CN115449471B

CN115449471B - Amplification structure, rapid nucleic acid detection chip, device and method

Info

Publication number: CN115449471B
Application number: CN202211406708.8A
Authority: CN
Inventors: 程鑫; 刘红均; 刘荣跃; 陈日飞; 林国洪
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology
Priority date: 2022-11-10
Filing date: 2022-11-10
Publication date: 2023-06-02
Anticipated expiration: 2042-11-10
Also published as: CN116731841A; CN116716180A; WO2024099079A1; CN115449471A

Abstract

The invention discloses an amplification structure, a rapid nucleic acid detection chip comprising the amplification structure, a device comprising the rapid nucleic acid detection chip, and a corresponding amplification method, a rapid nucleic acid detection method and a large-scale nucleic acid detection method. The amplification structure comprises a suspended film and a heating device, wherein the heating device is used for heating liquid drops on the suspended film. According to the micro-droplet-based rapid nucleic acid detection chip provided by the invention, the amplification of the sample to be detected in the heated droplet can be rapidly realized through the arrangement of the suspended film and the droplet.

Description

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

Technical Field

The invention belongs to the field of molecular amplification diagnosis in molecular biology, and particularly relates to an amplification structure, a rapid nucleic acid detection chip, a rapid nucleic acid detection device and a rapid nucleic acid detection method.

Background

The polymerase chain reaction (Polymerase chain reaction-PCR) can be used for nucleic acid replication and amplification, and the nucleic acid replication and amplification has a plurality of applications, such as being used for nucleic acid detection, which is an important field in biomolecule detection, and can realize accurate and quantitative detection of ultra-trace (even single nucleic acid molecule) nucleic acid through PCR amplification. PCR nucleic acid detection has wide application, including clinical disease diagnosis (such as diagnosis and efficacy evaluation of various infectious pathogenic microorganisms, prenatal and postnatal care 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 disease, bacillus anthracis, etc.), food safety detection (such as food-borne microorganisms, food allergens, transgenic foods, etc.), scientific research (such as quantitative research of related molecular biology of medicine, life sciences, agriculture and animal husbandry, etc.), etc. The application industries of the PCR nucleic acid quantitative detection technology comprise medical institutions, scientific research institutions, universities, disease control centers, inspection and quarantine offices, food enterprises, livestock enterprises and the like.

The Polymerase Chain Reaction (PCR) is the most common molecular biological technology for amplifying specific DNA fragments, and has the greatest characteristic of greatly increasing trace DNA fragments, combining with fluorescent probes, being applicable to detection of trace specific nucleic acid fragments, and being commonly used for detection of infectious pathogenic microorganisms, tumor analysis and genetic disease diagnosis. PCR has important application in detecting and diagnosing infectious diseases, and is a necessary technology for coping with large-scale colony epidemic situation (such as new coronavirus).

The conventional PCR amplification technology needs to be 95 percent ^o C-65 ^o C-72 ^o C, temperature circulation is carried out, and as a certain time is needed for temperature rise and temperature fall, the time of single circulation is needed to be 5-30 minutes. Typically, PCR is performed in 20-40 cycles, resulting in a PCR assay that takes several hours and is performed in a qualified detection institution laboratory. PCR detection has poor timeliness and needs professional detection unit operation, so that the PCR detection has a certain limit on the role of large-scale infectious epidemic prevention and control.

Specifically, in the existing PCR amplification technology, when temperature circulation is performed, besides temperature control of the liquid to be detected, a temperature control system inevitably heats sample supporting structures such as a substrate, so that a relatively long time is required for heating and cooling. Meanwhile, based on a milliliter-level liquid sample, when the concentration of the nucleic acid to be detected is relatively low, a certain waiting time is needed to enable the reaction to be sufficient. These factors result in a PCR cycle requiring about 5-30 minutes. Because of the multiple PCR cycles (20-40), the total time required for detection is relatively long (half an hour to several hours) and results cannot be obtained in real time (within minutes). The temperature cycling mode in PCR is commonly known as follows: (1) The thermoelectric chip is used for heating and refrigerating the liquid drops to realize temperature circulation, and the general temperature circulation needs more than 1 minute; (2) The liquid drop and the substrate are heated by adopting a traditional heating mode, the liquid drop and the substrate are naturally and cold, and the common temperature cycle needs about 5 minutes to 20 minutes; (3) Maintaining two high and low temperature regions on the substrate, circulating liquid back and forth between high and low temperature by microfluidic pipeline or droplet drive, wherein the temperature circulation is generally dependent on the speed of droplet drive, and the time can be Controlled within a few seconds. However, this approach requires precise manipulation of the droplets, is difficult to design and difficult to design, consumes relatively much power to maintain the two temperature zones, and has a droplet size of 95 ^o And C is very easy to generate bubbles when moving at high temperature, and brings great inconvenience to chip design and droplet control.

Disclosure of Invention

In view of the above-mentioned drawbacks 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.

Based on the technical problems, the invention provides an amplification structure which comprises a suspended film and a heating device, wherein the heating device is used for heating liquid drops on the suspended film.

Preferably, the liquid drop on the suspended film is wrapped with an anti-volatilization layer.

Preferably, the volatilization preventing layer is a non-volatile hydrophobic liquid film and/or a hydrophobic nano particle layer.

Preferably, when the nonvolatile hydrophobic liquid film is used as the volatilization preventing layer, the boiling point of the nonvolatile hydrophobic liquid film is higher than the boiling point of the liquid droplets.

Preferably, when the nonvolatile hydrophobic liquid film is used as the volatilization preventing layer, the nonvolatile hydrophobic liquid film is set as fluorine oil or silicone oil.

Preferably, the volatilization preventing layer is formed by self-assembling a surfactant on the surface of the liquid drop to form the volatilization preventing layer.

Preferably, a layer of suspended film is covered on the droplets on the suspended film, so that the droplets are sealed between the two layers of suspended films to form the anti-volatilization layer.

Preferably, the heating device is a microwave container, the suspended film and the liquid drops are placed in the microwave container, and the microwave container is used for heating the liquid drops.

Preferably, the heating device is arranged to heat the micro-needles, and the liquid drops on the suspended film are inserted through the heating micro-needles to heat.

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

Preferably, the heating device is a heating plate or a heating wire, and the heating plate or the heating wire is arranged below the suspended film for heating.

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

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

Preferably, the suspended film and the heating wire or the heating sheet are integrated to form a micro heater.

Preferably, the liquid drops on the suspended film and the area below the suspended film are fully filled by the anti-volatilization layer, so that the micro heater and the liquid drops on the micro heater can be fully coated.

Preferably, the micro-heater is suspended above the substrate, and at least two supporting conductive wires extend out of the micro-heater to be fixed on the substrate and form a micro-heater connecting terminal; the supporting conductive wire supports balance of the micro heater when suspended.

Preferably, a groove is formed in the substrate, the micro heater is arranged above the groove, and at least two supporting conductive wires extend out of the micro heater to the protruding edge of the groove to support the micro heater to hang above the groove; the supporting conductive wire extends to the protruding edge of the groove to be fixed, and the micro-heater connecting terminal is formed.

Preferably, four supporting conductive wires extend from the micro-heater to the protruding edges of the grooves to fix and form micro-heater connecting terminals, and the four supporting conductive wires support balance of the micro-heater when suspended.

Preferably, different electrical signals are input to the connection terminals, so that the micro-heater achieves rapid changes in temperature by heat conduction to the liquid droplets.

Preferably, the suspended film is provided with a hydrophobic or super-hydrophobic coating.

Preferably, the suspended film is one film or a composite film formed by a plurality of films selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene (p-xylene polymer) film and metal film.

Preferably, the heat radiator is further included.

Preferably, the heat dissipation device is one or more of thermoelectric cooling sheets, planar heat pipes or microfluidic pipeline fluid arranged on the suspended film.

The invention also provides a rapid nucleic acid detection chip comprising the amplification structure, which comprises the amplification structure. Preferably, the suspended film is provided with a reflection enhancing coating.

The invention also provides a rapid nucleic acid detection array chip, which comprises the rapid nucleic acid detection chip; the droplets are heated independently or in unison with each other in each rapid nucleic acid detection chip by a heating device to achieve rapid changes in temperature.

In addition, the invention also provides an amplification method: the method comprises the following steps:

setting a film in suspension;

placing a droplet containing an amplified sample on a suspended membrane;

the liquid drops are heated periodically, so that the circulation of the liquid drops at different temperatures is realized, and the amplification is realized;

Preferably, an anti-volatilization layer is arranged outside the liquid drop.

Preferably, the volatilization preventing layer is formed by one or more of the following methods: forming a volatilization-preventing layer by self-assembly of a surfactant on the surface of the liquid drop; covering a layer of hydrophobic nano particles on the surface of the liquid drop to form an anti-volatilization layer; and covering a layer of suspended film above the liquid drop to seal the liquid drop between the two layers of suspended films to form an anti-volatilization layer.

Preferably, the droplet periodic heating is realized by adopting one or more of the following heating methods: heating the lower part of the suspended film by using a heating wire or a heating sheet; or heating by inserting a microwave or ultrasonic probe into the droplet; and in a microwave oven in which the whole chip is placed, the droplets and the suspended film are heated in a non-contact manner by utilizing microwaves.

Preferably, the drop heating temperature is measured while periodically heating the drop.

Preferably, a temperature measuring resistance wire is used for heating and measuring the temperature of the liquid drop on the suspended film.

Preferably, the method for realizing the periodic heating of the liquid drops, realizing the circulation of the liquid drops at different temperatures and realizing the amplification comprises the following steps:

integrating the temperature measuring resistance wire with the suspended film to form a suspended micro-heater, wherein the liquid drop is arranged on the upper surface of the suspended film; coating the liquid drops on the micro heater with the anti-volatilization layer; and applying different electric signals to the suspended micro-heater to periodically heat the liquid drops, so that the liquid drops circulate at different temperatures to realize amplification.

Preferably, the rapid nucleic acid detection method further comprises the following steps:

placing the suspended micro-heater on a substrate with a groove, extending the suspended micro-heater out of at least two supporting conductive wires to the raised edge of the groove of the substrate, and supporting the micro-heater to be suspended above the groove; the supporting conductive wire extends to the protruding edge of the substrate groove to be fixed, so that a micro-heater connecting terminal is formed; the liquid drops and the area below the suspended film are filled completely by using an anti-volatilization layer, so that the micro heater and the liquid drops on the micro heater can be completely coated;

and applying different electrical signals to the micro-heater connecting terminals of the suspended micro-heater to periodically heat the liquid drops, so that the liquid drops circulate at different temperatures to realize amplification.

Preferably, the nonvolatile hydrophobic liquid film is used as the volatilization preventing layer, and the nonvolatile hydrophobic liquid film is set as fluorine oil or silicone oil.

Preferably, after the droplets are periodically heated, an additional heat dissipation device on the suspended film is used to dissipate heat from the droplets.

Preferably, the suspended film is used for radiating the liquid drop by adopting one or more modes of a hot-spot refrigerating sheet, a planar heat pipe or a microfluidic pipeline fluid.

Based on the amplification method, the invention also provides a rapid nucleic acid detection method, and the amplification method is used for amplifying the sample to be detected.

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

adding fluorescent markers into the liquid drops before amplification;

after amplification, the amplified liquid drop containing the fluorescent label is placed in a fluorescent detection device to finish fluorescent detection of nucleic acid;

and judging the detection result according to the fluorescence brightness of the amplified liquid drop.

The rapid nucleic acid detection method is preferable, wherein the liquid drop contains a sample to be detected, an amplification primer, enzyme, dNTP deoxyribonucleoside triphosphate, a template, a fluorescent probe and a buffer solution.

The rapid nucleic acid detection method is preferable to plate a reflection enhancing coating on the suspended film to enhance fluorescent reflection signals.

The invention also provides a rapid nucleic acid large-scale detection method, which is characterized in that the same sample or different samples are respectively divided into a plurality of liquid drops, and the plurality of liquid drops are detected simultaneously by using the rapid nucleic acid detection method.

The beneficial effects of the invention are as follows: (1) The invention provides an amplification structure and a corresponding rapid nucleic acid detection chip, and the amplification of a sample to be amplified in a heating liquid drop can be rapidly realized through the arrangement of a suspended film and the liquid drop. The liquid drop in-situ heating and cooling are adopted, the requirement on substrate heating is eliminated through the suspension film support, the liquid drop heating and cooling with the fastest speed can be realized, the liquid drop is not required to be driven, the chip design and operation are greatly simplified, and the usability and reliability are ensured. Meanwhile, the invention adopts micro liquid drops as a reaction container, so that the mass transfer and heat transfer speed is high, and the nucleic acid amplification reaction is fast; meanwhile, the liquid drops are placed on the suspended film, the unavoidable substrate heating in the temperature circulation process is reduced, the heating and cooling speeds are completed within 0.5 seconds, the time required by the existing PCR amplification temperature circulation is within 1-5 minutes, and the speed of the temperature circulation is increased by more than 100 times. Because the nucleic acid detection needs more than 20-30 times of PCR amplification, the total time of the nucleic acid detection can be shortened to within 1 minute from 30 minutes to several hours in the traditional method, the time required for the detection is greatly shortened, and the timeliness of the nucleic acid detection is greatly improved.

(2) The device has simple structure, each amplification structure and the rapid nucleic acid detection chip only detect single liquid drops, the single liquid drop detection capability is strong, and the detection cost is low: meanwhile, under the specific application scene, the cost of a single micro heater can be controlled to be about 1-10 yuan by adopting the MEMS processing technology, the dosage of reagents is greatly reduced by adopting micro liquid drops, the detection time is greatly shortened, and meanwhile, the detection cost is controlled to be extremely low, so that the method is suitable for large-scale popularization, such as timely and efficient nucleic acid screening in epidemic prevention and control; and (5) quick detection of pathogenic microorganisms for household use and the like.

(3) According to the amplification structure and the rapid nucleic acid detection chip provided by the invention, when amplification is carried out in liquid drops, the anti-volatilization layer is arranged on the liquid drops, so that aerosol pollution can be effectively prevented from being generated during nucleic acid amplification, and meanwhile, the amplification effect is ensured. Of course, the anti-volatilization layer can be formed in other ways, such as by self-assembling a film on the surface of the liquid drop by using an amphiphilic surfactant to reduce the volatilization of the liquid drop, or adding a high-boiling compatible solvent (such as glycol or polyethylene glycol) into the liquid drop, or covering a layer of hydrophobic nano particles on the surface of the liquid drop to form a liquid mark (liquid drop wrapped by a solid surface), or covering a layer of suspension film above the liquid drop to seal the liquid drop between two layers of suspension films, and the like, so as to prevent the volatilization of the liquid drop in the heating process. Because the cost of the chip and the reagent is low, the positive sample is sealed in the volatilization-proof layer such as oil drop after the amplification detection is finished for one-time use, and the pollution of the amplified nucleic acid molecules to the equipment can be effectively prevented.

(4) In the invention, a plurality of adaptive amplification heating devices aiming at the amplification structure and the rapid nucleic acid detection chip are also provided, and micro-heating resistance wires prepared by micro-nano processing are arranged on the suspended film; or heating by inserting a microwave (or ultrasonic) probe into the droplet. If all the liquid drops circulate in the same temperature range, the whole chip can be placed in a microwave oven, and the microwave can be utilized to heat all the liquid drops at the same time in a non-contact way.

(5) In order to enable the invention to rapidly realize the heating-cooling-heating cycle, the invention particularly provides that a cooling device, namely an additional heat dissipation device, such as a thermoelectric refrigeration sheet, or a planar heat pipe, or a microfluidic pipeline can be additionally arranged on the suspended film for fluid convection heat dissipation. The benefit of this arrangement is that the heating-cooling-heating cycle of the amplification process is achieved more quickly.

(6) The invention also provides a specific amplifying structure and a specific nucleic acid detecting chip structure, wherein a temperature measuring micro-resistance wire is arranged below the suspended film, and even the temperature measuring micro-resistance wire and the suspended film can be integrated together to form a micro-heater with a surface covered with the film, and the micro-heater is suspended and led out of more than two supporting conductive wires, such as four supporting conductive wires, so that the supporting of the suspended micro-heater is provided, and the supporting conductive wires are extended to form a micro-heater connecting terminal so as to provide electric signals for the suspended micro-heater for amplifying. The specific rapid nucleic acid detection structure provided by the invention can adopt MEMS processing technology to control the cost of a single micro heater to be about 1-10 yuan, and micro liquid drops are adopted to greatly reduce the dosage of reagents, so that the detection time is greatly shortened, and meanwhile, the detection cost is controlled to be extremely low.

In addition, the amplification structure, the rapid nucleic acid detection chip and the required device provided by the invention are compact and ultra-portable: the size of a single micro-heated chip is between 100 microns by 100 microns and 10 millimeters by 10 millimeters using MEMS processing techniques. The rapid nucleic acid detection is not only suitable for laboratory scenes, but also suitable for daily scenes such as families.

(7) According to the rapid nucleic acid detection chip provided by the invention, the suspended film is also provided with the hydrophobic or super-hydrophobic coating, so that the liquid drops are prevented from being spread flatly. The film may be coated with a reflection enhancing coating such as an Au, pt metal film, or a highly reflective multilayer dielectric film for enhancing the fluorescent reflected signal. The adaptive technical means can help the invention to further accelerate the nucleic acid detection speed, reduce the difficulty in rapid nucleic acid detection and improve the efficiency in nucleic acid detection.

(8) The invention also provides a rapid nucleic acid detection device, which integrates a plurality of rapid nucleic acid detection chips, and can integrate hundreds or thousands of micro-heater arrays on a certain substrate area, wherein each micro-heater in the array can rapidly perform thermal cycling operation on one liquid drop. The detection of a certain amount of nucleic acid is divided into a plurality of liquids to be detected simultaneously, so that rare nucleic acid copies are ensured not to be missed. In a large-scale micro-heater array, different samples to be tested can be subjected to thermal cycling on different micro-heaters, so that high-flux sample detection is realized; or for the same sample, different nucleic acid detection reagents are adopted to realize the simultaneous detection of multiple nucleic acids. The above manner can also be mixed and integrated, i.e. detection of multiple nucleic acids is performed simultaneously on multiple samples.

The amplification structure provided by the invention not only can be used for detecting nucleic acid, but also can be used for other scenes needing amplification. Taking the amplification structure for rapid nucleic acid detection as an example, more traditional nucleic acid detection is carried out in medical treatment, disease control or scientific research PCR laboratories, and epidemic situations and future diagnosis and treatment requirements require PCR nucleic acid detection to break through the limitation of the laboratories so as to adapt to more application scenes and even walk into families. The invention can realize the nucleic acid on-site detection (POCT), breaks through the application scene limitation under the characteristics of low cost, ultra portability, low power consumption and the like, enables high-efficiency energy-supply heating outpatient service, emergency treatment, customs, airports, exit and entry gateway and other high people flow gathering places to meet the requirement of quick screening of infectious diseases, and increases the coping capability and solving way of sudden public health events under multiple scenes. Rapid low cost nucleic acid detection is particularly an important means of current diagnosis, clinical treatment effect evaluation, crowd screening and epidemiological investigation when epidemic situation occurs for patients with novel coronavirus pneumonia (covd-19). The low-cost and rapid nucleic acid detection plays a role in field detection in the industries of livestock, agriculture and food. The disease-curing microorganism and biological warfare pathogen can be detected in the field or battlefield environment.

Drawings

FIG. 1 is a schematic diagram showing the structure of an amplification structure according to one embodiment of the present invention.

FIG. 2 is a schematic diagram of the structure of a hydrophobic liquid film evaporation preventing layer used for the liquid droplets in example 1 of the present invention.

FIG. 3 is a schematic diagram of the structure of the droplet of example 1 of the present invention using a surfactant to form the anti-volatilization layer.

Fig. 4 is a schematic structural view of a liquid droplet in example 1 of the present invention using hydrophobic nanoparticles to form a volatilization preventing layer.

Fig. 5 is a schematic diagram of the structure of the droplet according to embodiment 1 of the present invention for preventing volatilization by using a capping suspended film.

FIG. 6 is a schematic diagram showing the structures of examples 2-1 and 2-2 in example 2 of the present invention.

Fig. 7 is a schematic structural view of examples 2 to 3 in embodiment 2 of the present invention.

Fig. 8 is a schematic diagram of the structure of an integrated suspended thin film and heating device integrated micro-heater on the surface of a silicon wafer in embodiment 3 of the present invention.

Fig. 9 is a schematic structural view of the volatilization preventing layer in fig. 8.

FIG. 10 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 droplet, and the right side is a schematic diagram of a micro-heater structure with droplet and volatilization preventing layer.

FIG. 11 is a graph showing the relationship among the surface temperature, the heating voltage and the heating time of the micro-heater in example 3, wherein the horizontal axis represents the time required for heating, the vertical axis represents the heating temperature, and the order is the magnitude of the energizing voltage.

FIG. 12 is a graph of the time response of the drop-less microheater surface temperature ramp cycle of example 3, with the horizontal axis representing the time required for heating and the vertical axis representing the heating temperature.

FIG. 13 is a graph showing a temperature change when a droplet is placed on a micro-heater in example 3, wherein the horizontal axis represents a heating time required for heating and the vertical axis represents a heating temperature.

FIG. 14 is a graph showing the results of a group of experiments for amplifying nucleic acid of hepatitis B virus according to an embodiment of the present invention.

FIG. 15 is a graph showing the results of a control group for amplifying nucleic acid of hepatitis B virus according to an embodiment of the present invention.

FIG. 16 is a graph showing the amplification concentration versus the number of cycles of a control experiment for amplifying nucleic acid of hepatitis B virus according to an embodiment of the present invention.

FIG. 17 is a graph showing the results of a control experiment for amplifying a novel coronavirus (COVID-19) nucleic acid according to an embodiment of the present invention.

FIG. 18 is a graph showing a set of results of a control experiment for amplifying a novel coronavirus (COVID-19) nucleic acid according to an embodiment of the present invention.

FIG. 19 is a graph showing two experimental sets of results of a control experiment of novel coronavirus (COVID-19) nucleic acid amplification performed after 100-fold dilution in accordance with an embodiment of the present invention.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings:

In order to better illustrate the technical solution of the present invention, the present invention provides example 1:

example 1:

as shown in fig. 1, embodiment 1 provides a simple amplification structure in the present invention, and based on the amplification structure, a simple rapid nucleic acid detection chip may be formed, and may only include a suspended membrane 1 and a heating device (not shown in fig. 1), specifically for the preparation of the suspended membrane 1: a film is plated on the substrate 2, and when a part of the substrate 2 is taken out (such as a silicon substrate is etched by a dry method or a glass substrate is ablated by a laser), the film 1 is suspended to form a suspended film, so that a simple amplification structure is formed. And (3) placing a liquid drop 3 containing an amplified sample on the suspended film 1, and periodically heating, such as electrifying or heating the liquid drop by microwaves through the micro-resistance wire 5, so as to realize circulation of the liquid drop at different temperatures and realize PCR amplification. When the amplification structure is used for rapid nucleic acid detection, amplified droplets containing fluorescent markers are placed in a fluorescent detection device to complete fluorescent detection of nucleic acid, and the detection result is judged by recording the fluorescent brightness of the amplified droplets after each temperature cycle (amplification) to complete a melting curve of nucleic acid amplification.

Because the embodiment adopts liquid drops for amplification to form an amplification container, the liquid drops are small in size, the effect of rapid heating and temperature changing can be realized, the structure of the embodiment can be independently used for amplification to form an amplification structure, and fluorescent markers or probes can be added to realize rapid nucleic acid detection, so that the rapid nucleic acid detection chip is formed.

When the chip is used for preparing a rapid nucleic acid detection chip for nucleic acid detection, a sample to be detected, an amplification primer, enzyme, dNTPs (dNTPs), namely deoxyribonucleoside triphosphates, a template, a fluorescent probe and a buffer solution are contained in liquid drops, and the sample needs to be continuously heated and circulated in the amplification process.

In order to prevent the liquid drop from volatilizing in the heating process, an anti-volatilizing layer 4 needs to be disposed on the liquid drop, where the anti-volatilizing layer may be a hydrophobic liquid film, as shown in fig. 2, for example, the liquid drop may be wrapped by a high boiling point non-volatilizing oil film to form the anti-volatilizing layer 4, where the high boiling point refers to a liquid with a boiling point higher than that in the liquid drop, so as to avoid volatilizing the liquid in the protective layer during heating, and it is worth noting that the hydrophobic liquid film may be formed by self-assembling a surfactant on the surface of the liquid drop, as shown in fig. 3, for example, a amphiphilic surfactant is self-assembled on the surface of the liquid drop to form the anti-volatilizing layer 4, or a high boiling point compatible solvent (such as ethylene glycol or polyethylene glycol) is added into the liquid drop, as shown in fig. 4, or a layer of hydrophobic nano-particle is covered on the surface of the liquid drop to form the anti-volatilizing layer 4, as shown in fig. 5, or a layer of suspended film 1 is covered on the liquid drop to seal the liquid drop to form the anti-volatilizing layer between two suspended films 1, so as to form the anti-volatilizing layer, and thus the anti-volatilizing layer, and when the suspended film is covered on the suspended film, and the anti-volatilizing layer may be formed in a further way before the anti-volatilizing layer is formed.

Note that the volatilization-preventing layer 4 is not limited to the above-listed modes, and a better volatilization-preventing layer effect can be achieved by one or a combination of the modes during actual amplification or rapid nucleic acid detection.

As shown in FIGS. 14 to 16, the amplification control experiment of the single amplification structure or the rapid nucleic acid detection chip (the specific embodiment may be a micro heater) of the present invention was performed using hepatitis B virus inactivated virus, and as shown in FIG. 14, the rapid temperature cycle was performed for 4 seconds 95 using the scheme of the present invention ^o C,4 seconds 65 ^o C, a total of 8 seconds of PCR cycles, and the amplified fluorescence brightness was changed with the number of cycles as shown in FIG. 14. FIG. 15 is a blank of the present protocol without amplified virus, with the addition of the same amplification primers, enzyme, dNTPs, i.e., deoxyribonucleoside triphosphates, template, fluorescent probe, and buffer content of the experimental set. FIG. 16 is a graph of amplification concentration versus cycle number for a related control experiment, in which the abscissa axis represents relative fluorescence intensity and the ordinate axis represents the number of thermal cycles. It can be seen that a significant change in concentration can be detected after 30 cycles, thus taking only 4 minutes to achieve amplification and rapid nucleic acid detection of the virus sample.

As shown in FIGS. 17 to 19, in the single amplification structure or the rapid nucleic acid detection chip (a specific embodiment may be a micro-heater) provided by the present invention, a preliminary experiment control of rapid novel coronavirus (COVID-19) detection was performed using micro-droplets, and the same was performed for 4 seconds 95 ^o C,4 seconds 65 ^o C, PCR cycle amplification is performed every 8 seconds. FIG. 17 is a diagram of addition and implementationThe same new crown amplification primer, enzyme, dNTP, namely deoxyribonucleoside triphosphate, template, fluorescent probe and buffer solution are tested, but a new crown virus (COVID-19) amplification sample experimental blank control group is not put in, and the same amplification cycle treatment results are carried out on the blank control group; FIG. 18 is a graph showing the results of a control experiment in which a new coronavirus (COVID-19) amplification sample was placed, i.e., a group of experiments; FIG. 19 is a set of experimental results after 100-fold dilution of the positive standard at the concentration of the novel coronavirus (COVID-19) amplified sample of FIG. 18. As can be seen from FIGS. 17-19, under the scheme of the invention, the nucleic acid amplification is effectively and rapidly completed in one cycle of 8 seconds, and a relatively accurate detection result is obtained, and the experiment result shows that the obvious concentration change can be detected after 30 cycles, and only 4 minutes are required for 30 cycles.

It is noted that the experiment is a preliminary experiment, the subsequent PCR amplification temperature and reaction time can be further optimized, and the completion of one PCR cycle in 1 second is expected.

Example 2

In this example, the invention uses the chip of example 1 to realize the rapid nucleic acid detection chip heating mode:

in example 2-1, as shown in fig. 6, the heating and amplifying are performed by using a common heating plate 5 on the suspended film 1, and the heating plate 5 may be an integration of heating wires, or the metal plate contains heating wires, and this heating benefit is that the suspended film 1 is directly heated by using the heating plate 5, and a heating cycle in the heating process does not need to heat the substrate, so that the heating and cooling speed of the substrate do not need to be considered, and rapid heating and amplifying of the liquid drops on the suspended film 1 can be realized.

In example 2-2, a more concise heating and amplifying manner is provided herein, namely, the heating wire is arranged on the suspended film, so that the heating wire and the suspended film can be directly integrated together, and also referring to fig. 6, a suspended film with the heating wire, namely, a heating device suitable for rapid heating of liquid drops, is formed. In addition, the suspended film is directly heated by the heating wire, and a heating cycle in the heating process does not need to heat the substrate, so that the heating and cooling speeds of the substrate do not need to be considered, and the rapid heating and amplification of the liquid drop on the suspended film can be realized.

Examples 2 to 3

In examples 2-3, as shown in fig. 7, the heating micro-needle 6 is used, in this embodiment, the microwave (or ultrasonic) probe 6 is used to insert the droplet for heating, the suspended film is arranged to enable the heating to be irrelevant to the substrate, the droplet can be accurately and conveniently inserted for heating, the temperature blocking of the suspended film is removed, and the micro-needle heating enables the droplet to heat faster. The thermal cycling rate at the time of amplification further increases.

Examples 2 to 4

In examples 2-4, a simpler heating method is adopted, the heating speed is high, the method can be suitable for cyclic amplification of a plurality of liquid drops in the same temperature range, and in examples 2-4, microwaves placed in the whole amplification structure or the rapid nucleic acid detection chip are used for heating all liquid drops simultaneously in a non-contact manner, such as in a microwave oven. All droplets circulate in the same temperature range.

Examples 2 to 5

In examples 2-5, in other examples, such as examples 2-1 to 2-4, a temperature measuring device was provided on the heating device so that the heating process could be monitored in real time. Specifically, for example, a temperature measuring resistance wire can be used for example 2-2, and the temperature measuring resistance wire can be directly integrated on the suspended film, so that a heater capable of heating rapidly and measuring temperature in real time is formed, and the temperature can be measured simultaneously while heating circulation is performed on the suspended film rapidly.

In other examples, other means of temperature measurement during heating may be used to monitor the temperature of the chip in real time for the heating cycle.

Examples 2 to 6

In examples 2-6, in examples 2-1 to 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 thermal cycling, and specifically, an additional heat sink such as a thermoelectric cooling fin, or a planar heat pipe, or a microfluidic channel may be attached to the suspended film for fluid convection heat dissipation.

Example 3

In this embodiment, we provide a more specific amplification structure or rapid nucleic acid detection chip structure, and by experimental verification of this structure, rapid amplification of nucleic acids can be achieved.

Referring to fig. 8, a silicon nitride film is prepared by chemical plating (PECVD or LPCVD) on a silicon substrate, a metal micro heater (Ti/Au, or Ti/Pt, or Ti/Pd, etc.) is prepared on the film, and a micro heater is integrated by forming a suspended silicon nitride (suspended film 1) on the surface of the silicon wafer after wet or dry etching of the silicon substrate. As shown in fig. 9, a micro-heater is provided with a suspended film 1 and a heating device 5 integrated with the suspended film, liquid drops 4 of a sample to be measured are contained, the micro-heater is used for heating the liquid drops 4, and the temperature of the liquid drops is detected in real time through resistance measurement. Thus, a micro-heater with suspended film is formed. In order to prevent the liquid drop from volatilizing, the area below the suspended film is filled with fluorine oil or silicone oil, and the liquid drop to be tested is completely covered to form a liquid drop volatilizing prevention layer 4.

More specifically, as shown in fig. 10, a micro-heater 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 peripheral convex edge of the groove of the substrate, so that the supporting conductive wire can support the micro-heater and extend to the peripheral convex edge of the groove of the substrate to form a connecting terminal of the heater. The liquid drop with amplified sample is placed on the middle suspended silicon nitride micro-heater platform and is heated by electrifying the micro-heater electrode. The temperature of the micro-heater varies with the heating power. Because of suspending, the temperature can change rapidly during heating.

Similarly, the heater with the suspended film can be directly covered on the groove on the substrate, the part of the suspended film, which is close to the periphery of the groove of the substrate, is hollowed out, and only the middle micro-heater part is reserved and is electrified and heated through the micro-heater electrode. In other embodiments, other ways may be used to achieve suspended heating, as long as isolation of the micro-heater or suspended film from other portions of the substrate is maintained, i.e., within the scope of the present invention. The micro-heater structure in this embodiment may employ MEMS processing technology, where the cost of a single micro-heater is about 1-10 yuan, and the size of a single micro-heater is between 100 microns by 100 microns and 10 millimeters by 10 millimeters.

The structure of the embodiment can be singly used for amplifying to form an amplifying structure, and fluorescent labels or probes can be added to realize rapid nucleic acid detection, so that the rapid nucleic acid detection chip is formed.

The micro heater structure and the micro droplet nucleic acid detection in the embodiment greatly reduce the dosage of reagents, greatly shorten the detection time, simultaneously control the detection cost at an extremely low level, and are suitable for large-scale popularization, such as timely and efficient nucleic acid screening in epidemic prevention and control; and (5) quick detection of pathogenic microorganisms for household use and the like.

The structure in this embodiment is experimentally verified as shown in FIG. 11, in which the temperature on the suspended film can reach equilibrium rapidly at room temperature to 400 ^o And C. As shown in FIG. 12, with the micro-heater of the present structure, when the voltage on the micro-heater is applied to 0.8V, the temperature on the suspended film can reach 105 ^o C, around, and rise from room temperature to 105 ^o The time required for C is only 0.02 seconds. When the voltage on the micro-heater becomes 0, the suspended film is rapidly cooled from 105 ^o C takes only 0.02 seconds to room temperature. Therefore, the suspended micro-heater can complete one cycle of temperature cycle in 0.04 seconds. PCR nucleic acid detection requires placement of droplets on a suspended micro-heater. The time required for the temperature cycle increases due to the heat capacity of the droplet itself. Fig. 13 shows the temperature change when a droplet is placed on the suspended micro-heater in this embodiment. The droplets are PEG droplets ranging from room temperature to 120 volts when the micro-heater voltage is 1.5 volts ^o C takes 0.1 seconds; from 120 when the voltage on the micro-heater is 0 volts ^o C takes 0.37 seconds to cool to room temperature and the time required to complete a temperature cycle is 0.47 seconds. The aqueous liquid drop can resist to 170 under the coating of oily liquid ^o C is not volatilized, boiled and stably exists.

Example 4

In this embodiment, with the structure in embodiment 3, the micro heater is heated by a portable battery. The micro heater is used for heating the liquid drop, so that the micro heater is feasible, and basically only the liquid drop is heated, no extra power consumption is generated, and the energy consumption in the temperature cycle process is reduced to the minimum.

For 500 nanoliter drop to be tested, 60 times is completed ^o C-95 ^o The power consumption of the temperature cycle of C is only 0.0735J, and the temperature cycle is particularly suitable for portable equipment powered by batteries and is very suitable for detection in the field and on site.

Example 5

In this embodiment, a rapid nucleic acid detecting apparatus or a rapid amplification apparatus is provided, including a plurality of rapid nucleic acid detecting chips as described above; the droplets on the suspended film are heated independently or uniformly by a heating device in each rapid nucleic acid detection chip to realize rapid temperature change. Specifically, with the micro-heater rapid nucleic acid detection chip of example 3, each area is very small, normally about 1mm, so the micro-heater can be fabricated and is very suitable for 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 micro-heater array can be integrated on the same substrate with a certain area, or can be respectively arranged on different substrates for unified integration. Each micro-heater in the array can rapidly perform thermal cycling operation on one droplet. In a large-scale array, each micro-heater can heat the same liquid drop, so that detection of liquid drops with a certain volume is realized, for example, 100 nanoliters of each liquid drop can be divided into 1000 liquid drops, and 100 microliters of liquid to be detected can be placed on 1000 micro-heaters for parallel processing, so that rare nucleic acid copies are ensured to be not missed.

In addition, in the rapid nucleic acid detecting apparatus of the present embodiment, each rapid nucleic acid detecting chip such as a micro-heater can be independently energized to perform independent heating. Each rapid nucleic acid detection chip can also be used for placing different detection samples and nucleic acid detection occasions, so that each rapid nucleic acid detection chip can be used for thermally cycling different samples to be detected on different micro-heaters in a large-scale micro-heater array to realize high-flux sample detection; or for the same sample, different nucleic acid detection reagents are adopted to realize the simultaneous detection of multiple nucleic acids. The above manner can also be mixed and integrated, i.e. detection of multiple nucleic acids is performed simultaneously on multiple samples.

Example 6

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

placing liquid drops containing a sample to be amplified on a suspended film;

and (3) periodically heating the liquid drops to realize the circulation of the liquid drops at different temperatures and realize the amplification.

In this embodiment, the volatilization preventing layer is formed by one or several of the following methods: forming a volatilization-preventing layer by self-assembly of a surfactant on the surface of the liquid drop; covering a layer of hydrophobic nano particles on the surface of the liquid drop to form an anti-volatilization layer; and covering a layer of suspended film above the liquid drop to seal the liquid drop between the two layers of suspended films to form an anti-volatilization layer.

In this embodiment, the droplet periodic heating may be achieved by one or more of the following heating methods: heating the lower part of the suspended film by using a heating wire or a heating sheet; or heating by inserting a microwave or ultrasonic probe into the droplet; and in a microwave oven in which the whole chip is placed, the droplets and the suspended film are heated in a non-contact manner by utilizing microwaves.

The amplification method further comprises the steps of: the drop heating temperature is measured while periodically heating the drop. In this embodiment, a temperature measuring resistance wire may be used to heat and measure the temperature of the droplet on the suspended film. In other embodiments, other means may be used to heat the amplification and to measure the temperature.

Specifically, the method realizes the periodic heating of the liquid drops, realizes the circulation of the liquid drops at different temperatures, and comprises the following steps when the amplification is realized: integrating the temperature measuring resistance wire with the suspended film to form a suspended micro-heater, wherein the liquid drop is arranged on the upper surface of the suspended film; coating the liquid drops on the micro heater with the anti-volatilization layer; and applying different electric signals to the suspended micro-heater to periodically heat the liquid drops, so that the liquid drops circulate at different temperatures to realize amplification.

The amplification method in this embodiment can form a rapid nucleic acid detection chip by adding a fluorescent label or a fluorescent probe.

The amplified liquid drop containing the fluorescent marker is placed in a fluorescent detection device to finish fluorescent detection of nucleic acid;

Specifically, the liquid drop contains a sample to be detected, an amplification primer, enzyme, dNTP (deoxyribonucleoside triphosphate), a template, a fluorescent probe and a buffer solution. The outside of the liquid drop is provided with a volatilization preventing layer.

The following describes how to achieve rapid nucleic acid detection by a specific rapid nucleic acid detection chip, i.e., a micro-heater:

Placing the suspended micro-heater on a substrate with a groove, extending the suspended micro-heater out of at least two supporting conductive wires to the raised edge of the groove of the substrate, and supporting the micro-heater to be suspended above the groove; the supporting conductive wire extends to the protruding edge of the substrate groove to be fixed, so that a micro-heater connecting terminal is formed; and the nonvolatile hydrophobic liquid film is used as the nonvolatile layer, and is set as fluorine oil or silicone oil. And filling all the liquid drops and the area below the suspended film, so that the micro-heater and the liquid drops on the micro-heater can be completely coated.

And finally, applying different electric signals to the micro-heater connecting terminals of the suspended micro-heater to periodically heat the liquid drops, so that the liquid drops circulate at different temperatures to realize amplification.

And detecting fluorescence by using fluorescent markers in the liquid drops to obtain an amplification result.

In this embodiment and the foregoing embodiments, the suspended film is set to be one film or a composite film formed by several of silicon nitride, silicon oxide, carbon film, diamond film, parylene film, and metal film. In order to prevent the tension of the liquid drop on the suspension film from being destroyed to lead to dispersion of the sample, the suspension film is coated with a reflection enhancing coating film to enhance the fluorescence reflection signal.

On the basis of the scheme, in order to better and faster realize the heating cycle, after periodically heating the liquid drops, an additional heat dissipation device on the suspended film is adopted to dissipate heat of the liquid drops. Specifically, one or more modes of thermoelectric cooling sheets, planar heat pipes or microfluidic pipeline fluid are adopted on the suspended film to dissipate heat of the liquid drops.

In this embodiment, the rapid nucleic acid detection method and the rapid nucleic acid detection chip provided by the invention, such as a micro-heater, may be applied on a large scale, so as to realize a rapid nucleic acid large-scale detection method, in which the same sample or different samples are respectively divided into a plurality of droplets, and the plurality of droplets are detected simultaneously by using the rapid nucleic acid detection method.

The key point of the invention is that micro-droplets (nano-liter to micro-liter) are arranged on a suspended film, and a micro-heater or microwave heating is adopted to realize temperature circulation (65) within 0.5 seconds ^o C-95 ^o C) The droplets are covered with oil during circulation to avoid evaporation of the droplets. The invention adopts the in-situ heating and cooling of the liquid drops, eliminates the need of heating the substrate by the suspension film support, can realize the fastest liquid drop heating and cooling, does not need to drive the liquid drops, greatly simplifies the chip design and operation, and ensures the usability and reliability.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims

1. An amplification structure, characterized in that: the device comprises a suspended film and a heating device, wherein the heating device is used for heating liquid drops on the suspended film, and an anti-volatilization layer is wrapped on the liquid drops on the suspended film;

the heating device is arranged as a heating sheet or a heating wire, the heating sheet or the heating wire is arranged below the suspended film for heating, the suspended film and the heating wire or the heating sheet are integrated to form a micro-heater, the micro-heater further comprises a substrate, a groove is arranged on the substrate, the micro-heater is arranged above the groove, at least two supporting conductive wires extend out of the micro-heater to the raised edge of the groove, and the micro-heater is supported to be suspended above the groove; the supporting conductive wire extends to the protruding edge of the groove to be fixed, so that the micro-heater connecting terminal is formed; and different electric signals are input to the connecting terminal, so that the temperature of the micro heater is changed rapidly, heat is conducted to the liquid drop, and the rapid change of the liquid drop temperature is realized.

2. The amplification structure of claim 1, wherein: the anti-volatilization layer is arranged as a non-volatile hydrophobic liquid film and/or a hydrophobic nano particle layer.

3. The amplification structure of claim 2, wherein: when the nonvolatile hydrophobic liquid film is used as the volatilization preventing layer, the boiling point of the nonvolatile hydrophobic liquid film is higher than the boiling point of the liquid drop.

4. The amplification structure of claim 3, wherein: when the nonvolatile hydrophobic liquid film is used as the anti-volatilization layer, the nonvolatile hydrophobic liquid film is set as fluorine oil or silicone oil.

5. The amplification structure of claim 1, wherein: the volatilization preventing layer is formed by self-assembling a surface active agent on the surface of the liquid drop to form a film.

6. The amplification structure of claim 1, wherein: and covering a layer of suspension film above the liquid drops on the suspension film to seal the liquid drops between the two layers of suspension films so as to form the anti-volatilization layer.

7. The amplification structure of claim 1, wherein: and the liquid drops on the suspended film and the area below the suspended film are fully filled by the anti-volatilization layer, so that the micro heater and the liquid drops on the micro heater can be fully coated.

8. The amplification structure of claim 1, wherein: the heating device is provided with a temperature measuring device.

9. The amplification structure of claim 1, wherein: the heating plate or the heating wire is arranged as a heating and temperature measuring micro-resistance wire.

10. The amplification structure of claim 9, wherein: the number of the conductive wires is four.

11. The amplification structure of claim 1, wherein: the suspended film is provided with a hydrophobic or super-hydrophobic coating.

12. The amplification structure of claim 1, wherein: the suspended film is one film or a composite film formed by a plurality of films selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene film and metal film.

13. The amplification structure of claim 1, wherein: also comprises a heat dissipation device.

14. The amplification structure of claim 13, wherein: the heat dissipation device is one or more of thermoelectric cooling sheets, planar heat pipes or microfluidic pipeline fluid arranged on the suspended film.

15. A rapid nucleic acid detection chip, characterized in that: comprising an amplification structure according to any one of claims 1 to 14.

16. The rapid nucleic acid detection chip of claim 15, wherein: the suspended film is provided with a reflection enhancing coating.

17. A rapid nucleic acid detection device, characterized in that: comprising a plurality of rapid nucleic acid detection chips according to claim 16; the droplets on the suspended film are heated independently or uniformly by a heating device in each rapid nucleic acid detection chip to realize rapid temperature change.

18. An amplification method, characterized in that: amplification with the amplification structure of any one of claims 1 to 14, comprising the steps of:

setting a film in suspension;

placing a droplet containing an amplified sample on a suspended membrane;

the outside of the liquid drop is provided with an anti-volatilization layer;

the following heating method is adopted to realize the periodical heating of the liquid drops:

and heating the lower part of the suspended film by using a heating wire or a heating sheet.

19. The method of amplification of claim 18, wherein: the volatilization preventing layer is formed by adopting one or more of the following methods:

forming a volatilization-preventing layer by self-assembly of a surfactant on the surface of the liquid drop;

Covering a layer of hydrophobic nano particles on the surface of the liquid drop to form an anti-volatilization layer;

and covering a layer of suspended film above the liquid drop to seal the liquid drop between the two layers of suspended films to form an anti-volatilization layer.

20. The amplification method of claim 19, wherein: the drop heating temperature is measured while periodically heating the drop.

21. The amplification method of claim 20, wherein: and heating and measuring the temperature of the liquid drop on the suspended film by adopting a temperature measuring resistance wire.

22. The amplification method of claim 21, wherein: realize the periodic heating the liquid drop, realize the circulation of liquid drop under different temperatures, when realizing the expansion, include the following steps:

integrating the temperature measuring resistance wire with the suspended film to form a suspended micro-heater, wherein the liquid drop is arranged on the upper surface of the suspended film;

coating the liquid drops on the micro heater with the anti-volatilization layer;

and applying different electric signals to the suspended micro-heater to periodically heat the liquid drops, so that the liquid drops circulate at different temperatures to realize amplification.

23. The amplification method of claim 22, wherein: the method also comprises the following steps:

Placing the suspended micro-heater on a substrate with a groove, extending the suspended micro-heater out of at least two supporting conductive wires to the raised edge of the groove of the substrate, and supporting the micro-heater to be suspended above the groove; the supporting conductive wire extends to the protruding edge of the substrate groove to be fixed, so that a micro-heater connecting terminal is formed;

the liquid drops and the area below the suspended film are filled completely by using an anti-volatilization layer, so that the micro heater and the liquid drops on the micro heater can be completely coated;

24. The amplification method of claim 23, wherein: the nonvolatile hydrophobic liquid film is used as the anti-volatilization layer, and the nonvolatile hydrophobic liquid film is set as fluorine oil or silicone oil.

25. The amplification method of claim 19, wherein: the suspended film is one film or a composite film formed by a plurality of films selected from silicon nitride, silicon oxide, carbon film, diamond film, parylene film and metal film.

26. The amplification method of claim 19, wherein:

after the droplets are periodically heated, the droplets are cooled by an additional cooling device on the suspended film.

27. The method of amplification of claim 26, wherein: and radiating the liquid drop by adopting one or more modes of thermoelectric cooling sheets, planar heat pipes or microfluidic pipeline fluid on the suspended film.

28. A rapid nucleic acid detection method is characterized in that: amplification of a sample to be tested using the amplification method of any one of claims 19 to 27.

29. The rapid nucleic acid detection method of claim 28, wherein: also comprises the following steps

Adding fluorescent markers into the liquid drops before amplification;

30. The rapid nucleic acid detection method of claim 29, wherein: the liquid drop contains a sample to be detected, an amplification primer, enzyme, dNTP deoxyribonucleoside triphosphates, a template, a fluorescent probe and a buffer solution.

31. The rapid nucleic acid detection method of claim 30, wherein: and plating a reflection enhancing coating on the suspended film to enhance the fluorescence reflection signal.

32. A rapid nucleic acid large-scale detection method is characterized in that: the method for detecting a nucleic acid according to any one of claims 28 to 31, wherein the same sample or different samples are divided into a plurality of droplets, respectively, and the plurality of droplets are detected simultaneously.