CN115449471A - Amplification structure, rapid nucleic acid detection chip, device and method - Google Patents
Amplification structure, rapid nucleic acid detection chip, device and method Download PDFInfo
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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 rapid nucleic acid detection chip based on the micro-droplets, the amplification of a sample to be detected in the heated droplets can be rapidly realized through the arrangement of the suspended film and the droplets.
Description
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 device and a method.
Background
The Polymerase chain reaction (Polymerase chain reaction-PCR) can be used for copying and amplifying nucleic acid, and the copying and amplifying nucleic acid has various applications, such as being used in nucleic acid detection, wherein the nucleic acid detection is an important field in biomolecule detection, and the accurate and quantitative detection of ultra-micro (even single nucleic acid molecule) nucleic acid can be realized through the 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 and the like), animal disease detection (such as avian influenza, foot and mouth disease, swine fever, parasitic diseases, bacillus anthracis and the like), food safety detection (such as food-borne microorganisms, food allergens, transgenic foods and the like), scientific research (such as quantitative research of related molecular biology of medicine, life science, farming and pasturing and the like) and the like. The application industries of the PCR nucleic acid quantitative detection technology comprise medical institutions, scientific research institutions, colleges and universities, disease control centers, inspection and quarantine bureaus, food enterprises, animal husbandry enterprises and the like.
Polymerase Chain Reaction (PCR) is the most common molecular biology technology for amplifying and amplifying specific DNA fragments, and the PCR has the greatest characteristic of greatly increasing trace DNA fragments, can be used for detecting trace specific nucleic acid fragments by combining with a fluorescent probe, and is commonly used for detecting infected pathogenic microorganisms, tumor analysis and genetic disease diagnosis. PCR has important application in the detection and diagnosis of infectious diseases, and is a necessary technology for large-scale population epidemic situation (such as new coronavirus).
The traditional PCR amplification technology needs to be 95 o C-65 o C-72 o And C, temperature circulation is carried out, and the temperature rise and the temperature fall need a certain time, and the time of single circulation needs 5-30 minutes. Generally, 20-40 cycles of PCR expansion are needed, so that PCR detection needs several hours, and detection needs to be completed in a qualified laboratory of a detection institution. PCR detection has poor timeliness and needs professional detection unit operation, so that the function of the PCR detection in large-scale infectious epidemic prevention and control has certain limitation.
Specifically, in the conventional PCR amplification technology, in addition to the temperature-controlled liquid to be detected, the temperature control system inevitably heats the sample support structure such as the substrate during temperature cycling, which results in a long time for both temperature rise and temperature decrease. Meanwhile, based on a liquid sample with milliliter magnitude, when the concentration of nucleic acid to be detected is lower, certain waiting time is needed to ensure that the reaction is sufficient. These factors result in PCR cycles that take around 5-30 minutes. Because multiple PCR cycles (20-40) are required, 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 following temperature cycling modes are common in PCR: (1) The thermoelectric sheet is adopted to heat and refrigerate the liquid drops, so that temperature circulation is realized, and generally the 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 cooled, and the temperature circulation generally needs about 5-20 minutes; (3) The substrate is maintained with two temperature zones of high and low temperature, and liquid is circulated back and forth between the high and low temperature by a micro-fluidic pipeline or liquid drop driving, generally, the temperature circulation depends on the speed of the liquid drop driving, and the time can be controlled within a few seconds. However, this method requires precise operation of the droplets, the device design and difficulty are both large, the power consumption for maintaining the two temperature zones is large, and the droplets are at 95 deg.f o And C, bubbles are easily generated when the chip moves at high temperature, and great inconvenience is brought to chip design and liquid drop 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 above technical problem, the present 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 drops on the suspended film are wrapped with an anti-volatilization layer.
Preferably, the anti-volatilization layer is provided as a non-volatile hydrophobic liquid film and/or a hydrophobic nanoparticle layer.
Preferably, when the nonvolatile hydrophobic liquid film is used as the volatilization prevention layer, the boiling point of the nonvolatile hydrophobic liquid film is higher than that of the liquid droplets.
Preferably, when the non-volatile hydrophobic liquid film is used as the anti-volatilization layer, the non-volatile hydrophobic liquid film is set to be fluorine oil or silicone oil.
Preferably, the anti-volatilization layer is formed by surfactant self-assembly on the surface of the liquid drop to form the anti-volatilization layer.
Preferably, a layer of suspended film is covered above the liquid drops on the suspended film, so that the liquid drops 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 then the microwave container is utilized to heat the liquid drops.
Preferably, the heating device is arranged to heat the micro-needle, and the liquid drop inserted into the suspended film by the heating micro-needle is heated.
Preferably, the heating micro-needle is a micro-needle with a microwave or ultrasonic probe.
Preferably, heating device sets up to heating plate or heater strip, the heating plate perhaps the heater strip sets up the heating is carried out to unsettled film below.
Preferably, the heating device is provided with a temperature measuring device.
Preferably, the heating sheet 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 the micro-heater.
Preferably, the volatilization prevention layer is used for completely filling the liquid drops on the suspended film and the area below the suspended film, so that the micro-heater and the liquid drops on the micro-heater can be completely 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 the balance when the micro-heater is suspended.
Preferably, a groove is formed in the substrate, the micro heater is arranged above the groove, at least two supporting conductive wires extend out of the micro heater to reach 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, 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 for fixing and forming a micro-heater connecting terminal, and the four supporting conductive wires support the balance of the micro-heater when suspended.
Preferably, different electrical signals are input to the connection terminals, so that the micro-heater achieves a rapid change in temperature by conducting heat to the liquid droplets to achieve a rapid change in temperature.
Preferably, the suspended film is provided with a hydrophobic or super-hydrophobic coating.
Preferably, the suspended film is one or a composite film formed by several of silicon nitride, silicon oxide, a carbon film, a diamond film, a parylene film, a metal film.
Preferably, the device further comprises a heat dissipation device.
Preferably, the heat dissipation device is one or more of a thermoelectric refrigeration chip, a planar heat pipe or a 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 heating device is used for heating the liquid drops independently or mutually and uniformly in each rapid nucleic acid detection chip so as to realize rapid temperature change.
In addition, the present invention provides an amplification method comprising: the method comprises the following steps:
arranging a film in the air;
placing the droplet containing the amplified sample on a suspended film;
periodically heating the liquid drop to realize circulation of the liquid drop at different temperatures and realize amplification;
preferably, the liquid drop is externally provided with an anti-volatilization layer.
Preferably, the anti-volatilization layer is formed by one or more of the following methods: surfactant is used for self-assembly on the surface of the liquid drop to form an anti-volatilization layer; 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 drops to seal the liquid drops between the two layers of suspended films to form an anti-volatilization layer.
Preferably, the periodic heating of the liquid drops is realized 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 placing the whole chip in a microwave oven, and heating the liquid drops and the suspended film by utilizing microwaves in a non-contact manner.
Preferably, the droplet heating temperature is measured while periodically heating the droplet.
Preferably, a temperature measuring resistance wire is adopted to heat and measure the temperature of the liquid drops on the suspended film.
Preferably, the method realizes the periodic heating of the liquid drop, realizes the circulation of the liquid drop at different temperatures, and comprises the following steps of:
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; allowing the anti-volatilization layer to coat the droplets on the micro-heaters; different electric signals are applied to the suspended micro-heater to realize the periodic heating of the liquid drop, so that the liquid drop is circulated at different temperatures to realize the 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 at least two supporting conductive wires out of the suspended micro-heater 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 drop and the area below the suspended film are completely filled by an anti-volatilization layer, so that the micro heater and the liquid drop on the micro heater can be completely coated by the anti-volatilization layer;
and applying different electric signals to the micro-heater connecting terminal of the suspended micro-heater to realize periodic heating of the liquid drop, so that the liquid drop is circulated at different temperatures to realize 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 be fluorine oil or silicone oil.
Preferably, the suspended film is one or a composite film formed by several of silicon nitride, silicon oxide, a carbon film, a diamond film, a parylene film, a metal film.
Preferably, after the droplets are periodically heated, an additional heat dissipation device on the suspended film is used for dissipating heat of the droplets.
Preferably, one or more of a hot spot refrigerating sheet, a plane heat pipe or a micro-fluidic pipeline fluid is adopted on the suspended film to dissipate heat of the liquid drops.
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 is better, and further comprises the following steps:
adding a fluorescent label to the droplets prior to amplification;
after amplification, placing the amplified liquid drops containing the fluorescent markers in a fluorescence detection device to finish the fluorescence detection of the 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, and the liquid drop contains a sample to be detected, an amplification primer, an enzyme, dNTP deoxyribonucleoside triphosphate, a template, a fluorescent probe and a buffer solution.
The rapid nucleic acid detection method is better, and the suspended film is coated with the enhanced reflection coating film to enhance the fluorescence reflection signal.
The invention also provides a rapid large-scale nucleic acid detection method, which divides the same sample or different samples into a plurality of liquid drops respectively, and simultaneously detects the plurality of liquid drops by utilizing the rapid nucleic acid detection method.
The invention has the beneficial effects that: (1) The invention provides an amplification structure and a corresponding rapid nucleic acid detection chip, which can rapidly realize amplification of a sample to be detected amplified in a heated liquid drop through the arrangement of a suspended film and the liquid drop. The liquid drop in-situ heating and cooling are adopted, the requirement for heating the substrate is eliminated through the suspended film support, the liquid drop heating and cooling at the highest 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 the reliability are ensured. Meanwhile, the micro-droplets are used as a reaction vessel, 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 heating of the substrate inevitable in the temperature cycle process is reduced, the heating and cooling speed is completed within 0.5 second, the time required by the existing PCR amplification temperature cycle is within 1-5 minutes, and the temperature cycle speed 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 the traditional 30 minutes to several hours by adopting the invention, the time required by 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 a single liquid drop, the detection capability of the single liquid drop is strong, and the detection cost is low: meanwhile, in a specific application scene, the MEMS processing technology can be adopted, the cost of a single micro heater is controlled to be about 1-10 yuan, the using amount of reagents is greatly reduced by adopting micro liquid drops, the detection cost is controlled to be at an extremely low level while the detection time is greatly shortened, and the method is suitable for large-scale popularization, such as timely and efficient nucleic acid screening in epidemic situation prevention and control; domestic pathogenic microorganism fast-checking and the like.
(3) According to the amplification structure and the rapid nucleic acid detection chip provided by the invention, the liquid drop is amplified, and meanwhile, the anti-volatilization layer is arranged on the liquid drop, and can effectively prevent aerosol pollution generated during nucleic acid amplification and ensure the amplification effect. Certainly, the volatilization-preventing layer can be formed in other ways, such as self-assembling a amphiphilic surfactant on the surface of the droplet to form a film to reduce the volatilization of the droplet, adding a high-boiling compatible solvent (such as adding ethylene glycol or polyethylene glycol into an aqueous droplet), covering a layer of hydrophobic nanoparticles on the surface of the droplet to form a liquid martle (droplet wrapped by a solid surface), or covering a layer of suspended film above the droplet to seal the droplet between two layers of suspended films, and the like, so as to prevent the volatilization of the droplet in the heating process. Because the cost of the chip and the reagent is low, the chip and the reagent are disposable, and the positive sample is sealed in an anti-volatilization layer such as oil drops after amplification detection is finished, so that the pollution of amplified nucleic acid molecules to equipment can be effectively prevented.
(4) The invention also provides a plurality of adaptive amplification heating devices aiming at the amplification structure and the rapid nucleic acid detection chip, and micro heating resistance wires are prepared on the suspended film by micro-nano processing; or heating by inserting a microwave (or ultrasonic) probe into the droplet. If all droplets circulate in the same temperature range, the entire chip can also be placed in a microwave oven, and all droplets are heated simultaneously by microwaves without contact.
(5) In order to enable the invention to realize heating-cooling-heating circulation rapidly, the invention particularly provides that a cooling device, namely an additional heat dissipation device such as a thermoelectric refrigeration piece, or a plane heat pipe, or a microfluidic pipeline fluid convection heat dissipation device can be additionally arranged on the suspended film. The benefit of this arrangement is that the heating-cooling-heating cycle of the amplification process is achieved more rapidly.
(6) The invention also provides a specific amplification structure and a nucleic acid detection 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 the surface covered with the film, the micro heater is suspended and leads out more than two, such as four supporting conductive wires, so that the micro heater not only provides support for the suspended micro heater, but also extends to be a micro heater connecting terminal to provide electric signals for the suspended micro heater to amplify. The specific rapid nucleic acid detection structure provided by the invention can adopt an MEMS processing technology to control the cost of a single micro heater to be about 1-10 yuan, and adopts micro liquid drops to greatly reduce the dosage of reagents, so that the detection cost is controlled to be at an extremely low level while the detection time is greatly shortened.
Moreover, the amplification structure and the rapid nucleic acid detection chip provided by the invention are compact and ultra-portable with the required devices: the size of the individual micro-heating chips is between 100 microns by 100 microns and 10 mm by 10 mm 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 liquid drops are prevented from being spread out. The film can be coated with reflection enhancing coating film such as Au, pt metal film, or high reflection multi-layer dielectric film for enhancing fluorescence reflection signal. These adaptive technical means can help the invention 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, hundreds of micro-heater arrays can be integrated on a certain substrate area, and each micro-heater in the array can rapidly perform thermal cycle operation on one liquid drop. A certain amount of nucleic acid detection is divided into a plurality of liquids for simultaneous detection, so that rare nucleic acid copies are ensured not to be missed. In a large-scale micro-heater array, different samples to be detected can be thermally cycled on different micro-heaters, so that high-flux sample detection is realized; or different nucleic acid detection reagents are adopted for the same sample, so that the simultaneous detection of multiple nucleic acids is realized. The above method can also be mixed and integrated, i.e., a plurality of samples are simultaneously detected by a plurality of nucleic acids.
The amplification structure provided by the invention not only can be used for nucleic acid detection, but also can be used in other scenes needing amplification. Taking the amplification structure for rapid nucleic acid detection as an example, more traditional nucleic acid detection is developed in a PCR laboratory for medical treatment, disease control or scientific research, and epidemic situations and future diagnosis and treatment requirements require the PCR nucleic acid detection to break through the limitation of the laboratory to adapt to more application scenes, even to enter families. The invention can realize 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, efficiently enables high-efficiency people stream gathering places such as a fever outpatient service, an emergency call, customs, airports, entry and exit gateways and the like, meets the requirement of rapid infectious disease screening, and increases the coping capability and the solution way of emergent public health events in multiple scenes. The rapid low-cost nucleic acid detection is an important means for the accurate diagnosis, the evaluation of clinical treatment effect, the population screening in case of epidemic situation and the epidemiological investigation of the current novel coronavirus pneumonia (COVID-19) patients. The nucleic acid detection is low in cost and rapid, and simultaneously plays a role in field detection in the livestock raising, agriculture and food industries. Can detect disease-treating microorganisms and biological warfare pathogens in the field or battlefield and other environments.
Drawings
FIG. 1 is a schematic diagram of an amplification structure according to an embodiment of the present invention.
FIG. 2 is a schematic view of the structure of the anti-volatilization layer of the liquid drop adopting the hydrophobic liquid film in example 1 of the invention.
FIG. 3 is a schematic diagram of a structure of a droplet formed with a surfactant to form an anti-volatilization layer in example 1 of the present invention.
Fig. 4 is a schematic structural diagram of a droplet forming an anti-volatilization layer using hydrophobic nanoparticles according to example 1 of the present invention.
FIG. 5 is a schematic view of the structure of the embodiment 1 of the present invention in which the liquid drop is prevented from volatilizing by means of a cover-suspended film.
FIG. 6 is a schematic structural view of examples 2-1 and 2-2 in embodiment 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 structural diagram of an integrated flying thin film and heating device integrated micro-heater on the surface of a silicon wafer in example 3 of the present invention.
Fig. 9 is a schematic view showing the structure of the volatilization prevention layer in fig. 8.
Fig. 10 is a schematic diagram of an actual structure of a microheater according to example 3, in which the left side is a schematic diagram of a microheater without a droplet, and the right side is a schematic diagram of a microheater with a droplet and an anti-volatilization layer.
FIG. 11 is a graph showing the relationship among the surface temperature of the micro-heater, the heating voltage and the heating time in example 3, wherein the abscissa axis represents the heating time, the ordinate axis represents the heating temperature, and the order represents the magnitude of the energization voltage.
Fig. 12 is a time response graph of the surface temperature ramping cycle of the microheater without droplets in example 3, wherein the horizontal axis represents heating time and the vertical axis represents heating temperature.
Fig. 13 is a graph showing the temperature change when a droplet is placed on the microheater in example 3, wherein the horizontal axis shows the heating temperature when heating is required.
FIG. 14 is a graph showing the results of the hepatitis B virus nucleic acid amplification experiment performed according to the embodiment of the present invention.
FIG. 15 is a graph showing the results of the hepatitis B virus inactivated nucleic acid amplification control group according to the embodiment of the present invention.
FIG. 16 is a graph of concentration of amplification versus cycle number for a control experiment in which nucleic acid amplification of inactivated hepatitis B virus was performed according to an embodiment of the present invention.
FIG. 17 is a graph showing the results of a control experiment in which the novel coronavirus (COVID-19) nucleic acid amplification reaction was performed according to an embodiment of the present invention.
FIG. 18 is a graph showing the results of a control experiment in which the novel coronavirus (COVID-19) nucleic acid amplification was performed according to an embodiment of the present invention.
FIG. 19 is a graph of the results of a set of experiments in which a nucleic acid amplification control experiment for the novel coronavirus (COVID-19) was performed at 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:
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 simpler amplification structure in the present invention, and based on this amplification structure, a simple and fast nucleic acid detection chip can also be constituted, which can only include a suspended film 1 and a heating device (not shown in fig. 1), specifically for the preparation of the suspended film 1: a layer of 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 laser), the film 1 is suspended to form a suspended film, so that a simple amplification structure is formed. A liquid drop 3 containing an amplification sample is placed on the suspended film 1 and periodically heated, for example, the liquid drop is electrified or heated by microwaves through a micro resistance wire 5, so that the circulation of the liquid drop at different temperatures is realized, and the PCR amplification is realized. When the amplification structure is used for rapid nucleic acid detection, the amplified liquid drop containing the fluorescent label is placed in a fluorescence detection device to complete the fluorescence detection of the nucleic acid, and the melting curve of the nucleic acid amplification is completed by recording the fluorescence brightness of the liquid drop after each temperature cycle (amplification), so as to judge the detection result.
Because the embodiment adopts the liquid drop to amplify to form the amplification container, the liquid drop has small volume and can realize the effect of rapid heating and temperature changing, the structure of the embodiment can be independently used for amplifying to form an amplification structure, and can also be added with a fluorescent label or a probe to realize rapid nucleic acid detection, thereby becoming a rapid nucleic acid detection chip.
When the chip is used for nucleic acid detection to prepare a rapid nucleic acid detection chip, a sample to be detected, an amplification primer, an enzyme, dNTP (deoxyribose nucleotide triphosphate), a template, a fluorescent probe and a buffer solution are contained in the liquid drop, and the liquid drop needs to be heated and circulated continuously in the amplification process.
In order to prevent the liquid droplets from volatilizing during the heating process, an anti-volatilization layer 4 needs to be disposed on the liquid droplets, where the anti-volatilization layer may be a hydrophobic liquid film, as shown in fig. 2, for example, the liquid droplets may be wrapped by a high-boiling-point nonvolatile oil film to form the anti-volatilization layer 4, note that the high boiling point here refers to a liquid having a boiling point higher than that in the liquid droplets, and it is avoided that the liquid in the protective layer volatilizes during the heating process, and it is also worth noting that the hydrophobic liquid film herein may be a self-assembled film on the surface of the liquid droplets by using a surfactant, as shown in fig. 3, for example, a amphiphilic surfactant is self-assembled on the surface of the liquid droplets to form the anti-volatilization layer 4 to reduce the volatilization of the liquid droplets, or a high-boiling-point compatible solvent (such as ethylene glycol or polyethylene glycol is added to the aqueous liquid droplets) as shown in fig. 4, or a liquid droplet is formed by covering a layer of hydrophobic nanoparticles to form a liquid droplet covered on the surface, and then the anti-volatilization layer 4 is formed, as shown in fig. 5, or a suspended film is covered on the liquid droplets to form an anti-volatilization layer, and other ways.
Note that the anti-volatilization layer 4 is not limited to the above-mentioned modes, and during the actual amplification process or the rapid nucleic acid detection process, a better anti-volatilization layer effect can be achieved by one or a combination of the modes.
As shown in FIGS. 14 to 16, the single amplification structure or the rapid nucleic acid detection chip (in one embodiment, the micro heater) amplification control experiment using the inactivated hepatitis B virus of the present invention is performed, as shown in FIG. 14, using the protocol of the present invention to detect the inactivated hepatitis B virusRapid temperature cycling was performed for 4 seconds 95 o C,4 seconds 65 o C, 8 seconds in total, the PCR cycle, amplification fluorescence brightness with cycle number change as shown in figure 14. FIG. 15 shows the results of a blank control set of the protocol of the present invention with the same amplification primers, enzyme, dNTP, deoxyribonucleoside triphosphate, template, fluorescent probe, and buffer added to the experimental set, but without amplified virus. FIG. 16 is a graph of amplification concentration versus cycle number for a relevant control experiment, in which the axis of abscissa indicates relative fluorescence intensity and the axis of ordinate indicates the number of thermal cycles. It can be seen that after 30 cycles, a significant change in concentration can be detected, so that only 4 minutes are required to achieve amplification of the virus sample and rapid nucleic acid detection.
Referring to FIGS. 17 to 19, the preliminary experiment control of rapid neocoronavirus inactivation (COVID-19) detection using microdroplets on a single amplification structure or a rapid nucleic acid detection chip (in an embodiment, a micro heater) provided by the present invention is performed in the same manner as the above-mentioned preliminary experiment control for 4 seconds 95 o C,4 seconds 65 o And C, PCR cyclic amplification is carried out once every 8 seconds. FIG. 17 shows the results of an experimental blank with the same addition of new corona amplification primers, enzyme, dNTP, deoxyribonucleoside triphosphates, template, fluorescent probe and buffer as in the experimental group, but without the addition of a sample amplified with new coronavirus (COVID-19) and the same amplification cycle performed on the blank; FIG. 18 shows the results of experimental controls of an experimental group, which is a sample amplified by the addition of a new coronavirus (COVID-19); FIG. 19 is the results of two experimental runs following a 100-fold dilution of the positive standard at the concentration of the amplified sample of the novel coronavirus (COVID-19) of FIG. 18. As shown in FIGS. 17-19, under the scheme of the present invention, the nucleic acid amplification can be effectively and rapidly completed in one cycle of 8 seconds, and a relatively accurate detection result can be obtained, and the experiment result shows that the concentration can be detected after 30 cycles, and only 4 minutes is needed after 30 cycles.
It is worth noting that the experiment is a preliminary experiment, the PCR amplification temperature and the reaction time can be further optimized subsequently, and the completion of one PCR cycle within 1 second is expected.
Example 2
In this example, different examples are used to illustrate the heating method of the chip for rapid nucleic acid detection in the chip of example 1:
example 2-1, as shown in fig. 6, in this example, a common heating sheet 5 is used on the suspended film 1 for heating amplification, and the heating sheet 5 may be an integration of heating wires, or a metal sheet contains heating wires, which has the advantage that since the film 1 is suspended, the suspended film is directly heated by the heating sheet 5, and a heating cycle does not need to heat the substrate in the heating process, and therefore, the heating and cooling speed of the substrate does not need to be considered, and rapid heating amplification of droplets on the suspended film 1 can be achieved.
Example 2-2, in contrast to example 2-1, a more compact heating amplification method is provided, i.e., a heating wire is disposed on the suspended film, and the heating wire and the suspended film can be directly integrated together, and referring to fig. 6, a suspended film with a heating wire is formed, i.e., a heating device adapted for rapid heating of liquid droplets is formed. Similarly, because the heating wire is adopted to directly heat the suspended film, the substrate does not need to be heated in a heating process through one heating cycle, the heating and cooling speeds of the substrate do not need to be considered, and the rapid heating and amplification of the liquid drops on the suspended film can be realized.
Examples 2 to 3
In examples 2-3, as shown in fig. 7, we use a heating microneedle 6, in this embodiment, a microwave (or ultrasonic) probe 6 is used to insert into a liquid drop for heating, the suspension film is arranged to make the heating independent of the substrate, the liquid drop can be inserted and heated accurately and conveniently, the temperature resistance of the suspension film is removed, and the microneedle is heated to make the liquid drop heated more quickly. The thermal cycling rate is further increased during amplification.
Examples 2 to 4
In examples 2 to 4, a simpler heating method was used, and it was also found that the heating speed was high, and the method was suitable for cyclic amplification of a plurality of droplets in the same temperature range, and in examples 2 to 4, the entire amplification structure or the rapid nucleic acid detection chip was placed in a microwave such as a microwave oven, and all droplets were heated simultaneously by the microwave without contact. All droplets circulate within 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 to enable real-time monitoring of the heating process. Specifically, for example 2-2, a temperature measuring resistance wire can be used and directly integrated on the suspended film to form a heater capable of rapidly heating and measuring temperature in real time, and the temperature can be measured simultaneously while heating and circulating on the suspended film.
In other examples, a temperature measuring device during heating can be adopted in other ways to monitor the temperature of the chip in real time during heating circulation.
Examples 2 to 6
In examples 2-6, in examples 2-1 to 2-5, in addition to allowing the cooling of the heated droplets to cool naturally, a heat sink is provided on the fast nucleic acid chip to further accelerate the thermal cycle, and specifically, an additional heat sink such as a thermoelectric cooling plate, or a planar heat pipe, or a microfluidic channel may be added on the suspended membrane to dissipate heat by fluid convection.
Example 3
In this embodiment, we provide a more specific amplification structure or a rapid nucleic acid detection chip structure, and through experimental verification of the structure, rapid amplification of nucleic acid can be achieved.
Referring to fig. 8, a silicon nitride film is prepared on a silicon substrate by chemical vapor deposition (PECVD or LPCVD), a metal micro-heater (Ti/Au, ti/Pt, ti/Pd, or the like) is prepared on the film, and a suspended silicon nitride (suspended film 1) is formed on the surface of a silicon wafer after wet or dry etching of the silicon substrate, i.e., a micro-heater is integrated. Referring to fig. 9, the micro-heater has a suspended film 1 and a heating device 5 integrated with the suspended film, a droplet 4 containing a sample to be measured, heats the droplet 4 by using the micro-heater, and detects the temperature of the droplet in real time by resistance measurement. This forms a microheater with an overhanging film. In order to prevent the liquid drops from volatilizing, the area below the suspended film is filled with fluorine oil or silicon oil, and the liquid drops to be detected are completely covered to form a liquid drop volatilization prevention layer 4.
More specifically, as shown in fig. 10, the micro-heater with the 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 at the periphery of the groove of the substrate, so that the supporting conductive wire can support the micro-heater and extend to the raised edge at the periphery of the groove of the substrate to form a connecting terminal of the heater. The liquid drop with the amplified sample is placed on a silicon nitride micro-heater platform with the middle suspended, and is electrified and heated through a micro-heater electrode. The temperature of the micro-heater varies as the heating power varies. Because of suspension, the temperature can change rapidly during heating.
Similarly, the heater with the suspended film can directly cover the groove on the substrate, the part of the suspended film, which is protruded near the periphery of the groove of the substrate, is hollowed out, only the middle micro-heater part is reserved, and the micro-heater is electrified and heated through the micro-heater electrode. The effect can also be achieved, and in other embodiments, other modes can be adopted as long as the isolation of the micro-heater or the suspended film from other parts of the substrate is maintained, and the suspended heating is achieved, which is within the protection scope of the present invention. The micro-heater structure in this embodiment may be implemented using MEMS processing technology, where the cost of a single micro-heater is on the order of 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 independently used for amplification to form an amplification structure, and can also be added with a fluorescent label or a probe to realize rapid nucleic acid detection, so that the structure becomes a rapid nucleic acid detection chip.
By adopting the micro-heater structure and the micro-droplet nucleic acid detection in the embodiment, the dosage of the reagent is greatly reduced, the detection time is greatly shortened, and meanwhile, the detection cost is controlled to be extremely low, so that the micro-heater structure and the micro-droplet nucleic acid detection are suitable for large-scale popularization, such as timely and efficient nucleic acid screening in epidemic prevention and control; domestic pathogenic microorganism fast-checking and the like.
The structure of this embodiment is experimentally verified below, and as shown in fig. 11, when the voltage changes, the temperature on the suspended film can quickly reach equilibrium, from room temperature to 400 deg.f o C is changed. As shown in fig. 12, with the micro-heater of the present structure, when the voltage on the micro-heater is applied to 0.8 v,the temperature of the suspended film can reach 105 DEG o C or so, and rises from room temperature to 105 deg.C o The time required for C was only 0.02 seconds. When the voltage on the micro-heater becomes 0, the suspended film cools rapidly from 105 o Only 0.02 second is needed for C to reach room temperature. Therefore, the suspended micro-heater can complete a cycle of temperature cycling within 0.04 seconds. PCR nucleic acid detection requires placing droplets on a suspended microheater. The time required for temperature cycling increases due to the heat capacity of the droplets themselves. Fig. 13 shows the temperature change when a droplet is placed on the suspension microheater in this embodiment. The liquid drop is PEG liquid drop, and when the voltage of the micro-heater is 1.5V, the temperature is 120 to room temperature o C takes 0.1 second; when the voltage on the micro-heater is 0V, the secondary 120 o It took 0.37 seconds for C to cool to room temperature, and the time required to complete one temperature cycle was 0.47 seconds. The aqueous liquid drop can be tolerated to 170 percent under the coating of oily liquid o About C is non-volatile, non-boiling and stable.
Example 4
In this example, the structure of example 3 was modified by heating the micro-heater using a portable battery. Since the micro-heater basically only heats the liquid drops in the process of heating the liquid drops, no extra power consumption is caused, and the energy consumption in the temperature circulation process is reduced to the minimum degree, the micro-heater in the embodiment of the invention is feasible to be heated by adopting the battery.
For 500 nanoliters of the droplet to be measured, this is done once 60 o C-95 o The power consumption of the temperature cycle of C is only 0.0735 joule, and the method is particularly suitable for portable equipment powered by batteries and is very suitable for detection in the field.
Example 5
In this embodiment, a rapid nucleic acid detection device or a rapid amplification device is provided, which includes a plurality of rapid nucleic acid detection chips as described above; in each rapid nucleic acid detection chip, a heating device is independently or uniformly used for heating the liquid drops on the suspended film so as to realize rapid temperature change. Specifically, in the microheater chip for rapid nucleic acid detection in example 3, each area is very small, normally about 1mm, so the microheater can be made into a large-scale array, such as 2 × 2, 10 × 10, 100 × 100 array, and is very suitable 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, and can also be respectively arranged on different substrates to be integrated in a unified way. Each micro-heater in the array can rapidly perform thermal cycling operation on one liquid drop. In a large-scale array, each micro-heater can heat the same droplet to realize detection of a certain volume of droplets, for example, each droplet is 100 nanoliters, 100 microliters of liquid to be detected can be divided into 1000 droplets, and the droplets are placed on 1000 micro-heaters to be processed in parallel, so that rare nucleic acid copies are ensured not to be detected.
In addition, in the rapid nucleic acid detecting apparatus of this embodiment, each rapid nucleic acid detecting chip, such as a micro-heater, can be independently heated by independently applying power. Each rapid nucleic acid detection chip can also be provided with different detection samples and nucleic acid detection occasions, so that each rapid nucleic acid detection chip can realize high-flux sample detection by thermally cycling different samples to be detected on different micro-heaters in a large-scale micro-heater array; or different nucleic acid detection reagents are adopted for the same sample, so that the simultaneous detection of multiple nucleic acids is realized. The above method can also be mixed and integrated, that is, the detection of multiple nucleic acids can be performed on multiple samples at the same time.
Example 6
In this example, a specific amplification method is provided, comprising: arranging a film in the air;
placing the liquid drop containing the sample to be amplified on the suspended film;
and periodically heating the liquid drop to realize circulation of the liquid drop at different temperatures and realize amplification.
In this embodiment, the volatilization prevention layer is formed by one or more of the following methods: surfactant is used for self-assembly on the surface of the liquid drop to form an anti-volatilization layer; 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 drops to seal the liquid drops between the two layers of suspended films to form an anti-volatilization layer.
In this embodiment, the droplet periodic heating may be realized by one or more of the following heating methods: heating 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 placing the whole chip in a microwave oven, and heating the liquid drops and the suspended film by utilizing microwaves in a non-contact manner.
The amplification method further comprises the steps of: while periodically heating the droplet, a droplet heating temperature is measured. In this embodiment, a temperature measuring resistance wire may be used to heat and measure the temperature of the liquid drop on the suspended film. In other embodiments, other methods may be used to measure the temperature during the heating amplification.
Specifically, the method realizes the periodic heating of the liquid drop, realizes the circulation of the liquid drop at different temperatures, and comprises the following steps of: integrating the temperature measuring resistance wire and the suspended film to form a suspended micro-heater, wherein the liquid drop is arranged on the upper surface of the suspended film; allowing the anti-volatilization layer to coat the droplets on the microheater; different electric signals are applied to the suspended micro-heater to realize the periodic heating of the liquid drop, so that the liquid drop is circulated at different temperatures to realize the amplification.
The amplification method in this embodiment can form a rapid nucleic acid detection chip by adding a fluorescent label or a fluorescent probe.
Placing the amplified liquid drops containing the fluorescent markers in a fluorescence detection device to finish the fluorescence detection of the nucleic acid;
and judging the detection result according to the fluorescence brightness of the amplified liquid drop.
Specifically, the liquid drop contains a sample to be detected, an amplification primer, an enzyme, dNTP (deoxyribose nucleotide triphosphate), a template, a fluorescent probe and a buffer solution. And an anti-volatilization layer is arranged outside the liquid drop.
How to realize rapid nucleic acid detection is illustrated by a specific rapid nucleic acid detection chip, namely a micro-heater, as follows:
placing the suspended micro-heater on a substrate with a groove, extending at least two supporting conductive wires out of the suspended micro-heater 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 utilizing an anti-volatilization layer, wherein the non-volatile hydrophobic liquid film is used as the anti-volatilization layer, and the non-volatile hydrophobic liquid film is set to be fluorine oil or silicon oil. And filling all the liquid drops and the area below the suspended film so as to completely cover the micro-heaters and the liquid drops on the micro-heaters.
And finally, applying different electric signals to the micro-heater connecting terminal of the suspended micro-heater to realize periodic heating of the liquid drop, so that the liquid drop is circulated at different temperatures to realize amplification.
And detecting fluorescence by using the fluorescent label in the liquid drop to obtain an amplification result.
In this embodiment and the foregoing embodiments, the suspended thin film is a thin film or a composite thin film formed by several kinds of thin films selected from silicon nitride, silicon oxide, a carbon film, a diamond film, a parylene film, a metal film. In order to prevent the liquid drop tension on the suspended film from being damaged to cause sample dispersion, a reinforced reflection coating is coated on the suspended film to reinforce the fluorescence reflection signal.
On the basis of the scheme, in order to better and more quickly realize heating circulation, after the liquid drops are periodically heated, an additional heat dissipation device on a suspended film is adopted to dissipate heat of the liquid drops. Specifically, one or more of a thermoelectric refrigeration piece, a planar heat pipe or a microfluidic pipeline fluid is adopted on the suspended film to dissipate heat of the liquid drop.
In this embodiment, the rapid nucleic acid detection method and the rapid nucleic acid detection chip provided by the present invention, such as a micro-heater, can be applied in a large scale 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 simultaneously detected by the rapid nucleic acid detection method.
The key point of the invention is that micro-droplets (nano-liter to micro-liter) are placed on a suspended film, and a micro-heater or microwave is adopted for heating, so that the temperature cycle (65) within 0.5 second can be realized o C-95 o C) And the liquid drops are coated with oil in the circulating process to avoid volatilization of the liquid drops. The invention adopts the in-situ heating and cooling of the liquid drops, eliminates the need of heating the substrate through the suspended 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 the reliability.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (43)
1. An amplification construct, comprising: the heating device is used for heating liquid drops on the suspended film.
2. The amplification structure of claim 1, wherein: and the liquid drops on the suspended film are wrapped with an anti-volatilization layer.
3. The amplification structure of claim 2, wherein: the anti-volatilization layer is provided as a non-volatile hydrophobic liquid film and/or a hydrophobic nanoparticle layer.
4. The amplification structure of claim 3, wherein: when the nonvolatile hydrophobic liquid film is adopted as the volatilization prevention layer, the boiling point of the nonvolatile hydrophobic liquid film is higher than that of the liquid drop.
5. The amplification structure of claim 4, wherein: when the non-volatile hydrophobic liquid film is adopted as the anti-volatilization layer, the non-volatile hydrophobic liquid film is set to be fluorine oil or silicon oil.
6. The amplification structure of claim 2, wherein: the anti-volatilization layer is formed by surfactant self-assembling into a film on the surface of the liquid drop.
7. The amplification structure of claim 2, wherein: and covering a layer of suspended film above the liquid drops on the suspended film to seal the liquid drops between the two layers of suspended films so as to form the anti-volatilization layer.
8. The amplification structure of claim 1, wherein: the heating device is a microwave container, the suspended film and the liquid drops are placed in the microwave container, and then the microwave container is utilized to heat the liquid drops.
9. The amplification structure of claim 1, wherein: the heating device is arranged to heat the micro-needle, and the micro-needle is inserted into the liquid drop on the suspended film to heat the liquid drop.
10. The amplification structure of claim 9, wherein: the heating micro-needle is set as a micro-needle of a microwave or ultrasonic probe.
11. The amplification structure of claim 1, wherein: heating device sets up to heating plate or heater strip, the heating plate or the heater strip sets up heat below the unsettled film.
12. The amplification structure of claim 1, wherein: and the heating device is provided with a temperature measuring device.
13. The amplification structure of claim 11, wherein: the heating sheet or the heating wire is set as a heating and temperature measuring micro resistance wire.
14. The amplification structure of claim 11, wherein: the suspended film and the heating wire or the heating sheet are integrated to form the micro-heater.
15. The amplification structure of claim 14, wherein: the liquid drops on the suspended film and the area below the suspended film are completely filled by the anti-volatilization layer, so that the micro-heater and the liquid drops on the micro-heater can be completely coated by the anti-volatilization layer.
16. The amplification structure of claim 14, wherein: the micro heater is suspended above the substrate, and at least two supporting conductive wires extend out of the micro heater and are fixed on the substrate to form a micro heater connecting terminal; the supporting conductive wire supports the balance when the micro-heater is suspended.
17. The amplification structure of claim 16, wherein: the substrate is provided with a groove, 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, and the micro-heater connecting terminal is formed.
18. The amplification structure of claim 17, wherein: four supporting conductive wires extend from the micro heater to the protruding edges of the grooves to be fixed and form micro heater connecting terminals, and the four supporting conductive wires support balance of the micro heater during suspension.
19. The amplification structure of claim 16, wherein: different electric signals are input to the connection terminals, so that the micro-heater realizes rapid change of temperature through heat conduction to the liquid drops.
20. The amplification structure of claim 1, wherein: and a hydrophobic or super-hydrophobic coating is arranged on the suspended film.
21. The amplification structure of claim 1, wherein: the suspended film is one film or a composite film formed by several films of silicon nitride, silicon oxide, a carbon film, a diamond film, a parylene film and a metal film.
22. The amplification structure of claim 1, wherein: also comprises a heat dissipation device.
23. The amplification structure of claim 22, wherein: the heat dissipation device is one or more of a thermoelectric refrigeration piece, a planar heat pipe or a microfluidic pipeline fluid which are arranged on the suspended film.
24. A rapid nucleic acid detection chip is characterized in that: comprising an amplification structure as defined in any one of claims 1 to 23.
25. The rapid nucleic acid detection chip of claim 24, wherein: and the suspended film is provided with a reflection enhancing coating.
26. A rapid nucleic acid detection device, characterized in that: comprising a plurality of the rapid nucleic acid detecting chips according to claim 24 or 25; in each rapid nucleic acid detection chip, a heating device is independently or uniformly used for heating the liquid drops on the suspended film so as to realize rapid temperature change.
27. An amplification method, characterized in that: the method comprises the following steps:
arranging a film in the air;
placing the liquid drop containing the amplified sample on a suspended film;
and periodically heating the liquid drop to realize circulation of the liquid drop at different temperatures and realize amplification.
28. The amplification method of claim 27, wherein: and an anti-volatilization layer is arranged outside the liquid drops.
29. The amplification method according to claim 28, wherein: the anti-volatilization layer is formed by adopting one or more of the following methods:
surfactant is used for self-assembly on the surface of the liquid drop to form an anti-volatilization layer;
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 drops to seal the liquid drops between the two layers of suspended films to form an anti-volatilization layer.
30. The amplification method of claim 27, wherein: the periodic heating of the liquid drops 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 placing the whole chip in a microwave oven, and heating the liquid drops and the suspended film by utilizing microwaves in a non-contact manner.
31. The amplification method of claim 27, wherein: while periodically heating the droplet, a droplet heating temperature is measured.
32. The amplification method of claim 31, wherein: and heating and measuring the temperature of the liquid drop on the suspended film by using a temperature measuring resistance wire.
33. The amplification method of claim 32, wherein: the method realizes the periodic heating of the liquid drop, realizes the circulation of the liquid drop at different temperatures, and comprises the following steps when the amplification is realized:
integrating the temperature measuring resistance wire and 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 droplets on the microheater with an anti-volatilization layer;
different electric signals are applied to the suspended micro-heater to realize the periodic heating of the liquid drop, so that the liquid drop is circulated at different temperatures to realize the amplification.
34. The amplification method of claim 33, wherein: further comprising the steps of:
placing the suspended micro-heater on a substrate with a groove, extending at least two supporting conductive wires out of the suspended micro-heater 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 drop and the area below the suspended film are completely filled by an anti-volatilization layer, so that the micro heater and the liquid drop on the micro heater can be completely coated by the anti-volatilization layer;
and applying different electric signals to the micro-heater connecting terminal of the suspended micro-heater to realize periodic heating of the liquid drop, so that the liquid drop is circulated at different temperatures to realize amplification.
35. The amplification method of claim 34, wherein: and a non-volatile hydrophobic liquid film is adopted as the anti-volatilization layer, and is set to be fluorine oil or silicone oil.
36. The amplification method of claim 27, wherein: the suspended film is one film or a composite film formed by several films of silicon nitride, silicon oxide, a carbon film, a diamond film, a parylene film and a metal film.
37. The amplification method of claim 27, wherein:
after the droplets are periodically heated, an additional heat dissipation device on the suspended film is used for dissipating heat of the droplets.
38. The amplification method of claim 27, wherein: and one or more modes of a thermoelectric refrigerating sheet, a planar heat pipe or a microfluidic pipeline fluid are adopted on the suspended film to dissipate heat of the liquid drops.
39. A rapid nucleic acid detection method is characterized in that: amplification of a test sample using the amplification method of any one of claims 27 to 38.
40. The method for rapid nucleic acid detection according to claim 39, wherein: also comprises the following steps
Adding a fluorescent label to the droplets prior to amplification;
after amplification, placing the amplified liquid drops containing the fluorescent marker in a fluorescence detection device to finish the fluorescence detection of the nucleic acid;
and judging the detection result according to the fluorescence brightness of the amplified liquid drop.
41. The method of rapid nucleic acid detection according to claim 40, wherein: the liquid drop contains a sample to be detected, an amplification primer, an enzyme, dNTP deoxyribonucleoside triphosphate, a template, a fluorescent probe and a buffer solution.
42. The method for rapid nucleic acid detection according to claim 40, wherein: and plating a reflection enhancing coating on the suspended film to enhance the fluorescence reflection signal.
43. A rapid large-scale detection method of nucleic acid is characterized in that: dividing the same sample or different samples into a plurality of droplets, and simultaneously detecting the plurality of droplets by using the rapid nucleic acid detection method according to any one of claims 39 to 42.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211406708.8A CN115449471B (en) | 2022-11-10 | 2022-11-10 | Amplification structure, rapid nucleic acid detection chip, device and method |
| CN202310756256.4A CN116731841A (en) | 2022-11-10 | 2022-11-10 | Microneedle heating amplification structure, rapid nucleic acid detection chip, device and method |
| CN202310756262.XA CN116716180A (en) | 2022-11-10 | 2022-11-10 | Microwave heating amplification structure, rapid nucleic acid detection chip, device and method |
| PCT/CN2023/126623 WO2024099079A1 (en) | 2022-11-10 | 2023-10-26 | Amplification structure, and rapid nucleic acid detection chip, device and method |
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| CN202211406708.8A CN115449471B (en) | 2022-11-10 | 2022-11-10 | Amplification structure, rapid nucleic acid detection chip, device and method |
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| CN202310756256.4A Division CN116731841A (en) | 2022-11-10 | 2022-11-10 | Microneedle heating amplification structure, rapid nucleic acid detection chip, device and method |
| CN202310756262.XA Division CN116716180A (en) | 2022-11-10 | 2022-11-10 | Microwave heating amplification structure, rapid nucleic acid detection chip, device and method |
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| CN202310756256.4A Pending CN116731841A (en) | 2022-11-10 | 2022-11-10 | Microneedle heating amplification structure, rapid nucleic acid detection chip, device and method |
| CN202211406708.8A Active CN115449471B (en) | 2022-11-10 | 2022-11-10 | Amplification structure, rapid nucleic acid detection chip, device and method |
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| CN202310756256.4A Pending CN116731841A (en) | 2022-11-10 | 2022-11-10 | Microneedle heating amplification structure, rapid nucleic acid detection chip, device and method |
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| WO2024099079A1 (en) * | 2022-11-10 | 2024-05-16 | 南方科技大学 | Amplification structure, and rapid nucleic acid detection chip, device and method |
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| WO2024099079A1 (en) | 2024-05-16 |
| CN115449471B (en) | 2023-06-02 |
| CN116731841A (en) | 2023-09-12 |
| CN116716180A (en) | 2023-09-08 |
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