WO2024099079A1

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

Info

Publication number: WO2024099079A1
Application number: PCT/CN2023/126623
Authority: WO
Inventors: 程鑫; 刘红均; 刘荣跃; 陈日飞; 林国洪
Original assignee: 南方科技大学
Priority date: 2022-11-10
Filing date: 2023-10-26
Publication date: 2024-05-16
Also published as: CN115449471A; CN115449471B; CN116716180A; CN116731841A

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

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

Technical Field

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.

Background technique

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.

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).

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.

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.

technical problem

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.

Technical Solutions

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.

Preferably, the liquid droplets on the suspended membrane are coated with an anti-volatile layer.

Preferably, the anti-volatile layer is configured as a non-volatile hydrophobic liquid film and/or a hydrophobic nanoparticle layer.

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.

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.

Preferably, the anti-volatile layer is formed by self-assembly of a surfactant on the surface of the droplet to form a film.

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.

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.

Preferably, the heating device is configured to heat the microneedles, and the droplets on the suspended membrane are heated by inserting the heating microneedles.

Preferably, the heating microneedle is configured as a microwave or ultrasonic probe microneedle.

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.

Preferably, the heating device is provided with a temperature measuring device.

Preferably, the heating plate or heating wire is configured as a heating and temperature measuring micro-resistance wire.

Preferably, the suspended membrane is integrated with the heating wire or heating sheet to form a micro heater.

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.

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.

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.

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.

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.

Preferably, a hydrophobic or super-hydrophobic coating is provided on the suspended membrane.

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.

Preferably, a heat dissipation device is also included.

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.

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.

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.

In addition, 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;

Periodically heating the droplets to achieve circulation of the droplets at different temperatures to achieve amplification;

Preferably, an anti-volatile layer is disposed outside the droplet.

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.

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.

Preferably, when the droplets are periodically heated, the heating temperature of the droplets is measured.

Preferably, a temperature measuring resistance wire is used to heat and measure the temperature of the droplets on the suspended membrane.

Preferably, 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.

Preferably, 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.

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.

Preferably, after the droplets are periodically heated, an additional heat sink on the suspended membrane is used to dissipate the heat from the droplets.

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.

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.

The rapid nucleic acid detection method preferably further comprises the following steps

adding a fluorescent marker to the droplet before amplification;

After amplification, 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.

Beneficial Effects

(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) 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) 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) 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) 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) 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.

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) 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) 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.

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.

BRIEF DESCRIPTION OF THE DRAWINGS

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;

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;

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;

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;

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;

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.

Embodiments of the present invention

The present invention will be described in detail below through specific embodiments.

实施例1：Embodiment 1:

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.

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.

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.

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.

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.

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.

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.

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:

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:

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. 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.

Example 2-3

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.

Example 2-4

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.

Example 2-5

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.

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.

Example 2-6

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:

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.

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.

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.

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.

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.

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.

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:

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.

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:

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.

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.

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:

In this embodiment, a specific amplification method is provided, including: setting a film suspended in the air;

Place a droplet containing the sample to be amplified on a suspended membrane;

The droplets are heated periodically to achieve circulation of the droplets at different temperatures to achieve amplification.

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.

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.

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.

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.

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;

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.

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.

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.

The fluorescence is detected by using the fluorescent markers in the droplets to obtain the amplification results.

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.

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.

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.

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.

Industrial Applicability

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

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.
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.
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.
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.
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.
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.
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.
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.
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.
The amplification structure as described in claim 9 is characterized in that the heating microneedle is configured as a microwave or ultrasonic probe microneedle.
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.
The amplification structure as described in claim 1 is characterized in that a temperature measuring device is provided on the heating device.
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.
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.
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.
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.
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.
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.
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.
The amplification structure according to claim 1 is characterized in that a hydrophobic or super hydrophobic coating is provided on the suspended membrane.
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.
The amplification structure as described in claim 1 is characterized in that it also includes a heat dissipation device.
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.
A rapid nucleic acid detection chip, characterized by comprising an amplification structure as described in any one of claims 1 to 23.
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.
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.
An amplification method, characterized in that it comprises 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;

The droplets are heated periodically to achieve circulation of the droplets at different temperatures to achieve amplification.
The amplification method as described in claim 27 is characterized in that an anti-volatile layer is provided on the outside of the droplet.
The amplification method according to claim 27, characterized in that the anti-volatile layer is formed by one or more of the following methods:

Using 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;

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.
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:

Heating is performed by using a heating wire or a heating sheet under the suspended membrane;

Alternatively, a microwave or ultrasonic probe is inserted into the droplet for heating;

The entire chip is placed in a microwave oven, and microwaves are used to heat the droplets and the suspended film without contact.
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.
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.
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:

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;

Allowing the anti-volatile layer to cover the droplets on the micro-heater;

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.
The amplification method according to claim 33, characterized in that it also includes the following steps:

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;

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;

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 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.
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.
The amplification method according to claim 27, characterized in that:

After the droplets are periodically heated, an additional heat sink on the suspended membrane is used to dissipate the heat from the droplets.
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.
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.
The rapid nucleic acid detection method according to claim 39 is characterized in that: it also includes the following steps

adding a fluorescent marker to the droplet before amplification;

After amplification, 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 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.
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.
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.