TWI813125B - Method and device for nucleic acid amplification and method and device for nucleic acid detection - Google Patents

Abstract

The invention relates to a method and device for nucleic acid amplification and amplicon detection thereof. The nucleic acid amplification device comprises a reaction unit, an energy excitation unit, and an operation unit, and an auxiliary cooling unit. The reaction unit includes test analytes, a solution for nucleic acid amplification, and one or more solid phase carriers, each of which is functionalized with specific surface ligands for capturing target analytes and further nucleic acid amplification. By controlling the excitation energy outputs and the opening and closing sequence of the energy excitation unit, whereby an isolated environment is formed and an ultrafast thermal cycling cycle is generated around solid phase carriers. The present invention uses this device to perform a nucleic acid amplification method, and also uses a nucleic acid detection method and its device to detect amplicons on one or more solid phase carriers.

Description

Nucleic acid amplification method and device and nucleic acid detection method and device

The present invention relates to nucleic acid amplification methods and devices as well as nucleic acid detection methods and devices. In particular, it refers to a nucleic acid amplification method and a nucleic acid detection method that form an in-situ environment around a solid-phase carrier and perform amplification in the in-situ environment. Methods and devices for amplifying nucleic acids, as well as nucleic acid detection methods and devices after completing in-situ nucleic acid amplification.

Nucleic acid amplification tests (NAAT) have been widely used in molecular diagnosis, including infectious pathogen diagnosis, genetic testing, forensic medicine, agriculture and clinical medicine. Even if the substance to be tested contains a trace amount of the test substance, because the nucleic acid fragment to be detected has undergone nucleic acid amplification, the nucleic acid amplification test is highly sensitive and specific. Polymerase chain reaction (PCR) is a method for One of the target nucleic acid amplification test methods, its amplification to produce new deoxyribonucleic acid (DNA) strands requires three steps: (a) the temperature is raised to denature the double-stranded nucleic acid strand into single-stranded DNA (single- stranded DNA (abbreviated as: ssDNA) as a template; (b) the temperature drops so that the specific primer forms a bond with single-stranded DNA (ssDNA) through DNA sequence complementation; (c) new DNA is synthesized at the back end of the bonded primer by polymerase strands to produce double-stranded DNA (dsDNA) products. This is a temperature (thermal) cycle of PCR. The overall temperature includes 2~3 temperature intervals. The number of DNA molecules produced increases exponentially with circulation. However, traditional PCR instruments are generally large in size, expensive in equipment, high in energy consumption (using electric heating modules), and have long operation turnaround times (usually >1 hour).

In addition, isothermal Nucleic Acid Amplification Technologies (iNAATs), which have replaced traditional nucleic acid amplification tests (NAAT), have been developed, and their characteristics can amplify nucleic acid molecules at a constant and relatively moderate temperature. Compared with traditional nucleic acid amplification tests, iNAATs can significantly reduce the complex temperature (thermal) cycles, related complex equipment, and long operation time required for nucleic acid amplification. Since iNAATs do not require complex temperature (thermal) cycle design, simple instruments (such as heating plates, oven water baths) can be used to perform iNAATs reactions. Among various iNAATs methods/techniques, Loop-mediated isothermal amplification (LAMP) is the most widely used method, mainly because LAMP inhibits DNA polymerase (e.g., hemoglobin, immune Globulin IgG or IgM is well tolerated. In addition, LAMP nucleic acid amplification has high specificity, high sensitivity, and high amplification efficiency. This means that LAMP has the ability to be used from partially processed/or unprocessed biological samples. The potential to directly amplify target DNA or ribonucleic acid (RNA).

In addition, the paper titled "Loop-mediated isothermal amplification (LAMP)-review and classification of methods for sequence-specific detection" (published in Analytical Methods, publication date: February 14, 2020, paper publisher: Lisa Becherer , Nadine Borst, Mohammed Bakheit, Sieghard Frischmann, Roland Zengerle and Felix von Stetten), proposed that compared with traditional quantitative PCR (qPCR) or PCR, the sensitivity of LAMP-based nucleic acid amplification and detection is increased by 10 to 100 times.

In addition, the paper titled "Reduced False positives and Improved Reporting of Loop-Mediated Isothermal Amplification using Quenched Fluorescent primers" (published in Scientific Reports, publication date: May 14, 2019, paper published by: Patrick Hardinge & James AHMurray) mentioned that since 4 to 6 target-specific primers are used for nucleic acid amplification reactions, the specificity of LAMP should be better than traditional PCR using two primers. In addition, LAMP can generate more than 10 ⁹ DNA amplicons in 1 hour under isothermal conditions. By directly or indirectly detecting a large number of amplified DNA products or by-products of the LAMP reaction, it is possible to detect whether there is any detection in the sample. the existence of the subject matter. However, the high false positives and low discrimination of LAMP-based nucleic acid amplification and detection technology may limit its applicability in point-of-care testing.

The traditional PCR platform mainly uses electric heating elements (such as Peltier elements) to perform programmed heating and cooling to generate the temperature (thermal) cycle required for the polymerase chain reaction. This method uses heat conduction to allow Heat is transferred between the surface of the electric heating element and the reaction liquid, which often results in a long turnaround time due to its relatively slow temperature rise and fall rate (1.5~3°C/second). The paper titled "Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics" (published in Biosensors and Bioelectronics, publication date: June 18, 2019, paper publisher: Sang Hun Lee, Seung-min Park, Brian N. Kim, Oh Seok Kwon, Won-Yep Rho, Bong-Hyun Jun) proposed the key factors to achieve ultra-fast PCR, including rapid thermal cycling, low heat capacity materials, high thermal conductivity and high-speed polymerase. With the development of second-generation high-speed DNA polymerases, KAPA Biosystems, with its proprietary direct molecular evolution protein engineering technology, has screened a large number of DNA polymerase mutants through multiple rounds to screen out the second generation of high-speed DNA polymerases. The second generation KAPA2G Fast DNA polymerase has high-speed synthesis efficiency and its extension rate can be as high as 1Kb/second, which can significantly shorten the time required for DNA polymerase extension. In addition, in the past, in order to achieve rapid temperature (thermal) cycling, such as the capillary LightCycler ^® 2.0 Carousel-Based System (a rapid PCR detection instrument sold by Roche Diagnostics Co., Ltd.) or the microfluidic polymerase chain reaction, the reaction volume Limit it to nanoliters to microliters (nL~μL), thereby increasing the surface area to volume ratio to increase the effect of heat conduction, or using low heat capacity materials for heating, such as LightCycler® 2.0 Carousel-Based System uses air with low heat capacity to heat and cool to achieve rapid temperature (thermal) cycles. Although the overall nucleic acid amplification reaction can be completed in minutes, it is also accompanied by many technical problems, such as low amplicon yield. , Evaporation and bubble generation of trace reaction liquids, platform scalability and platform heat management. Therefore, rapid nucleic acid amplification is not just a simple implementation of rapid temperature (thermal) cycling, but a strategy for nucleic acid amplification that does not cause nucleic acid amplification reactions while achieving rapid temperature (thermal) cycling. produce adverse effects.

In response to the aforementioned problems, many research teams have studied and improved this problem, such as: Royal Institution for the Advancement of Learning McGill University, U.S. Invention Patent Publication No. US10,604,798B2 (Invention Name: HEATING MECHANISM FOR DNA AMPLIFICATION,EXTRACTION,OR STERILIZATION USING PHOTO-THERMAL NANOPARTICLES), the abstract mentions the use of photothermal nanoparticles in a contact and non-contact manner, under the stimulation of external light sources, to utilize this nano-level The heater is used to heat the reaction solution. Through this photothermal method, different temperature ranges are generated through external energy excitation with different energies for sterilization and photothermal lysis to achieve the extraction of nucleic acids and the amplification of specific nucleic acid fragments. Increase purpose. For example: The Regents of the University of California, US Patent Publication No. US11,130,993B2 (Invention Name: LED DRIVEN PLASMONIC HEATING APPARATUS FOR NUCLEIC ACIDS AMPLIFICATION), the abstract mentions a gold film with a specific thickness. The 3D structure reaction tank uses a plasma heating method generated by a light-emitting diode (LED; light-emitting diode) of a specific wavelength, thereby quickly generating the temperature (heat) required for the PCR reaction in the micro 3D structure reaction tank. ) loop. For example: University of Chicago, U.S.A. US11,045,874B2 invention patent publication number (invention name: BIPYRAMID-TEMPLATED SYNTHESIS OF MONODISPERSE NOBLE METAL NANOCRYSTALS), by synthesizing gold nanoparticles with a double pyramid configuration and generating plasmonic heating under the excitation of a light-emitting diode with a specific wavelength, PCR can be quickly generated in a micro reaction tank. The temperature (heat) cycle required for the reaction. The above methods all use precious metal materials and special configurations to convert light energy into thermal energy (reaction time) in the form of localized surface plasmon resonance (LSPR) under the excitation light of a specific wavelength light source. >100 picoseconds), such photothermal conversion characteristics can be regarded as a fast and immediate response. This nanoscale heater concept has a fast heating rate in the PCR thermal cycle and a uniform thermal field. By adjusting this nanoscale heater The concentration of particles and the energy intensity of incident light can easily achieve the purpose of temperature control. However, the above method still has some challenges and problems that need to be overcome, such as: (1) the evaporation problem of the reaction liquid in the micro reaction tank, (2) the cooling effect in the temperature (heat) cycle is not obvious, and external devices are still needed ( For example: fan) to achieve the purpose of cooling, (3) the incident light source required for localized surface plasmon resonance, its wavelength range may overlap with the spectrum of some organic fluorescent dyes, this phenomenon may limit its use in fluorescent Applications in light quantitative PCR.

In addition to the above-mentioned plasmonic heating method of photothermal nanoparticles, there is also a method of applying an alternating magnetic field to cause magnetic nanoparticles to generate heat, which has also been applied to PCR nucleic acid amplification technology. For example, the team of Xie Dabin of National Cheng Kung University, U.S. Invention Patent Publication No. US10,913,069B2 (invention name: METHOD AND DEVICE FOR POLYMERASE CHAIN REACTION). The patent request mentions a method for amplifying nucleic acids, including a reaction mixture containing target nucleic acids and a transition metal material in a reaction unit. Particle contact; and using electromagnetic radiation (EMR) with a frequency of approximately 200kHz to 500THz to irradiate transition metal material particles to generate the temperature (thermal) cycle required for PCR nucleic acid amplification. Similar commercial equipment such as Mic qPCR, which The world's first qPCR machine that uses electromagnetic heat as a heat source is a product manufactured and sold by Australian Bio Molecular Systems (BMS).

The above-mentioned heating methods using photothermal nanoparticles or magnetic nanoparticles, or traditional PCR using electric heating elements to heat the PCR reaction solution, overall, these heating methods belong to "volumetric heating" (volumetric heating) (that is, through Heating causes the temperature of the "whole reaction solution" to rise).

Different from this, the German molecular diagnostics company (GNA Biosolutions GmbH) uses original "Pulse Controlled Amplification (PCA) technology" to achieve ultra-fast PCR reactions. According to the literature name, it is "Ultra-fast PCR technologies for "point-of-care testing" paper (published in Journal of Laboratory Medicine, publication date: October 12, 2017, paper publisher: Lars Ullerich, Stephanie Campbell, Frank Krieg-Schneider, Federico Bürsgens and Joachim Stehr) and the United States US9,382,583B2 invention patent publication number (invention name: Method for the amplification of nucleic acids using heat transfer for nanoparticles, related application publications: DE102012201475B4, CN107604052A, EP2809806B1, WO2013113910A1) discloses this pulse-controlled nucleic acid amplification technology It is a technology that controls "localized heating" of an area. This technology integrates nanoparticles with photothermal conversion properties (primer functionalization), laser light sources, and large-volume reaction liquids for cooling purposes, through a short period of time. When pulsed laser light is irradiated (the interval time between laser light excitation is between 10 nanoseconds and 500 milliseconds), each time the laser light is irradiated on part of the gold nanoparticles, a special thermal radiation field required for the PCR reaction can be generated. This The thermal radiation field generated by this method is very rapid. Furthermore, this method only effectively heats the tiny environment of the surface area of the gold nanoparticles (containing their bound primers or amplicons), which (due to their high specific surface area) once the laser light stops illuminating the nanoparticles. Characteristics) will quickly cool to the temperature of the surrounding nucleic acid amplification reaction solution. During the overall PCR reaction process (that is, repeated temperature cycles), the temperature of the nucleic acid amplification reaction solution around the nanoparticles almost does not change. The above shows that PCA technology can quickly achieve the temperature required for nucleic acid amplification (such as PCR) (Thermal) circulation, this technology can not only greatly improve the evaporation problem of the reaction liquid that may be caused by the aforementioned "volume heating" (which will affect the accuracy of subsequent detection work), but also reduce the risk of traditional nucleic acid amplification (such as PCR). Cooling that requires special and precise control However, it still has its shortcomings, which will be explained in detail below.

In addition, in view of the aforementioned phenomenon of using "pulse controlled amplification technology" to create "regional local heating", this technology can not only use "photothermal particles" to achieve the above-mentioned local heating, but can also use other principles or mechanisms to produce "regional local heating". The phenomenon of "heating" is disclosed in the European invention patent publication No. EP3733292A1 of GNA Biosolutions (invention name: METHOD FOR CARRYING OUT A POLYMERASE CHAIN REACTION AND DEVICE FOR CARRYING OUT THE METHOD, related application publication: US2019/0249168A1 and the document name "Pulse -Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions" paper (published in PLOS NEGLECTED TROPICAL DISEASES, publication date: January 29, 2021, paper publisher: Katharina Müller, Sarah Daßen, Scott Holowachuk , Katrin Zwirglmaier, Joachim Stehr, Federico Buersgens, Lars Ullerich, Kilian Stoecker), which disclosed nucleic acid amplification technology using rapid energy pulse heating microcirculators (directly embedding micro-metal heating elements in the nucleic acid amplification reaction), through several When electric heating is applied in microseconds, the thermal radiation field generated instantly only occurs in the surface area of the miniature metal heating element (the designed nucleic acid amplification reaction area). After the above-mentioned energization state is released, the metal heating element will be instantly surrounded by The large-volume reaction liquid is cooled. By repeatedly controlling and generating the above-mentioned temperature field, this design can quickly generate the temperature (thermal) cycle required for nucleic acid amplification reactions (such as PCR), thereby achieving the purpose of rapid nucleic acid amplification.

It can be seen from the aforementioned patent cases and documents that GNA Biosolutions’ original “Pulse Controlled Amplification” (PCA) technology may realize ultra-fast nucleic acid amplification reactions (such as PCR) and subsequent nucleic acid amplification reactions. Molecular testing. Although this technology can significantly improve the transmission In traditional PCR reactions, the problem of reaction solution evaporation caused by "volume heating" can also reduce the need for special cooling designs or devices required in traditional nucleic acid amplification (such as PCR) processes.

However, this technology mainly dynamically controls the relative position of the laser light and the PCR reaction zone, so that the high-power laser beam passes through the PCR reaction zone at an interval of 10 nanoseconds to 500 milliseconds, thereby selectively and specifically irradiating the PCR Some gold nanoparticles in the reaction zone generate the temperature field required for the nucleic acid amplification reaction (as mentioned above). Basically, this kind of operation control has certain technical difficulties. In addition, the volume of PCR reaction solution (100~500μL) used for cooling purposes in PCA technology is relatively large, because the nucleic acid products in the PCA nucleic acid amplification reaction (for example: DNA) exists in the form of free single-stranded DNA (ssDNA) in the PCR reaction solution. Its method of detecting amplified nucleic acid molecules is as shown in European Invention Patent Publication No. EP2481817A1 (Invention Name: Process for detecting nucleic acids) As described in the invention patent publication No. EP3733292A1 and Müller et al. (2020), it is a polymer formed by detecting (primer functionalization) photothermal nanoparticles and amplifying free single-stranded DNA at 650 nanometers (nm ) spectral difference in wavelength, or it comes from the fluorescent signal generated by the TaqMan probe. Under the above nucleic acid detection method, using a large volume of PCR reaction solution (100~500μL) (due to the concentration dilution effect) will require more nucleic acid amplification times to accumulate nucleic acid amplification signals. This shortcoming will further affect the overall The time required for nucleic acid amplification and detection.

In addition, according to the PCA nucleic acid amplification method and device mentioned in the previous patent of GNA Biosolutions, there are still many parts that need improvement, which are summarized as follows:

1. Regional heating: Integrating suspended phase photothermal nanoparticles and large-volume PCR reaction solution, although it can achieve the "ultra-fast temperature (thermal) cycle" required for "ultra-fast nucleic acid amplification", and ultra-fast nucleic acid amplification During the process, the temperature of the PCR reaction solution does not change significantly. This feature can improve the problem of evaporation of the PCR reaction solution caused by the temperature (heat) cycle of the micro-PCR reaction solution in the "traditional rapid nucleic acid amplification" technology ( thus affecting the accuracy of detection). However, this technology mainly uses dynamic control of laser light The relative position to the PCR reaction zone allows the high-power laser beam to pass through the PCR reaction zone at an interval of 10 nanoseconds to 500 milliseconds, thereby selectively and specifically irradiating some of the gold nanoparticles in the PCR reaction zone. Generate the temperature field required for nucleic acid amplification reaction (as mentioned above). Basically, this kind of operation and control has certain technical difficulties.

2. GNA Biosolutions' previous patent case mentioned that the PCR used in the PCA nucleic acid amplification reaction adopts a two-step temperature program, which are 95°C required to denature the double-stranded DNA molecules and to bind the primers. and 65°C required for polymerase extension. Although this technology uses a large volume of PCR reaction solution as a cooling medium to reduce the gold nanoparticles from 95°C to 65°C, the temperature difference is only 30°C, so the cooling rate may not be as fast as claimed by the technology. The "instant cooling" effect.

3. This technology uses a large volume of PCR reaction solution as a cooling medium, so that the heated nanoparticles can be cooled down. However, the nucleic acid product of the PCA nucleic acid amplification reaction is produced in the PCR reaction solution in the form of free single-stranded DNA (ssDNA). Therefore, a large volume of the PCR reaction solution (i.e., dilution effect) will result in the need for more nucleic acid amplification. Increase the number of times to accumulate nucleic acid amplification signals. This shortcoming will further affect the time required for overall nucleic acid amplification and detection.

4. When faced with complex nucleic acid analytes: Traditional PCR nucleic acid amplification systems use double-primer designs. When faced with highly complex nucleic acid analytes, if some nucleic acid fragments have partial sequence similarities, they Partial heterozygous pairing will occur during the adhesion process, resulting in non-specific chimeric by-products, resulting in non-specific nucleic acid sequence amplification, which may lead to erroneous identification by point-of-care testing equipment.

Therefore, how to implement simple and rapid nucleic acid amplification detection while avoiding the above technical problems is still an issue that needs to be solved urgently.

In view of the problems of the prior art, one purpose of the present invention is to provide a simple and rapid operation and control to form the temperature (thermal) cycle required for a nucleic acid amplification reaction, and the nucleic acid amplification reaction will partially amplify nucleic acid molecules. Nucleic acid amplification method and device fixed on the surface of solid phase carrier. Another object of the present invention is that the nucleic acid on the surface of the solid phase carrier can be quickly purified, separated and concentrated, thereby facilitating the subsequent high specificity and high sensitivity detection and analysis of target nucleic acid molecules and methods and devices.

According to one object of the present invention, a nucleic acid amplification method is provided, which includes: a reaction unit including at least one analyte, a nucleic acid amplification reaction solution, and at least one solid phase carrier, wherein the reaction unit and its internal The test object, nucleic acid amplification reaction solution, and solid phase carrier are in a cooling environment, and the cooling environment has a cooling temperature; and the output of an external energy and its opening or closing timing are controlled, and at the same time, the cooling temperature of the cooling environment is used to form a process. One or more reaction temperature cycles required for nucleic acid amplification reactions, such that in each reaction temperature cycle, all solid-phase carriers are simultaneously excited by external energy and form an in-situ environment, or each solid-phase carrier stops receiving external energy and the original environment is formed. The in-situ environment gradually dissipates, allowing each solid-phase carrier, the analyte and the nucleic acid amplification reaction solution to perform a nucleic acid amplification reaction during the formation and dissipation of the in-situ environment, thereby producing an amplification reaction product.

Among them, the nucleic acid amplification reaction includes polymerase chain reaction (Polymerase Chain Reaction, PCR), ligase chain reaction (Ligase Chain Reaction, LCR) or isothermal nucleic acid amplification technology (Isothermal Nucleic Acid Amplification Technologies, iNAAT).

During the opening period of each reaction temperature cycle, all solid-phase carriers in the reaction unit are excited at the same time, so that an in-situ environment is formed around all solid-phase carriers, and amplification reactants are generated in each solid-phase carrier. A part of the amplification reaction product is retained in each solid phase carrier, and another part of the amplification reaction product is released into the nucleic acid amplification reaction solution, and during the shutdown period of each reaction temperature cycle During the period, the solid phase carrier is stopped to be excited, and the in-situ environment is gradually dissipated by the cooling temperature of the cooling environment.

Among them, the enzyme system can be a polymerase, and the polymerase further includes deoxyribonucleic acid polymerase (DNA polymerase), ribonucleic acid polymerase (RNA polymerase), and the enzyme can also be reverse transcriptase (RT), ribonuclease One or a combination of two or more (ribonuclease, RNase), helicases (helicases), DNA ligase (DNA ligase), can work synergistically with polymerase.

Among them, the external energy excitation method of each solid phase carrier includes contact excitation or non-contact excitation.

Among them, non-contact excitation includes photothermal excitation or magnetic excitation. The photothermal excitation system is one of the laser and LED arrays. The wavelength of light energy ranges from the visible spectrum to the near-infrared spectrum, and the wavelength range is 380 nanometers (nm) to 1.4 micrometers (μm).

Among them, the magnetic excitation system uses an alternating magnetic field to generate heat. The alternating magnetic field is formed by an alternating magnetic field generator, and the amplitude and frequency of the alternating magnetic field are set according to the field conditions of the temperature required for the solid-phase carriers to produce nucleic acid amplification reactions. Furthermore, the amplitude of the alternating magnetic field is 0.5~550kA/m and the frequency of the alternating magnetic field is 3~3,500kHz.

Among them, the contact excitation system is electric heat generation, the electric energy transmission can be electronic circuits and induced magnetic flux for electric energy transmission (wireless charging), and the electric heat generation system can be Joule heating, thermoelectric heating and surface acoustic waves. (surface acoustic waves; SAWs) generate heat.

Among them, the ratio of the total volume of all solid phase carriers to the volume of the nucleic acid amplification reaction solution is 1:200 to 1:1*10 ⁹ .

Among them, the size of each solid phase carrier is 8 to 2,000,000 nm, and the preferred size of the solid phase carrier for photothermal excitation and magnetovariation excitation for heat generation is 8 to 1,000 nm; the preferred size of the solid phase carrier for electrothermal excitation is 8 to 1,000 nm. 1,000~2,000,000 nanometers.

Among them, each solid phase carrier system is one of sphere, ellipse, disk, star, rod, square, anisotropic protrusion, nanoshell, nanocage, double triangular pyramid, and microfilament. One or any combination of two or more.

Wherein, each solid phase carrier may be one of, or any combination of two or more, suspended in the reaction unit or each solid phase carrier fixed to the inner wall of the cavity of the reaction unit.

Among them, the cooling temperature range is -10~50℃.

Among them, the reaction unit or the nucleic acid amplification reaction solution can be pre-cooled to the cooling temperature in advance, or be housed in an external auxiliary cooling unit. The reaction unit or the nucleic acid amplification reaction solution can be pre-cooled, or the external auxiliary cooling unit One or more reaction temperature cycles are performed while maintaining the cooling temperature.

Wherein, the external auxiliary cooling unit is one or a combination of any two or more of ice, polymeric water-absorbent resin, chemical endothermic reactant and refrigeration chip. The high-molecular polymer water-absorbent resin is carboxymethyl cellulose, and the chemical endothermic reactant is the endothermic reaction caused by ammonium nitrate dissolving or urea dissolving in water.

Wherein, the substances to be tested are cells, organelles, bacteria, viruses, protists or combinations thereof, and each solid phase carrier system includes a multifunctional body, at least one enrichment ligand and at least one amplification ligand, wherein each The enrichment ligands are connected to the surface of the multifunctional body, and each enrichment ligand provides binding to the test substance. The amplification ligands are connected to the surface of the multifunctional body, and each amplification ligand provides binding to biological substances. The biological substance is DNA or ribonucleic acid released from the substance to be tested, or in the amplification preparation. After replication, the amplification reaction product is released into the nucleic acid amplification reaction solution. Wherein, the enrichment ligand can be an antibody, aptamer, oligonucleotide, protein, polysaccharide or a combination thereof. Among them, the amplification ligand can be an aptamer, an oligonucleotide, and has a functional group modification for bonding to a solid-phase carrier, which can be modified with a primary amine, biotin, thiol group, or a combination thereof; adding oligonucleotides The specificity and stability modification of acid capture of biological substances can be modified by locked nucleic acid (LNA), phosphorothioates, morpholino, or a combination thereof.

Among them, the substances to be tested are cells, organelles, bacteria, viruses, protists or combinations thereof. And part of the solid phase carrier system includes a enrichment body and at least one enrichment ligand, wherein each enrichment ligand is connected to the surface of the enrichment body, and each enrichment ligand provides binding to the analyte, and another A part of the solid phase carrier system includes an amplification body and at least one amplification ligand. Each amplification ligand is connected to the surface of the amplification body, and each amplification ligand provides biological substances released by binding to the test substance. The biological substance is DNA or ribonucleic acid released from the substance to be tested, or an amplification reaction product that is released into the amplification reaction solution after the amplification ligand is replicated. Among them, the enriched ontology is equivalent to the multifunctional ontology that only has enriched ligands. Among them, the amplification body is equivalent to the multifunctional body only having the amplification ligand.

The substance to be tested is a free DNA or ribonucleic acid substance, and each solid-phase carrier system includes an amplification body and at least one amplification ligand, and each amplification ligand is connected to the surface of the amplification body, And each amplification ligand provides binding to the analyte.

According to one object of the present invention, a rapid nucleic acid amplification system is provided, including a reaction unit, an auxiliary cooling unit, an external energy excitation unit, and an integrated driver; wherein the reaction unit is provided to accommodate a reaction solution, a test object, and nucleic acid amplification The reaction solution and at least one solid phase carrier; wherein the auxiliary cooling unit can be a nucleic acid amplification reaction solution that has been pre-cooled to a cooling temperature, or the reaction unit can be continuously cooled by an external auxiliary cooling unit arranged around the reaction unit, and make the nucleic acid amplification reaction solution Maintained at cooling temperature; the external energy excitation unit provides a solid-phase carrier to stimulate heat generation; the integrated driver is used to control the energy output, opening and closing timing of the external energy excitation unit, and can be based on the external energy corrector It performs energy output with the temperature detection unit and provides feedback control to the external auxiliary cooling unit to form one or more reaction temperature cycles required for nucleic acid amplification reactions.

According to another object of the present invention, a nucleic acid detection method is provided. After the nucleic acid amplification method is completed, the nucleic acid detection is carried out in the following steps. An operating unit is used to assist in purification, separation and concentration of the nucleic acid bound to the solid phase carrier. The amplification reaction product uses a detection module to identify one or any of the optical changes, thermal sensing changes, electrochemical changes, magnetic changes or electrical quality changes produced on the amplification reaction product on the solid phase carrier. A combination of two or more. The detection module further identifies the amplification reaction product bound to the solid phase carrier.

Wherein, the method for producing optical changes in the amplification reaction product includes mixing a nucleic acid label into the amplification reaction product and using a photometer to detect the light intensity, or using an enzyme-binding immunosorbent assay to detect the amplification reaction product. Optical changes or chemiluminescence changes, or spectral changes produced by the combination of the solid-phase carrier and the amplification reactant.

Among them, the detection module is a nucleic acid lateral flow test strip detection or an immune lateral flow test strip, which is used to detect the optical changes produced on the amplification reaction product.

Among them, the nucleic acid lateral flow test strip detection or immune lateral flow test strip detection is supplemented by thermal sensor detection, surface plasmon resonance spectroscopy, or any combination thereof to enhance the nucleic acid lateral flow test strip detection or immune lateral flow test strip detection. Reaction sensitivity of lateral flow test strip assays.

Wherein, the method for producing electrochemical formula changes on the amplification reaction product includes one or a combination of electrochemical detection coupled with enzyme-binding immunosorbent assay, electrical impedance spectroscopy (EIS) detection.

Among them, the method for producing magnetic changes in the amplification reaction product includes AC magnetic introduction instrument detection and giant magnetoresistance detection or a combination of the two.

Wherein, the method for producing changes in electrical mass on the amplification reaction product is a quartz crystal microbalance.

According to another object of the present invention, a rapid nucleic acid detection device is provided, which includes an operation unit and a detection module. The operation unit assists in purification, separation, and concentration of solid phase carriers, and the detection module identifies amplified nucleic acids on the solid phase carrier. One or a combination of any two or more of optical changes, thermal sensing changes, electrochemical changes, magnetic changes or electrical mass changes produced on the reactants is used to detect the amplification reactants bound to the solid-phase carrier. .

In summary, the present invention is different from existing nucleic acid amplification methods and has the following advantages:

(1) Simply by controlling the characteristics of external energy excitation (such as energy size and frequency) and the cooling effect in the reaction unit, the nucleic acid molecules required for amplification can be rapidly generated in the in-situ environment on the surface of the solid phase carrier. Temperature cycling, thereby achieving the purpose of rapid nucleic acid amplification.

(2) The nucleic acid amplification reaction solution itself is first lowered to the cooling temperature, or the reaction unit is placed in a cooling environment through an auxiliary cooling unit, which can inhibit non-target primer adhesion and amplification of non-target nucleic acids, thus increasing the target nucleic acid molecules. Amplified specificity.

(3) The space on the surface of the solid-phase carrier is limited. Therefore, in a relatively short operating time (or a relatively small number of nucleic acid amplification cycles), the immobilized amplified nucleic acid molecules on the surface of the solid-phase carrier can achieve saturated state, which will facilitate subsequent detection of amplified nucleic acid molecules.

(4) If the solid phase carrier has strong magnetism, the amplification reaction products produced by nucleic acid amplification on the solid phase carrier can be further purified and concentrated through the magnetic field operation of the operating unit to further purify and concentrate these fixed phase carriers. This technical characteristic will be beneficial to the detection of subsequent amplification reactions (for example, improving the detection efficiency).

(5) Since the entire nucleic acid amplification reaction solution is maintained at a low temperature, it not only avoids the problems of evaporation and bubble generation of the nucleic acid amplification reaction solution, but also has the advantage of inhibiting or reducing non-specific nucleic acid amplification.

10:Reaction unit

11:Integrated drive

12: Energy excitation unit

121:Laser collimator

122:Laser launcher

13:External auxiliary cooling unit

14: Temperature detection unit

15: Operating unit

16:External energy corrector

17:Detection module

171:Immune lateral flow test strip

1711:Test line

1712:Control line

172:Laser composite infrared thermal sensor

20:Object to be tested

21:Biological substances

30: Nucleic acid amplification reaction solution

301:Introduction

3011: Fluorescent Marker

302:Polymerase

40:Solid phase carrier

401:Magnetic core

402:Middle layer

403: Nano gold shell

41:Multifunctional body

42:Amplified ontology

43: Enrichment Ontology

50: In situ environment

60:Amplification reaction product

70: Enriched ligands

71:Amplification ligand

72:Polyethylene glycol

73:Bovine serum albumin

S10~S90: steps

S100~S400: steps

Figure 1(a) is a schematic diagram of rapid temperature rise according to one embodiment of the present invention; Figure 1(b) is a schematic diagram of rapid temperature reduction according to one embodiment of the present invention; Figure 2 is a schematic diagram of the structure and surface characteristics of the solid phase carrier in one embodiment of the present invention. Schematic diagram; Figure 3 is a schematic structural diagram of a nucleic acid amplification system according to an embodiment of the present invention; Figure 4(a) is a suspension state of the solid phase carrier mixed into the nucleic acid amplification reaction solution in the reaction unit according to one embodiment of the present invention. Schematic diagram; Figure 4(b) shows that in one embodiment of the present invention, the solid phase carrier is semi-fixed to the bottom of the reaction tank in a bonded manner in the reaction unit, and bonded to the inner wall of the cavity of the reaction unit in a semi-suspended manner; Figure 4(c) is a schematic diagram of the solid phase carrier being fixed on the inner wall of the cavity of the reaction unit in an embodiment of the invention; Figure 5 is an embodiment of the invention, using a solid phase carrier with photothermal conversion capability for water cooling A schematic diagram of the structure of a nucleic acid amplification system based on the formula on-bead polymerase chain reaction; Figure 6 is a schematic diagram of an operating unit for purifying a solid-phase carrier according to an embodiment of the present invention; Figures 7(a) to (e) are an implementation of the present invention. Schematic diagram of the first reaction process of water-cooled on-bead PCR in the example; Figure 7(f)~(g) shows the second reaction process after completing Figure 7(a)~(e) Schematic diagram; Figure 7(h)~(k) is a schematic diagram of the third reaction process after completing Figure 7(f)~(g); Figure 7(l) is a schematic diagram of completing multiple reaction processes; Figure 8 is a schematic diagram of the nucleic acid of the present invention Flow chart of amplification method and detection method; Figure 9(a) is a schematic diagram of a primer pair for an isothermal cyclic amplification reaction (LAMP) according to an embodiment of the present invention. Figure 9(b)~(e) is a schematic diagram of Figure 9( a) Schematic diagram of pulsed water-cooled on-bead isothermal circular amplification reaction (LAMP); Figure 10(a) shows an embodiment of the present invention. The solid-phase carrier excites for 60 seconds and then stops excitation for 120 seconds and repeats continuously for 10 seconds. A schematic diagram of the temperature measurement of photothermal conversion; Figure 10(b) is a schematic diagram of the temperature measurement of a solid-phase carrier excited at different powers in one embodiment of the present invention; Figure 11(a) is a schematic diagram of gold nanoparticles used in one embodiment of the present invention. Magnetic Gold Nanoshells (MGNs) micro-scale suspension, the relationship between the laser intensity applied to it and the photothermal conversion temperature of the nano-gold magnetic shell; Figure 11(b) shows the micro-scale gold nanoshell suspension at night Infrared thermography images after excitation with different powers; Figure 12(a) is a schematic diagram of the appearance of one embodiment of the auxiliary cooling unit of the present invention; Figure 12(b) is the auxiliary cooling unit of Figure 12(a) giving nucleic acid amplification The enhanced reaction solution contains a single phase under 100 external laser excitation cycles (400 milliwatt (mW)/0.16 square centimeters (cm ² ), irradiation for 1.25 seconds; off for 0.5 seconds; 150mW/0.16cm ² for 7.5 seconds). The overall temperature change of the nucleic acid amplification reaction solution of the nanogold magnetic shell; Figure 13(a) is a schematic diagram before detection of the polymerase chain reaction on water-cooled beads using immune lateral flow test strips according to one embodiment of the present invention; Figure 13(b) is a schematic diagram of the detection results of water-cooled bead polymerase chain reaction using immune lateral flow test strips according to an embodiment of the present invention; Figure 14(a) is a schematic diagram of Figure 13(b) using plasma A schematic diagram of the positive reaction of the immune lateral flow test strip using plasma thermal sensing to detect the detection result of solid-phase carrier nucleic acid amplification; Figure 14(b) shows the analysis of Figure 13(b) using plasma thermal sensing Immune lateral flow test strip to detect the negative reaction of the solid-phase carrier nucleic acid amplification test result; Figure 15(a) shows an embodiment of the present invention using an 808nm wavelength laser to perform water-cooled on-bead polymerase chaining Reaction, with laser cycle time cycle (400mW/0.16cm ² irradiation for 1.25 seconds, 150mW/0.16cm ² irradiation for 7.5 seconds), under different laser cycle time cycles (10~50 cycles), the results on the nanogold magnetic shell A schematic diagram of the detection results of the amplification reaction detected with an immune lateral flow test strip; Figure 15(b) shows an embodiment of the present invention using a 808 nm wavelength laser to perform a water-cooled bead polymerase chain reaction. Under the optimal laser Excitation conditions and the shortest laser cycle time (400mW/0.16cm ² irradiation for 1.25 seconds, 150mW/0.16cm ² irradiation for 7.5 seconds), different laser cycle times (10~30 times), on the nanogold magnetic shell A schematic diagram of the electrophoresis analysis results of the amplification reaction product and the amplification reaction product remaining in the nucleic acid amplification reaction solution; Figure 16(a) is a schematic diagram of the electrophoresis analysis results after the test substance is a simple sample and subjected to a traditional polymerase chain reaction; Figure 16(b) is a schematic diagram of the detection results when the analyte is a simple sample and is tested using a water-cooled bead polymerase chain reaction followed by an immune lateral flow test strip; Figure 17(a) is a complex sample where the analyte is ( A schematic diagram of the electrophoresis analysis results after a traditional polymerase chain reaction (mixed nucleic acids of various pathogenic bacteria and E. coli nucleic acid samples); Figure 17(b) shows the complex sample to be tested after a water-cooled bead polymerase chain reaction. Schematic diagram of the detection results using immune lateral flow test strips; Figure 18(a) shows a suspension of mixed enrichment entities and amplification entities in one embodiment of the present invention, using 808nm wavelength laser with different laser powers After the photothermal lysis reaction of bacterial cells is carried out by excitation, a schematic diagram of the detection results of the colony formation test is shown; Figure 18(b) is a suspension of mixed enriched entities and amplified entities in one embodiment of the present invention, using 808nm wavelength laser with different After photothermal cleavage with laser power, a water-cooled on-bead polymerase reaction is performed, and a schematic diagram of the detection results is performed with an immune lateral flow test strip; Figure 19(a) shows the mixing of two functional magnetic properties in an embodiment of the present invention. Nano-gold magnetic shell, in which Escherichia coli suspension is used as the test object, the detection efficiency and the overall operation flow of water-cooled polymerase chain reaction on water-cooled beads are evaluated by laser excitation; Figure 19(b) is an embodiment of the present invention The suspension of the enriched entity and the amplified entity is mixed in the suspension, in which the Escherichia coli suspension is used as the test substance, and the immune lateral flow test strip is used to evaluate the laser-excited water-cooled on-bead polymerase chain reaction (on-bead PCR). Schematic diagram of sensitivity test results; Figure 20(a) is an image of the immune lateral flow test strip in Figure 19(b) located at the test line. Image analysis software (Image J) was used to perform pixel intensity analysis and a schematic diagram of image quantification. .

Figure 20(b) shows the thermal image of the immune lateral flow test strip under different 808nm laser energy excitation (24mW and 140mW), using an infrared lens to focus on the test line, and the thermal image minus the environment A quantitative graph of the temperature difference that rises after a background temperature.

Figure 21(a) is a schematic diagram of the temperature changes of laser-excited water-cooled polymerase chain reaction on beads at different PCR reaction starting temperatures.

Figure 21(b) is a schematic diagram of the efficiency evaluation of laser-excited water-cooled polymerase chain reaction on beads at different PCR reaction starting temperatures.

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but are not used to limit the present invention.

The present invention is a nucleic acid amplification method that provides a test object 20, a nucleic acid amplification reaction solution 30, and at least one solid phase carrier 40 inside a reaction unit 10, and the reaction unit 10 is in a cooling environment that reaches the required cooling temperature. The temperature system is -10~50℃, the preferred cooling temperatures are 15-30℃, 30-50℃, the preferred cooling temperatures are -10~0℃, 2~15℃, by controlling the operating conditions of external energy excitation ( For example: energy and frequency), coupled with the cooling effect provided by the cooling environment of the reaction unit 10, an in-situ environment 50 is formed inside the reaction unit 10 around the surface of the solid phase carrier 40, and in-situ nucleic acid amplification is performed in the in-situ environment 50. The temperature (thermal) cycle required for the reaction causes an in-situ nucleic acid amplification to be performed inside the reaction unit 10 on the solid-phase carrier 40 during each temperature cycle to produce the amplification reactant 60 . It should be noted here that the temperature (thermal) cycle required to perform the nucleic acid amplification reaction inside the reaction unit 10 refers to the different temperatures required to denature the nucleic acid molecules, bind the primers, and extend the polymerase. However, in actual implementation, It is not limited to this. The size of each solid phase carrier 40 is 8 to 2,000,000 nanometers (nm), among which the preferred size of the solid phase carrier 40 for photothermal and magnetic heating is 8 to 1,000 nanometers; the preferred size of the solid phase carrier 40 for electrothermal heating is is 1,000~2,000,000 nanometers.

For the definition of the "in-situ environment 50" of the present invention, please refer to Figure 1. When the solid phase carrier 40 inside the reaction unit 10 is in an ideal and evenly distributed state, by controlling the operating conditions of external energy excitation (Figure 1(a)), and the cooling temperature of the cooling environment in the reaction unit 10, the size of the outward radiation range of the thermal radiation field of the solid phase carrier 40 can be adjusted, and the thermal radiation fields on the surface of the solid phase carrier 40 are incompatible with each other. Overlapping each other forms an independent nucleic acid amplification reaction space, or is called the regional heating range of the solid phase carrier 40 (shown in FIG. 1(b) ), which is referred to as the in-situ environment 50 in the present invention. In addition, when the excitation of external energy stops instantaneously, the cooling temperature of the reaction unit 10 is used to enable the solid phase carrier 40 to quickly achieve the purpose of cooling.

In addition, the definition of "in-situ nucleic acid amplification reaction" in the present invention means that the nucleic acid amplification reaction occurs in the in-situ environment 50 on the surface of the solid phase carrier 40, and the amplification reaction product 60 will be fixed on the solid phase. On the surface of the carrier 40, the amplification reactants 60 include but are not limited to amplified nucleic acid molecules, and a portion of the amplification reactants 60 produced by the in-situ nucleic acid amplification reaction remain on each solid phase carrier 40. The other part of the amplification reaction product 60 releases the amplification reaction product 60 into the nucleic acid amplification reaction solution 30 .

In the present invention, the nucleic acid amplification reaction solution 30 includes free primers 301, nucleotides, enzymes and reaction additives. The enzyme system can be polymerase 302. The polymerase 302 further includes deoxyribonucleic acid polymerase (DNA polymerase) and ribonucleic acid polymerase (RNA polymerase). The enzyme can also be reverse transcriptase (RT) or ribonuclease. One or a combination of two or more (ribonuclease, RNase), helicases (helicases), DNA ligase (DNA ligase), can work synergistically with polymerase.

In the present invention, the nucleic acid amplification reaction may be a polymerase chain reaction (Polymerase Chain Reaction, PCR), a ligase chain reaction (Ligase Chain Reaction, LCR), or an isothermal nucleic acid amplification reaction (Isothermal Nucleic Acid Amplification Technologies). , iNAATs), Among the many iNAATs methods, the isothermal loop amplification reaction (Loop-mediated isothermal Amplification, LAMP) is preferred, in which a 2-step temperature (thermal) cycle program is used in the polymerase chain reaction method, in which the temperatures used for nucleic acid denaturation are located at 85~95℃, the temperature for primer binding and polymerase extension is 60~65℃; the temperature for isothermal circular amplification reaction is 60~65℃.

In one embodiment of the present invention, referring to Figure 2, the solid phase carrier 40 includes a multifunctional body 41, at least one enrichment ligand 70 and at least one amplification ligand 71, wherein the enrichment ligand 70 is connected to the multifunctional body 41, the enrichment ligand 70 can be an antibody, an aptamer, an oligonucleotide, a protein, a polysaccharide, or a combination thereof, wherein the amplification ligand 71 is connected to the surface of the multifunctional body 41, and the amplification ligand 71 can Aptamers and oligonucleotides have functional group modifications for bonding with solid-phase carriers, which can be modified with primary amines, biotin, thiol groups, proteins, polysaccharides or combinations thereof; increasing the number of oligonucleotides and The specificity and stability modification of biological substance capture can be modified by locked nucleic acid (LNA), phosphorothioates (phosphorothioates), morpholino (morpholino) or a combination thereof. However, when the present invention is actually implemented, Not limited to this.

The enrichment ligand 70 selected in this embodiment can match the aforementioned analyte 20. The analyte 20 referred to here can be a cell, an organelle, a bacterium, a virus, a protist, or a combination thereof. The enrichment ligand 70 specifically binds the analyte 20 and adsorbs the analyte 20 on the enrichment ligand 70 of the solid-phase carrier 40 .

In this embodiment, at least one amplification ligand 71 is provided on the multifunctional body 41, which can bind to the free primer in the nucleic acid amplification reaction solution 30. Furthermore, the amplification ligand 71 can be bound to the biological substance 21. The biological substance 21 can be a nucleic acid molecule of the test object 20 released at high temperature, or can be released into the nucleic acid after the amplification ligand is replicated. Amplification reaction product 60 of amplification reaction solution 30. In addition, the amplification reaction product 60 can be combined with the labeled primer 301 in the nucleic acid amplification reaction solution 30. The label in the labeled primer 301 is a nucleic acid label, where the nucleic acid label includes but is not limited to radioactive isotopes (such as : ³ H, ¹⁴ C and ³² P, etc.), digoxin (DIG), biotin (biotin), fluorescent label 3011 (for example: fluorescent isothiocyanate (FITC), Dik Texas Red, Cy2, Cy3, Cy5, Cy7, rhodamine B, etc.) and luminescent substances (for example: acridinyl ester (2',6'-DiMethylcarbonylphenyl-10-sulfopropylacridiniuM-9-carboxylate 4'-NHS Ester)), but when the present invention is actually implemented, it is not limited to this.

In this embodiment, the surface of the solid phase carrier 40 has been processed by at least one or more procedures to form functionalized enrichment ligands 70 and amplification ligands 71 with extremely high specificity, thereby targeting trace amounts. The analyte 20 is analyzed.

In another embodiment of the present invention, the analyte 20 can be a cell, an organelle, a bacterium, a virus, a protist, or a combination thereof, and a portion of each solid phase carrier 40 includes an enrichment body and at least one enrichment compound. body 70, and another part of each solid phase carrier 40 includes an amplification body 42 and at least one amplification ligand 71, wherein the enrichment body is connected to the enrichment ligand 70, and the enrichment ligand 70 provides contact with the test substance 20 is matched and combined, the amplification body 42 is connected to the amplification ligand 71, and the amplification ligand 71 provides the biological substance 21 released by binding to the test object 20, or is bound to the amplification ligand 71 and then released to the Amplification reaction product 60 of nucleic acid amplification reaction solution 30.

In yet another embodiment of the present invention, the substance to be tested 20 may be free DNA or ribonucleic acid substances. In addition, free nucleic acid substances include body fluid free nucleic acid substances, tumor free nucleic acid substances or combinations thereof. Each solid phase carrier 40 includes an amplification body 42 and at least one amplification ligand 71. The amplification body 42 is connected to the amplification ligand 71. , and the amplification ligand 71 provides binding to the test substance 20, or binds to the amplification reaction product that is released into the nucleic acid amplification reaction solution 30 after the amplification ligand 71 is copied.

In various embodiments of the present invention, in order to enable the solid-phase carrier 40 to adapt to different external energy excitation methods, different external energy excitation methods for the solid-phase carrier 40 include instant heating through photothermal conversion or high-frequency induction electromagnetic methods. , where the surface structure of the solid phase carrier 40 can be treated with plasma Heat, or the surface layer of the solid phase carrier 40 is an organic photothermal coating, which can be heated by light energy; or the solid phase carrier 40 is a magnetic solid phase carrier 40, which can be applied to the area of the magnetic solid phase carrier 40 Heating, by placing the magnetic solid phase carrier 40 in an external magnetic field whose frequency should be within the radio frequency range (alternating magnetic field) where the amplitude of the alternating magnetic field is 0.5~550kA/m and the frequency of the alternating magnetic field is 3~3,500kHz. , to achieve the required temperature range for nucleic acid denaturation, primer binding and polymerase extension.

Furthermore, the multifunctional body 41, the enrichment body, and the amplification body 42 of the solid phase carrier 40 can be of different types. In one embodiment of the present invention, the solid phase carrier 40 is excited by "photothermal". In other words, the solid phase carrier 40 can be implemented in the form of a spherical body made of metal or composite materials, including a magnetic core 401, and an intermediate support layer 402 that stabilizes and maintains the outer photothermal conversion layer structure, where The support layer can be a silicon shell, and its overall configuration can be spherical, elliptical, disc-shaped, star-shaped, rod-shaped, square, anisotropically convex (such as nanostar), nanoshell, nanometer One of cage, double triangular pyramid, microfilament, or a combination of any two or more forms is not limited to this form. ; The magnetic core 401 contains transition metals and must also include their oxides, such as ferrous oxide (FeO), iron oxide (Fe ₂ O ₃ ), ferric oxide (Fe ₃ O ₄ ), iron oxide (hydroxide) ( FeO(OH)), ferrous hydroxide (Fe(OH) ₂ ), ferric hydroxide (Fe(OH) ₃ ), cobalt monoxide (CoO), cobalt oxy(hydroxide) (CoO(OH)), The present invention is not limited to the group consisting of cobalt tetroxide (Co ₃ O ₄ ) and its derivatives and mixtures. The surface of the solid phase carrier 40 is made of a noble metal layer with a regional surface plasmon resonance (LSPR) effect, such as It is composed of gold, silver, palladium, platinum or their combination or an organic layer with photothermal conversion efficiency. For example, it can be incorporated with cyanine with near-infrared (Near Infrared Spectroscopy, NIR) absorption spectrum. Materials such as polypyrrole or graphene.

Please refer to Figure 3. The present invention provides a rapid nucleic acid amplification system, which includes a reaction unit 10, an integrated driver 11, an energy excitation unit 12, and an auxiliary cooling unit. Among them, the reaction unit 10 is provided to accommodate the nucleic acid amplification reaction solution 30 and a plurality of solid phase carriers 40, wherein the solid phase carriers 40 are suspended in The nucleic acid amplification reaction solution 30 (Fig. 4(a)) is either bonded to the inner wall of the cavity of the reaction unit 10 in a semi-suspended manner (Fig. 4(b)) or fixed to the inner wall of the cavity of the reaction unit 10 (Fig. 4(c) )), the auxiliary cooling unit can be in the following forms:

1. The nucleic acid amplification reaction solution 30 (volume of at least 100 μL) is the auxiliary cooling unit. It first uses the external auxiliary cooling unit 13 to cool the nucleic acid amplification reaction solution 30 to the cooling temperature, and then introduces it into the reaction unit 10, so that the reaction unit The nucleic acid amplification reaction solution 30 within 10 serves as an auxiliary cooling unit. In this form, after external energy is provided, the nucleic acid amplification reaction solution 30 will gradually heat up and continue to maintain the cooling temperature during the reaction. Therefore, the nucleic acid amplification reaction The efficiency is worse than the auxiliary cooling unit described below, but the overall operation time is still shorter than the traditional polymerase chain reaction nucleic acid amplification reaction.

2. The nucleic acid amplification reaction solution 30 and the external auxiliary cooling unit 13 together form an auxiliary cooling unit. The external auxiliary cooling unit 13 is arranged around the reaction unit 10 (as shown in Figure 5), and the external auxiliary cooling unit 13 continues to respond to the reaction. The surrounding of the unit 10 provides a cooling temperature, so that the temperature of the nucleic acid amplification reaction solution 30 in the reaction unit 10 drops and maintains the cooling temperature, and the external auxiliary cooling unit 13 can further be a temperature detection unit 14 (for example: K-type thermocouple) Temperature changes of the external auxiliary cooling unit 13 are detected in real time and feedback control of the temperature is implemented, so that the nucleic acid amplification reaction solution 30 inside the reaction unit 10 is continuously at a cooling temperature, so that the nucleic acid amplification reaction can be performed quickly.

In addition, the external auxiliary cooling unit 13 can be made of ice or high-molecular polymer water-absorbing resin (such as carboxymethyl cellulose) or cooling chip (such as Peltier Cooler) with large specific heat capacity. The external auxiliary cooling unit 13 can also be selected with water (0.5 milliliters (mL) ~ 1mL) or can be selected with water containing, for example, ammonium nitrate (Molar heat of solution (△Hsol) = 26.2 kilojoules (KJ)), urea (Molar heat of solution) △Hsol=15KJ) is a chemical endothermic reaction caused by its crystal dissolving in water.

Referring to FIG. 3 , the energy excitation unit 12 directly and efficiently provides external energy to the solid-phase carrier 40 and converts it into thermal energy in a contact or non-contact energy transfer manner. When this energy excites the unit The moment the element 12 stops providing energy to the solid phase carrier 40, the cooling temperature provided by the auxiliary cooling unit is used to quickly dissipate the heat of the solid phase carrier 40 through the interaction and regulation between the aforementioned energy excitation unit 12 and the auxiliary cooling unit. , the present invention can quickly form the temperature (thermal) cycle required for the nucleic acid amplification reaction in the in-situ environment 50 around the solid-phase carrier 40, and complete the nucleic acid amplification in the form of in-situ nucleic acid amplification on the surface of the solid-phase carrier 40. amplification reaction.

In the present invention, the contact excitation system is electrothermal heat generation. The electric energy transmission can be carried out by electronic circuits and induced magnetic flux (wireless charging). The electrothermal heat generation system can be Joule heating (Joule heating) or thermoelectric heating (Thermoelectric heating). Generate heat with surface acoustic waves (SAWs).

In the present invention, the non-contact excitation method of the solid phase carrier 40 is preferably a plasma heating method. As shown in FIG. 3 , the energy excitation unit 12 is composed of an integrated driver 11 (for example, including a control-operated energy excitation unit 12 Driven by a computer and LabVIEW driver software), the energy excitation output of the energy excitation unit 12 (for example, an infrared fiber-coupled laser) and its on/off timing are controlled, so that the solid phase carrier 40 in the reaction unit 10 generates regional heat. Radiation field changes.

Furthermore, the energy excitation unit 12 can excite the solid-phase carrier 40 in a non-contact manner, and can use self-photothermal excitation and heating, as shown in Figure 5. For example, the energy excitation unit 12 is a laser collimator 121 and a laser emitter. 122 is composed of an LED diode light source, or the energy excitation unit 12 is a magnetic excitation and heating method (for example, a high-frequency magnetic field generator), which is not limited in the actual implementation of the present invention. The energy excitation unit 12 mainly heats the solid-phase carrier 40 itself and the in-situ environment 50 in the nearby region (the distance is measured in hundreds of nanometers (nm) to hundreds of micrometers (μm)).

In the present invention, the rapid nucleic acid amplification system further includes a temperature detection unit 14 (for example: K-type thermocouple) to detect the temperature feedback of the external auxiliary cooling unit 13; the nucleic acid amplification reaction is performed during the auxiliary cooling. Under the balanced control of the heating of the unit and the energy excitation unit 12 of the solid-phase carrier 40, the temperature (thermal) cycle required for the polymerase chain reaction is quickly realized, and the nucleic acid amplification reaction is quickly completed through this.

In the present invention, the rapid nucleic acid amplification system further includes an operating unit 15. The operating unit 15 can be a permanent magnet, an electromagnet, or a combination thereof. The present invention is not limited thereto; simple magnetic operations can be performed through the operating unit 15. , purifying and concentrating (enriching) the solid phase carrier 40 in the reaction unit 10, which will facilitate subsequent detection and analysis of the amplification reactant 60 bound to the solid phase carrier 40 (for example: enhancing the amplification of the amplification reaction). Increase the detection signal of reactant 60), which is not limited by the present invention. As shown in FIG. 6 , in the subsequent operation of concentrating (enriching) the amplification reaction product 60 , the magnetic solid-phase carrier 40 is simply magnetically attracted through the operating unit 15 , so that the solid-phase carrier 40 and the nucleic acid amplification agent 40 can be combined with each other. The reaction solution 30 is separated. This purification and separation can not only remove interfering substances in the detection of back-end nucleic acid molecules (for example, non-specific amplified nucleic acid products or free primers 301 and polymerase 302, etc.), but also concentrate (rich) through this tool. The operation of the set) can further improve the efficacy of subsequent nucleic acid molecule detection (for example, detection sensitivity).

It must be noted here that the present invention can simplify the temperature control equipment and achieve temperature control by only using a single energy excitation unit 12 and an external energy calibrator 16 , where the external energy calibrator can be a light intensity meter. By controlling the energy excitation unit 12 The energy intensity and energy excitation time are controlled to control the temperature around the solid phase carrier 40, so that an in-situ environment 50 is generated around all the solid phase carriers 40. The present invention can reduce the temperature of the environment as shown in GNA patent US9,382,583B2 invention patent publication number (invention title: As disclosed in Method for the amplification of nucleic acids using heat transfer for nanoparticles), a two-dimensional mirror scanner deflects laser in two dimensions, focusing the laser selectively and specifically on PCR samples from 10 nanoseconds to 500 milliseconds. steps on the nanoparticles.

In one embodiment of the present invention, the substance to be tested 20 is free DNA or ribonucleic acid material or a biological substance 21 released from the substance to be tested 20 , and the solid phase carrier 40 is an amplification body 42 , Please refer to Figure 7(a). Each amplification ligand 71 is connected to the surface of the amplification body 42, and the amplification ligand 71 binds to the test object 20 or the biological substance 21. Please refer to Figure 7(b). When the external energy source excites the solid-phase carrier 40 to generate an in-situ environment 50, the polymerase 302 in the nucleic acid amplification reaction solution 30 is used to perform the first nucleic acid amplification ligand 71. The amplification reaction produces amplification reactant 60. As shown in Figure 7(c), the external energy is turned off briefly, and the thermal field around the solid carrier 40 is quickly cooled by the auxiliary cooling unit, causing the in-situ environment 50 to dissipate. See Figure 7(d), and the thermal field is again The external energy excitation is turned on again to generate the in-situ environment 50 for denaturation reaction, so that the test substance 20 and the amplification reaction substance 60 are separated. Please refer to Figure 7(e). When the external energy is turned off briefly, the free primer 301 with the fluorescent label 3011 in the nucleic acid amplification reaction solution 30 is combined with the amplification reaction product 60, and the original test substance 20 is then combined with the amplification reaction product 60. Another amplification ligand 71 on the solid support 40 is bound.

Please refer to Figure 7 (f) ~ (g), the external energy excitation is turned on, and the polymerase 302 uses the test substance 20 or the biological material 21 to be immobilized on the amplification ligand 71 on the solid carrier 40 as an amplification reaction product. 60 is used as a template, and another nucleic acid amplification reaction is performed, and an amplification reaction product 60 is produced. Figure 7 (h) ~ (k) shows another cycle in which the excitation of the energy excitation unit 12 is turned on and off, and another cycle is performed. The nucleic acid amplification reaction shown in Figure 7(l) goes through multiple cycles, and the amplification reaction product 60 carried on the solid phase carrier 40 reaches saturation.

In one embodiment of the present invention, for "photothermal" excitation of the solid phase carrier 40, the surface of the solid phase carrier 40 is preferably made of gold nanoshell 403. The gold nanoshell 403 contains a magnetic core. The gold nanoshell 403 and There is a support layer 402 between the magnetic cores 401, which is collectively referred to as the "nano-gold magnetic shell" below. The photothermal excitation system is either a laser or an LED array or a combination of both. The preferred external energy source is laser. The main wavelength range is from the visible spectrum to the near-infrared spectrum (380nm~1.4μm). In addition, the different concentrations of nano-gold magnetic shell suspensions all show that they have the characteristics of absorbing the near-infrared spectrum region (750nm~1.4μm), and the better one Under 808nm laser excitation, the surface of the gold nanomagnetic shell can produce localized surface plasmon resonance (LSPR), thereby producing a plasma heating effect.

In another embodiment of the present invention, the solid-phase carrier 40 is excited by "magnetic change". The solid-phase carrier 40 includes a magnetic core 401. Under an external alternating current magnetic field, the induced heating effect of the solid-phase carrier 40 is mainly attributed to the nanostructure. The magnetization reversal includes the Neel relaxation mechanism (Néel relaxation; the magnetic moment rotation of the solid phase carrier 40 and the Brown relaxation mechanism (Brown relaxation; the physical rotation of the solid phase carrier 40 in the water medium). This heating ability depends on Based on the characteristics of nanostructures, such as average size, configuration, magnetization and magnetic anisotropy, as well as the amplitude (Hac) and frequency (f) of the external alternating magnetic field, where the alternating magnetic field is formed by an alternating magnetic field generator , the amplitude and frequency of the alternating magnetic field are set based on the field conditions required for each solid phase carrier 40 to produce the temperature required for nucleic acid amplification. Furthermore, the amplitude of the alternating magnetic field is 0.5~550 kiloamps/meter (kA /m) and the frequency of the alternating magnetic field is 3~3,500 kilohertz (kHz).

Furthermore, the solid phase carrier 40 can be a stable colloidal single-phase magnetic solid phase carrier 40 dispersed in the nucleic acid amplification reaction solution 30 in the form of a suspension, where the single phase refers to a single dispersed suspension, and when the solid phase carrier 40 When the size reaches the nanometer level, a stable colloidal single-phase solution will be formed according to the potential on the surface; the solid phase carrier 40 can be a strong magnetic or superparamagnetic iron oxide solid phase carrier 40 (Superparamagnetic iron oxide Nanoparticles; SPIONs), especially magnetic Iron ore (Fe ₃ O ₄ ), maghemite (γ-Fe ₂ O ₃ ), or spinel structure ferrite nanomaterials Spinel ferrite containing MIIFe ₂ O ₄ (where MII =Co ²⁺ , Ni ²⁺ , Zn ²⁺ , Mn ^2+, etc.), the addition of transition metals, such as Ni, Co, etc. mixed into ferrite nanomaterials, can obtain a larger saturation magnetization value. The stable effective magnetic anisotropy (Keff) and stronger magnetization are lost under the external alternating magnetic field.

The size of the solid-phase carrier 40 excited by magnetism is between 8 and 1,000 nm. In addition to the common sphere, the configuration also includes nanocube, nanooctahedron, and rod. , disk, hollow sphere, star or tetrapod, etc., not limited to these form. With the amplitude (Hac) and frequency (f) of the external alternating magnetic field, a wide range of frequencies (3~3,500kHz) and field amplitudes (0.5~550kA/m) can be used to generate the temperature required for nucleic acid amplification using the solid-phase carrier 40. Field condition setting.

In the above embodiments, the surface of the multifunctional body 41, the enrichment body or the amplification body 42 of the solid phase carrier 40 further includes filling material. The filling material is used to prevent the solid phase carrier 40 from aggregating and increase the stability of the water solution. In addition to inhibiting the adsorption of polymerase, the filling material also prevents the adsorption of non-specific proteins and primers 301 to the solid phase carrier 40. The filling material can be polyethylene glycol (polyethylene glycol) with a molecular weight of 5 to 10 kilodaltons (kDa). , PEG) 72 and bovine serum albumin (BSA) 73 , and the enrichment ligand or amplification ligand 71 forms a stable covalent bond with the functional group provided by the crosslinker and the filling material. , in this embodiment, the cross-linked covalent bonding between the carboxyl group, the amine group, and the thiol group is preferred.

As shown in Figure 8, the nucleic acid amplification reaction method of the present invention can be a water-cooled polymerase chain reaction on beads or an isothermal cyclic amplification reaction on water-cooled beads. The water-cooled method refers to Using the aforementioned auxiliary cooling unit to cool down, on-bead refers to performing an in-situ nucleic acid amplification reaction on the solid phase carrier 40, which includes the following steps:

In step S10, a sample is mixed with a plurality of solid phase carriers 40, wherein the sample contains at least one analyte 20, so that the plurality of solid phase carriers 40 capture at least one analyte 20, and then step S20 is performed.

Step S20, in a reaction unit 10, use the operation unit 15 to clean and remove impurities, and concentrate (enrich) the analyte 20, if the at least one analyte is a free DNA or ribonucleic acid substance , then proceed directly to step S51, if not, continue to step S30.

Step S30, at least one energy excitation unit 12 provides an external energy source to the plurality of solid phase carriers 40, so that the temperature of the plurality of solid phase carriers 40 rises to the cleavage temperature of the cleavable test object 20 (for example: 80~95°C ), continue to step S40.

In step S40, the at least one analyte 20 is cleaved to release a biological substance 21 (for example, DNA or ribonucleic acid), and then step S50 is performed.

Step S50, control the energy excitation unit 12 to provide an external energy source to the plurality of solid phase carriers 40 until reaching a nucleic acid hybridization temperature, so that the plurality of solid phase carriers 40 are combined with the at least one biological substance 21, and through this operation Unit 15 is used to purify, separate and enrich the plurality of solid phase carriers 40, and then proceed to step S60.

Step S51, control the energy excitation unit 12 to provide an external energy source to the plurality of solid phase carriers 40 until reaching a nucleic acid hybridization temperature, so that the plurality of solid phase carriers 40 are combined with the at least one analyte 20, and pass through the plurality of solid phase carriers 40. The operation unit 15 is used to purify, separate and enrich the plurality of solid phase carriers 40, and then proceed to step S60.

Step S60, add the pre-cooled nucleic acid amplification reaction solution 30 (the temperature of the pre-cooled nucleic acid amplification reaction solution 30 is between -10~4°C) into the reaction unit 10, and combine it with the external auxiliary cooling unit 13 , if the water-cooled on-bead polymerase chain reaction is performed, the process proceeds directly to step S70; if the water-cooled on-bead isothermal circular amplification reaction is performed, the process proceeds directly to step S71.

Step S70, control the energy excitation unit 12 to provide external energy to the plurality of solid phase carriers 40, so that the in-situ environment 50 of the solid phase carrier 40 reaches the nucleic acid denaturation (90~95°C) required for the polymerase chain reaction and primer adhesion/ The polymerase extension temperature (60~65°C) is used to perform an in-situ nucleic acid amplification reaction on the plurality of solid-phase carriers 40 through at least one enzyme. To enter the detection stage, proceed to step S80.

Step S71, control the energy excitation unit 12 to provide external energy to the plurality of solid-phase carriers 40, so that the in-situ environment 50 around the surface reaches the temperature required for the isothermal cyclic amplification reaction (60~65°C), by at least An enzyme performs an in-situ nucleic acid amplification reaction on the plurality of solid phase carriers 40. If the detection stage is to be entered, step S80 is continued.

In step S80, the amplification body 42 and the supernatant are separated through the operation unit 15.

Step S90, a detection module 17 is used to identify the signal change on the amplification reaction product 60 located on the amplification body 42, by detecting the color change, luminescence change or the nucleic acid label on the amplification reaction product 60. its combination.

In one embodiment, in the water cooling on-bead Loop-mediated isothermal amplification reaction of the present invention, the solid phase carrier 40 is introduced with at least one pair of specific ones on the surface. The primer 301 serves as the amplification body 42 of the amplification ligand 71 (shown in Figure 9(a)). In this embodiment, at least one pair of specific primers 301 includes three pairs of primers 301 (FIP, BIP, LF, LB, B3 and F3), causing it to perform a water-cooled isothermal circular amplification reaction on beads on the amplification body 42. When the external energy source is turned off, the specific primer 301 on the amplification body 42 will capture the target through DNA hybridization. Nucleic acid sequence, which is the analyte 20 or biological material 21 (shown in Figure 9(b)). When the external energy source is turned on, the amplification body 42 generates a temperature range suitable for isothermal circular amplification reaction. Polymerase 302 performs preliminary extension (shown in Figure 9(c)), and then when the external energy source is turned off, the temperature is rapidly lowered to stop the isothermal circular amplification reaction (shown in Figure 9(d)), Figure 9(b) ~ After multiple cycles of the step cycle in Figure 9(d), the amplification reactant 60 carried on the amplification body 42 reaches saturation (shown in Figure 9(e)). After the reaction is completed, in addition to the amplification body 42 itself, the amplification reaction product 60 can be concentrated (enriched) and purified through the operation unit 15. This purification and separation operation by the amplification body 42 also assists in specificity. Amplification reagent 60 of the sex detection (LAMP/RT-LAMP) reaction. The nucleic acid fragment to be amplified only contains true positive amplified fragments between the 3' ends of FIP and BIP to improve the specificity of LAMP/RT-LAMP product detection and reduce the occurrence of false positives.

t1：代表熱量從一個奈米顆粒以平均奈米顆粒距離擴散到下一個奈米顆粒所需的時間；s1：則代表比例因子，s1為當固相載體40照射臨界激發間隔t1時，產生熱力場熱流擴散的距離的量度，當處於固相載體40的局部加熱時，s1小於或等於1；|X|則代表平均每個固相載體40彼此間的距離；D則代表固相載體40之間介質的熱擴散係數。 In addition, according to GNA Biosolutions' US Patent No. US9,382,583 B2, the occurrence of "local heating" of the photothermal solid phase carrier 40 is based on the following conditions. When the energy excitation interval is shorter or equal to the critical excitation interval t1, The critical excitation interval t1 can be expressed by the following equation:

t1: represents the time required for heat to diffuse from one nanoparticle to the next nanoparticle at the average nanoparticle distance; s1: represents the scaling factor, s1 is the thermal force generated when the solid phase carrier 40 irradiates the critical excitation interval t1 A measure of the distance of field heat flow diffusion, when the solid phase carrier 40 is locally heated, s1 is less than or equal to 1; |X| represents the average distance between each solid phase carrier 40; D represents the distance between the solid phase carriers 40 Thermal diffusion coefficient of the intermediary.

According to the conditions disclosed in the aforementioned patent, the operation must use a two-dimensional mirror scanner to scan light through the nucleic acid amplification reaction containing photothermal particles at a frequency of 10 nanoseconds (ns) to 500 milliseconds (milliseconds, ms). Liquid 30, therefore in the overall implementation of the aforementioned patent, it is necessary to accurately control the above actions in order to accurately control the exposure time of photothermal particles, which is a key factor for the success of this technology.

On the other hand, the present invention uses a relatively easy-to-implement method to control the solid-phase carrier 40 to receive external energy excitation to generate a regional thermal radiation field phenomenon. First, the present invention uses the solid-phase carrier 40 suspended in the nucleic acid amplification reaction solution 30. The ratio of the total volume of the solid phase carrier 40 to the volume of the nucleic acid amplification reaction solution 30 is 1:200 to 1:1*10 ⁹ , and the preferred volume ratio is 1:1*10 ⁴ to 1:1*10 ⁸ . This means that the above-mentioned distance | Solid phase carrier 40 to produce zone heating phenomenon.

Furthermore, the nucleic acid amplification reaction solution 30 is placed in the external auxiliary cooling unit 13, and the external auxiliary cooling unit 13 provides a cooling temperature environment to greatly confine the in-situ environment 50 generated by the solid phase carrier 40, so that it can be evenly dispersed. The ideal state does not overlap with the in situ environment 50 created by other solid supports 40 . In this way, the present invention does not need to control the relative movement relationship between the energy excitation unit 12 and the reaction unit 10. And a relatively long excitation time (between one second to tens of seconds) can be used to excite all the suspended or immobilized solid phase carriers 40 in the reaction unit 10. In addition, each time the energy excitation unit 12 is driven, there is no moving energy excitation. The relative position of the unit 12 and the reaction unit 10 also causes obvious differences between the present invention and the US invention patent of GNA Biosolutions (US 9,382,583 B2 publication number).

In addition, the solid phase carrier 40 of the present invention, due to its high body surface area ratio (as mentioned above), can not only be used as an efficient stage for separation of the amplification reaction product 60 of the test object 20, but also can be used as a These solid-phase carriers 40 are used to remove nucleic acid amplification reaction inhibitors that may exist in the test object 20. In addition, the in-situ nucleic acid amplification reaction is performed in the in-situ environment 50 formed on the surface of the solid-phase carrier 40. Since the solid-phase carrier The space that the surface of 40 can carry the immobilized amplification reactant 60 is limited. Therefore, under a relatively short operation time (or a relatively small number of nucleic acid amplification cycles), the amplification reaction on the surface of the solid phase carrier 40 The substance 60 can reach a saturated state, which will facilitate subsequent detection of the amplification reaction substance 60 .

Please refer to FIG. 10 . In the embodiment of the present invention, the nanogold magnetic shell selected for the solid phase carrier 40 has good photothermal conversion characteristics and stability, and the light energy absorption effect of the laser light wavelength is between 780 and 1,000 nm. Preferably, the preferred range is between 800 and 950nm. In this embodiment, a gold nanoshell is added to the trace nucleic acid amplification reaction solution 30 (20 μL) to form a trace single-phase gold nanoshell suspension, in which the concentration of the gold nanoshell suspension is 2.9*10 ¹⁰ particles/ml, and the 808nm laser was selected for photothermal conversion and stability experiments. As shown in Figure 10(a), the integrated driver 11 is used to drive the 808nm fiber-coupled laser for 10 consecutive laser cycles. Cycle (laser power: 400 milliwatts (mW)/0.16 square centimeters (cm ² ); first on for 60 seconds; then off for 120 seconds) to excite this trace amount of single-phase nanometer gold magnetic shell suspension, this trace amount of single-phase nanometer gold magnetic shell suspension The maximum photothermal conversion temperature of the gold magnetic shell suspension can reach 62.29±1.06℃, and its coefficient of variation is 1.70%. This result shows that this nanometer gold magnetic shell has stable photothermal conversion characteristics and will not cause solidification due to high-power laser irradiation. The configuration of the phase carrier 40 changes. As shown in Figure 10(b), the trace single-phase gold nanomagnetic shell suspension in Figure 10(a) was excited with 808nm lasers of different powers. The different powers used included 200mW/0.16cm ² and 250mW/ 0.16cm ² , 300mW/0.16cm ² , 400mW/0.16cm ² , 500mW/0.16cm ² , 600mW/0.16cm ² and 700mW/0.16cm ² , and also includes a blank sample solution that does not contain nano-gold magnetic shell with 1 Watt (W)/0.16cm ² laser excitation as a control group, a trace amount of single-phase gold nanomagnetic shell suspension was locally photothermally heated by laser to a stable microscale directly measured temperature. The results show that the nanogold magnetic shell It has good photothermal conversion characteristics, and the temperature at which the photothermal conversion reaches a stable state is directly proportional to the input laser power.

Referring to Figure 11, in an embodiment of the present invention, a trace amount of gold nanoparticle magnetic shell suspension (20 μL) is measured in a micro-scale manner. The temperature at which the gold nanoparticle magnetic shell suspension is photothermally heated to a stable state is used to establish a laser The relationship between the power intensity and the micro-scale environmental temperature around the gold nano-magnetic shell is used to estimate the relationship between the temperature area of the in-situ environment of the gold nano-magnetic shell 50 and the laser wattage through micro-scale measurements. Figure 11(a) shows the photothermal conversion temperature of 20 μL of nanogold magnetic shell suspension with a concentration of 2.9*10 ¹⁰ particles/ml tested under different laser powers as described in Figure 10(b). Micro-scale direct measurement was used to establish the photothermal conversion and heating curve of the nanogold magnetic shell in the stable state under different laser powers (the linear equation is y=0.0778x+44.126; R ² =0.9104). The temperature area selected in the upper box in Figure 11(a) is the temperature range (85~95°C) required for cell lysis and DNA denaturation; the temperature area selected in the lower box in Figure 11(a) is an isothermal ring. The temperature range (55~65°C) required for primer binding and polymerase extension operations in the amplification reaction (LAMP). Figure 11(b) shows the reaction unit 10 of the nanogold magnetic shell suspension in the stable state under different 808nm laser powers (200mW/0.16cm ² , 250mW/0.16cm ² , 400mW/0.16cm ² ). Infrared thermography, the upper part of Figure 11(b) is the result of irradiating the blank sample solution with 808nm laser power (200mW/0.16cm ² , 250mW/0.16cm ² , 400mW/0.16cm ² ), the temperature is between 27.9~ Between 29°C and 29°C, the center temperature of the suspension containing nanogold magnetic shell is 51.6°C when it is excited by 200mW/0.16cm ² laser to a stable state, and the center temperature is 63.9°C when it is excited by 250mW/0.16cm ² laser to a stable state. , 400mW/0.16cm ² laser excitation to a core temperature of 79.1°C in a stable state.

Please refer to Figure 12, which is an embodiment of the auxiliary cooling unit of the present invention. The nucleic acid amplification reaction solution 30 containing the single-phase gold nanomagnetic shell is subjected to 100 laser excitation cycles (one cycle: first 400 mW /0.16cm ² irradiation for 1.25 seconds; then turned off for 0.5 seconds; then 150mW/0.16cm ² irradiation for 7.5 seconds), the temperature change of the nucleic acid amplification reaction solution 30 containing a single-phase gold nanomagnetic shell suspension, Figure 12(a) The structure of the auxiliary cooling unit in this embodiment is composed of a large volume of nucleic acid amplification reaction solution 30 (100 μL) and its external auxiliary cooling unit 13. In this embodiment, the external auxiliary cooling unit 13 uses ice with a large specific heat capacity (volume 550 μL of water frozen into ice), surrounding the reaction unit 10 containing the nucleic acid amplification reaction solution 30 to assist and maintain the temperature of the nucleic acid amplification reaction solution 30 in the nucleic acid amplification reaction unit 10 At cooling temperature (2~10℃). Figure 12(b) shows the evaluation of the particle number of the fixed gold nanomagnetic shell (5.8*10 ⁸ particle number), using 30 volumes of different nucleic acid amplification reaction solutions (20 μL and 100 μL) to disperse and suspend the above gold nanomagnetic shell. , and divided into groups according to whether the external auxiliary cooling unit 13 as shown in Figure 12(a) is used, and a blank sample solution containing only nucleic acid amplification reaction solution was added as a control group, and 100 external laser excitation cycles were performed on the above groups. After a cycle (one cycle: first 400mW/0.16cm ² , irradiate for 1.25 seconds; then turn off for 0.5 seconds; then irradiate at 150mW/0.16cm ² for 7.5 seconds), the temperature of the nucleic acid amplification reaction solution 30 in each group changes, and at the same time Evaluate whether the above combination of operating conditions can maintain the temperature of the nucleic acid amplification reaction solution 30 at the cooling temperature (2~10°C) under how many external laser excitation cycles, and its temperature changes will not be significantly affected by the external Changes due to changes in the number of laser excitation cycles; the grouping includes 100 μL of nanogold magnetic shell suspension plus an external auxiliary cooling unit, 100 μL of blank sample solution plus an external auxiliary cooling unit 13, and 100 μL of nanogold magnetic shell suspension. There is no external auxiliary cooling unit and the 20 microliter nano-gold magnetic shell suspension has no external auxiliary cooling unit, where the laser cycle period corresponds to the Y-axis on the right, and the Y-axis on the right corresponds to the laser power (mW); this embodiment The results show (Figure 12(b)) that when 100 μL of nucleic acid amplification reaction solution 30 is used to disperse and suspend the single-phase gold nanomagnetic shell, and an external auxiliary cooling unit 13 (volume of ice: 550 μL) is used, 65 times Under the external laser excitation cycle (one cycle: 400mW/0.16cm ² , irradiation for 1.25 seconds; off for 0.5 seconds; 150mW/0.16cm ² for 7.5 seconds of irradiation), corresponding to the left Y-axis, the left Y-axis is the nucleic acid amplification reaction solution 30 The temperature (°C) is maintained at the cooling temperature (2~10°C). This result also shows that under this design and operating conditions, the heating method of the nanogold magnetic shell when stimulated by laser belongs to the "local heating" mode. That is, the heating is limited to the in-situ environment 50, and the average temperature of the entire nucleic acid amplification reaction solution 30 is still maintained at the cooling temperature (2~10°C).

Figure 13 shows an embodiment of the present invention, using an immune lateral flow test strip 171 to detect the amplification reaction product 60 produced by the polymerase chain reaction on water-cooled beads. As shown in Figure 13(a), after completing the water-cooled bead polymerase chain reaction using laser excitation, the nanogold magnetic shell is purified and concentrated (enriched) through the magnetic operating unit 15, in which the gold nanoparticles are The shell is combined with the amplification reaction product 60 produced by the in-situ nucleic acid amplification reaction, which can remove the nucleic acid molecule products produced by the non-specific nucleic acid amplification reaction and other substances in the nucleic acid amplification reaction solution 30 (for example: primer 301, polymerization enzyme 302, etc.), and at the same time, the amplification reaction product 60 produced by the specific in-situ nucleic acid amplification reaction can be concentrated (enriched). After re-suspending the purified and concentrated gold nanomagnetic shell in TBST buffer, rapid detection was performed with immune lateral flow test strip 171, as shown in Figure 13(a) and Figure 13(b). The amplification reactant 60 is bound to the gold magnetic shell, and the amplification reactant 60 is bound to a nucleic acid label. The nucleic acid label in this embodiment is a primer 301 with a fluorescent isothiocyanate (FITC) label. , and then use the anti-fluorescent isothiocyanate antibody bound to the test line (test line) 1711 on the lateral flow test strip to identify and capture the nucleic acid-labeled nucleic acid bound to the amplification reaction 60. The rice gold magnetic shell forms a visually detectable mark (for example: forming a line on the test line 1711). This reaction can be completed within 3 to 5 minutes. The control line is used as a quality control for conventional immune lateral flow test strips 171 purpose.

Figure 14 is another embodiment of the present invention, which uses plasma thermal sensing technology to interpret the test results of the immune lateral flow test strip 171 (as shown in Figure 13(b)). This technology can increase the number of traditional immune cells. lateral flow The sensitivity of test strip 171 detection. As shown in Figure 14(a), the amplification reaction 60 on the nanogold magnetic shell is combined with a fluorescent isothiocyanate (FITC)-labeled primer 301, which is then combined with a primer 301 on the lateral flow test strip. The anti-fluorescent isothiocyanate antibody on the test line 1711 is used to identify and capture the nano-gold magnetic shell that binds the amplification reaction 60 and carries the nucleic acid-labeled primer 301. The nano-gold magnetic shell uses laser composite infrared rays The excitation of the thermal sensor 172 generates a heat (field) source, and this heat (field) source is further displayed in the form of thermal imaging, whereby the plasma thermal sensing technology can increase the detection results of the immune lateral flow test strip 171 Sensitivity of interpretation. As shown in FIG. 14(b), the immune lateral flow test strip 171 of FIG. 13(b) is analyzed by plasma thermal sensing to detect nucleic acid amplification, but there is no solid phase carrier 40 in the test line 1711. Schematic diagram of a captured negative reaction.

Please refer to Figure 15, which is an embodiment of the present invention, using 808nm laser with a laser cycle (one cycle: first 400mW/ ^0.16cm2 irradiation for 1.25 seconds, and then 150mW/ ^0.16cm2 irradiation for 7.5 seconds) against Escherichia coli ( E.coli ATCC35218A) malB gene fragment, in which the malB gene fragment is the detection target for routine detection of polymerase chain reaction of E. coli. Water-cooled bead polymerase chain reaction is performed at different laser cycle times (10 to 50 times). cycle), perform an in-situ nucleic acid amplification reaction on a gold nanomagnetic shell. After completion, use immune lateral flow test strip 171 and urea agar gel electrophoresis to sequentially analyze and detect the polymerase chain reaction on water-cooled beads. The result of nucleic acid amplification performed on the solid phase carrier 40 and the nucleic acid amplification reaction solution 30 . As shown in Figure 15(a), using immune lateral flow test strip 171 to detect different laser cycle tests (10 to 50 cycles), the in-situ nucleic acid amplification reaction on the nanogold magnetic shell will increase with the As the number of laser cycles increases, the signal intensity of the test line 1711 on the immune lateral flow test strip 171 also gradually increases, which illustrates the super amplification formed in a short laser cycle, that is, The problem of low-amount nucleic acid amplification loss during the nucleic acid amplification reaction cycle can be made up by increasing the number of laser cycles. As shown in Figure 15(b), denatured agar colloid made of urea agar gum is used for electrophoresis to analyze the supernatant remaining after the nanogold magnetic shell that has completed the in-situ nucleic acid amplification reaction is separated by the operating unit 15 , after collecting the DNA contained in the supernatant through DNA precipitation, it is heated with high-concentration urea to denature the DNA, and then conducts urea gel electrophoresis analysis to observe whether amplification reaction product 60 remains in the supernatant. The purified malB -FITC fragment is the amplification product of the malB gene labeled with FITC and is used as a positive control group for the amplification reaction 60. The B3-FITC calibration primer is the B3 primer labeled with FITC and is used as a positive control for the primer. group, the results showed that in the supernatant separated after different laser cycles (10 to 30 cycles), only the free primer 301 was detected, and there was no amplification reaction product 60. This result is also It indirectly proves that the nucleic acid amplification reaction occurs on the nanogold magnetic shell.

Please refer to Figure 16. In one embodiment of the present invention, under simple sample conditions, the performance of nucleic acid amplification and detection is compared with the traditional polymerase chain reaction and the water-cooled bead polymerase chain reaction performed with 808 nm laser excitation. The difference is that the simple samples use the following control groups and the DNA of microbial strains as individual samples, including no template control (NTC), Acinetobacter baumannii ( Acinetobacter baumannii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae, Staphylococcus epidermidis , Staphylococcus aureus (American Type Culture Collection (ATCC): BAA977), Streptococcus agalactiae , Enterococcus faecalis , Mycobacterium abscessus , Candida albicans , Aspergillus niger and Escherichia coli (ATCC: 35218), and use the malB gene of Escherichia coli ( malB gene) fragment was used as the detection target, and was used with agar gel electrophoresis for sensitivity testing and specificity testing. As shown in Figure 16(a), in the traditional polymerase chain reaction detection, the same ⁴⁰ thermal cycles (one thermal cycle: first 95°C, 15 Seconds; then 60°C, 30 seconds), the sensitivity test and specificity test of nucleic acid amplification and detection of malB gene were carried out. The results showed that the sensitivity of traditional detection polymerase chain reaction for detecting simple samples ranged from 10 to 10 ² copy number (upper picture in Figure 16(a)), and the detection specificity is good (lower picture in Figure 16(a)), and only E. coli samples show detection signals. Similarly, as shown in Figure 16(b), in a water-cooled bead polymerase chain reaction, a simple sample of the same E. coli was used for malB gene detection, and the genomic DNA content of the E. coli added initially They are 0.00675 picogram (pg) (1 copy number of malB gene), 0.0675pg (10 copy number of malB gene), 0.675pg (10 ² copy number of malB gene), 6.75pg (10 ³ copy number of malB gene). malB gene), 67.5pg (10 ⁴ copy number of malB gene), by using an immune lateral flow test strip 171 and an anti-fluorescent isothiocyanate antibody combined with the test line 1711 to detect the gold nanoparticles The results of the fluorescent isothiocyanate-labeled primer 301 bound to the amplification reaction 60 of the malB gene bound to the shell showed that its detection sensitivity was also between 10 and 10 ² copy numbers.

Please refer to Figure 17, which shows the difference in nucleic acid amplification and detection efficiency between traditional polymerase chain reaction and water-cooled bead polymerase chain reaction using 808nm laser excitation under complex sample conditions. Complex samples Contains the genomic DNA of the aforementioned simple sample E. coli ( Escherichia coli , ATCC: 35218), and a mixture of genomic DNA of five other common human pathogenic microbial strains, including Acinetobacter baumannii ( Acinetobacter baumannii ), Green Pseudomonas aeruginosa , Staphylococcus aureus (ATCC: BAA977), Streptococcus agalactiae and Enterococcus faecalis , among which the genomic DNA content of the initially added E. coli The copy numbers of 0, ¹ , 10, 10 2, 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ are used as groups. In addition, 6.75 nanogram (ng) (10 ⁶ copy number) above 5 is added to each group. A mixture of genomic DNA of common human pathogenic microbial strains, as shown in Figure 17(a), in the traditional polymerase chain reaction detection, using complex samples, in the same 40 thermal cycles (one thermal cycle: first Under the operating settings of 95°C, 15 seconds; and 60°C, 30 seconds), the sensitivity test of nucleic acid amplification and detection of the malB gene was performed. The results showed that the sensitivity of traditional polymerase chain reaction detection was reduced to 10 ⁵ copy numbers. To achieve specific identification, the sensitivity of detection is significantly reduced. However, as shown in Figure 17(b), the embodiment of the present invention uses laser excitation to perform a water-cooled bead polymerase chain reaction, and subsequently uses an immune lateral flow test strip 171 to detect the nanogold magnetic shell. The detection sensitivity of the amplified nucleic acid molecules is still maintained at 10 to 10 ² copy number under the conditions of complex samples such as the above. This result shows that the nucleic acid amplification of water-cooled bead polymerase chain reaction using laser excitation is as effective as The detection sensitivity will not be affected by complex nucleic acid analytes 20.

Please refer to Figure 18. In one embodiment of the present invention, an experiment is performed using a suspension of a mixed enrichment body and amplification body 42. The enrichment body and the amplification body 42 are both nanogold magnetic shells. The enrichment system is After capturing Escherichia coli, purifying and separating it through the magnetic operation unit 15, and then photothermally lysing the analyte 20 captured on the enrichment body, wherein the analyte 20 is Escherichia coli (ATCC: 35218). The mixed liquid causes E. coli to release biological substances 21, and the amplification body 42 is used to capture the biological substances 21 released by E. coli. As shown in Figure 18(a), the suspension of the enrichment body and the amplification body 42 are mixed, and the antibodies on the enrichment body that can specifically recognize the O antigen and the K antigen of E. coli are used as enrichment ligands to amplify The body 42 has an F3 primer 301 ( malB F3 primer) that can specifically hybridize the E. coli-specific malB gene fragment as the amplification ligand 71. As shown in the upper part of Figure 18(a), 20 μL of mixed rich Collect the suspension of the body and the amplified body 42, mix it with 1 μL of E. coli mixture and 19 μL of TBST buffer containing 1% BSA, wherein 1 μL of the E. coli mixture contains 8* ¹⁰³ colony forming units (colony). -forming unit (CFU), mix and react at room temperature (25°C) for 20 minutes, purify and concentrate the enriched body and amplified body 42 through the magnetic operating unit 15, and then rinse with TBST buffer to complete After washing, the operation unit 15 is used to concentrate and remove the TBST buffer used for washing, and then the washed enriched body and amplification body 42 are resuspended in 20 μL of TBST buffer to achieve low background and high specificity to capture the large intestine. bacilli, and if the above steps of this embodiment are performed only with a suspension containing only the amplified entity, E. coli cannot be effectively captured; as shown in the lower part of Figure 18(a), the enrichment containing captured E. coli The suspension of the bulk was excited with an 808nm laser and continued to be excited for 5 minutes at different laser powers. The laser power groups included 0mW/0.16cm ² , 300mW/0.16cm ² , 400mW/0.16cm ² and 500mW/0.16cm ² , 1 μL of each component contains a suspension containing the enriched body of E. coli, and then spread it on LB solid medium, and judge the efficiency of photothermal lysis of E. coli by the formation of bacterial cells. If new cells cannot be generated, The bacterial cells have been lysed, and the results show that continuous stimulation at 400mW/ ^0.16cm2 for 5 minutes can lyse E. coli. As shown in Figure 18(b), the biological material 21 decomposed by photothermal cracking of Escherichia coli (ATCC35218) is used to capture the malB gene fragment contained in the biological material 21 through the amplification ligand 71, and through laser excitation, water-cooled beads are used. The polymerase chain reaction was carried out and then evaluated using the immune lateral flow test strip 171. The results showed that under the photothermal lysis of 400mW/0.16cm ² for 5 minutes, the photothermal lysis of E. coli was successfully achieved and the biological substances were released 21. The malB gene fragment contained in the biological substance 21 is captured by the amplification body 42 and is excited by laser to perform a water-cooled bead polymerase chain reaction, which can be detected on the test line 1711 on the immune lateral flow test strip 171 signal.

Please refer to Figure 19, which is an experiment of mixing the suspension of the enrichment body and the amplification body 42 in an embodiment of the present invention. The enrichment body system is a nanogold magnetic shell, and the enrichment ligand system is specifically identifiable. Antibodies against the O and K antigens of Escherichia coli, in which the amplification body 42 is a nanogold magnetic shell, and the amplification ligand 71 is the F3 primer 301 ( malB F3 primer) that can recognize the malB gene, and is used in different bacterial colonies Forming units of Escherichia coli (ATCC: 35218) were used as the test substance 20, in which different colony forming units were measured by absorbance at 600nm wavelength and grouped, including 0CFU, 8CFU, 8*10CFU, 8*10 ² CFU and 8*10 ³ CFU, evaluate the laser excitation for water-cooled bead polymerase chain reaction, and then perform the immune lateral flow test strip 171 detection, test the sensitivity of the immune lateral flow test strip 171 and the time required for the overall process . As shown in Figure 19(a), the overall operation process of this integrated platform includes four key procedures, namely "capture and concentration of the target analyte 20", "photothermal cleavage of the analyte 20 and capture of the released biological substances". 21", "Water-cooled on-bead polymerase chain reaction using laser excitation", and "Immune lateral flow test strip 171 detection".

In the step of "capture and concentration of the target analyte 20", 5 μL of the suspension of the enriched entity, 15 μL of the suspension of the amplification entity 42, 1 μL of the analyte 20 and 19 μL of TBST containing 1% bovine serum albumin Buffer mixing reaction, shaking and mixing at 25°C for 20 minutes, allowing the amplification ligand 71 on the amplification body 42 to capture E. coli, and then using the magnetic operation unit 15 to concentrate (enrich) the body and amplify 42 and remove the supernatant, rinse with TBST buffer to remove impurities and remove the TBST buffer, which can be completed within 25 minutes.

In the step of "photothermally cleavage the analytes and capture the released biological substances 21", the enriched body and the amplified body 42 after completing the step of "capturing and concentrating the target analytes 20" and washed with TBST buffer are used. Resuspend in 20 μL of TBST buffer and continue to irradiate with 808 nm laser at 400 mW for 5 minutes for photothermal cleavage, so that the test substance 20 releases the biological substance 21 and is captured by the amplification ligand 71 on the amplification ligand body. Then, the magnetic operating unit 15 is used to concentrate the enrichment body and the amplification body 42 and the supernatant is removed, and then 100 μL of the nucleic acid amplification reaction solution 30 is added and resuspended.

In the step of "carrying out water-cooled on-bead polymerase chain reaction using laser excitation", the rich nucleic acid amplification reaction solution 30 is resuspended in the step of "photothermal cleavage of substrates and capturing the released biological substances 21". Set the main body and the amplification body 42, and perform 40 laser cycles with 808nm laser (one cycle: first irradiate 400mW/0.16cm ² for 1.25 seconds, then irradiate 150mW/0.16cm ² for 7.5 seconds) to complete the water-cooled bead coating The polymerase chain reaction is followed by using a magnetic operation unit 15 to concentrate the enriched body and amplification body 42 and remove the supernatant, and then add 20 μL of TBST buffer for resuspension, which can be completed within 6 minutes.

In the "Immune Lateral Flow Test Strip 171 Detection" step, the enrichment body and amplification body 42 will be carried out after completing the "water-cooled bead polymerase chain reaction with laser excitation" step and resuspended in TBST buffer. The immune lateral flow test strip 171 is used for detection. The signal reaction of the test line 1711 on the immune lateral flow test strip 171 can be directly observed with the naked eye, or the laser composite infrared thermal sensor 172 can be used for detection. It can be detected within 3 to 5 seconds. Minutes to complete. The entire process can be completed within 45 minutes.

As shown in Figure 19(b), the system will complete each step of Figure 19(a), complete laser excitation, perform water-cooled bead polymerase chain reaction and detect with immune lateral flow test paper strip 171, and the result will be displayed in 8CFU The signal response of the group immune lateral flow test strip 171 can be visually observed, which means that the sensitivity of the detection falls on the E. coli containing 8 CFU in each reaction.

Please refer to Figure 20, which is a comparison of the detection sensitivity of the plasma thermal sensing combined with immune lateral flow test strip 171 and the general immune lateral flow test strip 171 according to another embodiment of the present invention. As shown in Figure 19(b), the image of the immune lateral flow test strip 171 at the test line 1711 is analyzed using image analysis software (Image J). The quantified image results are as shown in Figure 20(a). shows that the tested E. coli colony-forming units are 0 CFU, 8 CFU, 8*10 CFU, 8*10 ² CFU and 8*10 ³ CFU. Although the sensitivity of the image analysis detection falls on the E. coli containing 8 CFU in each reaction, The inspection of E. coli containing 8CFU~8*10CFU in the reaction is difficult to visually identify and judge. In the embodiment of the present invention, the method of integrating plasmon thermal sensing and immune lateral flow test strip 171 can greatly improve the judgment. As shown in Figure 20 (b, upper picture), the immune lateral flow test strip 171 is excited with 808nm laser energy of 24mW and 140mW respectively, and the laser composite infrared thermal sensor 172 is focused on the test line 1711 The thermal imaging image presented by the upper detection; as shown in Figure 20(b), the lower image) is shown in Figure 20(b), the upper image) after being excited with 808nm laser energy of 24mW and 140mW for thermal imaging to measure the temperature. A quantitative chart of the rising temperature difference (△T(℃)) obtained after deducting the background temperature (18.2°C) in the environment, and adding three standard deviations to the average of the rising temperature difference measured in the 0CFU group as The boundary of the detection limit. The temperature difference above this is judged as positive. The results show that when excited by 808nm laser power of 24mW and 140mW respectively, the rising temperature difference of the thermal image clearly shows whether it is 24mW or 140mW. With 808nm laser power excitation, the detection sensitivity falls within the range of 8 CFU of E. coli contained in each reaction.

In order to further understand the impact of laser excitation on the performance of water-cooled bead polymerase chain reaction when polymerase chain reaction is performed in different cooling temperature ranges, whether an ice pack is attached, whether the ice pack is enlarged, and nucleic acid amplification 30 volumes of the reaction solution were divided into groups, and the temperature difference (△T(℃)) of the nucleic acid amplification reaction solution rising 30 times under different conditions was tested. The number of nanogold magnetic shells in each group was 2.8*10 ⁸ particles. Including O1 (with ice pack, 100 μL nucleic acid amplification reaction solution 30), O2 (with ice pack, 100 μL nucleic acid amplification reaction solution 30), Non-1 (without ice pack, 100 μL nucleic acid amplification reaction solution 30), Non-2 (without ice pack, 100 μL nucleic acid amplification reaction solution 30) Ice pack, 100 μL nucleic acid amplification reaction solution 30), Non(2x)-1 (no ice pack, 200 μL nucleic acid amplification reaction solution 30), Non(2x)-2 (no ice pack, 200 μL nucleic acid amplification reaction solution 30), E1 (with extra large ice pack, 100 μL nucleic acid amplification reaction solution 30), E2 (with extra large ice pack, 100 μL nucleic acid amplification reaction solution 30), of which the ice pack contains 550 μL of water frozen into ice, of which the large ice pack contains 2,200 μL The water was frozen into ice. Each group performed laser excitation to perform a water-cooled bead polymerase chain reaction, and then quantified by bead-based ELISA. 30 pairs of nucleic acid amplification reaction solutions of the groups were evaluated. The effect of laser excitation on the performance of water-cooled on-bead polymerase chain reaction, please refer to Figure 21(a), which is a 190nm nanogold magnetic shell suspension with laser cycling at different starting temperatures as grouped above. The cycle (first 690 mW irradiation for 1.25 seconds, and then 360 mW irradiation for 7.5 seconds) was cycled 50 times, and a thermocouple was used to detect the temperature change of the nucleic acid amplification reaction solution 30 in real time. Please refer to Figure 21(b). The nucleic acid amplification reaction solution of each group was quantified by bead-based ELISA. 30 Evaluation of the efficiency of water-cooled bead polymerase chain reaction for laser excitation , the Y-axis on the left corresponds to the scatter line, which represents the scatter line diagram of the rising temperature change of the nucleic acid amplification reaction solution 30 after performing laser excitation to perform a water-cooled bead polymerase chain reaction as shown in Figure 21(b). The temperature difference when O1 rises is 11.2℃ (3.8~15℃), the temperature difference when O2 rises is 10.1℃ (2.1~12.2℃), and the temperature difference when Non-1 rises is 16.8℃ (13.5~30.3℃). Among them, the temperature difference when Non-2 rises is 20.1℃ (12.6~32.7℃), among which the temperature difference when Non(2x)-1 rises is 14.2℃ (17.2~31.4℃), among which the temperature difference when Non(2x)-2 rises is 14.8℃ (17.5~32.3℃), the temperature difference when E1 rises is 9.8℃ (1.1~10.9℃), and the temperature difference when E2 rises is 6.7℃ (-8.7~-2℃); the Y-axis on the right corresponds to the straight square The figure represents the 450nm absorbance value of the bead enzyme immunoassay method to quantitatively evaluate the 808nm laser-excited water-cooled bead polymerase chain reaction efficiency. The absorbance value of O1 is 0.147, and the absorbance value of O2 rise is 0.168, where The absorbance value of Non-1 is 0.132, among which the absorbance value of Non-2 is 0.120, among which the absorbance value of Non(2x)-1 is 0.128, among which the absorbance value of Non(2x)-2 is 0.138, among which the absorbance value of E1 is 0.148, of which the absorbance value of E2 is 0.195. The larger the absorbance value, the better the overall nucleic acid amplification efficiency.

In an embodiment of the present invention, a water-cooled on-bead polymerase chain reaction is stimulated by laser, in which a laser cycle is used to control the temperature cycle process around the solid-phase carrier 40, including a plurality of cycle reaction periods, and each cycle reaction period includes nucleic acid molecules in sequence. The denaturation reaction period, the primer adhesion period and the polymerase extension period are used to denature the nucleic acid molecules for 0.5 to 5 seconds, preferably 1 to 2.5 seconds, and the laser power is 400 to 800mW. The nucleic acid molecules The temperature during the denaturation reaction is 85~95°C, preferably 95°C. The primer adhesion period and the polymerase extension period are 2 to 15 seconds, preferably 5 to 15 seconds, the laser power is 100 to 400 mW, and the temperature during the primer adhesion period and polymerase extension period is 55 to 65°C. Preferably 60℃. In addition, the cycle time during each cycle reaction is the time of the laser irradiation cycle process, which is 5 to 15 minutes.

The present invention is a nucleic acid detection method. Each of the aforementioned solid phase carriers 40 is magnetic, and after in-situ nucleic acid amplification is completed on each solid phase carrier 40 to generate the amplification reaction product 60, the nucleic acid detection is performed in the following steps: using an operating unit 15. Assist in the purification, separation and concentration of the amplification reactants 60 produced on each solid phase carrier 40; use the detection module 17 to identify the optical changes and heat transfer generated on each amplification reactant 60 located on each solid phase carrier 40. One or a combination of any two or more of inductive changes, electrochemical changes, magnetic changes or electrical quality changes.

In the present invention, the method for producing optical changes in each amplification reaction product 60 includes combining the amplification reaction product 60 with a primer 301 with a nucleic acid label, and using a photometer to detect the light intensity, where the nucleic acid label can be a fluorescent The optical label 3011 can detect the fluorescence intensity generated by the fluorescent label 3011 through a fluorescence meter, or the amplification reaction product 60 can detect the optical change or chemiluminescence change produced by using an enzyme-binding immunosorbent analysis method, or the solid-phase carrier 40 Spectral changes produced by binding to amplification reagent 60.

In the present invention, the optical changes produced on the amplification reaction product 60 are based on the optical changes produced by nucleic acid lateral flow test strip detection or immune lateral flow test paper strip 171 detection, further supplemented by thermal sensing. detection, surface plasmon resonance spectrum or a combination thereof to enhance the sensitivity of the nucleic acid lateral flow test strip detection or the immune lateral flow test strip 171 detection.

In the present invention, the method for producing magnetic changes in the amplification reaction product 60 includes AC magnetic introduction instrument detection and giant magnetoresistive detection or a combination of the two.

In the present invention, the method for producing an electrochemical formula change on the amplification reaction product 60 includes electrochemical detection coupled with an enzyme-conjugated immunosorbent assay, electrical impedance spectroscopy (EIS) detection, or a combination of the two.

In the present invention, the method for producing changes in electrical mass on the amplification reaction product 60 is a quartz crystal microbalance (QCM).

According to the above, the present invention uses a variety of experiments to prove: external energy stimulates the rapid hot and cold temperature cycle process on the solid phase carrier 40, and the amplification reaction product 60 produced by the in-situ nucleic acid amplification reaction on the back-end rapid solid phase carrier 40 The detection, including the establishment of the temperature required for nucleic acid amplification by external energy excitation on the solid phase carrier 40, and the discussion of the optimal and shortest external energy excitation (energy output and frequency period of irradiation), all prove that this case can be used in The purpose of amplifying nucleic acids and back-end detection is completed in a fast and stable state. In addition, operable implementation examples are also provided for sample pre-processing of the test object 20, and can be combined with rapid nucleic acid amplification and detection at the back-end. link to achieve the purpose of rapid nucleic acid testing. Therefore, the present invention can be applied to Point of Care Testing (POCT) based on nucleic acid detection. In summary, the present invention has the following advantages:

1. Using a solid phase carrier 40 with a high body surface area ratio, a combination of ligands (for example: antibodies, aptamers, oligonucleotides, proteins, polysaccharides, or its combination).

2. By controlling the relationship between the conditions of external energy excitation and the low-temperature reaction solution, the present invention can quickly create in-situ lysis of cells, capture of target nucleic acids, and in-situ nucleic acids in the in-situ environment 50 on the surface of the solid-phase carrier 40 The temperature conditions required for the amplification reaction.

3. Due to the above-mentioned in-situ environment 50 on the surface of the solid-phase carrier 40, the in-situ nucleic acid amplification reaction is limited to the surface of the solid-phase carrier 40. The cooling temperature of the nucleic acid amplification reaction solution 30 around the solid phase carrier 40 not only provides In addition to the rapid cooling of the solid phase carrier 40, the surrounding solution environment at a cooling temperature will also inhibit the amplification reaction of non-target nucleic acids. This feature will be beneficial to the specificity of nucleic acid amplification and detection (ie, nucleic acid molecule detection). In addition, this technical feature can also form a physical (such as temperature field) separation between nucleic acid amplification and detection, forming a process similar to virtual emulsion polymerase chain reaction or droplet digital polymerase chain reaction (Droplet Digital PCR, ddPCR). ), this phenomenon makes the nucleic acid amplification and detection reactions less susceptible to the complexity of the nucleic acid to be tested.

4. The overall low-temperature nucleic acid amplification reaction solution 30 can greatly improve the technical problems of evaporation and bubble generation of the nucleic acid amplification reaction solution 30 in traditional miniaturized nucleic acid amplification equipment.

5. The present invention can realize the temperature (thermal) cycle required for the nucleic acid amplification reaction in an ultra-fast manner, coupled with the in-situ solid-phase nucleic acid amplification reaction, and the subsequent purification, separation and concentration of the amplified nucleic acid molecules, which makes The overall nucleic acid amplification and detection time is significantly reduced.

6. The present invention greatly simplifies temperature control equipment and instruments.

7. Simplify the pretreatment of the test substance 20 and the detection of the amplification reaction product 60: the magnetic solid phase carrier 40 can be applied to the purification and concentration (enrichment) of the test substance 20, biological material 21, amplification reaction product 60, etc. ), in addition, the solid phase carrier 40 can also be used to subsequently use the immune lateral flow test strip 171 to perform signal substances for amplified nucleic acid detection (for example, visual images or heat detected through plasma thermal sensing. image), which will facilitate the preprocessing of the test substance 20 and the collection and detection of the amplification reaction substance 60 respectively.

The above brief description of the present invention is intended to provide a basic explanation of several aspects and technical features of the present invention. The Summary of the Invention is not a detailed description of the invention, and therefore its purpose is not to specifically enumerate key or important elements of the invention, nor to define the scope of the invention. It is merely to present several concepts of the invention in a concise manner.

10: Reaction unit 12: Energy excitation unit 13: External auxiliary cooling unit 30: Nucleic acid amplification reaction solution 40: Solid phase carrier 50: In situ environment

Claims (21)

A nucleic acid amplification method, including: a reaction unit including at least one test object, a nucleic acid amplification reaction solution, and at least one solid phase carrier, wherein the reaction unit and the test object inside the reaction unit, the nucleic acid amplification reaction solution, and the The amplification reaction solution and the solid phase carrier are in a cooling environment, the cooling environment has a cooling temperature; and the output of an external energy and the timing of opening or closing are controlled, and at the same time, the cooling temperature of the cooling environment is used to form nucleic acid amplification One or more reaction temperature cycles required for the reaction, such that in each reaction temperature cycle, all the solid phase carriers are simultaneously stimulated by external energy to form an in-situ environment, or each solid phase carrier stops receiving external energy. causing the in-situ environment to gradually dissipate, allowing each of the solid-phase carrier, the analyte and the nucleic acid amplification reaction solution to perform a nucleic acid amplification reaction during the formation and dissipation of the in-situ environment, thereby producing an amplification reaction product; During the start of each reaction temperature cycle, all the solid phase carriers in the reaction unit are simultaneously excited, so that the in-situ environment is formed around all the solid phase carriers, and amplification is generated in each solid phase carrier. Amplification reactants, a part of the amplification reactants is retained in each solid phase carrier, and another part of the amplification reactants is released into the nucleic acid amplification reaction solution, and during the shutdown period of each reaction temperature cycle , stop exciting the solid phase carrier, and use the cooling temperature of the cooling environment to gradually dissipate the in-situ environment. The nucleic acid amplification method described in claim 1, wherein the ratio of the total volume of the solid phase carriers to the volume of the nucleic acid amplification reaction solution is 1:200 to 1:1*10 ⁹ . In the nucleic acid amplification method as described in claim 1, the size of each solid-phase carrier further ranges from 8 to 2,000,000 nm. The nucleic acid amplification method as described in claim 1, wherein the cooling temperature is -10~50°C. The nucleic acid amplification method of claim 1, wherein the reaction unit or the nucleic acid amplification reaction solution can be pre-cooled to a cooling temperature or housed in an external auxiliary cooling unit, and the pre-cooled reaction unit or external The auxiliary cooling unit performs one or more reaction temperature cycles while maintaining the cooling temperature. The nucleic acid amplification method of claim 1, wherein the analyte is a cell, an organelle, a bacterium, a virus, a protist or a combination thereof. The nucleic acid amplification method of claim 6, wherein each solid phase carrier system includes: a multifunctional body; at least one enrichment ligand, each enrichment ligand is connected to the surface of the multifunctional body, and each enrichment ligand A collection ligand provides binding to the analyte; at least one amplification ligand, each amplification ligand is connected to the surface of the multifunctional body, and each amplification ligand provides binding to a biological substance. The nucleic acid amplification method of claim 7, wherein the biological substance is DNA or ribonucleic acid released from the analyte, or is released into the nucleic acid amplification reaction after the amplification ligand is replicated liquid amplification reaction. The nucleic acid amplification method of claim 6, wherein part of the solid phase carrier system includes: an enrichment body; at least one enrichment ligand, each of the enrichment ligands is connected to the surface of the enrichment body, and each The enriched ligand provides binding to the analyte; the other part of the solid phase carrier system includes: an amplification body; At least one amplification ligand, each amplification ligand is connected to the surface of the amplification body, and each amplification ligand provides biological substances released by binding to the analyte. The nucleic acid amplification method of claim 9, wherein the biological substance is DNA or ribonucleic acid released from the test object, or is released into the nucleic acid amplification reaction solution after the amplification ligand is replicated amplification reaction product. The nucleic acid amplification method of claim 1, wherein the analyte is free DNA or ribonucleic acid material. The nucleic acid amplification method of claim 11, wherein each solid-phase carrier system includes: an amplification body; at least one amplification ligand, each of the amplification ligands is connected to the surface of the amplification body, and each of the Amplification ligands are provided to bind the analyte. A rapid nucleic acid amplification system includes: a reaction unit that accommodates a reaction solution, a test object, a nucleic acid amplification reaction solution and at least one solid phase carrier; an auxiliary cooling unit that can be pre-cooled in advance The nucleic acid amplification reaction solution reaches a cooling temperature, or is formed by an external auxiliary cooling unit arranged around the reaction unit. The external auxiliary cooling unit continues to cool the reaction unit and amplifies the nucleic acid. The reaction liquid maintains the cooling temperature; an external energy excitation unit is used to provide the solid phase carrier to stimulate heat generation; an integrated driver is used to control the energy output, opening and closing timing of the external energy excitation unit, and can be controlled based on an external energy excitation unit. The energy calibrator and a temperature detection unit perform energy output to the external The auxiliary cooling unit performs feedback control to form one or more reaction temperature cycles required for nucleic acid amplification reactions; in each reaction temperature cycle, all the solid-phase carriers are simultaneously excited by external energy to form an in-situ environment, or the solid-phase carrier stops receiving external energy so that the in-situ environment gradually dissipates, so that the solid-phase carrier, the test substance and the nucleic acid amplification reaction solution proceed in the process of the formation and dissipation of the in-situ environment. The nucleic acid amplification reaction produces amplification reactants, part of the amplification reactants is retained in each solid phase carrier, and another part of the amplification reactants is released into the nucleic acid amplification reaction solution. A nucleic acid detection method, after completing the nucleic acid amplification method of any one of claims 1 to 12, nucleic acid detection is carried out in the following steps: using an operating unit to assist in purification, separation and concentration of the nucleic acid bound to the solid phase carrier The amplification reaction product; using a detection module to identify one of the optical changes, thermal sensing changes, electrochemical changes, magnetic changes or electrical quality changes produced on the amplification reaction product located on the solid phase carriers Or any combination of two or more. The nucleic acid detection method as described in claim 14, wherein the method for producing optical changes on the amplification reaction product includes mixing a nucleic acid label into the amplification reaction product and using a photometer to detect the light intensity, or the amplification reaction product An enzyme-binding immunosorbent assay is used to detect the optical changes or chemiluminescence changes produced, or the spectral changes produced by the combination of the solid-phase carrier and the amplification reaction product. As for the nucleic acid detection method described in claim 15, the optical change method produced on the amplification reaction product, the detection module is a nucleic acid lateral flow test strip detection or an immune lateral flow test strip, used to Detect the resulting optical changes. The nucleic acid detection method as described in claim 16, wherein the nucleic acid lateral flow test strip detection or immune lateral flow test strip detection is supplemented by thermal sensor detection, surface plasmon resonance spectroscopy, or any combination thereof to enhance the Reaction sensitivity of nucleic acid lateral flow test strip test or immune lateral flow test strip test. The nucleic acid detection method as described in claim 14, wherein the method for producing an electrochemical formula change on the amplification reaction product includes one of electrochemical detection coupled with an enzyme-binding immunosorbent analysis method and electrical impedance spectroscopy (EIS) detection. One or a combination of both. The nucleic acid detection method according to claim 14, wherein the method for producing magnetic changes in the amplification reaction product includes AC magnetic introduction instrument detection and giant magnetoresistance detection or a combination of any two. The nucleic acid detection method of claim 14, wherein the method for producing changes in electrical mass on the amplification reaction product is a quartz crystal microbalance. A rapid nucleic acid detection device, including: an operating unit to assist in the purification, separation and concentration of the amplification reaction product as described in claims 1 to 12; a detection module to identify the optical signals generated on the amplification reaction product One or a combination of any two or more changes, thermal sensing changes, electrochemical changes, magnetic changes or electrical mass changes.