present invention, by means of the arrangement of the suspended thin film and the droplets, amplification of a sample to be detected in the heated droplets can be rapidly achieved.

Description

扩增结构、快速核酸检测芯片、装置与方法Amplification structure, rapid nucleic acid detection chip, device and method 技术领域Technical Field
本发明属分子生物学中分子扩增诊断领域,具体涉及一种扩增结构、快速核酸检测芯片、装置与方法。The present invention belongs to the field of molecular amplification diagnosis in molecular biology, and specifically relates to an amplification structure, a rapid nucleic acid detection chip, a device and a method.
背景技术Background technique
聚合酶链式反应(Polymerase chain reaction - PCR)可以将核酸复制扩增,核酸复制扩增有多种应用,如用在核酸检测上,核酸检测是生物分子检测中的一个重要领域,通过PCR扩增可实现对超微量(甚至单个核酸分子)核酸的准确和定量检测。PCR核酸检测有广泛应用,包括临床疾病诊断(如各种传染性病原微生物诊断和疗效评价、优生优育检测、肿瘤标志物及瘤基因检测、遗传基因检测等)、动物疾病检测(如禽流感、口蹄疫、猪瘟、寄生虫病、炭疽芽孢杆菌等)、食品安全检测(如食源微生物、食品过敏源、转基因食品等)、科学研究(如医学、生命科学、农牧等相关分子生物学定量研究)等。PCR核酸定量检测技术的应用行业包括医疗机构、科研院所、高校、疾控中心、检验检疫局、食品企业、畜牧企业等。Polymerase chain reaction (PCR) can replicate and amplify nucleic acids. Nucleic acid replication and amplification have many applications, such as nucleic acid detection. Nucleic acid detection is an important field in biomolecular detection. PCR amplification can achieve accurate and quantitative detection of ultra-trace amounts of nucleic acids (even single nucleic acid molecules). PCR nucleic acid detection has a wide range of applications, including clinical disease diagnosis (such as diagnosis and efficacy evaluation of various infectious pathogenic microorganisms, eugenics detection, tumor marker and tumor gene detection, genetic gene detection, etc.), animal disease detection (such as avian influenza, foot-and-mouth disease, swine fever, parasitic diseases, anthrax Bacillus, etc.), food safety testing (such as foodborne microorganisms, food allergens, genetically modified foods, etc.), scientific research (such as medicine, life sciences, agriculture and animal husbandry and other related molecular biology quantitative research), etc. The application industries of PCR nucleic acid quantitative detection technology include medical institutions, scientific research institutes, universities, disease control centers, inspection and quarantine bureaus, food companies, livestock companies, etc.
聚合酶链式反应(PCR)是一种最常见可用于放大扩增特定的DNA片段的分子生物学技术, PCR的最大特点是能将微量的DNA片段大幅增加,结合荧光探针,可用于微量特定核酸片段的检测,常见用于感染性病原微生物检测,肿瘤分析及遗传病诊断。PCR在传染性疾病的检测、诊断上有重要应用,是应对大规模群体性疫情(如新冠病毒)的必备技术。Polymerase chain reaction (PCR) is the most common molecular biology technique that can be used to amplify specific DNA fragments. The biggest feature of PCR is that it can significantly increase trace amounts of DNA fragments. Combined with fluorescent probes, it can be used to detect trace amounts of specific nucleic acid fragments. It is commonly used in infectious pathogenic microorganism detection, tumor analysis, and genetic disease diagnosis. PCR has important applications in the detection and diagnosis of infectious diseases and is an essential technology for responding to large-scale mass epidemics (such as the new coronavirus).
传统PCR扩增技术需在95oC-65oC-72oC 之间进行温度循环,由于升降温需要一定的时间,单次循环的时间需5-30分钟。一般PCR需进行20-40次循环扩展,导致PCR检测需要几个小时的时间,且检测需在有资质的检测机构实验室内完成。PCR检测时效性差、且需要专业检测单位操作,导致其在大规模传染性疫情防控中的作用存在一定局限。Traditional PCR amplification technology requires temperature cycling between 95oC-65oC-72oC. Since it takes a certain amount of time to raise and lower the temperature, a single cycle takes 5-30 minutes. Generally, PCR requires 20-40 cycles of expansion, which means that PCR testing takes several hours, and the test must be completed in a qualified testing agency laboratory. PCR testing has poor timeliness and requires professional testing units to operate, which leads to certain limitations in its role in large-scale infectious disease prevention and control.
具体而言,现有PCR扩增技术,在进行温度循环时,除了温控待测液体,温控系统不可避免的加热衬底等样品支撑结构,导致升温和降温都需要比较长的时间。同时,基于毫升量级的液体样本,在待测核酸浓度比较低时,需要一定的等待时间使得反应充分。这些因素导致PCR循环需要的时间在5-30分钟左右。因为需要多次PCR循环(20-40次),检测所需的总时间比较长(半个小时到几个小时),无法实时(几分钟内)出结果。PCR中的温度循环方式常见有以下几种:(1)采用热电片对液滴加热和制冷,实现温度循环,一般温度循环需要1分钟以上;(2)采用传统加热方式对液滴和衬底加热,液滴和衬底自然冷确,一般温度循环需要5分钟-20分钟左右;(3)在衬底上维持高低温两个温区,通过微流控管道或者液滴驱动将液体在高低温之间来回循环,一般温度循环取决于液滴驱动的速度,时间可以控制在几秒以内。但此种方式需要对液滴进行精确操作,器件设计和难度均较大,维持两个温区的功耗较大,且液滴在95oC高温下运动时极易产生气泡,为芯片设计、液滴操控均带来很大的不方便。Specifically, in the existing PCR amplification technology, in addition to temperature control of the liquid to be tested, the temperature control system inevitably heats the sample support structure such as the substrate during temperature cycling, resulting in a relatively long time for both heating and cooling. At the same time, based on the milliliter-level liquid sample, when the concentration of the nucleic acid to be tested is relatively low, a certain waiting time is required to allow the reaction to be sufficient. These factors result in the time required for PCR cycling being about 5-30 minutes. Because multiple PCR cycles (20-40 times) are required, the total time required for detection is relatively long (half an hour to several hours), and the results cannot be obtained in real time (within a few minutes). The following are common temperature cycling methods in PCR: (1) Using thermoelectric chips to heat and cool the droplets to achieve temperature cycling. Generally, the temperature cycle takes more than 1 minute; (2) Using traditional heating methods to heat the droplets and substrates, the droplets and substrates cool naturally. Generally, the temperature cycle takes about 5 minutes to 20 minutes; (3) Maintaining two high and low temperature zones on the substrate, and circulating the liquid back and forth between high and low temperatures through microfluidic channels or droplet drive. Generally, the temperature cycle depends on the speed of the droplet drive, and the time can be controlled within a few seconds. However, this method requires precise manipulation of the droplets, and the device design and difficulty are relatively high. The power consumption of maintaining two temperature zones is high, and bubbles are easily generated when the droplets move at a high temperature of 95oC, which brings great inconvenience to chip design and droplet manipulation.
技术问题technical problem
有鉴于现有技术的上述缺陷,本发明所要解决的技术问题是如何实现快速稳定又方便地核酸扩增。In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to achieve rapid, stable and convenient nucleic acid amplification.
技术解决方案Technical Solutions
基于上述技术问题,本发明提供了一种扩增结构,包括悬空薄膜和加热装置,所述加热装置用于加热悬空薄膜上的液滴。Based on the above technical problems, the present invention provides an amplification structure, including a suspended membrane and a heating device, wherein the heating device is used to heat droplets on the suspended membrane.
较优的,在悬空薄膜上的所述液滴上包裹有防挥发层。Preferably, the liquid droplets on the suspended membrane are coated with an anti-volatile layer.
较优的,所述防挥发层设置为非挥发性疏水液膜和/或疏水纳米颗粒层。Preferably, the anti-volatile layer is configured as a non-volatile hydrophobic liquid film and/or a hydrophobic nanoparticle layer.
较优的,当采用所述非挥发性疏水液膜作为所述防挥发层时,所述非挥发性疏水液膜的沸点高于所述液滴沸点。Preferably, when the non-volatile hydrophobic liquid film is used as the anti-volatile layer, the boiling point of the non-volatile hydrophobic liquid film is higher than the boiling point of the liquid droplets.
较优的,采用所述非挥发性疏水液膜作为所述防挥发层时,所述非挥发性疏水液膜设置为氟油或硅油。Preferably, when the non-volatile hydrophobic liquid film is used as the anti-volatile layer, the non-volatile hydrophobic liquid film is set to be fluorine oil or silicone oil.
较优的,所述防挥发层由表面活性剂在所述液滴表面自组装成膜形成所述防挥发层。Preferably, the anti-volatile layer is formed by self-assembly of a surfactant on the surface of the droplet to form a film.
较优的,在悬空薄膜上的所述液滴上方加盖一层悬空薄膜使液滴封存在两层悬空薄膜之间以形成所述防挥发层。Preferably, a layer of suspended film is added above the liquid droplets on the suspended film so that the liquid droplets are sealed between two layers of suspended films to form the anti-volatile layer.
较优的,所述加热装置设置为微波容器,所述悬空薄膜及液滴置于所述微波容器中,再利用微波容器对液滴进行加热。Preferably, the heating device is configured as a microwave container, the suspended film and the liquid droplets are placed in the microwave container, and the liquid droplets are heated by the microwave container.
较优的,所述加热装置设置为加热微针,通过所述加热微针插入悬空薄膜上的所述液滴进行加热。Preferably, the heating device is configured to heat the microneedles, and the droplets on the suspended membrane are heated by inserting the heating microneedles.
较优的,所述加热微针设置为微波或者超声波探头微针。Preferably, the heating microneedle is configured as a microwave or ultrasonic probe microneedle.
较优的,所述加热装置设置为加热片或者加热丝,所述加热片或者所述加热丝设置在所述悬空薄膜下方进行加热。Preferably, the heating device is configured as a heating plate or a heating wire, and the heating plate or the heating wire is disposed under the suspended membrane for heating.
较优的,所述加热装置上设置有测温装置。Preferably, the heating device is provided with a temperature measuring device.
较优的,所述加热片或者加热丝设置为加热及测温微电阻丝。Preferably, the heating plate or heating wire is configured as a heating and temperature measuring micro-resistance wire.
较优的,所述悬空薄膜与所述加热丝或者加热片集成形成微加热器。Preferably, the suspended membrane is integrated with the heating wire or heating sheet to form a micro heater.
较优的,利用防挥发层将悬空薄膜上的液滴以及所述悬空薄膜下方区域全部填充,使其能够将微加热器以及所述微加热器上的液滴全部包覆。Preferably, the anti-volatile layer is used to fill the liquid droplets on the suspended membrane and the area below the suspended membrane, so that the anti-volatile layer can completely cover the micro-heater and the liquid droplets on the micro-heater.
较优的,所述微加热器悬空于衬底上方,所述微加热器上延伸出至少两根支撑导电线至所述衬底上固定并形成微加热器连接端子;所述支撑导电线支撑所述微加热器悬空时的平衡。Preferably, the microheater is suspended above the substrate, and at least two supporting conductive wires extend from the microheater to be fixed on the substrate and form microheater connection terminals; the supporting conductive wires support the balance of the microheater when it is suspended.
较优的,所述衬底上设置有凹槽,所述微加热器设置在所述凹槽的上方,所述微加热器上延伸出至少两根支撑导电线至所述凹槽的凸起边缘,支撑所述微加热器在所述凹槽上方悬空;所述支撑导电线延伸至所述凹槽的凸起边缘固定,形成所述微加热器连接端子。Preferably, a groove is provided on the substrate, the microheater is arranged above the groove, and at least two supporting conductive wires extend from the microheater to the raised edge of the groove to support the microheater to be suspended above the groove; the supporting conductive wires extend to the raised edge of the groove and are fixed to form the microheater connecting terminal.
较优的,从所述微加热器上延伸出四根支撑导电线至所述凹槽的凸起边缘固定并形成微加热器连接端子,所述四根支撑导电线支撑所述微加热器悬空时的平衡。Preferably, four supporting conductive wires extend from the microheater to the raised edge of the groove to be fixed and form a microheater connection terminal, and the four supporting conductive wires support the balance of the microheater when it is suspended in the air.
较优的,对所述连接端子输入不同电信号,使得所述微加热器其实现温度的快速变化以热传导至液滴实现温度的快速变化。Preferably, different electrical signals are input to the connection terminals, so that the micro-heater can achieve rapid temperature changes so as to conduct heat to the droplets to achieve rapid temperature changes.
较优的,所述悬空薄膜上设置有疏水或者超疏水镀层。Preferably, a hydrophobic or super-hydrophobic coating is provided on the suspended membrane.
较优的,所述悬空薄膜设置为氮化硅、氧化硅、碳膜、金刚石膜、parylene派瑞林(对二甲苯聚合物)膜、金属膜中的一种薄膜或者几种形成的复合薄膜。Preferably, the suspended membrane is configured to be a thin film selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene (paraxylene polymer) film, metal film, or a composite film formed by the above-mentioned thin films.
较优的,还包括散热装置。Preferably, a heat dissipation device is also included.
较优的,所述散热装置为设置于所述悬空薄膜上的热电制冷片、平面热管或者微流控管道流体中的其中一种或者几种。Preferably, the heat dissipation device is one or more of a thermoelectric cooling sheet, a planar heat pipe or a microfluidic pipeline fluid arranged on the suspended membrane.
本发明还提供一种包括扩增结构的快速核酸检测芯片,包括前述的扩增结构。较优的,所述悬空薄膜上设置有增强反射镀膜。The present invention also provides a rapid nucleic acid detection chip including an amplification structure, including the aforementioned amplification structure. Preferably, an enhanced reflection coating is provided on the suspended membrane.
本发明还提供一种快速核酸检测阵列芯片,包括如前所述的快速核酸检测芯片;每一快速核酸检测芯片中独立或者相互统一地利用加热装置对所述液滴进行加热以实现温度的快速变化。The present invention also provides a rapid nucleic acid detection array chip, including the rapid nucleic acid detection chip as described above; in each rapid nucleic acid detection chip, a heating device is used independently or uniformly to heat the droplets to achieve rapid temperature changes.
除此之外,本发明还提供一种扩增方法:包括以下步骤:In addition, the present invention also provides an amplification method, comprising the following steps:
设置一薄膜悬空;A film is set to be suspended in the air;
将含有扩增样本的液滴置于悬空薄膜上;A droplet containing the amplified sample is placed on a suspended membrane;
周期性加热所述液滴,实现液滴在不同温度下的循环,实现扩增;Periodically heating the droplets to achieve circulation of the droplets at different temperatures to achieve amplification;
较优的,所述液滴外部设置有防挥发层。Preferably, an anti-volatile layer is disposed outside the droplet.
较优的,采用以下的一种或者几种方法形成所述防挥发层:用表面活性剂在液滴表面自组装成膜形成防挥发层;在所述液滴表面覆盖一层疏水纳米颗粒形成防挥发层;在所述液滴上方加盖一层悬空薄膜将液滴封存在两层悬空薄膜之间形成防挥发层。Preferably, the anti-volatile layer is formed by one or more of the following methods: using a surfactant to self-assemble into a film on the surface of the droplet to form an anti-volatile layer; covering the surface of the droplet with a layer of hydrophobic nanoparticles to form an anti-volatile layer; adding a layer of suspended film above the droplet to seal the droplet between two layers of suspended film to form an anti-volatile layer.
较优的,采用以下一种或者几种加热方法实现液滴周期性加热:在悬空薄膜下方利用加热丝或者加热片进行加热;或者使用微波或者超声波探针插入所述液滴进行加热;将整个芯片放置的微波炉中,利用微波对所述液滴以及悬空薄膜无接触加热。Preferably, one or more of the following heating methods are used to achieve periodic heating of the droplets: heating with a heating wire or a heating plate under the suspended film; or inserting a microwave or ultrasonic probe into the droplets for heating; placing the entire chip in a microwave oven and using microwaves to heat the droplets and the suspended film without contact.
较优的,在周期性加热所述液滴时,对液滴加热温度进行测量。Preferably, when the droplets are periodically heated, the heating temperature of the droplets is measured.
较优的,采用测温电阻丝对所述悬空薄膜上的液滴进行加热测温。Preferably, a temperature measuring resistance wire is used to heat and measure the temperature of the droplets on the suspended membrane.
较优的,实现周期性加热所述液滴,实现液滴在不同温度下的循环,实现扩增时,包括以下步骤:Preferably, the droplets are heated periodically to achieve circulation of the droplets at different temperatures and to achieve amplification, comprising the following steps:
将所述测温电阻丝与所述悬空薄膜进行集成,形成一悬空微加热器,所述液滴置于所述悬空薄膜上表面;使所述防挥发层包覆所述微加热器上的液滴;对所述悬空微加热器施加不同电信号,实现周期性加热所述液滴,使得液滴在不同温度下进行循环,实现扩增。The temperature measuring resistance wire is integrated with the suspended film to form a suspended microheater, and the droplets are placed on the upper surface of the suspended film; the anti-volatile layer covers the droplets on the microheater; different electrical signals are applied to the suspended microheater to periodically heat the droplets, so that the droplets circulate at different temperatures to achieve amplification.
较优的,本快速核酸检测方法还包括以下步骤:Preferably, the rapid nucleic acid detection method further comprises the following steps:
将所述悬空微加热器置于一具有凹槽的衬底上,将所述悬空微加热器延伸出至少两根支撑导电线至所述衬底凹槽的凸起边缘,支撑所述微加热器在所述凹槽上方悬空;所述支撑导电线延伸至所述衬底凹槽的凸起边缘固定,形成微加热器连接端子;利用防挥发层将所述液滴以及所述悬空薄膜下方区域全部填充,使其能够将微加热器以及所述微加热器上的液滴全部包覆;The suspended microheater is placed on a substrate with a groove, and at least two supporting conductive wires are extended from the suspended microheater to the raised edge of the substrate groove to support the microheater to be suspended above the groove; the supporting conductive wires are extended to the raised edge of the substrate groove and fixed to form a microheater connection terminal; the droplets and the area below the suspended film are completely filled with an anti-volatile layer so that the microheater and the droplets on the microheater are completely covered;
对所述悬空微加热器的所述微加热器连接端子施加不同电信号,实现周期性加热所述液滴,使得液滴在不同温度下进行循环,实现扩增。Different electrical signals are applied to the microheater connection terminals of the suspended microheater to periodically heat the droplets, so that the droplets circulate at different temperatures to achieve amplification.
较优的,采用所述非挥发性疏水液膜作为所述防挥发层,所述非挥发性疏水液膜设置为氟油或硅油。Preferably, the non-volatile hydrophobic liquid film is used as the anti-volatilization layer, and the non-volatile hydrophobic liquid film is set to fluorine oil or silicone oil.
较优的,所述悬空薄膜设置为氮化硅、氧化硅、碳膜、金刚石膜、parylene派瑞林(对二甲苯聚合物)膜、金属膜中的一种薄膜或者几种形成的复合薄膜。Preferably, the suspended membrane is configured to be a thin film selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene (paraxylene polymer) film, metal film, or a composite film formed by the above-mentioned thin films.
较优的,在周期性加热所述液滴之后,采用悬空薄膜上的额外散热装置对所述液滴进行散热。Preferably, after the droplets are periodically heated, an additional heat sink on the suspended membrane is used to dissipate the heat from the droplets.
较优的,在所述悬空薄膜上采用热点制冷片、平面热管或者微流控管道流体中的其中一种或者几种方式对所述液滴进行散热。Preferably, one or more of the following methods, including a hot spot cooling sheet, a planar heat pipe or a microfluidic channel fluid, is used on the suspended membrane to dissipate heat from the droplets.
基于前述扩增方法,本发明还提供一种快速核酸检测方法,使用前述扩增方法进行待测样本扩增。Based on the aforementioned amplification method, the present invention also provides a rapid nucleic acid detection method, which uses the aforementioned amplification method to amplify the sample to be tested.
该快速核酸检测方法,较优的,还包括以下步骤The rapid nucleic acid detection method preferably further comprises the following steps
扩增前在所述液滴中加入荧光标记;adding a fluorescent marker to the droplet before amplification;
扩增后将含荧光标记的扩增后液滴,置于荧光检测装置中,完成核酸的荧光检测;After amplification, the amplified droplets containing fluorescent markers are placed in a fluorescence detection device to complete the fluorescence detection of nucleic acids;
根据扩增后液滴的荧光亮度,判定检测结果。The detection result is determined based on the fluorescence brightness of the droplets after amplification.
该快速核酸检测方法,较优的,所述液滴内含有待测样本、扩增引物、酶、dNTP脱氧核糖核苷三磷酸、模板、荧光探针和缓冲液。The rapid nucleic acid detection method is preferably such that the droplets contain a sample to be tested, an amplification primer, an enzyme, dNTP deoxyribonucleoside triphosphates, a template, a fluorescent probe and a buffer.
该快速核酸检测方法,较优的,在所述悬空薄膜上镀上增强反射镀膜以增强荧光反射信号。This rapid nucleic acid detection method is preferably performed by coating the suspended film with an enhanced reflection coating to enhance the fluorescent reflection signal.
本发明还提供一种快速核酸大规模检测方法,将同一样本或者不同样本分别分成多个液滴,利用如前述的快速核酸检测方法同时对多个液滴进行检测。The present invention also provides a rapid large-scale nucleic acid detection method, which divides the same sample or different samples into multiple droplets, and uses the aforementioned rapid nucleic acid detection method to simultaneously detect the multiple droplets.
有益效果Beneficial Effects
(1)本发明提供了扩增结构以及相应的快速核酸检测芯片,通过悬空薄膜和液滴的设置,能够快速实现加热液滴中扩增待测样本的扩增。采用液滴原位加热和降温,通过悬空薄膜支撑,消除对衬底加热的需要,可实现最快速度的液滴升降温,无需驱动液滴,大大简化芯片设计和操作,确保易用性、可靠性。同时,本发明采用微液滴作为反应容器,传质、传热速度块,核酸扩增反应快;同时将液滴置于悬空薄膜上,减少温度循环过程中不可避免的衬底加热,使得升温和降温的速度在0.5秒以内完成,而现有PCR扩增温度循环所需时间在1-5分钟以内,将温度循环的速度提升100倍以上。因核酸检测需要PCR扩增20-30次以上,采用本发明可将核酸检测的总时间从传统的30分钟到几个小时,缩短到1分钟以内,极大地缩短检测所需时间,极大地提升核酸检测的时效性。(1) The present invention provides an amplification structure and a corresponding rapid nucleic acid detection chip. Through the arrangement of suspended membranes and droplets, the amplification of the sample to be tested in the heated droplets can be quickly realized. The droplets are heated and cooled in situ, and the need for substrate heating is eliminated through the support of the suspended membrane. The fastest droplet heating and cooling can be achieved without driving the droplets, which greatly simplifies the chip design and operation, ensuring ease of use and reliability. At the same time, the present invention uses micro droplets as reaction containers, with fast mass transfer and heat transfer speeds, and fast nucleic acid amplification reactions; at the same time, the droplets are placed on the suspended membrane to reduce the inevitable substrate heating during the temperature cycle, so that the heating and cooling speed is completed within 0.5 seconds, while the time required for the existing PCR amplification temperature cycle is within 1-5 minutes, and the temperature cycle speed is increased by more than 100 times. Since nucleic acid detection requires PCR amplification for more than 20-30 times, the total time of nucleic acid detection can be shortened from the traditional 30 minutes to several hours to less than 1 minute by using the present invention, greatly shortening the time required for detection and greatly improving the timeliness of nucleic acid detection.
(2)本发明装置结构简单,每个扩增结构以及快速核酸检测芯片仅检测单个液滴,单个液滴检测能力强,检测成本低:同时,具体应用的场景下,我们可以采用MEMS加工技术,将单个微加热器的成本控制在1-10元左右,采用微液滴极大地减少试剂的用量,在极大的缩短检测时间的同时,将检测成本控制在极低水平,适合大规模推广,比如疫情防控中及时高效的核酸筛查;家庭用病原微生物快查等应用。(2) The device of the present invention has a simple structure. Each amplification structure and rapid nucleic acid detection chip only detects a single droplet. The single droplet has strong detection capabilities and low detection costs. At the same time, in specific application scenarios, we can use MEMS processing technology to control the cost of a single microheater to around 1-10 yuan. The use of microdroplets greatly reduces the amount of reagents used, greatly shortening the detection time while controlling the detection cost to an extremely low level. It is suitable for large-scale promotion, such as timely and efficient nucleic acid screening in epidemic prevention and control; rapid detection of pathogenic microorganisms for household use, and other applications.
(3)本发明提供的扩增结构和快速核酸检测芯片,在液滴中进行扩增的同时,在液滴上设置防挥发层,防挥发层可以有效防止在核酸扩增时产生气溶胶污染,同时也保证扩增的效果,这里的防挥发层可以是多种方式形成,如采用高沸点的非挥发性疏水液膜,如硅油或者氟油,此时,核酸扩增是在封闭的油相中完成,可有效防止气溶胶的形成。当然,防挥发层可以采用其他方式形成,如由双亲表面活性剂在液滴表面自组装成膜减少液滴挥发,或者在液滴中加入高沸点相容溶剂(如水性液滴中加入乙二醇或聚乙二醇),或者在液滴表面覆盖一层疏水纳米颗粒形成liquid marble(固态表面包裹的液滴), 或者在液滴上方加盖一层悬空薄膜将液滴封存在两层悬空薄膜之间等方式防止液滴在加热过程的挥发。因为芯片和试剂的成本低,为一次性使用,扩增检测结束后阳性样本封存在防挥发层如油滴中,可有效防止扩增后的核酸分子对设备的污染。(3) The amplification structure and rapid nucleic acid detection chip provided by the present invention, while amplification is performed in the droplets, an anti-volatile layer is set on the droplets. The anti-volatile layer can effectively prevent aerosol pollution during nucleic acid amplification, and also ensure the effect of amplification. The anti-volatile layer here can be formed in a variety of ways, such as using a high-boiling point non-volatile hydrophobic liquid film, such as silicone oil or fluoro oil. At this time, nucleic acid amplification is completed in a closed oil phase, which can effectively prevent the formation of aerosols. Of course, the anti-volatile layer can be formed in other ways, such as by amphiphilic surfactants self-assembling into a film on the surface of the droplets to reduce the volatilization of the droplets, or adding a high-boiling point compatible solvent to the droplets (such as adding ethylene glycol or polyethylene glycol to aqueous droplets), or covering the surface of the droplets with a layer of hydrophobic nanoparticles to form liquid marble (droplets wrapped in a solid surface), or adding a layer of suspended film above the droplets to seal the droplets between two layers of suspended films to prevent the droplets from volatilizing during the heating process. Because the chips and reagents are low-cost and disposable, after the amplification test, the positive samples are sealed in an anti-volatile layer such as oil droplets, which can effectively prevent the amplified nucleic acid molecules from contaminating the equipment.
(4)本发明中,还提供了多种针对本扩增结构和快速核酸检测芯片的适应性扩增加热装置,在悬空薄膜上利用微纳加工制备的微加热电阻丝;或者使用微波(或者超声波)探针插入液滴进行加热。如果所有液滴在同一温度范围内循环,还可以将整个芯片放置的微波炉中,利用微波对所有液滴无接触同时加热。(4) The present invention also provides a variety of adaptive amplification heating devices for the amplification structure and rapid nucleic acid detection chip, using micro-heating resistor wires prepared by micro-nano processing on a suspended membrane; or using microwave (or ultrasonic) probes to insert into droplets for heating. If all droplets circulate within the same temperature range, the entire chip can also be placed in a microwave oven, and microwaves can be used to heat all droplets simultaneously without contact.
(5)为了使得本发明能够快速实现加热-冷却-加热循环,本发明特别地提出在悬空薄膜可以加装冷却装置即额外散热装置,如热电制冷片,或者平面热管,或微流控管道流体对流散热。这样设置的好处是更加快速实现扩增过程的加热-冷却-加热循环。(5) In order to enable the present invention to quickly realize the heating-cooling-heating cycle, the present invention specifically proposes that a cooling device, i.e., an additional heat dissipation device, such as a thermoelectric cooling sheet, or a planar heat pipe, or a microfluidic channel fluid convection heat dissipation can be installed on the suspended membrane. The advantage of such a configuration is that the heating-cooling-heating cycle of the amplification process can be realized more quickly.
(6)本发明同时提出一种具体的扩增结构和核酸检测芯片结构,将悬空薄膜下方设置测温微电阻丝,甚至可将测温微电阻丝与悬空薄膜共同集成,形成一个表面覆盖薄膜的微加热器,微加热器悬空并引出两根以上,如四根支撑导电线,既为悬空微加热器提供支撑,又延伸成为微加热器连接端子,为悬空微加热器提供电信号进行扩增。本发明提供的具体快速核酸检测结构,可以采用MEMS加工技术,将单个微加热器的成本控制在1-10元左右,采用微液滴极大地减少试剂的用量,在极大的缩短检测时间的同时,将检测成本控制在极低水平,本发明提出的具体核酸检测芯片结构,能够有效地快速实现核酸扩增,极大增加扩增效率,同时生产成本低,对于核酸控制过程好控制,由于加热器和悬空薄膜固定,只需要提供不同的电信号就能够实现快速扩增,操作非常方便,相对于现有的扩增方法和设备而言,不需要对液滴进行精确操作,器件设计简单,也不需要维持两个温区的功耗,优势极大。(6) The present invention also proposes a specific amplification structure and a nucleic acid detection chip structure, wherein a temperature measuring micro-resistance wire is arranged under the suspended membrane, and the temperature measuring micro-resistance wire and the suspended membrane can even be integrated together to form a micro-heater with a surface covered with a thin film, and the micro-heater is suspended and leads out two or more, such as four supporting conductive wires, which not only provide support for the suspended micro-heater, but also extend to become a micro-heater connection terminal, and provide an electrical signal for the suspended micro-heater to amplify. The specific rapid nucleic acid detection structure provided by the present invention can use MEMS processing technology to control the cost of a single micro-heater to about 1-10 yuan, and use micro-droplets to greatly reduce the amount of reagents, while greatly shortening the detection time, and controlling the detection cost to an extremely low level. The specific nucleic acid detection chip structure proposed by the present invention can effectively and quickly realize nucleic acid amplification, greatly increase the amplification efficiency, and has a low production cost. The nucleic acid control process is well controlled. Since the heater and the suspended membrane are fixed, only different electrical signals need to be provided to achieve rapid amplification, and the operation is very convenient. Compared with the existing amplification methods and devices, there is no need to accurately operate the droplets, the device design is simple, and there is no need to maintain the power consumption of two temperature zones, which has great advantages.
并且,本发明提出的扩增结构和快速核酸检测芯片和需要的装置紧凑,超便携:采用MEMS加工技术,单个微加热芯片的尺寸在100微米*100微米到10毫米*10毫米之间。让快速核酸检测不仅仅适用于实验室场景,更能够适用于家庭等日常场景。In addition, the amplification structure and rapid nucleic acid detection chip proposed by the present invention and the required device are compact and ultra-portable: using MEMS processing technology, the size of a single micro-heating chip is between 100 microns * 100 microns and 10 mm * 10 mm, making rapid nucleic acid detection not only suitable for laboratory scenarios, but also for daily scenarios such as homes.
(7)本发明提供的快速核酸检测芯片,在悬空薄膜上还设置疏水或者超疏水镀层,防止液滴平铺摊开。薄膜上可镀有增强反射镀膜如Au、Pt金属膜,或高反射的多层介质膜,用于增强荧光反射信号。这些适应性技术手段,能够帮助本发明进一步加快核酸检测速度,减少快速核酸检测中的难度,提高核酸检测中的效率。(7) The rapid nucleic acid detection chip provided by the present invention also has a hydrophobic or super-hydrophobic coating on the suspended film to prevent the droplets from spreading out. The film can be coated with an enhanced reflection coating such as Au, Pt metal film, or a highly reflective multilayer dielectric film to enhance the fluorescent reflection signal. These adaptive technical means can help the present invention further speed up the nucleic acid detection speed, reduce the difficulty in rapid nucleic acid detection, and improve the efficiency in nucleic acid detection.
(8)本发明还提供一种快速核酸检测装置,将多个快速核酸检测芯片进行集成,在一定衬底面积上,可以集成成百上千个微加热器阵列,阵列中每个微加热器可以快速对一个液滴进行热循环操作。将一定量的核酸检测分成多个液体同时检测,确保稀少核酸拷贝不漏检。在大规模微加热器阵列中,也可以在不同微加热器上热循环不同的待测样本,实现高通量的样本检测;或者对同一个样本,采用不同的核酸检测试剂,实现多种核酸的同时检测。以上方式还可以混合集成,即对多个样本同时进行多种核酸的检测。(8) The present invention also provides a rapid nucleic acid detection device, which integrates multiple rapid nucleic acid detection chips. On a certain substrate area, hundreds or thousands of microheater arrays can be integrated, and each microheater in the array can quickly perform thermal cycling operations on a droplet. A certain amount of nucleic acid detection is divided into multiple liquids for simultaneous detection to ensure that rare nucleic acid copies are not missed. In a large-scale microheater array, different samples to be tested can also be thermally cycled on different microheaters to achieve high-throughput sample detection; or different nucleic acid detection reagents can be used for the same sample to achieve simultaneous detection of multiple nucleic acids. The above methods can also be mixed and integrated, that is, multiple nucleic acids can be detected on multiple samples at the same time.
本发明提出的扩增结构,不仅仅可以用于核酸检测,还可以用于其他需要进行扩增的场景。以该扩增结构用于快速核酸检测为例,传统核酸检测更多的是在医疗、疾控或科研PCR实验室开展,而疫情及未来的诊疗需求,需要PCR核酸检测突破实验室的限制,去适应更多的应用场景,甚至是走入家庭。本发明可实现核酸现场检测(POCT),在低成本、超便携、低功耗等特点下突破应用场景限制,高效赋能发热门诊、急诊、海关、机场、出入境关口等高人流聚集地,满足传染病快速筛查的需求,增加多场景下的突发公共卫生事件的应对能力和解决途径。快速低成本核酸检测特别是当下新型冠状病毒肺炎(COVID-19)患者确诊、临床治疗效果评估、发生疫情时人群筛查及流行病学调查的重要手段。低成本、快速核酸检测同时在畜牧、农业、食品行业中发挥现场检测作用。在野外或战场等环境下可检测治病微生物和生物战病原体。The amplification structure proposed in the present invention can be used not only for nucleic acid detection, but also for other scenarios that require amplification. Taking the amplification structure for rapid nucleic acid detection as an example, traditional nucleic acid detection is more carried out in medical, disease control or scientific research PCR laboratories, and the epidemic and future diagnosis and treatment needs require PCR nucleic acid detection to break through the limitations of the laboratory to adapt to more application scenarios, and even go into the home. The present invention can realize nucleic acid on-site detection (POCT), break through the application scenario limitations under the characteristics of low cost, ultra-portability, and low power consumption, and efficiently enable fever clinics, emergency departments, customs, airports, entry and exit checkpoints and other high-traffic gathering places to meet the needs of rapid screening of infectious diseases, and increase the response capabilities and solutions to public health emergencies in multiple scenarios. Rapid and low-cost nucleic acid detection is an important means for the diagnosis of patients with new coronavirus pneumonia (COVID-19), clinical treatment effect evaluation, population screening and epidemiological surveys when an epidemic occurs. Low-cost and rapid nucleic acid detection also plays a role in on-site detection in animal husbandry, agriculture, and food industries. Pathogenic microorganisms and biological warfare pathogens can be detected in the wild or on the battlefield.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是本发明一具体实施方式的扩增结构示意图;FIG1 is a schematic diagram of an amplification structure according to a specific embodiment of the present invention;
图2是本发明实施例1中液滴采用疏水性液膜防挥发层结构示意图;FIG2 is a schematic diagram of the structure of the anti-volatile layer of the hydrophobic liquid film used for the droplets in Example 1 of the present invention;
图3是本发明实施例1中液滴采用表面自组织活性剂形成防挥发层的结构示意图;3 is a schematic diagram of the structure of the anti-volatile layer formed by the surface self-organizing active agent in the droplet in Example 1 of the present invention;
图4是本发明实施例1中液滴采用疏水纳米颗粒形成防挥发层的结构示意图;FIG4 is a schematic diagram of the structure of the anti-volatile layer formed by the droplets using hydrophobic nanoparticles in Example 1 of the present invention;
图5是本发明实施例1中液滴采用加盖悬空薄膜的方式进行防挥发的结构示意图;5 is a schematic diagram of the structure of preventing the droplets from volatilizing by covering them with suspended films in Example 1 of the present invention;
图6是本发明实施例2中实例2-1、2-2结构示意图;FIG6 is a schematic diagram of the structures of Examples 2-1 and 2-2 in Embodiment 2 of the present invention;
图7是本发明实施例2中实例2-3的结构示意图;FIG7 is a schematic diagram of the structure of Example 2-3 in Example 2 of the present invention;
图8是本发明实施例3中硅片表面上的集成悬空薄膜和加热装置集成微加热器的结构示意图;FIG8 is a schematic diagram of the structure of an integrated suspended membrane and a heating device integrated micro-heater on the surface of a silicon wafer in Example 3 of the present invention;
图9是图8中防挥发层的结构示意图;FIG9 is a schematic structural diagram of the anti-volatile layer in FIG8;
图10是实施例3中一实际微加热器结构示意图,其中左边是无液滴的微加热器结构示意图,右边是加液滴和防挥发层的微加热器结构示意图;FIG10 is a schematic diagram of an actual micro-heater structure in Example 3, wherein the left side is a schematic diagram of a micro-heater structure without droplets, and the right side is a schematic diagram of a micro-heater structure with droplets and an anti-volatile layer;
图11是实施例3中微加热器表面温度、加热电压与加热时间之间的关系,其中横轴为加热所需用时,纵轴为加热温度,阶数为通电电压大小;FIG11 is a diagram showing the relationship between the surface temperature of the micro-heater, the heating voltage and the heating time in Example 3, wherein the horizontal axis is the time required for heating, the vertical axis is the heating temperature, and the order is the magnitude of the power-on voltage;
图12是实施例3中无液滴的微加热器表面温度升降循环的时间响应曲线图,其中横轴为加热所需用时,纵轴为加热温度;12 is a time response curve of the temperature rise and fall cycle of the surface of the micro-heater without droplets in Example 3, wherein the horizontal axis is the time required for heating and the vertical axis is the heating temperature;
图13是实施例3中微加热器上放置液滴时的温度变化曲线图,其中横轴为加热所需用时,纵轴为加热温度;13 is a temperature change curve diagram when a droplet is placed on the micro-heater in Example 3, wherein the horizontal axis is the time required for heating and the vertical axis is the heating temperature;
图14是本发明具体实施例进行乙肝灭活病毒核酸扩增实验组结果图;14 is a graph showing the results of a hepatitis B virus inactivated nucleic acid amplification experiment group according to a specific embodiment of the present invention;
图15是本发明具体实施例进行乙肝灭活病毒核酸扩增对照组结果图;15 is a graph showing the results of a control group of hepatitis B inactivated virus nucleic acid amplification performed in a specific embodiment of the present invention;
图16是本发明具体实施例进行乙肝灭活病毒核酸扩增对照实验的扩增浓度-循环次数的曲线图;16 is a graph showing the amplification concentration-cycle number of a control experiment for amplification of inactivated hepatitis B virus nucleic acid in a specific embodiment of the present invention;
图17是本发明具体实施例进行新冠灭活病毒(COVID-19)核酸扩增对照实验对照组结果图;FIG17 is a graph showing the results of a control group of a novel coronavirus inactivated virus (COVID-19) nucleic acid amplification control experiment performed in a specific embodiment of the present invention;
图18是本发明具体实施例进行新冠灭活病毒(COVID-19)核酸扩增对照实验实验一组结果图;FIG18 is a set of result graphs of a control experiment of nucleic acid amplification of the inactivated coronavirus (COVID-19) according to a specific embodiment of the present invention;
图19是本发明具体实施例进行新冠灭活病毒(COVID-19)核酸扩增对照实验实验组一稀释100倍后的实验二组结果图。Figure 19 is a graph showing the results of Experiment 2 after the first experimental group was diluted 100 times in a control experiment of nucleic acid amplification of the inactivated coronavirus (COVID-19) in a specific embodiment of the present invention.
本发明的实施方式Embodiments of the present invention
下面将通过具体实施例对本发明进行详细说明。The present invention will be described in detail below through specific embodiments.
实施例1:Embodiment 1:
如图1所示,实施例1提供了一种本发明中较为简易的扩增结构,基于本扩增结构,同样可以构成一种简易的快速核酸检测芯片,可以只包括悬空薄膜1和加热装置(图1未示出),具体对于悬空薄膜1的制备:在衬底2上镀上一层薄膜,将衬底2一部分取出时(比如干法刻蚀硅衬底,或激光烧蚀玻璃衬底),薄膜1悬空形成悬空薄膜,就形成一个简易的扩增结构。将一个含有扩增样本的液滴3置于悬空薄膜1上,周期性加热,如通过微电阻丝5通电或微波加热液滴,实现液滴在不同温度下的循环,实现PCR扩增。当把该扩增结构用于快速核酸检测时,将含荧光标记的扩增后液滴,置于荧光检测装置中,完成核酸的荧光检测,通过记录每次温度循环(扩增)后液滴的荧光亮度,完成核酸扩增的融熔曲线,判定检测结果。As shown in FIG1 , Example 1 provides a relatively simple amplification structure in the present invention. Based on this amplification structure, a simple rapid nucleic acid detection chip can also be formed, which can only include a suspended membrane 1 and a heating device (not shown in FIG1 ). Specifically, for the preparation of the suspended membrane 1, a thin film is plated on the substrate 2. When a part of the substrate 2 is taken out (such as dry etching of a silicon substrate, or laser ablation of a glass substrate), the thin film 1 is suspended to form a suspended membrane, thus forming a simple amplification structure. A droplet 3 containing an amplified sample is placed on the suspended membrane 1, and periodically heated, such as by energizing a micro-resistance wire 5 or heating the droplet with a microwave, to achieve the circulation of the droplet at different temperatures and achieve PCR amplification. When the amplification structure is used for rapid nucleic acid detection, the amplified droplet containing a fluorescent marker is placed in a fluorescence detection device to complete the fluorescence detection of nucleic acid. By recording the fluorescence brightness of the droplet after each temperature cycle (amplification), the melting curve of nucleic acid amplification is completed to determine the detection result.
由于本实施例采用液滴进行扩增,形成扩增容器,液滴体积小,能够实现快速加热变温的效果,本实施例的结构,可以单独用于扩增形成一种扩增结构,也能够加入荧光标记或者探针实现快速的核酸检测,成为一种快速核酸检测芯片。Since this embodiment uses droplets for amplification to form an amplification container, the droplets are small in size and can achieve the effect of rapid heating and temperature change. The structure of this embodiment can be used alone for amplification to form an amplification structure, and fluorescent markers or probes can be added to achieve rapid nucleic acid detection, becoming a rapid nucleic acid detection chip.
当用于核酸检测制成快速核酸检测芯片时,液滴内部含有待测样本、扩增引物、酶、dNTP即脱氧核糖核苷三磷酸、模板、荧光探针和缓冲液,在扩增过程中,需要不断对其进行加热循环。When used for nucleic acid detection to make a rapid nucleic acid detection chip, the droplet contains the sample to be tested, amplification primers, enzymes, dNTPs (deoxyribonucleoside triphosphates), templates, fluorescent probes and buffer. During the amplification process, it needs to be continuously heated and circulated.
为防止液滴在加热过程中挥发,需要在液滴上设置防挥发层4,这里的防挥发层可以是疏水类液膜,如图2,例如液滴可用高沸点的非挥发油膜形成防挥发层4包裹,,注意这里的高沸点,是指沸点高于液滴中的液体,避免在加热时,防护层的液体也进行挥发,另外值得注意的是这里的疏水类液膜,可以是采用表面活性剂在液滴表面自组装成膜,如图3,例如用双亲表面活性剂在液滴表面自组装成膜形成防挥发层4减少液滴挥发,或者在液滴中加入高沸点相容溶剂(如水性液滴中加入乙二醇或聚乙二醇),如图4,或者在液滴表面覆盖一层疏水纳米颗粒形成liquid marble(固体表面包覆的液滴)进而形成防挥发层4, 如图5,或者在液滴上方加盖一层悬空薄膜1将液滴封存在两层悬空薄膜1之间形成防挥发的效果,进而形成防挥发层,在加盖悬空薄膜时,可以在液滴上同时进行油封形成防挥发层4或者前述其他方式进一步防止挥发。In order to prevent the droplets from volatilizing during the heating process, it is necessary to set an anti-volatile layer 4 on the droplets. The anti-volatile layer here can be a hydrophobic liquid film, as shown in Figure 2. For example, the droplets can be wrapped with a high-boiling point non-volatile oil film to form an anti-volatile layer 4. Note that the high boiling point here refers to a boiling point higher than that of the liquid in the droplets to prevent the liquid in the protective layer from volatilizing during heating. It is also worth noting that the hydrophobic liquid film here can be a surfactant that self-assembles on the surface of the droplets, as shown in Figure 3. For example, an amphiphilic surfactant is used to self-assemble on the surface of the droplets to form an anti-volatile layer 4 to reduce the volatilization of the droplets, or a high-boiling point compatible solvent is added to the droplets (such as adding ethylene glycol or polyethylene glycol to aqueous droplets), as shown in Figure 4, or a layer of hydrophobic nanoparticles is covered on the surface of the droplets to form a liquid marble (liquid droplets coated on a solid surface) to form an anti-volatile layer 4, as shown in FIG5, or a layer of suspended film 1 is added above the liquid droplets to seal the liquid droplets between two layers of suspended films 1 to form an anti-volatile effect, thereby forming an anti-volatile layer. When the suspended film is added, oil sealing can be performed on the liquid droplets at the same time to form an anti-volatile layer 4, or the aforementioned other methods can be used to further prevent volatility.
注意这里的防挥发层4不仅仅限于以上列举的几种方式,实际扩增过程中或者快速核酸检测过程中,也可以通过这几种方式中一种或者几种组合,实现更好的防挥发层效果。Note that the anti-volatile layer 4 here is not limited to the several methods listed above. In the actual amplification process or rapid nucleic acid detection process, one or a combination of these methods can also be used to achieve a better anti-volatile layer effect.
如图14至图16,利用乙肝灭活病毒进行本发明单扩增结构或快速核酸检测芯片(具体的实施方式可以是微加热器)的扩增对照实验,如图14,利用本发明方案对乙肝灭活病毒进行快速温度循环,4秒95oC,4秒65oC,共计 8秒一次PCR循环,扩增荧光亮度随循环次数变化如图14。图15是加入实验组相同扩增引物、酶、dNTP即脱氧核糖核苷三磷酸、模板、荧光探针和缓冲液等内容物,但无扩增病毒的本发明方案空白对照组结果。图16是相关对照实验的扩增浓度-循环次数的曲线,其中横坐标轴表示相对荧光强度,纵坐标轴表示热循环次数。可以看到,30次循环后浓度明显变化可进行检测,因此仅需要4分钟即可实现该病毒样本的扩增以及快速核酸检测。As shown in Figures 14 to 16, the amplification control experiment of the single amplification structure or rapid nucleic acid detection chip of the present invention (the specific implementation method can be a microheater) is carried out using the inactivated hepatitis B virus. As shown in Figure 14, the inactivated hepatitis B virus is subjected to rapid temperature cycling using the scheme of the present invention, 4 seconds at 95oC, 4 seconds at 65oC, a total of 8 seconds per PCR cycle, and the amplification fluorescence brightness changes with the number of cycles as shown in Figure 14. Figure 15 is the result of the blank control group of the scheme of the present invention in which the same amplification primers, enzymes, dNTPs, i.e., deoxyribonucleoside triphosphates, templates, fluorescent probes, and buffers as in the experimental group are added, but there is no amplification virus. Figure 16 is a curve of amplification concentration-cycle number of related control experiments, in which the horizontal axis represents the relative fluorescence intensity and the vertical axis represents the number of thermal cycles. It can be seen that the concentration changes significantly after 30 cycles and can be detected, so it only takes 4 minutes to achieve the amplification of the virus sample and rapid nucleic acid detection.
如图17至图19,在本发明提供的单扩增结构或快速核酸检测芯片(具体的实施方式可以是微加热器)上利用微液滴进行快速新冠灭活病毒(COVID-19)检测初步实验对照,同样的进行4秒95oC,4秒65oC, 每8秒一次PCR循环扩增。图17是加入与实验组相同的新冠扩增引物、酶、dNTP即脱氧核糖核苷三磷酸、模板、荧光探针和缓冲液,但不放入新冠病毒(COVID-19)扩增样本的实验空白对照组并对其空白对照组进行了相同扩增循环处理的结果;图18是放入新冠病毒(COVID-19)扩增样本即实验一组的实验对照结果;图19是在图18的新冠病毒(COVID-19)扩增样本浓度下的阳性标准稀释100倍之后的实验二组实验结果。由图17-图19看出,在本发明的方案下,有效快速在8秒一次循环里完成核酸扩增,并取得较为准确的检测结果,由该实验结果可以看到,30次循环后浓度明显变化可进行检测,采用30次循环只需要4分钟。As shown in Figures 17 to 19, a preliminary experimental control of rapid COVID-19 inactivated virus (COVID-19) detection is performed using microdroplets on a single amplification structure or rapid nucleic acid detection chip provided by the present invention (a specific embodiment can be a microheater), and the same PCR cycle amplification is performed for 4 seconds at 95oC, 4 seconds at 65oC, and once every 8 seconds. Figure 17 is the result of adding the same COVID-19 amplification primers, enzymes, dNTPs, i.e., deoxyribonucleoside triphosphates, templates, fluorescent probes, and buffer as the experimental group, but without the COVID-19 amplification sample, and the blank control group is subjected to the same amplification cycle treatment; Figure 18 is the experimental control result of the COVID-19 amplification sample, i.e., the experimental group 1; Figure 19 is the experimental result of the experimental group 2 after the positive standard at the COVID-19 amplification sample concentration of Figure 18 was diluted 100 times. As can be seen from Figures 17 to 19, under the scheme of the present invention, nucleic acid amplification is effectively and quickly completed in one cycle of 8 seconds, and relatively accurate detection results are obtained. From the experimental results, it can be seen that the concentration changes significantly after 30 cycles and can be detected, and 30 cycles only take 4 minutes.
值得注意的是,本实验为初步实验,后续可将PCR扩增温度和反应时间可进一步优化,有望实现1秒完成一次PCR循环。It is worth noting that this experiment is a preliminary one, and the PCR amplification temperature and reaction time can be further optimized in the future, and it is expected that one PCR cycle can be completed in 1 second.
实施例2:Embodiment 2:
本实施例中,采用不同的实例说明本发明采用实施例1中的芯片中实现快速核酸检测芯片的加热方式:In this embodiment, different examples are used to illustrate the heating method of the rapid nucleic acid detection chip implemented in the chip in Example 1 of the present invention:
实例2-1,如图6,本实例中采用在悬空薄膜1上采用普通的加热片5来进行加热扩增,加热片5可以是加热丝的集成,或者是金属片中含有加热丝,这种加热好处是由于薄膜1悬空,直接利用加热片5对悬空薄膜进行加热,加热过程中一个加热循环不需要对衬底进行加热,因此并不需要考虑衬底的加热和冷却速度,可以实现在悬空薄膜1上的液滴快速加热扩增。Example 2-1, as shown in Figure 6, in this example, an ordinary heating plate 5 is used on the suspended film 1 to perform heating amplification. The heating plate 5 can be an integration of heating wires, or a metal plate containing heating wires. The advantage of this heating method is that since the film 1 is suspended, the suspended film can be directly heated by the heating plate 5. During the heating process, the substrate does not need to be heated in one heating cycle, so there is no need to consider the heating and cooling speed of the substrate, and rapid heating amplification of droplets on the suspended film 1 can be achieved.
实例2-2,相对于实例2-1,这里提供一种更加简洁的加热扩增方式,即在悬空薄膜上设置加热丝,可以直接将加热丝与悬空薄膜集成在一起,同样参见图6,形成一种带有加热丝的悬空薄膜,即适应性用于液滴快速加热的加热器件。同样的,由于采用加热丝对悬空薄膜直接进行加热,加热过程中一个加热循环不需要对衬底进行加热,因此并不需要考虑衬底的加热和冷却速度,可以实现在悬空薄膜上的液滴快速加热扩增。Example 2-2, relative to Example 2-1, here is a more concise heating amplification method, that is, a heating wire is set on the suspended film, and the heating wire can be directly integrated with the suspended film, also see Figure 6, to form a suspended film with a heating wire, that is, a heating device that is adaptable to rapid heating of droplets. Similarly, since the suspended film is directly heated by the heating wire, the substrate does not need to be heated during a heating cycle during the heating process, so there is no need to consider the heating and cooling speed of the substrate, and rapid heating amplification of droplets on the suspended film can be achieved.
实例2-3Example 2-3
在实例2-3中,如图7,我们使用加热微针6,本实施例中采用微波(或者超声波)探针6插入液滴进行加热,,悬空薄膜的设置使得加热与衬底无关,能够准确方便地对液滴进行插入加热,除去了悬空薄膜的温度阻却,微针加热使得液滴加热更快。扩增时热循环速度进一步增加。In Example 2-3, as shown in FIG7 , we use a heating microneedle 6. In this embodiment, a microwave (or ultrasonic) probe 6 is inserted into the droplet for heating. The setting of the suspended membrane makes the heating independent of the substrate, and the droplet can be inserted and heated accurately and conveniently. The temperature barrier of the suspended membrane is removed, and the microneedle heating makes the droplet heat faster. The thermal cycle speed is further increased during amplification.
实例2-4Example 2-4
实例2-4中,采用一种更加简便的加热方法,同时满足加热速度快,且能够适用于多个液滴在同一温度范围内循环扩增,在实例2-4中,整个扩增结构或者快速核酸检测芯片放置的微波中如放置在微波炉中,利用微波对所有液滴无接触同时加热。所有液滴在同一温度范围内循环。In Example 2-4, a simpler heating method is used, which satisfies the requirement of fast heating speed and can be applied to multiple droplets for cyclic amplification within the same temperature range. In Example 2-4, the entire amplification structure or rapid nucleic acid detection chip is placed in a microwave, such as a microwave oven, and microwaves are used to heat all droplets simultaneously without contact. All droplets circulate within the same temperature range.
实例2-5Example 2-5
实例2-5中,在其他实例中,如实例2-1至2-4加热装置上设置测温装置,使得加热过程能够实时监测。具体而言,例如对于实例2-2可以采用测温电阻丝,直接集成到悬空薄膜上,形成既能够快速加热又能够实时测温的加热器,快速对悬空薄膜上进行加热循环的同时能够同时测温。In Example 2-5, in other examples, a temperature measuring device is provided on the heating device, such as in Examples 2-1 to 2-4, so that the heating process can be monitored in real time. Specifically, for example, for Example 2-2, a temperature measuring resistance wire can be used and directly integrated into the suspended membrane to form a heater that can both quickly heat and measure temperature in real time, and can quickly perform a heating cycle on the suspended membrane while measuring temperature at the same time.
其他实例中,可以采用其他方式采用加热时的测温装置,对于芯片加热循环进行实时温度监控。In other examples, other methods may be used to use a temperature measuring device during heating to perform real-time temperature monitoring of the chip heating cycle.
实例2-6Example 2-6
实例2-6中,在实例2-1至实例2-5的实例中,除了让加热的液滴的冷却自然冷却之外,快速核酸芯片上设置散热装置以进一步加快热循环,具体而言,悬空薄膜上可以附加额外散热装置,如热电制冷片,或者平面热管,或微流控管道流体对流散热。In Example 2-6, in the examples of Example 2-1 to Example 2-5, in addition to allowing the heated droplets to cool naturally, a heat sink is provided on the rapid nucleic acid chip to further accelerate the thermal cycle. Specifically, an additional heat sink can be attached to the suspended membrane, such as a thermoelectric cooler, or a planar heat pipe, or a microfluidic channel fluid convection heat dissipation.
实施例3:Embodiment 3:
本实施例中,我们提供一种更加具体的扩增结构或者快速核酸检测芯片结构,通过对该结构进行实验验证,能够实现对核酸快速的扩增。In this embodiment, we provide a more specific amplification structure or a rapid nucleic acid detection chip structure. By experimentally verifying the structure, rapid amplification of nucleic acids can be achieved.
如图8,在硅衬底上,化学镀膜(PECVD或LPCVD)制备氮化硅薄膜,薄膜上制备金属微加热器(Ti/Au, 或者Ti/Pt,或者Ti/Pd等),硅衬底湿法或干法刻蚀后在硅片表面形成悬空氮化硅(悬空薄膜1)即集成一个微加热器。如图9,微加热器上具有悬空薄膜1,和悬空薄膜集成的加热装置5,含有待测样本的液滴4,利用微加热器加热液4,并通过电阻测量实时探测液滴温度。这样就形成了一个带有悬空薄膜的微加热器。为防止液滴挥发,用氟油或硅油填充悬空薄膜下方的区域,并将待测液滴全部覆盖,形成液滴防挥发层4。As shown in Figure 8, a silicon nitride film is prepared by chemical plating (PECVD or LPCVD) on a silicon substrate, and a metal microheater (Ti/Au, or Ti/Pt, or Ti/Pd, etc.) is prepared on the film. After wet or dry etching of the silicon substrate, a suspended silicon nitride (suspended film 1) is formed on the surface of the silicon wafer, that is, a microheater is integrated. As shown in Figure 9, the microheater has a suspended film 1, and a heating device 5 integrated with the suspended film, and a droplet 4 of a sample to be tested is contained. The liquid 4 is heated by a microheater, and the temperature of the droplet is detected in real time by resistance measurement. In this way, a microheater with a suspended film is formed. In order to prevent the droplets from volatilizing, fluorine oil or silicone oil is used to fill the area below the suspended film, and the droplets to be tested are completely covered to form a droplet anti-evaporation layer 4.
更具体的,如图10,将带有悬空薄膜的微加热器放置于一带有凹槽的衬底中间,由加热器向衬底凹槽周边凸起边缘延伸支撑导电线,使得支撑导电线能够支撑起微加热器,并延伸至衬底凹槽周边凸起边缘形成加热器的连接端子。带有扩增样品的液滴置于中间悬空的氮化硅微加热器平台上,通过微加热器电极通电加热。微加热器的温度随加热功率变化而变化。因为悬空,加热时温度可快速变化。More specifically, as shown in FIG10 , a microheater with a suspended film is placed in the middle of a substrate with a groove, and a supporting conductive wire is extended from the heater to the raised edge of the substrate groove so that the supporting conductive wire can support the microheater and extend to the raised edge of the substrate groove to form a connecting terminal of the heater. A droplet with an amplified sample is placed on the suspended silicon nitride microheater platform in the middle and is heated by powering the microheater electrode. The temperature of the microheater changes with the heating power. Because it is suspended, the temperature can change rapidly during heating.
同样的,也可先将带有悬空薄膜的加热器直接覆盖于衬底上的凹槽,对悬空薄膜靠近衬底凹槽周边凸起的部分进行镂空,仅保留中间微加热器部分,通过微加热器电极通电加热。也可以实现效果,在其他实施例中,可以采用其他方式,只要保持微加热器或者悬空薄膜与衬底其他部分的隔离,实现悬空加热,即在本发明保护的范围内。本实施例中的微加热器结构可以采用MEMS加工技术,单个微加热器的成本在1-10元左右,采用MEMS加工技术,单个微加热器的尺寸在100微米*100微米到10毫米*10毫米之间。Similarly, the heater with the suspended film can be directly covered on the groove on the substrate, and the raised part of the suspended film near the periphery of the substrate groove can be hollowed out, leaving only the middle microheater part, and heating it by energizing the microheater electrode. The effect can also be achieved. In other embodiments, other methods can be used. As long as the microheater or the suspended film is isolated from other parts of the substrate, suspended heating is achieved, which is within the scope of protection of the present invention. The microheater structure in this embodiment can adopt MEMS processing technology. The cost of a single microheater is about 1-10 yuan. Using MEMS processing technology, the size of a single microheater is between 100 microns * 100 microns and 10 mm * 10 mm.
本实施例的结构,可以单独用于扩增形成一种扩增结构,也能够加入荧光标记或者探针实现快速的核酸检测,成为一种快速核酸检测芯片。The structure of this embodiment can be used alone for amplification to form an amplification structure, and can also be added with fluorescent markers or probes to achieve rapid nucleic acid detection, thereby becoming a rapid nucleic acid detection chip.
采用本实施例中的微加热器结构和微液滴核酸检测极大地减少试剂的用量,在极大的缩短检测时间的同时,将检测成本控制在极低水平,适合大规模推广,比如疫情防控中及时高效的核酸筛查;家庭用病原微生物快查等应用。The use of the micro-heater structure and micro-droplet nucleic acid detection in this embodiment greatly reduces the amount of reagents used, greatly shortens the detection time, and controls the detection cost at an extremely low level, which is suitable for large-scale promotion, such as timely and efficient nucleic acid screening in epidemic prevention and control; rapid detection of pathogenic microorganisms for household use, and other applications.
以下通过实验对本实施例中的结构进行实验验证,如图11所示,当电压变化时,悬空薄膜上的温度可快速达到平衡,在室温到400oC之间变化。如图12,采用本结构的微加热器,微加热器上的电压加到0.8伏时,悬空薄膜上的温度可达到105oC左右,并且从室温上升到105oC所需的时间仅为0.02秒。当微加热器上电压变为0时,悬空薄膜快速冷却,从105oC到室温仅需0.02秒。因此,悬空微加热器可以在0.04秒的时间内,完成一个周期的温度循环。PCR核酸检测需要在悬空微加热器上放置液滴。因液滴本身有热容,温度循环所需时间增长。如图13,是本实施例中悬空微加热器上放置液滴时的温度变化。液滴为PEG液滴,当微加热器电压为1.5伏时,从室温到120oC耗时0.1秒;当微加热器上电压为0伏时,从120oC冷却到室温耗时0.37秒,完成一个温度循环所需的时间为0.47秒。水性液滴在油性液体包覆下,可耐受至170oC左右不挥发、不沸腾,稳定存在。The structure of this embodiment is experimentally verified by experiments as follows. As shown in FIG11 , when the voltage changes, the temperature on the suspended film can quickly reach equilibrium and change between room temperature and 400°C. As shown in FIG12 , when the voltage on the microheater of this structure is added to 0.8 volts, the temperature on the suspended film can reach about 105°C, and the time required to rise from room temperature to 105°C is only 0.02 seconds. When the voltage on the microheater becomes 0, the suspended film cools rapidly, and it only takes 0.02 seconds to go from 105°C to room temperature. Therefore, the suspended microheater can complete a cycle of temperature cycling within 0.04 seconds. PCR nucleic acid testing requires placing droplets on the suspended microheater. Because the droplets themselves have heat capacity, the time required for the temperature cycle increases. As shown in FIG13 , this is the temperature change when droplets are placed on the suspended microheater in this embodiment. The droplets are PEG droplets. When the voltage of the microheater is 1.5 volts, it takes 0.1 seconds to cool from room temperature to 120°C. When the voltage on the microheater is 0 volts, it takes 0.37 seconds to cool from 120°C to room temperature. The time required to complete a temperature cycle is 0.47 seconds. When the aqueous droplets are coated with oily liquids, they can withstand temperatures of around 170°C without evaporation or boiling, and exist stably.
实施例4:Embodiment 4:
本实施例中,对于实施例3中的结构,采用便携式电池对该微加热器进行加热。由于微加热器加热液滴过程中,基本上只对液滴加热,无额外功耗,温度循环过程中的能量消耗降到最低程度,因此,采用电池对本发明实施例中的微加热器进行加热,是可行的。In this embodiment, for the structure in Embodiment 3, a portable battery is used to heat the micro heater. Since the micro heater basically only heats the droplets during the process of heating the droplets, without additional power consumption, the energy consumption during the temperature cycle is reduced to a minimum, therefore, it is feasible to use a battery to heat the micro heater in the embodiment of the present invention.
对于500纳升的待测液滴,完成一次60oC-95oC的温度循环的功耗仅为0.0735焦耳,特别适合于电池供电的便携式设备,非常适合在野外、现场开展检测。For a 500 nanoliter droplet to be tested, the power consumption to complete a 60oC-95oC temperature cycle is only 0.0735 joules, which is particularly suitable for battery-powered portable devices and is very suitable for testing in the field.
实施例5:Embodiment 5:
本实施例中,提供一种快速核酸检测装置或者快速扩增装置,包括若干个如前面所述的快速核酸检测芯片;每一快速核酸检测芯片中独立或者统一地利用加热装置对悬空薄膜上的液滴进行加热以实现温度的快速变化。具体而言,以实施例3中的微加热器快速核酸检测芯片而言,每个面积非常小,正常在1mm左右,因此微加热器可制成且非常适合制作成大规模阵列,比如2*2, 10*10, 100*100阵列,用于多个样本、多种核酸的高通量并行检测。In this embodiment, a rapid nucleic acid detection device or rapid amplification device is provided, including several rapid nucleic acid detection chips as described above; in each rapid nucleic acid detection chip, a heating device is used independently or uniformly to heat the droplets on the suspended film to achieve rapid temperature changes. Specifically, for the microheater rapid nucleic acid detection chip in Example 3, each area is very small, normally around 1 mm, so the microheater can be made and is very suitable for making into large-scale arrays, such as 2*2, 10*10, 100*100 arrays, for high-throughput parallel detection of multiple samples and multiple nucleic acids.
这里的微加热器阵列,可以集成在同一个具有一定面积的衬底上,也可以分别设置在不同衬底上统一集成。阵列中每个微加热器可以快速对一个液滴进行热循环操作。在大规模阵列中,每个微加热器可以加热同一种液滴,实现对一定体积量的液滴的检测,比如每个液滴100纳升,100微升的待测液体可以分成1000个液滴,放置在1000个微加热器上并行处理,确保稀少核酸拷贝不漏检。The microheater array here can be integrated on the same substrate with a certain area, or it can be set separately on different substrates for unified integration. Each microheater in the array can quickly perform thermal cycling operations on a droplet. In a large-scale array, each microheater can heat the same droplet to detect a certain volume of droplets. For example, each droplet is 100 nanoliters, and 100 microliters of the liquid to be tested can be divided into 1,000 droplets and placed on 1,000 microheaters for parallel processing to ensure that rare nucleic acid copies are not missed.
此外,本实施例中的快速核酸检测装置中,每个快速核酸检测芯片如微加热器,均可通过独立的通电,以进行独立加热。每个快速核酸检测芯片,也可以放置不同的检测样本和核酸检测时机,因此,每个在大规模微加热器阵列中,可以在不同微加热器上热循环不同的待测样本,实现高通量的样本检测;或者对同一个样本,采用不同的核酸检测试剂,实现多种核酸的同时检测。以上方式还可以混合集成,即对多个样本同时进行多种核酸的检测。In addition, in the rapid nucleic acid detection device of this embodiment, each rapid nucleic acid detection chip, such as a microheater, can be independently powered on for independent heating. Each rapid nucleic acid detection chip can also be placed with different test samples and nucleic acid detection opportunities. Therefore, in each large-scale microheater array, different samples to be tested can be thermally cycled on different microheaters to achieve high-throughput sample detection; or different nucleic acid detection reagents can be used for the same sample to achieve simultaneous detection of multiple nucleic acids. The above methods can also be mixed and integrated, that is, multiple nucleic acids can be detected simultaneously on multiple samples.
实施例6:Embodiment 6:
本实施例中,提供具体的扩增方法,包括:设置一薄膜悬空;In this embodiment, a specific amplification method is provided, including: setting a film suspended in the air;
将含有待扩增样本的液滴置于悬空薄膜上;Place a droplet containing the sample to be amplified on a suspended membrane;
周期性加热所述液滴,实现液滴在不同温度下的循环,实现扩增。The droplets are heated periodically to achieve circulation of the droplets at different temperatures to achieve amplification.
本实施例中采用以下的一种或者几种方法形成所述防挥发层:用表面活性剂在液滴表面自组装成膜形成防挥发层;在所述液滴表面覆盖一层疏水纳米颗粒形成防挥发层;在所述液滴上方加盖一层悬空薄膜将液滴封存在两层悬空薄膜之间形成防挥发层。In this embodiment, the anti-volatile layer is formed by one or more of the following methods: using a surfactant to self-assemble into a film on the surface of the droplet to form an anti-volatile layer; covering the surface of the droplet with a layer of hydrophobic nanoparticles to form an anti-volatile layer; adding a layer of suspended film above the droplet to seal the droplet between two layers of suspended film to form an anti-volatile layer.
本实施例中,可以采用以下一种或者几种加热方法实现液滴周期性加热:在悬空薄膜下方利用加热丝或者加热片进行加热;或者使用微波或者超声波探针插入所述液滴进行加热;将整个芯片放置的微波炉中,利用微波对所述液滴以及悬空薄膜无接触加热。In this embodiment, one or more of the following heating methods can be used to achieve periodic heating of the droplets: heating with a heating wire or a heating plate under the suspended film; or inserting a microwave or ultrasonic probe into the droplets for heating; placing the entire chip in a microwave oven and using microwaves to heat the droplets and the suspended film without contact.
扩增方法还包括以下步骤:在周期性加热所述液滴时,对液滴加热温度进行测量。本实施例中,可以采用测温电阻丝对所述悬空薄膜上的液滴进行加热测温。其他实施例中,也可采用其他的方式,在加热扩增时进行加热测温。The amplification method further includes the following steps: when the droplets are periodically heated, the heating temperature of the droplets is measured. In this embodiment, a temperature measuring resistance wire can be used to heat and measure the temperature of the droplets on the suspended membrane. In other embodiments, other methods can also be used to perform heating and temperature measurement during heating amplification.
具体的,实现周期性加热所述液滴,实现液滴在不同温度下的循环,实现扩增时,包括以下步骤:将所述测温电阻丝与所述悬空薄膜进行集成,形成一悬空微加热器,所述液滴置于所述悬空薄膜上表面;使所述防挥发层包覆所述微加热器上的液滴;对所述悬空微加热器施加不同电信号,实现周期性加热所述液滴,使得液滴在不同温度下进行循环,实现扩增。Specifically, to achieve periodic heating of the droplets, circulation of the droplets at different temperatures, and amplification, the following steps are included: integrating the temperature measuring resistance wire with the suspended film to form a suspended micro-heater, and placing the droplets on the upper surface of the suspended film; allowing the anti-volatile layer to cover the droplets on the micro-heater; applying different electrical signals to the suspended micro-heater to achieve periodic heating of the droplets, so that the droplets circulate at different temperatures and amplification is achieved.
本实施例中的扩增方法可以通过加入荧光标记或者荧光探针形成快速核酸检测芯片。The amplification method in this embodiment can form a rapid nucleic acid detection chip by adding fluorescent markers or fluorescent probes.
含荧光标记的扩增后液滴,置于荧光检测装置中,完成核酸的荧光检测;The amplified droplets containing fluorescent markers are placed in a fluorescence detection device to complete the fluorescence detection of nucleic acids;
根据扩增后液滴的荧光亮度,判定检测结果。The detection result is determined based on the fluorescence brightness of the droplets after amplification.
具体的,液滴内含有待测样本、扩增引物、酶、dNTP即脱氧核糖核苷三磷酸、模板、荧光探针和缓冲液。所述液滴外部设置有防挥发层。Specifically, the droplet contains a sample to be tested, an amplification primer, an enzyme, dNTP (deoxyribonucleoside triphosphate), a template, a fluorescent probe and a buffer. An anti-volatile layer is disposed outside the droplet.
以下通过一种具体的快速核酸检测芯片即微加热器来说明如何实现快速核酸检测:The following uses a specific rapid nucleic acid detection chip, namely a microheater, to illustrate how to achieve rapid nucleic acid detection:
将所述悬空微加热器置于一具有凹槽的衬底上,将所述悬空微加热器延伸出至少两根支撑导电线至所述衬底凹槽的凸起边缘,支撑所述微加热器在所述凹槽上方悬空;所述支撑导电线延伸至所述衬底凹槽的凸起边缘固定,形成微加热器连接端子;利用防挥发层,采用所述非挥发性疏水液膜作为所述防挥发层,所述非挥发性疏水液膜设置为氟油或硅油。将所述液滴以及所述悬空薄膜下方区域全部填充,使其能够将微加热器以及所述微加热器上的液滴全部包覆。The suspended microheater is placed on a substrate with a groove, and at least two supporting conductive wires of the suspended microheater are extended to the raised edge of the substrate groove to support the microheater to be suspended above the groove; the supporting conductive wires are extended to the raised edge of the substrate groove and fixed to form a microheater connection terminal; an anti-volatile layer is used, and the non-volatile hydrophobic liquid film is used as the anti-volatile layer, and the non-volatile hydrophobic liquid film is set to fluorine oil or silicone oil. The droplets and the area below the suspended film are completely filled so that the microheater and the droplets on the microheater can be completely covered.
最后,对所述悬空微加热器的所述微加热器连接端子施加不同电信号,实现周期性加热所述液滴,使得液滴在不同温度下进行循环,实现扩增。Finally, different electrical signals are applied to the microheater connection terminals of the suspended microheater to periodically heat the droplets, so that the droplets circulate at different temperatures to achieve amplification.
利用液滴中的荧光标记,对荧光进行检测,获得扩增结果。The fluorescence is detected by using the fluorescent markers in the droplets to obtain the amplification results.
本实施例以及前述的实施例中,所描述的悬空薄膜设置为氮化硅、氧化硅、碳膜、金刚石膜、parylene派瑞林(对二甲苯聚合物)膜、金属膜中的一种薄膜或者几种形成的复合薄膜。为了让悬空薄膜上的液滴张力不被破坏导致样本弥散,在所述悬空薄膜上镀上增强反射镀膜以增强荧光反射信号。In this embodiment and the aforementioned embodiments, the suspended membrane is configured as a thin film selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene (paraxylene polymer) film, metal film, or a composite film formed by the above. In order to prevent the droplet tension on the suspended membrane from being destroyed and causing sample diffusion, an enhanced reflection coating is applied on the suspended membrane to enhance the fluorescence reflection signal.
在以上方案的基础上,为了更好更快地实现加热循环,在周期性加热所述液滴之后,采用悬空薄膜上的额外散热装置对所述液滴进行散热。具体而言,在所述悬空薄膜上采用热电制冷片、平面热管或者微流控管道流体中的其中一种或者几种方式对所述液滴进行散热。On the basis of the above scheme, in order to achieve a better and faster heating cycle, after the droplets are periodically heated, an additional heat sink on the suspended membrane is used to dissipate heat from the droplets. Specifically, one or more of the following methods, including thermoelectric cooling sheets, planar heat pipes, or microfluidic channel fluids, are used on the suspended membrane to dissipate heat from the droplets.
本实施例中还可以将前述快速核酸检测方法以及本发明提供的快速核酸检测芯片如微加热器大规模应用,实现一种快速核酸大规模检测方法,其将同一样本或者不同样本分别分成多个液滴,利用前面所述的快速核酸检测方法同时对多个液滴进行检测。In this embodiment, the aforementioned rapid nucleic acid detection method and the rapid nucleic acid detection chip provided by the present invention, such as a microheater, can also be applied on a large scale to realize a rapid nucleic acid large-scale detection method, which divides the same sample or different samples into multiple droplets, and uses the aforementioned rapid nucleic acid detection method to simultaneously detect multiple droplets.
本发明的关键点是将微液滴(纳升到微升)置于悬空薄膜上,采用微加热器或微波加热,可实现0.5秒以内的温度循环(65oC-95oC),循环过程中液滴覆油以避免液滴挥发。本发明采用液滴原位加热和降温,通过悬空薄膜支撑,消除对衬底加热的需要,可实现最快速度的液滴升降温,无需驱动液滴,大大简化芯片设计和操作,确保易用性、可靠性。The key point of the present invention is to place micro droplets (nanoliter to microliter) on a suspended film, and use a micro heater or microwave heating to achieve a temperature cycle within 0.5 seconds (65oC-95oC). During the cycle, the droplets are covered with oil to prevent the droplets from volatilizing. The present invention uses in-situ heating and cooling of the droplets, and through the support of the suspended film, eliminates the need for substrate heating, and can achieve the fastest droplet heating and cooling without driving the droplets, greatly simplifying chip design and operation, ensuring ease of use and reliability.
工业实用性Industrial Applicability
以上所述仅为本发明的较佳实施例,凡依本发明权利要求范围所做的均等变化与修饰,皆应属本发明权利要求的涵盖范围。The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims (43)

  1. 一种扩增结构,其特征在于:包括悬空薄膜和加热装置,所述加热装置用于加热悬空薄膜上的液滴。An amplification structure, characterized in that it comprises a suspended membrane and a heating device, wherein the heating device is used to heat droplets on the suspended membrane.
  2. 如权利要求1所述的扩增结构,其特征在于:在悬空薄膜上的所述液滴上包裹有防挥发层。The amplification structure as described in claim 1 is characterized in that: the droplets on the suspended film are wrapped with an anti-volatile layer.
  3. 如权利要求2所述的扩增结构,其特征在于:所述防挥发层设置为非挥发性疏水液膜和/或疏水纳米颗粒层。The amplification structure according to claim 2, characterized in that the anti-volatile layer is configured as a non-volatile hydrophobic liquid film and/or a hydrophobic nanoparticle layer.
  4. 如权利要求3所述的扩增结构,其特征在于:当采用所述非挥发性疏水液膜作为所述防挥发层时,所述非挥发性疏水液膜的沸点高于所述液滴沸点。The amplification structure as described in claim 3 is characterized in that: when the non-volatile hydrophobic liquid film is used as the anti-volatile layer, the boiling point of the non-volatile hydrophobic liquid film is higher than the boiling point of the droplets.
  5. 如权利要求4所述的扩增结构,其特征在于:采用所述非挥发性疏水液膜作为所述防挥发层时,所述非挥发性疏水液膜设置为氟油或硅油。The amplification structure as described in claim 4 is characterized in that: when the non-volatile hydrophobic liquid film is used as the anti-volatility layer, the non-volatile hydrophobic liquid film is set to fluorine oil or silicone oil.
  6. 如权利要求2所述的扩增结构,其特征在于:所述防挥发层由表面活性剂在所述液滴表面自组装成膜形成。The amplification structure as described in claim 2 is characterized in that: the anti-volatile layer is formed by surfactant self-assembling into a film on the surface of the droplet.
  7. 如权利要求2所述的扩增结构,其特征在于:在悬空薄膜上的所述液滴上方加盖一层悬空薄膜使液滴封存在两层悬空薄膜之间以形成所述防挥发层。The amplification structure as claimed in claim 2 is characterized in that: a layer of suspended film is added above the droplets on the suspended film so that the droplets are sealed between two layers of suspended films to form the anti-volatile layer.
  8. 如权利要求1所述的扩增结构,其特征在于:所述加热装置设置为微波容器,所述悬空薄膜及液滴置于所述微波容器中,再利用微波容器对液滴进行加热。The amplification structure as described in claim 1 is characterized in that: the heating device is configured as a microwave container, the suspended film and the droplets are placed in the microwave container, and the microwave container is used to heat the droplets.
  9. 如权利要求1所述的扩增结构,其特征在于:所述加热装置设置为加热微针,通过所述加热微针插入悬空薄膜上的所述液滴进行加热。The amplification structure as described in claim 1 is characterized in that: the heating device is configured to heat the microneedle, and the droplet on the suspended film is heated by inserting the heating microneedle.
  10. 如权利要求9所述的扩增结构,其特征在于:所述加热微针设置为微波或者超声波探头微针。The amplification structure as described in claim 9 is characterized in that the heating microneedle is configured as a microwave or ultrasonic probe microneedle.
  11. 如权利要求1所述的扩增结构,其特征在于:所述加热装置设置为加热片或者加热丝,所述加热片或者所述加热丝设置在所述悬空薄膜下方进行加热。The amplification structure as described in claim 1 is characterized in that: the heating device is configured as a heating plate or a heating wire, and the heating plate or the heating wire is arranged under the suspended film for heating.
  12. 如权利要求1所述的扩增结构,其特征在于:所述加热装置上设置有测温装置。The amplification structure as described in claim 1 is characterized in that a temperature measuring device is provided on the heating device.
  13. 如权利要求11所述的扩增结构,其特征在于:所述加热片或者加热丝设置为加热及测温微电阻丝。The amplification structure as described in claim 11 is characterized in that: the heating plate or heating wire is configured as a heating and temperature measuring micro-resistance wire.
  14. 如权利要求11所述的扩增结构,其特征在于:所述悬空薄膜与所述加热丝或者加热片集成形成微加热器。The amplification structure as described in claim 11 is characterized in that the suspended membrane is integrated with the heating wire or the heating plate to form a micro heater.
  15. 如权利要求14所述的扩增结构,其特征在于:利用防挥发层将悬空薄膜上的液滴以及所述悬空薄膜下方区域全部填充,使其能够将微加热器以及所述微加热器上的液滴全部包覆。The amplification structure as described in claim 14 is characterized in that: the droplets on the suspended film and the area below the suspended film are completely filled with the anti-volatile layer, so that it can completely cover the microheater and the droplets on the microheater.
  16. 如权利要求14所述的扩增结构,其特征在于:所述微加热器悬空于衬底上方,所述微加热器上延伸出至少两根支撑导电线至所述衬底上固定并形成微加热器连接端子;所述支撑导电线支撑所述微加热器悬空时的平衡。The amplification structure as described in claim 14 is characterized in that: the microheater is suspended above the substrate, and at least two supporting conductive wires extend from the microheater to be fixed on the substrate and form microheater connection terminals; the supporting conductive wires support the balance of the microheater when it is suspended.
  17. 如权利要求16所述的扩增结构,其特征在于:所述衬底上设置有凹槽,所述微加热器设置在所述凹槽的上方,所述微加热器上延伸出至少两根支撑导电线至所述凹槽的凸起边缘,支撑所述微加热器在所述凹槽上方悬空;所述支撑导电线延伸至所述凹槽的凸起边缘固定,形成所述微加热器连接端子。The amplification structure as described in claim 16 is characterized in that: a groove is provided on the substrate, the microheater is arranged above the groove, at least two supporting conductive wires extend from the microheater to the raised edge of the groove, supporting the microheater to be suspended above the groove; the supporting conductive wires extend to the raised edge of the groove and are fixed to form the microheater connection terminal.
  18. 如权利要求17所述的扩增结构,其特征在于:从所述微加热器上延伸出四根支撑导电线至所述凹槽的凸起边缘固定并形成微加热器连接端子,所述四根支撑导电线支撑所述微加热器悬空时的平衡。The amplification structure as described in claim 17 is characterized in that: four supporting conductive wires extend from the microheater to the raised edge of the groove to be fixed and form a microheater connection terminal, and the four supporting conductive wires support the balance of the microheater when it is suspended in the air.
  19. 如权利要求16所述的扩增结构,其特征在于:对所述连接端子输入不同电信号,使得所述微加热器其实现温度的快速变化以热传导至液滴实现温度的快速变化。The amplification structure as described in claim 16 is characterized in that different electrical signals are input to the connecting terminals so that the microheater can achieve rapid temperature changes by conducting heat to the droplets to achieve rapid temperature changes.
  20. 如权利要求1所述的扩增结构,其特征在于:所述悬空薄膜上设置有疏水或者超疏水镀层。The amplification structure according to claim 1 is characterized in that a hydrophobic or super hydrophobic coating is provided on the suspended membrane.
  21. 如权利要求1所述的扩增结构,其特征在于:所述悬空薄膜设置为氮化硅、氧化硅、碳膜、金刚石膜、parylene膜、金属膜中的一种薄膜或者几种形成的复合薄膜。The amplification structure as described in claim 1 is characterized in that: the suspended film is set to be a film selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene film, metal film, or a composite film formed by several of them.
  22. 如权利要求1所述的扩增结构,其特征在于:还包括散热装置。The amplification structure as described in claim 1 is characterized in that it also includes a heat dissipation device.
  23. 如权利要求22所述的扩增结构,其特征在于:所述散热装置为设置于所述悬空薄膜上的热电制冷片、平面热管或者微流控管道流体中的其中一种或者几种。The amplification structure as described in claim 22 is characterized in that: the heat dissipation device is one or more of a thermoelectric cooling sheet, a planar heat pipe or a microfluidic pipeline fluid arranged on the suspended membrane.
  24. 一种快速核酸检测芯片,其特征在于:包括如权利要求1至23任一项所述的扩增结构。A rapid nucleic acid detection chip, characterized by comprising an amplification structure as described in any one of claims 1 to 23.
  25. 如权利要求24所述的快速核酸检测芯片,其特征在于:所述悬空薄膜上设置有增强反射镀膜。The rapid nucleic acid detection chip as described in claim 24 is characterized in that an enhanced reflection coating is provided on the suspended membrane.
  26. 一种快速核酸检测装置,其特征在于:包括若干个如权利要求24或 25所述的快速核酸检测芯片;每一快速核酸检测芯片中独立或者统一地利用加热装置对悬空薄膜上的液滴进行加热以实现温度的快速变化。A rapid nucleic acid detection device, characterized in that it comprises a plurality of rapid nucleic acid detection chips as described in claim 24 or 25; each rapid nucleic acid detection chip independently or uniformly utilizes a heating device to heat the droplets on the suspended film to achieve rapid temperature changes.
  27. 一种扩增方法,其特征在于:包括以下步骤:An amplification method, characterized in that it comprises the following steps:
    设置一薄膜悬空;A film is set to be suspended in the air;
    将含有扩增样本的液滴置于悬空薄膜上;A droplet containing the amplified sample is placed on a suspended membrane;
    周期性加热所述液滴,实现液滴在不同温度下的循环,实现扩增。The droplets are heated periodically to achieve circulation of the droplets at different temperatures to achieve amplification.
  28. 如权利要求27所述的扩增方法,其特征是:所述液滴外部设置有防挥发层。The amplification method as described in claim 27 is characterized in that an anti-volatile layer is provided on the outside of the droplet.
  29. 如权利要求27所述的扩增方法,其特征是:采用以下的一种或者几种方法形成防挥发层:The amplification method according to claim 27, characterized in that the anti-volatile layer is formed by one or more of the following methods:
    用表面活性剂在液滴表面自组装成膜形成防挥发层;Using surfactant to self-assemble into a film on the surface of the droplet to form an anti-volatile layer;
    在所述液滴表面覆盖一层疏水纳米颗粒形成防挥发层;Covering the surface of the droplet with a layer of hydrophobic nanoparticles to form an anti-volatile layer;
    在所述液滴上方加盖一层悬空薄膜将液滴封存在两层悬空薄膜之间形成防挥发层。A layer of suspended film is added above the liquid droplets to seal the liquid droplets between two layers of suspended films to form an anti-volatile layer.
  30. 如权利要求27所述的扩增方法,其特征在于:采用以下一种或者几种加热方法实现液滴周期性加热:The amplification method according to claim 27, characterized in that: periodic heating of the droplets is achieved by using one or more of the following heating methods:
    在悬空薄膜下方利用加热丝或者加热片进行加热;Heating is performed by using a heating wire or a heating sheet under the suspended membrane;
    或者使用微波或者超声波探针插入所述液滴进行加热;Alternatively, a microwave or ultrasonic probe is inserted into the droplet for heating;
    将整个芯片放置的微波炉中,利用微波对所述液滴以及悬空薄膜无接触加热。The entire chip is placed in a microwave oven, and microwaves are used to heat the droplets and the suspended film without contact.
  31. 如权利要求27所述的扩增方法,其特征在于:在周期性加热所述液滴时,对液滴加热温度进行测量。The amplification method as described in claim 27 is characterized in that: when the droplets are periodically heated, the heating temperature of the droplets is measured.
  32. 如权利要求31所述的扩增方法,其特征在于:采用测温电阻丝对所述悬空薄膜上的液滴进行加热测温。The amplification method as described in claim 31 is characterized in that: a temperature measuring resistance wire is used to heat and measure the temperature of the droplets on the suspended film.
  33. 如权利要求32所述的扩增方法,其特征在于:实现周期性加热所述液滴,实现液滴在不同温度下的循环,实现扩增时,包括以下步骤:The amplification method according to claim 32, characterized in that: periodically heating the droplets to achieve circulation of the droplets at different temperatures, and when achieving amplification, comprising the following steps:
    将所述测温电阻丝与所述悬空薄膜进行集成,形成一悬空微加热器,所述液滴置于所述悬空薄膜上表面;Integrate the temperature measuring resistance wire with the suspended membrane to form a suspended micro heater, and place the liquid droplet on the upper surface of the suspended membrane;
    使防挥发层包覆所述微加热器上的液滴;Allowing the anti-volatile layer to cover the droplets on the micro-heater;
    对所述悬空微加热器施加不同电信号,实现周期性加热所述液滴,使得液滴在不同温度下进行循环,实现扩增。Different electrical signals are applied to the suspended micro-heater to periodically heat the droplets, so that the droplets circulate at different temperatures to achieve amplification.
  34. 如权利要求33所述的扩增方法,其特征在于:还包括以下步骤:The amplification method according to claim 33, characterized in that it also includes the following steps:
    将所述悬空微加热器置于一具有凹槽的衬底上,将所述悬空微加热器延伸出至少两根支撑导电线至所述衬底凹槽的凸起边缘,支撑所述微加热器在所述凹槽上方悬空;所述支撑导电线延伸至所述衬底凹槽的凸起边缘固定,形成微加热器连接端子;The suspended microheater is placed on a substrate having a groove, and at least two supporting conductive wires are extended from the suspended microheater to the raised edge of the groove of the substrate to support the microheater to be suspended above the groove; the supporting conductive wires are extended to the raised edge of the groove of the substrate and fixed to form a microheater connection terminal;
    利用防挥发层将所述液滴以及所述悬空薄膜下方区域全部填充,使其能够将微加热器以及所述微加热器上的液滴全部包覆;The anti-volatile layer is used to fill the liquid droplets and the area below the suspended film so that the anti-volatile layer can completely cover the micro-heater and the liquid droplets on the micro-heater;
    对所述悬空微加热器的所述微加热器连接端子施加不同电信号,实现周期性加热所述液滴,使得液滴在不同温度下进行循环,实现扩增。Different electrical signals are applied to the microheater connection terminals of the suspended microheater to periodically heat the droplets, so that the droplets circulate at different temperatures to achieve amplification.
  35. 如权利要求34所述的扩增方法,其特征在于:采用非挥发性疏水液膜作为所述防挥发层,所述非挥发性疏水液膜设置为氟油或硅油。The amplification method as described in claim 34 is characterized in that a non-volatile hydrophobic liquid film is used as the anti-volatility layer, and the non-volatile hydrophobic liquid film is set to fluorine oil or silicone oil.
  36. 如权利要求27所述的扩增方法,其特征在于:所述悬空薄膜设置为氮化硅、氧化硅、碳膜、金刚石膜、parylene膜、金属膜中的一种薄膜或者几种形成的复合薄膜。The amplification method as described in claim 27 is characterized in that: the suspended film is set to be a film selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene film, metal film, or a composite film formed by several of them.
  37. 如权利要求27所述的扩增方法,其特征在于:The amplification method according to claim 27, characterized in that:
    在周期性加热所述液滴之后,采用悬空薄膜上的额外散热装置对所述液滴进行散热。After the droplets are periodically heated, an additional heat sink on the suspended membrane is used to dissipate the heat from the droplets.
  38. 如权利要求27所述的扩增方法,其特征在于:在所述悬空薄膜上采用热电制冷片、平面热管或者微流控管道流体中的其中一种或者几种方式对所述液滴进行散热。The amplification method as described in claim 27 is characterized in that: one or more of the following methods are used on the suspended membrane to dissipate heat for the droplets: a thermoelectric cooler, a planar heat pipe, or a microfluidic channel fluid.
  39. 一种快速核酸检测方法,其特征是:使用如权利要求27至38任一项所述扩增方法进行待测样本扩增。A rapid nucleic acid detection method, characterized in that: the sample to be tested is amplified using the amplification method as described in any one of claims 27 to 38.
  40. 如权利要求39所述的快速核酸检测方法,其特征是:还包括以下步骤The rapid nucleic acid detection method according to claim 39 is characterized in that: it also includes the following steps
    扩增前在所述液滴中加入荧光标记;adding a fluorescent marker to the droplet before amplification;
    扩增后将含荧光标记的扩增后液滴,置于荧光检测装置中,完成核酸的荧光检测;After amplification, the amplified droplets containing fluorescent markers are placed in a fluorescence detection device to complete the fluorescence detection of nucleic acids;
    根据扩增后液滴的荧光亮度,判定检测结果。The detection result is determined based on the fluorescence brightness of the droplets after amplification.
  41. 如权利要求40所述的快速核酸检测方法,其特征在于:所述液滴内含有待测样本、扩增引物、酶、dNTP脱氧核糖核苷三磷酸、模板、荧光探针和缓冲液。The rapid nucleic acid detection method as described in claim 40 is characterized in that the droplet contains a sample to be tested, an amplification primer, an enzyme, a dNTP deoxyribonucleoside triphosphate, a template, a fluorescent probe and a buffer.
  42. 如权利要求40所述的快速核酸检测方法,其特征在于:在所述悬空薄膜上镀上增强反射镀膜以增强荧光反射信号。The rapid nucleic acid detection method as described in claim 40 is characterized in that: an enhanced reflection coating is coated on the suspended film to enhance the fluorescent reflection signal.
  43. 一种快速核酸大规模检测方法,其特征在于:将同一样本或者不同样本分别分成多个液滴,利用如权利要求39至42任一项所述的快速核酸检测方法同时对多个液滴进行检测。A rapid large-scale nucleic acid detection method, characterized in that the same sample or different samples are divided into multiple droplets, and the multiple droplets are detected simultaneously using the rapid nucleic acid detection method as described in any one of claims 39 to 42.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101917784A (en) * 2010-09-10 2010-12-15 中国科学院上海微系统与信息技术研究所 Three-dimensional micro heater with groove-shaped heating film region and manufacturing method thereof
CN107754962A (en) * 2017-11-22 2018-03-06 南方科技大学 A kind of Digital Microfluidic droplet drive device and driving method
US20200376493A1 (en) * 2017-04-21 2020-12-03 Essenlix Corporation Molecular manipulation and assay with controlled temperature (ii)
CN115449471A (en) * 2022-11-10 2022-12-09 南方科技大学 Amplification structure, rapid nucleic acid detection chip, device and method

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014008518A1 (en) * 2012-07-10 2014-01-16 Lexogen Gmbh Flexible dna sensor carrier and method
CA3009080C (en) * 2016-03-10 2023-10-10 Pioneer Hi-Bred International, Inc. Light-mediated polymerase chain reaction amplification and product detection system and methods of use
US10543466B2 (en) * 2016-06-29 2020-01-28 Digital Biosystems High resolution temperature profile creation in a digital microfluidic device
AU2020336448A1 (en) * 2019-08-27 2022-03-24 Volta Labs, Inc. Methods and systems for droplet manipulation
US11660602B2 (en) * 2019-08-28 2023-05-30 Mgi Holdings Co., Limited Temperature control on digital microfluidics device

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101917784A (en) * 2010-09-10 2010-12-15 中国科学院上海微系统与信息技术研究所 Three-dimensional micro heater with groove-shaped heating film region and manufacturing method thereof
US20200376493A1 (en) * 2017-04-21 2020-12-03 Essenlix Corporation Molecular manipulation and assay with controlled temperature (ii)
CN107754962A (en) * 2017-11-22 2018-03-06 南方科技大学 A kind of Digital Microfluidic droplet drive device and driving method
CN115449471A (en) * 2022-11-10 2022-12-09 南方科技大学 Amplification structure, rapid nucleic acid detection chip, device and method
CN116716180A (en) * 2022-11-10 2023-09-08 南方科技大学 Microwave heating amplification structure, rapid nucleic acid detection chip, device and method
CN116731841A (en) * 2022-11-10 2023-09-12 南方科技大学 Microneedle heating amplification structure, rapid nucleic acid detection chip, device and method

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