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

Abstract

The present invention relates to a nucleic acid amplification method and an apparatus thereof, and a nucleic acid detection method and an apparatus thereof. The nucleic acid amplification apparatus includes a reaction unit, an energy excitation unit, an operation unit, and an auxiliary cooling unit. The reaction unit includes a test substance, one or more solid phase carriers, and a nucleic acid amplification reaction solution. Each solid phase carrier has a specific surface ligand functionalized for purification and separation of the target test substance and nucleic acid amplification reaction. By controlling the energy output and opening and closing timing of the energy excitation unit, a reaction temperature (thermal) cycle is generated, during which an in-situ environment is formed around each solid phase carrier, which reacts in the in-situ environment to generate an amplification reaction product. The present invention is to carry out a nucleic acid amplification method using the above-mentioned apparatus, and to detect an amplification reaction product using a nucleic acid detection method and an apparatus thereof.

Description

[Technical field]

[0001] The invention disclosed in this disclosure relates to a method and device for amplifying nucleic acid, and a method and device for detecting nucleic acid, and in particular to a method and device for amplifying nucleic acid by forming an in-situ environment around a solid support. Furthermore, the invention disclosed in this disclosure relates to a method and device for detecting nucleic acid after completion of nucleic acid amplification in the in-situ environment. [Background technology]

[0002] Nucleic acid amplification tests (NAAT) are widely used in molecular diagnostics, including infectious agent diagnosis, genetic testing, forensic medicine, agriculture, and clinical medicine. Nucleic acid amplification tests are highly sensitive and specific, even when the analyte is present in a trace amount in the test material, because the nucleic acid fragments to be detected are amplified. Polymerase chain reaction (PCR) is a common method used to amplify target nucleic acids. A total of three steps, from step (a) to step (c), are required for amplification to generate a new deoxyribonucleic acid (DNA) strand. In step (a), the temperature is increased to denature the double-stranded nucleic acid strand into single-stranded DNA (ssDNA) using the double-stranded nucleic acid strand as a template. In step (b), the temperature is decreased, and specific primers are attached to the single-stranded DNA (ssDNA) through the complementarity of the DNA sequence. In step (c), a new DNA strand is synthesized by polymerase at the rear end of the bound primer, generating a double-stranded DNA (dsDNA) product. This is the temperature (thermal) cycle of PCR, the total temperature contains 2 to 3 temperature intervals, and the number of DNA molecules produced increases exponentially with the cycle. However, traditional PCR instruments are generally large in size, expensive in equipment, have high energy consumption (using electric heating modules), and long operation time (usually more than 1 hour).

[0003] In addition, existing isothermal nucleic acid amplification technologies (iNAATs) can replace traditional nucleic acid amplification tests (NAATs) because they can amplify nucleic acid molecules at a constant and relatively mild temperature. Compared with traditional nucleic acid amplification tests, iNAATs can significantly reduce the complex temperature (thermal) cycles, associated complex equipment, and long operation times required for nucleic acid amplification. Because iNAATs do not require complex temperature (thermal) cycle designs, iNAATs reactions can be performed using simple equipment such as heating plates and oven-water baths. Among the various iNAATs methods or technologies, the reason why loop-mediated isothermal amplification (LAMP) is the most widely used is that LAMP is highly resistant to substances that inhibit DNA polymerase (such as hemoglobin, immunoglobulin IgG, or IgM). In addition, LAMP nucleic acid amplification has the characteristics of high specificity, high sensitivity, and high amplification efficiency. This means that LAMP has the potential to directly amplify target DNA or ribonucleic acid (RNA) from partially processed or unprocessed biological samples.

[0004] Compared to traditional quantitative PCR (qPCR) or PCR, LAMP-based nucleic acid amplification and detection sensitivity is improved by 10-100 times, according to the paper titled "Loop-Mediated Isothermal Amplification (LAMP) - A Review and Classification of Sequence-Specific Detection Methods." The paper was published in Analytical Methods on February 14, 2020. The authors are Lisa Becherer, Nadine Borst, Mohammed Bakheit, Sieghard Frischmann, Roland Zengerle, and Felix von Stetten.

[0005] Furthermore, according to the contents of the paper titled "Reduced False Positives and Enhanced Reporting of Loop-Mediated Isothermal Amplification using Quenched Fluorescent primers," compared with conventional PCR using two primers, the specificity of LAMP is superior when using 4 to 6 target-specific primers in the nucleic acid amplification reaction. In addition, LAMP can generate more than 109 DNA amplicons within an hour under isothermal conditions, and the presence of the target in the sample can be confirmed by directly or indirectly detecting a large amount of DNA amplification products and by-products of the LAMP reaction. However, LAMP-based nucleic acid amplification and detection technology has a high false positive rate and low discrimination power, which may limit its application in point-of-care testing. The above paper was published in Scientific Reports on May 14, 2019 by Patrick Hardinge and James AH Murray.

[0006] Conventional PCR platforms mainly use electric heating elements such as Peltier elements to perform programmed heating and cooling to generate the temperature (thermal) cycles required for polymerase chain reaction. This method involves conducting heat between the surface of an electric heater and the reaction solution, but it can take a long time because the heating and cooling rates are relatively slow (1.5 to 3 °C / sec). According to the contents of the paper titled "Emergence of ultrafast nucleic acid amplification technology for next-generation molecular diagnostics," the key elements for realizing ultrafast PCR include rapid thermal cycles, low heat capacity materials, high thermal conductivity, and fast polymerization enzymes. The above paper was published in Biosensors and Bioelectronics on June 18, 2019. The authors are Sang Hun Lee, Seung-min Park, Brian N. Kim, Oh Seok Kwon, Won-Yep Rho, and Bong-Hyun Jun). With the development of second-generation fast DNA polymerases, KAPA Biosystems used its proprietary direct molecular evolution protein engineering technology to screen a large number of DNA polymerase variants in multiple rounds and selected KAPA2G Fast DNA polymerase with second-generation fast synthesis efficiency. The extension speed of this method can reach 1Kb / sec, which significantly shortens the time required for DNA polymerase extension. In addition, to achieve rapid temperature (thermal) cycling, existing technologies use capillary LightCycle 2.0 Carousel-Based System (a rapid PCR detection device sold by Roche Diagnostics Co., Ltd.) or microfluidic polymerase chain reaction. The reaction volume is limited to a few nanoliters to microliters (nL~μL), which increases the surface area-to-volume ratio and enhances the effect of heat conduction. In addition, existing technologies also use materials with low heat capacity for heating. For example, the LightCycler 2.0 carousel-based system achieves rapid temperature (thermal) cycling by using low thermal capacity air for heating and cooling.However, although this method can complete the entire nucleic acid amplification reaction within a few minutes, it also causes many technical problems such as low amplicon yield, evaporation and bubble generation of trace amounts of reaction solution, platform scalability, and difficulty in thermal management. Therefore, rapid nucleic acid amplification is not just about achieving rapid temperature (thermal) cycling, but also a nucleic acid amplification strategy that can achieve rapid temperature (thermal) cycling without adversely affecting the nucleic acid amplification reaction.

[0007] Many research teams have proposed ways to improve the above problems. For example, the Royal Institution for the Advancement of Learning McGill University's US Patent Publication No. US10,604,798B2 (Invention title: HEATING MECHANISM FOR DNA AMPLIFICATION, EXTRACTION, OR STERILIZATION USING PHOTO-THERMAL NANOPARTICLES) proposes the use of photothermal nanoparticles. This nanoscale heater is used to heat the reaction solution in a contact or non-contact manner under the stimulation of an external light source. In this photothermal method, different temperature ranges are generated by external energy excitation with different energies, which are used for sterilization and photothermal lysis to achieve nucleic acid extraction and amplification of specific nucleic acid fragments. The Regents of the University of California's US Patent Publication No. US11,130,993B2 (Invention title: LED-driven plasmonic heating device for nucleic acid amplification) abstract describes it as a 3D structure of a gold film of a specific thickness. A plasma heating method using light-emitting diodes (LEDs) of a specific wavelength is adopted to rapidly generate the temperature (heat) cycle required for PCR reaction in a micro-structured reaction chamber. Alternatively, in the University of Chicago's US Patent Publication No. US11,045,874B2 (Invention name: BIPYRAMID-TEMPLATED SYNTHESIS OF MONODISPERSE NOBLE METAL NANOCRYSTALS), a synthetic gold nanoparticle with a double pyramid structure is used to heat the plasma generated by excitation of a light-emitting diode of a specific wavelength, thereby rapidly generating the temperature (heat) cycle required for PCR reaction in a micro-reaction chamber. All of the above methods use precious metal materials and special configurations to convert light energy into heat energy (reaction time exceeds 100 picoseconds) in the form of localized surface plasmon resonance (LSPR) under the excitation light of a specific wavelength light source. This photothermal conversion characteristic can be regarded as a fast and real-time response.This nanoscale heater concept has a fast heating rate in PCR thermal cycling, and temperature control can be easily achieved by adjusting the concentration of nanoparticles and the energy intensity of the incident light. However, the above method still has some challenges and problems that need to be overcome. For example, the first is the problem of evaporation of the reaction solution in the micro reaction tank. Second, the cooling effect in temperature (thermal) cycling is not obvious, and an external device (such as a fan) is still required to achieve the cooling effect. Third, the wavelength range of the incident light source required for localized surface plasmon resonance may overlap with the spectrum of some organic fluorescent dyes, which may limit its application in fluorescent quantitative PCR.

[0008] In addition to the above-mentioned plasmonic heating method of photothermal nanoparticles, there is also a method of applying an alternating magnetic field to heat magnetic nanoparticles, which is also used in PCR nucleic acid amplification technology. For example, in the US invention patent publication number US10,913,069B2 (Invention name: METHOD AND DEVICE FOR POLYMERASE CHAIN ​​REACTION) by the (▲Xie ▲Da ▼Bin) team of National Cheng Kung University, a method of amplifying nucleic acids is described in the patent claim. The method includes contacting a reaction mixture containing target nucleic acid with particles of transition metal material in a reaction unit, and irradiating the transition metal material particles using electromagnetic radiation (EMR) with a frequency of about 200kHz to 500THz to generate the temperature (heat) cycle required for PCR nucleic acid amplification. Among existing technologies, Australian Bio Molecular Systems (BMS) manufactured and sold the world's first qPCR device called "Mic qPCR" that uses electromagnetic heat as a heat source.

[0009] The heating methods using photothermal nanoparticles or magnetic nanoparticles mentioned above, as well as the method of heating the PCR reaction solution using electric heating components in conventional PCR methods, are all "volumetric heating" (i.e., heating increases the temperature of the entire reaction solution).

[0010] In contrast, a German molecular diagnostics company (GNA Biosolutions GmbH) has realized ultrafast PCR reactions using its own "Pulse Controlled Amplification (PCA) technology". This pulse-controlled nucleic acid amplification technology is a technology that controls the "local heating" of an area, and was published in a paper titled "Ultrafast PCR Technology for Point-of-Care Testing" (published in the Journal of Laboratory Medicine on October 12, 2017, by Lars Ullerich, Stephanie Campbell, Frank Krieg-Schneider, Federico Buersgens, and Joachim Stehr) and US Patent No. US9,382,583B2 (the invention title is a method for amplifying nucleic acids using heat transfer of nanoparticles, and related applications are DE102012201475B4, CN107604052A, EP2809806B1, and WO2013113910A 1). This technology uses nanoparticles with photothermal conversion properties (primer-functionalized), a laser light source, and a large amount of reaction solution for cooling, and is performed by short-term laser light pulse irradiation (laser light excitation interval is 10 nanoseconds to 500 milliseconds). Each time the laser light irradiates a part of the gold nanoparticles, a special thermal radiation field required for PCR reaction is generated. The thermal radiation field generated by this method is very fast. In addition, this method effectively heats only the small surface area environment of the gold nanoparticles containing the bound primers or amplicons. When the laser light stops irradiating the nanoparticles, these nanoparticles are rapidly cooled (due to their high specific surface area characteristics) to the temperature of the surrounding nucleic acid amplification reaction solution. During the entire PCR reaction process (i.e., repeated temperature cycles), the temperature of the surrounding nucleic acid amplification reaction solution in which the nanoparticles are present hardly changes. From the above, it was found that the PCA technology can quickly realize the temperature (heat) cycles required for nucleic acid amplification such as PCR.This technology not only significantly improves the problem of evaporation of reaction fluids (which affects the accuracy of subsequent detection work) that can be caused by the aforementioned "volumetric heating," but also reduces the need for specialized and precisely controlled cooling devices (such as fans and temperature feedback control) in traditional nucleic acid amplification (e.g., PCR) processes, but it still has disadvantages that will be described in detail below.

[0011] In addition, regarding the phenomenon of realizing "local heating" using the aforementioned "pulse-controlled amplification technology", this technology not only uses "photothermal particles" to realize the above-mentioned local heating, but can also use other principles or mechanisms to generate the "local heating" phenomenon. GNA Biosolutions' European Patent Publication No. EP3733292A1 (the invention title is a method for performing a polymerase chain reaction and an apparatus for performing the method, the related application publication is US2019 / 0249168A1) and a paper titled "Pulse-controlled amplification - a new powerful tool for on-site diagnosis under resource-limited conditions" (published in PLOS NEGLECTED TROPICAL DISEASES, published on January 29, 2021, by Katharina Mueller, Sarah Daβen, Scott Holowachuk, Katrin Zwirglmaier, Joachim Stehr, Federico Buersgens, Lars Ullerich, Kilian The paper published in the journal Stoecker uses a high-speed energy pulse heating microcirculator to generate nucleic acid amplification technology (embedding a small metal heating component directly into the nucleic acid amplification reaction), which instantly generates a thermal radiation field only on the surface area of ​​the small metal heating element (designed nucleic acid amplification reaction area) by heating electricity in a few microseconds, and after the above-mentioned current is removed, the metal heating element is instantly cooled by the large amount of reaction liquid present around it. This design can rapidly generate the temperature (heat) cycle required for nucleic acid amplification reaction (such as PCR) by repeatedly controlling and generating the above-mentioned temperature field, thereby achieving the purpose of rapid nucleic acid amplification.

[0012] As can be seen from the above-mentioned patent and non-patent documents, GNA Biosolutions' proprietary "Pulse Controlled Amplification Technology" (Pulse Controlled Amplification, PCA) can realize ultrafast nucleic acid amplification reactions (such as PCR) and subsequent nucleic acid molecule detection. This technology can significantly improve the problem of evaporation of reaction solutions caused by "volumetric heating" in conventional PCR reactions, and can also reduce the need for special cooling designs and devices required in conventional nucleic acid amplification (such as PCR) processes.

[0013] However, this technology mainly involves dynamically controlling the relative positions of the laser light and the PCR reaction zone so that the high-power laser light passes through the PCR reaction zone at intervals of 10 nanoseconds to 500 milliseconds. This selectively and specifically irradiates a portion of the gold nanoparticles in the PCR reaction zone, generating the temperature field required for the nucleic acid amplification reaction (as mentioned above). Basically, there are technical difficulties in this kind of operation control. In addition, the amount of target PCR reaction solution (100-500 μL) used to cool the PCA technology is relatively large. And the nucleic acid product (such as DNA) of the PCA nucleic acid amplification reaction exists in the form of free single-stranded DNA (ssDNA) in the PCR reaction solution. The detection method of the amplified nucleic acid molecule is as follows: For example, according to European Patent Publication No. EP2481817A1 (Invention Title: Method for Detecting Nucleic Acids) and European Patent Publication No. EP3733292A1 and Muller et al. (2020), the polymer formed by (primer-functionalized) photothermal nanoparticles and amplified free single-stranded DNA is detected, and the detection of the amplified nucleic acid molecule is realized by the spectral difference at a wavelength of 650 nanometers (nm) or the fluorescent signal generated by the TaqMan probe. In the above nucleic acid detection method, if a large amount of PCR reaction solution (100-500 μL) is used (due to the concentration dilution effect), more nucleic acid amplification time is required to accumulate the nucleic acid amplification signal, and this disadvantage further affects the time required for overall nucleic acid amplification and detection.

[0014] Furthermore, there are still many areas that need improvement regarding the PCA nucleic acid amplification method and apparatus described in the aforementioned GNA Biosolutions patent document, which can be summarized as follows.

[0015] The existing local heating method uses photothermal nanoparticles in a suspended phase and a large amount of PCR reaction solution. This method can realize the "ultrafast temperature (thermal) cycle" required for "ultrafast nucleic acid amplification", and the temperature of the PCR reaction solution does not change significantly during the ultrafast nucleic acid amplification process. This can improve the problem of evaporation of the PCR reaction solution caused by the "conventional fast nucleic acid amplification" technology during the temperature (thermal) cycle of the micro PCR reaction solution (affecting the detection accuracy). However, this technology mainly dynamically controls the relative position of the laser light and the PCR reaction zone so that the high-power laser light passes through the PCR reaction zone at intervals of 10 nanoseconds to 500 milliseconds. This allows selective and specific irradiation of some of the gold nanoparticles in the PCR reaction zone, generating the temperature field required for the nucleic acid amplification reaction (as mentioned above). Basically, there are technical difficulties in this kind of operation control.

[0016] Regarding the aforementioned GNA Biosolutions patent case, the PCR used in the PCA nucleic acid amplification reaction employs a two-step temperature program, one of which is 95°C required to denature double-stranded DNA molecules, and the other is 65°C required for primer binding and polymerase extension. This technology uses a large amount of PCR reaction solution as a cooling medium to lower the temperature of the gold nanoparticles from 95°C to 65°C, but the temperature difference is only 30°C, so the cooling speed may not be as good as the claimed "real-time cooling" effect.

[0017] In this technique, a large amount of PCR reaction solution is used as a cooling medium to cool the heated nanoparticles. However, the nucleic acid product of the PCA nucleic acid amplification reaction is generated in the PCR reaction solution in the form of free single-stranded DNA (ssDNA), so the amount of PCR reaction solution is large (i.e., dilution effect). This disadvantage increases the number of times to accumulate the nucleic acid amplification signal, further affecting the time required for overall nucleic acid amplification and detection.

[0018] Traditional PCR nucleic acid amplification uses a double primer design. When faced with highly complex nucleic acid test materials, if some nucleic acid fragments have partial sequence similarity, partial hybrid pairing will occur during the binding process, resulting in the generation of non-specific chimeric by-products, which will lead to the amplification of non-specific nucleic acid sequences, ultimately leading to erroneous judgments such as fixed-point testing devices. Summary of the Invention [Problem to be solved by the invention]

[0019] Therefore, in order to realize simple and rapid nucleic acid amplification and detection, it is currently an urgent task to overcome the above technical problems. [Means for solving the problem]

[0020] In order to solve the above problems in the prior art, the present invention provides a nucleic acid amplification method and device that is easy to operate and control, and can rapidly form the temperature (thermal) cycle required for the nucleic acid amplification reaction. Furthermore, during the nucleic acid amplification reaction, a portion of the amplified nucleic acid molecule is immobilized on the surface of a solid phase carrier. In addition, the detection and analysis method and device of the present invention can rapidly purify, separate, and concentrate the nucleic acid located on the surface of the solid phase carrier, thereby improving the specificity and sensitivity of the subsequent target nucleic acid molecule.

[0021] The nucleic acid amplification method of the present invention includes disposing at least one test substance, at least one nucleic acid amplification reaction solution, and at least one solid phase carrier in a reaction unit, forming a cooling environment in the reaction unit and its interior, and maintaining a predetermined cooling temperature; Controlling the output of external energy and its opening and closing timing, while forming one or more reaction temperature cycles required for nucleic acid amplification reaction to occur through the predetermined cooling temperature of the cooling environment, in which all the solid phase carriers are excited simultaneously by the external energy to form an in-situ environment in each reaction temperature cycle, or each solid phase carrier stops receiving the external energy and the in-situ environment gradually dissipates; The solid phase carrier, the test substance, and the nucleic acid amplification reaction solution carry out a nucleic acid amplification reaction in the formation and disappearance of an in situ environment to generate an amplification reaction product.

[0022] Nucleic acid amplification reactions include Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), or Isothermal Nucleic Acid Amplification Technologies (iNAAT).

[0023] In the nucleic acid amplification method of the present invention, during the start of each reaction temperature cycle of the nucleic acid amplification method, all the solid phase carriers in the reaction unit are excited simultaneously, a field environment is formed around each solid phase carrier, the amplification reaction products are generated on each solid phase carrier, a portion of the amplification reaction products is retained on each solid phase carrier, while another portion of the amplification reaction products is released into the nucleic acid amplification reaction solution; and further, during the shutdown period of each reaction temperature cycle, the excitation of each solid phase carrier is stopped, and each field environment is gradually dissipated by the cooling temperature of the cooling environment.

[0024] The enzyme system may be a polymerase, which may be a DNA polymerase or an RNA polymerase. Additionally, the enzyme may be one or more combinations of a reverse transcriptase (RT), a ribonuclease (RNase), a helicase, and a DNA ligase (DNA ligase), which may act synergistically with the polymerase.

[0025] The method by which the external energy excites each solid support is contact excitation or non-contact excitation.

[0026] Non-contact excitation can be photothermal or magnetic excitation. Photothermal excitation is one of the laser and LED arrays, where the wavelength of the light energy spans the visible to near infrared spectrum, with wavelengths ranging from 380nm to 1.4μm.

[0027] In magnetic excitation, an alternating magnetic field is used to generate heat. The alternating magnetic field is generated by an alternating magnetic field generator, and the amplitude and frequency of the alternating magnetic field are set according to the field conditions, such as the temperature required for the solid phase carrier to cause a nucleic acid amplification reaction. The amplitude of the alternating magnetic field is 0.5 to 550 kA / m, and the frequency of the alternating magnetic field is 3 to 3500 kHz.

[0028] Contact excitation is electrical heating, and electrical energy can be transmitted (wireless charging) through electronic circuits and induced magnetic flux. Electrical heating can be generated by Joule heating, thermoelectric heating, or surface acoustic waves (SAW).

[0029] The ratio of the total volume of all the solid phase carriers to the volume of the nucleic acid amplification reaction solution is 1:200 to 1:1×10 9 .

[0030] The size of the solid phase carrier is 8 to 2,000,000 nm. In the case of heat generation by photothermal excitation and magnetic excitation, the preferred size of the solid phase carrier is 8 to 1,000 nm, and in the case of electrothermal excitation, the preferred size of the solid phase carrier is 1,000 to 2,000,000 nm.

[0031] Each solid support may be one or any combination of two or more of the following: a sphere, an ellipsoid, a disk, a star, a rod, a square, an anisotropic protrusion, a nanoshell, a nanocage, a double triangular pyramid, a microfilament.

[0032] Each solid support may be suspended within the reaction unit or fixed to the inner wall of the cavity of the reaction unit, in different embodiments it may be one of them or a combination thereof.

[0033] The cooling temperature range is -10 to 50°C.

[0034] The reaction unit or the nucleic acid amplification reaction solution may be pre-cooled to the cooling temperature or may be placed in an external auxiliary cooling unit. One or more reaction temperature cycles are performed while the cooling temperature is maintained by the pre-cooled reaction unit, the pre-cooled nucleic acid amplification reaction solution, or the external auxiliary cooling unit.

[0035] The external auxiliary cooling unit is ice, a polymeric water-absorbing resin, a chemical endothermic reactant, a cooling chip, or a combination of any two or more. The polymeric water-absorbing resin is carboxymethyl cellulose. The chemical endothermic reactant is an endothermic reaction caused by ammonium nitrate dissolved in water or urea dissolved in water.

[0036] The analyte is a cell, an organelle, a bacterium, a virus, a protist, or a combination thereof. Each solid support includes a multifunctional body, at least one enrichment ligand, and at least one amplification ligand. Each enrichment ligand is bound to the surface of the multifunctional body and binds to the analyte. Each amplification ligand is bound to the surface of the multifunctional body and binds to the biological material. The biological material is deoxyribonucleic acid or ribonucleic acid released from the analyte, or an amplification reaction product released into the nucleic acid amplification reaction solution after the amplification ligand is replicated. The enrichment ligand is an antibody, a nucleic acid aptamer, an oligonucleotide, a protein, a polysaccharide, or a combination thereof. The amplification ligand is a nucleic acid aptamer, an oligonucleotide, and has a functional group modification for binding to the solid support, such as a primary amine, a biotin, a thiol group modification, or a combination thereof. Modifications that increase the specificity and stability of the capture of the oligonucleotide and the biological material, such as locked nucleic acid (LNA), phosphorothioate, morpholino modification, or a combination thereof.

[0037] The analyte is a cell, an organelle, a bacterium, a virus, a protist, or a combination thereof. A part of the solid support includes an enrichment body and at least one enrichment ligand, each of which is bound to the surface of the enrichment body and binds to the analyte. Another part of the solid support includes an amplification body and at least one amplification ligand, each of which is bound to the surface of the amplification body to bind to a biological substance released by the analyte. The biological substance is deoxyribonucleic acid or ribonucleic acid released from the analyte, or an amplification reaction product released into the amplification reaction solution after the amplification ligand is replicated. The enriched ontology corresponds to a multifunctional body having only the enriched ligand. The amplification body corresponds to a multifunctional body having only the amplification ligand.

[0038] The analyte is free DNA or RNA material. Each solid support contains an amplification body and at least one amplification ligand, each amplification ligand bound to the surface of the amplification body and capable of binding the analyte.

[0039] The rapid nucleic acid amplification system of the present invention includes a reaction unit, an auxiliary cooling unit, an external energy excitation unit, and an integrated driver. The reaction unit accommodates a reaction solution, a test substance, a nucleic acid amplification reaction solution, and at least one solid phase carrier. The auxiliary cooling unit is an external auxiliary cooling unit arranged around the reaction unit, which cools the nucleic acid amplification reaction solution to a cooling temperature in advance, or the reaction unit, and the external auxiliary cooling unit continues to cool the reaction unit so that the nucleic acid amplification reaction solution maintains the pre-cooled temperature. The external energy excitation unit excites the solid phase carrier to generate heat. The integrated driver controls the energy output and opening / closing timing of the external energy excitation unit, executes the energy output based on an external energy calibrator and a temperature detection unit, and feedback controls the external auxiliary cooling unit to form one or more reaction temperature cycles required for the nucleic acid amplification reaction.

[0040] In the nucleic acid amplification method of the present invention, after the nucleic acid amplification method is completed, nucleic acid detection is performed in the following manner: the amplification reaction product bound to the solid phase carrier is purified, separated, and concentrated by an operation unit; a detection module is utilized to detect one or any combination of two or more of optical changes, thermosensing changes, electrochemical changes, magnetic changes, or mass changes caused by the amplification reaction product bound to the solid phase carrier; the detection module further identifies the amplification reaction product bound to the solid phase carrier.

[0041] Methods for producing an optical change in the amplification reaction product include one or more of mixing a nucleic acid label with the amplification reaction product and detecting light intensity using a photometer, detecting an optical or chemiluminescent change produced in the amplification reaction product using an enzyme-linked immunosorbent assay, or a spectral change produced by binding of the amplification reaction product to the solid phase support.

[0042] The method of producing an optical change in the amplification reaction product includes a nucleic acid lateral flow strip or a lateral flow immunoassay strip in which the detection module is used to detect the optical change produced.

[0043] In addition to the nucleic acid lateral flow strip or the lateral flow immunoassay strip, the nucleic acid detection method uses thermal sensor detection, surface plasmon resonance spectroscopy, or any combination thereof to increase the sensitivity of detection of the nucleic acid lateral flow strip or the lateral flow immunoassay strip.

[0044] The method for electrochemically altering the amplification reaction product is one or a combination of electrochemical detection, enzyme-linked immunosorbent assay, and electrical impedance spectroscopy (EIS) detection.

[0045] The method for producing a magnetic change in the amplification reaction product is one or a combination of detection of frequency-dependent AC magnetic susceptibility by AC magnetometry and giant magnetoresistance measurement.

[0046] The method of generating a change in the electrical mass of the amplification reaction product is carried out using a quartz crystal microbalance.

[0047] The rapid nucleic acid detection device of the present invention includes an operation unit and a detection module, and can detect the amplification reaction products bound to the solid phase carrier. The operation unit purifies, separates, and concentrates the amplification reaction products. The detection module detects any combination of one or more of optical, thermosensitive, electrochemical, magnetic, or mass changes occurring in the amplification reaction products.

[0048] I understand the above. The present invention is different from existing nucleic acid amplification methods and has the following advantages:

[0049] First, by simply controlling the characteristics of the external energy excitation (such as the magnitude and frequency of the energy) and the cooling effect of the reaction unit, the temperature cycle required for the amplification of nucleic acid molecules can be rapidly generated in the in situ environment on the solid support surface, thereby achieving the goal of rapid nucleic acid amplification.

[0050] Second, by lowering the nucleic acid amplification reaction solution itself to a cooling temperature or placing the reaction apparatus in a cooled environment via an auxiliary cooling device, binding of non-target primers and amplification of non-target nucleic acids can be suppressed, improving the nucleic acid amplification reaction and improving the specificity of the amplification of target nucleic acid molecules.

[0051] Third, the amplified nucleic acid molecules immobilized on the solid support surface can reach saturation in a relatively short operation time (or with a relatively small number of nucleic acids) because they are limited by the space on the solid support surface, which is useful for the subsequent detection of the amplified nucleic acid molecules.

[0052] Fourth, if the solid phase carrier has strong magnetism, the amplification reaction product generated by nucleic acid amplification immobilized on the solid phase carrier can be further purified and concentrated by the magnetic field operation of the operation unit. This technical feature is beneficial for the subsequent detection of the amplification reaction product (e.g., improved detection efficiency).

[0053] Fifth, since the entire nucleic acid amplification reaction solution is kept at a low temperature, problems such as evaporation of the nucleic acid amplification reaction solution and generation of bubbles can be avoided, and non-specific nucleic acid amplification can be suppressed or reduced. [Brief description of the drawings]

[0054] [Figure 1(a)] FIG. 1(a) is a schematic diagram of rapid temperature rise according to one embodiment of the present invention. [Figure 1(b)] FIG. 1(b) is a schematic diagram of rapid cooling according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic diagram of the structure and surface characteristics of a solid support in one embodiment of the present invention. [Diagram 3] FIG. 3 is a schematic diagram of a nucleic acid amplification system according to one embodiment of the present invention. [Figure 4(a)] FIG. 4(a) is a schematic diagram showing a suspension state of solid phase carriers mixed in a nucleic acid amplification reaction solution in a reaction unit according to one embodiment of the present invention. [Figure 4(b)] FIG. 4(b) shows an embodiment of the present invention in which the solid phase carrier is semi-fixed by adhesion to the bottom of the reaction vessel in the reaction unit and is adhered in a semi-floating state to the inner wall of the cavity of the reaction unit. [Figure 4(c)] FIG. 4(c) is a schematic diagram of a solid support fixed to the inner wall of the cavity of a reaction unit in one embodiment of the present invention. [Diagram 5] FIG. 5 is a schematic diagram of a nucleic acid amplification system that uses a solid support with photothermal transduction capabilities to perform a water-cooled on-bead polymerase chain reaction, according to one embodiment of the present invention. [Figure 6] FIG. 6 is a schematic diagram of an operational unit for purifying a solid support according to one embodiment of the present invention. [Figure 7(a)-7(e)] 7(a) to 7(e) are schematic diagrams of the first reaction step of water-cooled on-bead polymerase chain reaction (on-bead PCR) in one embodiment of the present invention. [Figure 7(f)-7(g)]7(f)-7(g) are schematic diagrams of a second reaction step after completion of FIGS. 7(a)-7(e). [Figures 7(h)-7(k)] 7(h)-7(k) are schematic diagrams of the third reaction step after completion of 7(f)-7(g). [Figure 7(l)] FIG. 7(l) is a schematic showing the completion of multiple reactions. [Figure 8] FIG. 8 is a flow chart of the nucleic acid amplification and detection methods of the present invention. [Figure 9(a)] FIG. 9(a) is a schematic diagram of a primer pair for loop-mediated isothermal amplification reaction (LAMP) according to one embodiment of the present invention. [Figure 9(b)-9(e)] 9(b)-9(e) are schematic diagrams of pulsed water-cooled loop-mediated isothermal amplification (LAMP) on beads using FIG. 9(a). [Figure 10(a)] FIG. 10(a) is a schematic diagram showing photothermal conversion temperature measurement of a solid support in one embodiment of the present invention, in which a cycle of excitation for 60 seconds followed by a pause for 120 seconds is repeated 10 times. [Figure 10(b)] FIG. 10(b) is a schematic diagram of temperature measurement of a solid support excited with different powers in one embodiment of the present invention. [Figure 11(a)] FIG. 11(a) illustrates the relationship between the laser intensity applied to a microscale suspension of Magnetic Gold Nanoshells (MGNs) and the photothermal conversion temperature of the MGNs in one embodiment of the present invention. [Figure 11(b)] Figure 11(b) shows infrared thermal images of the microscale magnetic gold nanoshell suspension after excitation with different powers. [Figure 12(a)] FIG. 12(a) is a schematic diagram showing the appearance of one embodiment of the auxiliary cooling unit of the present invention. [Figure 12(b)]FIG. 12(b) is an illustration of the overall temperature change of a nucleic acid amplification reaction solution containing single-phase magnetic gold nanoshells when the auxiliary cooling unit of FIG. 12(a) is subjected to 100 external laser excitation cycles (irradiation at 400 mW / 0.16 cm2 for 1.25 seconds, 0.5 seconds off, irradiation at 150 mW / 0.16 cm2 for 7.5 seconds). [Figure 13(a)] FIG. 13(a) is a schematic diagram of a water-cooled bead-polymerase chain reaction pre-detection using an immune lateral flow test strip according to one embodiment of the present invention. [Figure 13(b)] FIG. 13(b) is a schematic diagram of the detection results of water-cooled bead-polymerase chain reaction using an immune lateral flow test strip according to one embodiment of the present invention. [Figure 14(a)] FIG. 14(a) is a schematic diagram of the positive reaction in FIG. 13(b) using plasma thermal sensing to analyze the immuno-lateral flow test strip to detect the detection result of solid phase carrier nucleic acid amplification. [Figure 14(b)] FIG. 14(b) is a schematic diagram of the negative reaction of FIG. 13(b) using plasma thermal sensing to analyze the immuno-lateral flow test strip to detect nucleic acid amplification on the solid phase carrier. [Figure 15(a)] FIG. 15(a) shows the use of lateral flow immunoassay strips to detect the amplification reaction products immobilized on MGNs at different photon cycles (i.e., 10 to 50 cycles) of water-cooled on-bead polymerase chain reaction with an 808 nm wavelength laser, where each photon cycle comprises irradiation at 400 mW / 0.16 cm2 for 1.25 s and irradiation at 150 mW / 0.16 cm2 for 7.5 s. Schematic diagram of the results. [Figure 15(b)] Figure 15(b) shows an example of the present invention using an 808 nm wavelength laser to perform water-cooled on-bead polymerase chain reaction under optimal laser excitation conditions and shortest photon cycles (irradiation at 400 mW / 0.16 cm2 for 1.25 s and irradiation at 150 mW / 0.16 cm2 for 7.5 s), and shows a schematic diagram of the electrophoretic analysis results of the amplification reaction products on the magnetic gold nanoshell and the amplification reaction products remaining in the nucleic acid amplification reaction solution at different photon cycles (10 to 30 times). [Figure 16(a)] FIG. 16(a) is a schematic diagram of an electrophoretic analysis of a simple sample (analyte) after performing a conventional polymerase chain reaction. [Figure 16(b)] FIG. 16(b) is a schematic diagram of the detection results of a simple sample (analyte) tested using an immuno-lateral flow test strip after performing water-cooled bead polymerase chain reaction. [Figure 17(a)] FIG. 17(a) is a schematic diagram of the electrophoretic analysis results of a complex sample (mixed with nucleic acids from multiple pathogenic bacteria and an E. coli nucleic acid sample) subjected to conventional polymerase chain reaction. [Figure 17(b)] FIG. 17(b) is a schematic diagram showing the detection results obtained by subjecting a test substance (complex sample) to a water-cooled bead polymerase chain reaction and detecting the same with an immune lateral flow test strip. [Figure 18(a)] FIG. 18(a) is a schematic diagram of the test results of using an 808 nm wavelength laser with different laser powers to excite a suspension of mixed enrichment and amplification bodies in one embodiment of the present invention to perform a photothermal lysis reaction of bacterial cells and subsequently form colonies. [Figure 18(b)] FIG. 18(b) is a schematic diagram of the test results using an 808 nm wavelength laser with different laser powers to induce photothermal lysis of a suspension of mixed enrichment and amplification bodies, followed by water-cooled on-bead polymerase chain reaction, using an immuno-lateral flow test strip in one embodiment of the present invention. [Figure 19(a)] Figure 19(a) shows the mixing of two functional magnetic gold nanoshells in one embodiment of the present invention, and is a schematic diagram of using E. coli suspension as the test substance to evaluate the detection efficiency and overall operation process of water-cooled bead polymerase chain reaction using laser excitation. [Figure 19(b)] Figure 19(b) is a schematic diagram showing a mixed suspension of enrichment and amplification bodies in an embodiment of the present invention, in which an E. coli suspension is used as the test substance to evaluate the detection results of the sensitivity of laser-induced water-cooled on-bead polymerization (on-bead PCR) using an immune lateral flow test strip. [Figure 20(a)]FIG. 20(a) is an image of the immuno-lateral flow test strip of FIG. 19(b) placed at the test line, pixel intensity analysis was performed using image analysis software (Image J), ​​and a schematic of image quantification is shown. [Figure 20(b)] Figure 20(b) shows the thermal imaging images using infrared lens focused to test the on-line detection by exciting the immuno-lateral flow test strip with different 808 nm laser energies (24 mW and 140 mW). [Figure 20(c)] FIG. 20(c) is a graph quantitatively illustrating the temperature difference that occurs after subtracting the background temperature in the environment from the thermal image of FIG. 20(b). [Figure 21(a)] FIG. 21(a) is a schematic diagram of the temperature change of the laser-induced water-cooled bead polymerase chain reaction at different PCR reaction initiation temperatures. [Figure 21(b)] FIG. 21(b) is a schematic diagram of the performance evaluation of laser-induced water-cooled bead polymerase chain reaction at different PCR reaction initiation temperatures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Hereinafter, the embodiments of the present invention will be described in detail. The objects, features, advantages, and ideas of the present invention will be clear to those skilled in the art from the description of this specification, and those skilled in the art can easily reproduce the present invention from the description of this specification. The embodiments of the invention described below show preferred embodiments of the present invention, and are shown for illustrative or explanatory purposes, and do not limit the present invention thereto. It will be clear to those skilled in the art that various changes and modifications can be made based on the description of this specification within the intent and scope of the present invention disclosed in this specification.

[0056] The nucleic acid amplification method according to one embodiment of the present invention includes a test substance 20, a nucleic acid amplification reaction solution 30, and at least one solid support 40 in a reaction unit 10. The cooling environment of the reaction unit 10 is maintained at a predetermined cooling temperature, which is −10 to 50° C. The preferred cooling temperatures are 15 to 30° C. and 30 to 50° C. The preferred cooling temperatures are −10 to 0° C. and 2 to 15° C. In particular, an in-situ environment 50 is formed around the surface of the solid support 40 by controlling the operating conditions of the external energy excitation (e.g., energy and frequency) to bring about a cooling effect in the cooling environment of the reaction unit 10. In addition, in the in-situ environment 50, a temperature (thermal) cycle required for the nucleic acid amplification reaction is further performed, so that the solid support 40 undergoes nucleic acid amplification at its position (in-situ) in each temperature cycle, generating an amplification reaction product 60. Here, the temperature (thermal) cycle required to perform the nucleic acid amplification reaction in the reaction unit 10 refers to the different temperatures required to denature the nucleic acid molecules, bind the primers, and extend the polymerase. The temperature (thermal) cycle is not limited to this. The size of each solid phase carrier 40 is 8 to 2,000,000 nanometers (nm), but the preferred size of the solid phase carrier 40 for photothermal and magnetic heating is 8 to 1,000 nanometers, and the solid phase carrier 40 for electric heating is 1,000 to 2,000,000 nanometers.

[0057] As shown in FIG. 1(a) and FIG. 1(b), the definition of the "in-situ environment 50" in the present invention is that when the solid phase carrier 40 is uniformly distributed in the reaction unit 10 under ideal conditions, the operating conditions of the external energy excitation (shown in FIG. 1(a)) and the cooling temperature of the cooling environment in the reaction unit 10 are controlled to control the size of the outward radiation range of the thermal radiation field of the solid phase carrier 40, so that the thermal radiation fields on the surface of the solid phase carrier 40 do not overlap with each other and form an independent nucleic acid amplification reaction space. Alternatively, the in-situ environment 50 of the present invention can also be called the local heating range of the solid phase carrier 40 (shown in FIG. 1(b)). In addition, when the excitation of the external energy is stopped instantaneously, the solid phase carrier 40 can be rapidly cooled depending on the cooling temperature of the reaction unit 10.

[0058] In addition, the so-called "in-situ nucleic acid amplification reaction" in the present invention means that a nucleic acid amplification reaction occurs in the in situ environment 50 on the surface of the solid phase carrier 40, and an amplification reaction product 60 is immobilized on the surface of the solid phase carrier 40. The amplification reaction product 60 includes, but is not limited to, an amplified nucleic acid molecule, and a portion of the amplification reaction product 60 generated by the in situ nucleic acid amplification reaction remains in each solid phase carrier 40, and another portion of the amplification reaction product 60 is released into the nucleic acid amplification reaction solution 30.

[0059] In one embodiment of the present invention, the nucleic acid amplification reaction solution 30 includes free primers 301, nucleotides, enzymes, and reaction additives. The enzyme system may be a polymerase 302, which may be a DNA polymerase or an RNA polymerase. Additionally, the enzyme may be one or more combinations of reverse transcriptase (RT), ribonuclease (RNase), helicase, DNA ligase (DNA ligase), and may act synergistically with the polymerase.

[0060] In one embodiment of the present invention, the nucleic acid amplification reaction includes a polymerase chain reaction (PCR), a ligase chain reaction (LCR), or an isothermal nucleic acid amplification technology (iNAAT). Among the many iNAAT methods, a preferred one is loop-mediated isothermal amplification (LAMP). A two-step temperature (thermal) cycling process is used in the polymerase chain reaction method, in which the temperature used for denaturing the nucleic acid is 85-95°C, the temperature for primer binding and polymerase extension is 60-65°C, and the temperature for the loop-mediated isothermal amplification reaction is 60-65°C.

[0061] Referring to FIG. 2, in one embodiment of the present invention, the solid support includes a multifunctional body 41, at least one enrichment ligand 70, and at least one amplification ligand 71. Each enrichment ligand 70 is attached to the surface of the multifunctional body 41. The enrichment ligand 70 is an antibody, a nucleic acid aptamer, an oligonucleotide, a protein, a polysaccharide, or a combination thereof. The amplification ligand 71 is attached to the surface of the multifunctional body 41 and is a nucleic acid aptamer, an oligonucleotide, and has a functional group modification for binding to the solid support, such as a primary amine, biotin, a thiol group modification, a protein, a polysaccharide, or a combination thereof. Modifications that enhance the specificity and stability of capturing oligonucleotides and biological substances are locked nucleic acids (LNA), phosphorothioates, morpholino modifications, or combinations thereof. However, the present invention is not limited thereto.

[0062] The enrichment ligand 70 selected in this embodiment can match the analyte 20. The analyte 20 can be a cell, an organelle, a bacterium, a virus, a protist, or a combination thereof. After specific binding, the analyte 20 is adsorbed to the enrichment ligand 70 on the solid support 40.

[0063] In this embodiment, at least one amplification ligand 71 is provided on the multifunctional body 41, and can bind to free primers in the nucleic acid amplification reaction solution 30. The amplification ligand 71 can also bind to a biological material 21, which can be, for example, a nucleic acid molecule of the test substance 20 released by high temperature, or an amplification reaction product 60 released into the nucleic acid amplification reaction solution 30 after the amplification ligand is replicated. The amplification reaction product 60 can also bind to a primer 301 having a label in the nucleic acid amplification reaction solution 30, and the label in the primer 301 is a nucleic acid label. Examples of nucleic acid markers include, but are not limited to, radioisotopes (e.g., 3H, 14C, 32P, etc.), digoxin (DIG), biotin, fluorescent labels (e.g., fluorescein isothiocyanate (FITC), Texas Red, Cy2, Cy3, Cy5, Cy7, rhodamine B, etc.), and luminescent substances (e.g., acridinium ester (2',6'-DiMethylcarbonylphenyl-10-sulfopropylacridinium-9-carboxylate 4'-NHS Ester)).

[0064] In this embodiment, the surface of the solid support 40 is treated by at least one or more procedures to form highly specific functionalized enrichment ligands 70 and amplification ligands 71, thereby analyzing trace amounts of the target analyte 20.

[0065] In another embodiment of the present invention, the analyte 20 is a cell, a cellular organelle, a bacterium, a virus, a protist, or a combination thereof. A portion of the solid support 40 includes an entrapping body and at least one enrichment ligand 70, and another portion of the solid support 40 includes an amplification body 42 and at least one amplification ligand 71. The enrichment ligand 70 is bound to the surface of the entrapping body, and the enrichment ligand 70 is used to bind the analyte 20. The amplification ligand 71 is bound to the surface of the amplification body 42, and the amplification ligand 71 is used to bind the biological material 21 released from the analyte 20. Alternatively, the amplification ligand 71 is replicated and binds to the amplification reaction product 60 released in the nucleic acid amplification reaction solution 30.

[0066] In yet another embodiment of the present invention, the test substance 20 is, for example, free DNA or ribonucleic acid material. Furthermore, the free nucleic acid includes free cell nucleic acid, free tumor nucleic acid, or a combination thereof. Each solid phase support 40 includes an amplification body 42 and at least one amplification ligand 71. The amplification ligand 71 is connected to the amplification body 42 and binds to the test substance 20. Alternatively, the amplification ligand 71 is replicated and binds to the amplification reaction product released into the nucleic acid amplification reaction solution 30.

[0067] In various embodiments of the present invention, the solid support 40 is excited with different external energies, such as photothermal conversion or high-frequency induction electromagnetic methods for real-time heating. In one embodiment of the present invention, plasma is used to heat the surface structure of the solid support 40. In another embodiment of the present invention, light energy is used to heat an organic photothermal coating as the surface layer of the solid support 40. In another embodiment of the present invention, the magnetic solid support 40 is heated by local heating. That is, the magnetic solid support 40 is placed in an external magnetic field, the amplitude of the alternating magnetic field is controlled in the range of 0.5 to 550 kA / m, and the frequency of the alternating magnetic field is controlled in the high-frequency range of 3 to 3,500 kHz (alternating magnetic field), to achieve the temperature range required for denaturation of nucleic acid, adhesion of primer, and extension of polymerase.

[0068] In one embodiment of the present invention, in the case of the solid support 40 excited by "photothermal", the body of the solid support 40 is, for example, a sphere formed of a metal or composite material, and includes a magnetic core 401 and an intermediate support layer 402. The intermediate support layer 402 is used to stabilize and maintain the structure of the outer photothermal conversion layer, and may be a silicon shell. The overall shape of the intermediate support layer 402 may be a sphere, an ellipse, a disk, a star, a rod, a square, an anisotropic convex surface (such as a nanostar), a nanoshell, a nanocage, or a double triangular pyramid. The overall shape of the intermediate support layer 402 is a combination of one or more of these microfilaments, but the present invention is not limited to this form. The magnetic core 401 includes a transition metal or its oxide, such as ferrous oxide (FeO), ferric oxide (Fe2O3), triiron tetroxide (Fe3O4), ferric oxide hydroxide (FeO(OH)), ferrous hydroxide (Fe(OH)2), ferric hydroxide (Fe(OH)3), cobalt monoxide (CoO), cobalt oxide hydroxide (CoO(OH)), tricobalt tetroxide (Co3O4). However, the present invention is not limited to the group consisting of derivatives and mixtures thereof. The surface of the solid support 40 is composed of a noble metal layer (such as gold, silver, palladium, platinum, or a combination thereof) having a localized surface plasmon resonance (LSPR) effect, or an organic layer having light-to-heat conversion efficiency. Cyanine, polypyrrole, or graphene with a near infrared (Near Infrared Spectroscopy, NIR) absorption spectrum can also be added to the surface of the solid support 40.

[0069] As shown in FIG. 3, a high-speed nucleic acid amplification system according to an embodiment of the present invention includes a reaction unit 10, an integrated driver 11, an energy excitation unit 12, and an auxiliary cooling unit. The reaction unit 10 accommodates a nucleic acid amplification reaction solution 30 and a plurality of solid-phase carriers 40. The solid-phase carriers 40 are suspended in the nucleic acid amplification reaction solution 30 (FIG. 4(a)), or are bonded in a semi-suspended state to the inner wall of the cavity of the reaction unit 10 (FIG. 4(b)), or are fixed to the cavity of the wall of the reaction unit 10 (FIG. 4(c)). In an embodiment of the present invention, the auxiliary cooling unit may be in the following form.

[0070] First, the nucleic acid amplification reaction solution 30 (volume 100 μL or more) is used as an auxiliary cooling unit. Specifically, the external auxiliary cooling unit 13 cools the nucleic acid amplification reaction solution 30 to a cooling temperature, and then is introduced into the reaction unit 10, and the nucleic acid amplification reaction solution 30 in the reaction unit 10 is used as an auxiliary cooling unit. In this way, after external energy is applied, the nucleic acid amplification reaction solution 30 gradually rises in temperature and continues to maintain the cooling temperature during the reaction, so that the efficiency of the nucleic acid amplification reaction is worse than that of the auxiliary cooling unit described below, but the overall operation time is still shorter than that of the conventional nucleic acid amplification reaction by polymerase chain reaction.

[0071] Secondly, the auxiliary cooling unit is composed of the nucleic acid amplification reaction solution 30 and the external auxiliary cooling unit 13. The external auxiliary cooling unit 13 is arranged around the reaction unit 10 (as shown in FIG. 5) and continuously provides a cooling temperature around the reaction unit 10 so that the temperature of the nucleic acid amplification reaction solution 30 in the reaction unit 10 decreases (i.e., the cooling temperature is maintained). In addition, a temperature detection unit 14 (e.g., a K-type thermocouple) that detects the temperature change of the external auxiliary cooling unit 13 in real time and feedback controls the temperature can be provided. The acid amplification reaction solution 30 in the reaction unit 10 is always kept at the cooling temperature, allowing for rapid nucleic acid amplification reaction.

[0072] The external auxiliary cooling unit 13 may be, for example, ice or a polymeric water-absorbing resin (such as carboxymethyl cellulose) having a large specific heat capacity, or a cooling chip (such as a Peltier cooler). In another embodiment of the present invention, the external auxiliary cooling unit 13 may also contain water (0.5 mL to 1 mL) or ammonium nitrate (molar heat of solution (ΔHsol) = 26.2 KJ), or urea (molar heat of solution ΔHsol) = 15 KJ). These crystals dissolve in water and cause a chemical endothermic reaction.

[0073] As shown in Fig. 3, the energy excitation unit 12 directly and efficiently provides external energy to the solid phase carrier 40 and converts it into heat energy through contact or non-contact energy transfer. When the energy excitation unit 12 stops supplying energy to the solid phase carrier 40, the auxiliary cooling unit with a cooling temperature rapidly dissipates the heat of the solid phase carrier 40. Therefore, the present invention can rapidly form a temperature (heat) cycle required for the nucleic acid amplification reaction in the in-situ environment 50 around the solid phase carrier 40 through the interaction and regulation between the energy excitation unit 12 and the auxiliary cooling unit, and complete the nucleic acid amplification reaction on the surface of the solid phase carrier 40 by the method of in situ nucleic acid amplification.

[0074] In an embodiment of the present invention, the contact excitation is electrothermal heating, and the power transfer can be achieved by electronic circuits and inductive magnetic flux (wireless charging). The generation of electrical heat can be Joule heating, thermoelectric heating, or heating by surface acoustic waves (SAWs).

[0075] In the embodiment of the present invention, the method for exciting the solid support 40 in a non-contact manner is preferably a plasma heating method. As shown in Fig. 3, an integrated driver 11 (e.g., including a computer and LabVIEW driver software for controlling the operation of the energy excitation unit 12) drives the energy excitation unit 12. In this manner, the output energy of the energy excitation unit 12 (e.g., an infrared fiber-coupled laser) and the timing of its opening and closing are controlled, and a local thermal radiation field change is generated in the solid support 40 in the reaction unit 10.

[0076] In an embodiment of the present invention, the energy excitation unit 12 excites the solid support 40 in a non-contact manner, such as photothermal excitation or heating. As shown in FIG. 5, the energy excitation unit 12 is composed of a laser collimator 121 and a laser emitter 122, and may be an LED diode light source. In another embodiment of the present invention, the energy excitation unit 12 is a magnetic excitation heating type (e.g., a high-frequency magnetic field generator), but the present invention is not limited thereto. The energy excitation unit 12 mainly heats the solid support 40 itself and the in-situ environment 50 in its vicinity (the distance is from several hundred nanometers (nm) to several hundred micrometers (μm)).

[0077] In an embodiment of the present invention, the high-speed nucleic acid amplification system further includes a temperature detection unit 14 (e.g., a K-type thermocouple) for detecting the temperature feedback of the external auxiliary cooling device 13. By controlling the auxiliary cooling unit and the heating of the energy excitation unit 12 of the solid-phase support 40 in a well-balanced manner, the temperature (thermal) cycle required for the polymerase chain reaction is rapidly realized, and the nucleic acid amplification reaction is rapidly completed.

[0078] In an embodiment of the present invention, the high-speed nucleic acid amplification system further includes an operation unit 15. The operation unit 15 can be composed of a permanent magnet, an electromagnet, or a combination thereof, but the present invention is not limited thereto. In an embodiment of the present invention, the unit 15 is used to perform a simple magnetic operation for purifying and concentrating (concentrating) the solid phase carrier 40 in the reaction unit 10, which is useful for the subsequent detection and analysis of the amplification reaction product 60 bound to the solid phase carrier 40 (e.g., enhancing the detection signal of the amplification reaction product 60), but the present invention is not limited thereto. As shown in FIG. 6, in the subsequent concentrating (concentrating) operation of the amplification reaction product 60, the solid phase carrier 40 and the nucleic acid amplification reaction solution 30 are separated simply by magnetically attracting the magnetic solid phase carrier 40 via the operation unit 15. This purification and separation not only removes interfering substances (e.g., non-specific amplification nucleic acid products or free primers 301 and polymerase 302, etc.) in the detection of back-end nucleic acid molecules, but also further improves the detection efficiency (e.g., detection sensitivity) of the subsequent nucleic acid molecules through this concentration operation.

[0079] It should be particularly noted here that the present invention can simplify the temperature control device. That is, only a single energy excitation unit 12 and an external energy calibrator 16 can be used for temperature control. The external energy calibrator is, for example, a light intensity meter, and changes the temperature around the solid-phase carrier 40 by controlling the energy intensity and energy excitation time of the energy excitation unit 12, thereby generating an in situ environment 50 around all the solid-phase carriers. As a result, the present invention can reduce the step of deflecting laser light two-dimensionally using a two-dimensional mirror scanner and selectively and specifically focusing the laser on a portion of the nanoparticles in the PCR sample for 10 nanoseconds to 500 milliseconds, as disclosed in GNA's invention patent number US9,382,583B2 (the invention title is a method for amplifying nucleic acid using heat transfer of nanoparticles).

[0080] In one embodiment of the invention, the analyte 20 is free DNA, ribonucleic acid material, or biological material 21 released from the analyte 20, and the solid support 40 is an amplification entity 42. As shown in FIG. 7(a), each amplification ligand 71 is bound to the surface of the amplification body 42, and the amplification ligand 71 is bound to the test substance 20 or biological substance 21. As shown in FIG. 7(b), when an 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 carry out a first nucleic acid amplification in the amplification ligand 71, and an amplification reaction produces an amplification reaction product 60. As shown in FIG. 7(c), the external energy source is temporarily turned off, and the thermal field around the solid carrier 40 is rapidly cooled by the auxiliary cooling unit, and the in-situ environment 50 dissipates. As shown in FIG. 7(d), the external energy source is turned on again to form the in-situ environment 50 again to cause a denaturation reaction, and separate the test substance 20 and the amplification reaction product 60. As shown in Figure 7(e), the external energy source is temporarily shut off, causing the free primer 301 having a fluorescent label 3011 in the nucleic acid amplification reaction solution 30 to bind to the amplification reaction product 60, and the test substance 20 binds to another amplification ligand 71 on the solid support 40.

[0081] As shown in Figures 7(f)-(g), after turning on the external energy source, the polymerase 302 immobilizes the analyte 20 or biological material 21 on the amplification ligand 71 on the solid support 40, and further nucleic acid amplification reaction is carried out using the amplification reaction product 60 as a template to produce the amplification reaction product 60. Figures 7(h)-(k) show cycles in which the energy excitation unit 12 is turned on and off to carry out another nucleic acid amplification reaction. Figure 7(l) shows that after multiple cycles, the amplification reaction product 60 supported on the solid support 40 reaches saturation.

[0082] In one embodiment of the present invention, the solid support 40 is excited by "photothermality", and the surface of the solid support 40 is a gold nanoshell 403 (or also called "magnetic gold nanoshell"). The gold nanoshell 403 has a magnetic core, and between the gold nanoshell 403 and the magnetic core 401 there is an intermediate layer 402. In one embodiment of the present invention, the photothermal excitation is either a laser, an LED array, or a combination of both. In one embodiment of the present invention, the external energy source is a laser, and the laser wavelength is mainly in the visible spectrum to near infrared spectrum (380 nm to 1.4 μm). Furthermore, in an embodiment of the present invention, it is shown that the magnetic gold nanoshell suspensions of different concentrations all have the property of absorbing in the near infrared spectral region (750 nm to 1.4 μm). In another embodiment of the present invention, a localized surface plasmon resonance (LSPR) effect can be generated on the surface of the magnetic gold nanoshell under 808 nm laser excitation, thereby generating a plasma heating effect.

[0083] In another embodiment of the present invention, the solid support 40 is excited by a "magnetic change", and the solid support 40 includes a magnetic core 401. When the solid support 40 is exposed to an external AC magnetic field, the induction heating effect is mainly due to the magnetization reversal of the nanostructure, including the Neel relaxation mechanism (magnetic moment rotation of the solid support 40) and the Brownian relaxation mechanism (physical rotation of the solid support 40 in an aqueous medium). This heating ability depends on the characteristic nanostructure, such as the average size, composition, magnetization, and magnetic anisotropy, as well as the amplitude (Hac) and frequency (f) of the applied alternating magnetic field. The alternating magnetic field is formed by an alternating magnetic field generator, and its amplitude and frequency are set according to the field conditions that generate the temperature required for nucleic acid amplification of each solid support 40. In addition, the amplitude of the alternating magnetic field is 0.5 to 550 kiloamperes per meter (kA / m), and the frequency of the alternating magnetic field is 3 to 3500 kilohertz (kHz).

[0084] In another embodiment of the present invention, the solid support 40 may be a stable colloidal single-phase magnetic solid support 40 dispersed in the nucleic acid amplification reaction solution 30 in the form of a suspension. Single phase refers to a single dispersed suspension, and when the size of the solid support 40 reaches the nanoscale, a stable colloidal single-phase solution is formed due to the electric potential of its surface. In one embodiment of the present invention, the solid support 40 is, for example, a ferromagnetic or superparamagnetic iron oxide solid support 40 (Superparamagnetic iron oxide Nanoparticles, SPIONs), particularly magnetite (Fe3O4), maghemite (γ-Fe2O3). In another embodiment of the present invention, the solid support 40 is, for example, a spinel structure ferrite nanomaterial, for example, a spinel ferrite containing MIIFe2O4 (where MII=Co2+, Ni2+, Zn2+, Mn2+, etc.). In another embodiment of the present invention, the solid support 40 may be composed of a ferrite nanomaterial doped with a transition metal (e.g., Ni, Co, etc.), which provides a larger saturation magnetization value, a stable effective magnetic anisotropy (Keff), and a stronger magnetization loss under an external AC magnetic field.

[0085] In another embodiment of the present invention, the size of the solid support 40 excited by the magnetic change is 8 to 1000 nm, and its shape may be, but is not limited to, a sphere, a nanocube, a nanooctahedron, a rod, a disk, a hollow sphere, a star, a tetrapod, etc. The amplitude (Hac) and frequency (f) of the external AC magnetic field can be adjusted in a wide range of frequencies (3 to 3,500 kHz) and magnetic field amplitudes (0.5 to 550 kA / m).

[0086] In the above embodiment, the surface of the multifunctional body 41, enrichment or amplification body 42 of the solid support 40 further includes a packing material. The packing material is used to prevent aggregation of the solid support 40, increase the stability of the aqueous solution, and inhibit the adsorption of the polymerase. The packing material also prevents non-specific proteins and primers 301 from adsorbing to the solid support 40. In addition, the packing material may be polyethylene glycol (PEG) 72 with a molecular weight of 5 to 10 kilodaltons (kDa) and bovine serum albumin (BSA) 73. The enrichment or amplification ligand 71 uses a crosslinker to form a stable covalent bond with the functional groups provided by the packing material, such as the covalent bond between the carboxyl group, the amine group, and the thiol group.

[0087] As shown in Fig. 8, the nucleic acid amplification reaction method of the present invention refers to two reactions: water-cooled on-beads polymerase chain reaction or water-cooled on-beads loop-mediated isothermal amplification reaction. In particular, the water-cooled type refers to using the above-mentioned auxiliary cooling device for cooling, and the bead type refers to performing an in-situ nucleic acid amplification reaction on a solid support 40. The nucleic acid amplification reaction method of the present invention includes the following steps.

[0088] In step S20, in the reaction unit 10, the operation unit 15 is used to wash and remove impurities and concentrate (enrich) the test substance 20 if at least one analyte is free DNA or if ribonucleic acid material is present, proceed directly to step S51, otherwise proceed to step S30.

[0089] In step S30, at least one energy excitation unit 12 provides an external energy source to the multiple solid supports 40, and the temperature of the multiple solid supports 40 increases to the thermal dissociation temperature of the test substance 20 (e.g., 80 to 95°C), and the process proceeds to step S40.

[0090] In step S40, at least one test substance 20 is thermally dissociated to release biological material 21 (eg, DNA or ribonucleic acid), and then step S50 is performed.

[0091] In step S50, the energy excitation unit 12 is adjusted to provide an external energy source to the plurality of solid phase carriers 40 until a nucleic acid hybridization temperature is reached, and the plurality of solid phase carriers 40 bind with at least one biological material 21. The operation unit 15 purifies, separates and concentrates the plurality of solid phase carriers 40, and then step S60 is performed.

[0092] In step S51, the energy excitation unit 12 is adjusted to provide an external energy source to the plurality of solid phase carriers 40 until the nucleic acid hybridization temperature is reached, and the plurality of solid phase carriers 40 bind to at least one analyte 20. The operation unit 15 purifies, separates, and concentrates the plurality of solid phase carriers 40, and then step S60 is performed.

[0093] In step S60, a pre-cooled nucleic acid amplification reaction solution 30 (the temperature of the pre-cooled nucleic acid amplification reaction solution 30 is -10°C to 4°C) is added to the reaction unit 10, and the external auxiliary cooling unit 13 is connected to the reaction unit 10. If a water-cooled on-bead polymerase chain reaction is performed, proceed directly to step S70, and if a water-cooled on-bead loop-mediated isothermal amplification reaction is performed, proceed directly to step S71.

[0094] In step S70, the energy excitation unit 12 is adjusted to provide external energy to the multiple solid phase carriers 40, causing the in-situ environment 50 around the solid phase carriers 40 to reach the nucleic acid denaturation temperature (90-95°C) and primer binding / polymerase extension temperature (60-65°C) required for the polymerase chain reaction, and further, an in-situ nucleic acid amplification reaction is carried out on the multiple solid phase carriers 40 via at least one enzyme. To enter the detection stage, proceed to step S80.

[0095] In step S71, the energy excitation unit 12 is adjusted to provide external energy to the multiple solid phase carriers 40, so that the in-situ environment 50 reaches a temperature (60 to 65°C) required for the loop-mediated isothermal amplification reaction, and then an in-situ nucleic acid amplification reaction is carried out on the multiple solid phase carriers 40 via at least one enzyme. To enter the detection stage, proceed to step S80. To enter the detection stage, proceed to step S80.

[0096] In step S80, the amplification body 42 and the supernatant are separated via the operation unit 15.

[0097] In step S90, the detection module 17 is used to identify a signal change in the amplification reaction product 60 located on the amplification body 42 and label the color change, emission change, or combination thereof generated by detecting the nucleic acid on the amplification reaction product 60.

[0098] In one embodiment, the water-cooled on-beads loop-mediated isothermal amplification reaction of the present invention introduces at least one pair of specific primers 301 on the surface of the solid support 40. The primers 301 function as the amplification body 42 of the amplification ligand 71 (shown in FIG. 9(a)). In this embodiment, the at least one pair of specific primers 301 includes three pairs of primers 301 (FIP, BIP, LF, LB, B3 and F3). The water-cooled on-beads loop-mediated isothermal amplification reaction is carried out on the amplification body 42. When the external energy source is turned off, the specific primers 301 on the amplification body 42 capture the target nucleic acid sequence through DNA hybridization. When the nucleic acid sequence is the analyte 20 or biological material 21 (shown in FIG. 9(b)) and the external energy source is turned on, the amplification body 42 generates a temperature range suitable for the loop-mediated isothermal amplification reaction, and preliminary extension is performed by the polymerase 302 (shown in FIG. 9(c)). Then, when the external energy source is turned off, the temperature drops rapidly and the loop-mediated isothermal amplification reaction is stopped (as shown in FIG. 9(d)). After multiple cycles of the steps of FIG. 9(b) to FIG. 9(d), the amplification reaction product 60 carried on the amplification body 42 reaches saturation (as shown in FIG. 9(e)). After the reaction is completed, the amplified amplification reaction product 60 can be concentrated (enriched) and purified through the operation unit 15 in addition to the amplification body 42. This purification and separation operation by the amplification body 42 is also useful for specific detection of the amplification reaction product 60 of the reaction (LAMP / RT-LAMP). The amplified nucleic acid fragments contain only true positive amplified fragments between the 3' ends of FIP and BIP, which improves the specificity of LAMP / RT-LAMP product detection and reduces the occurrence of false positives.

[0099] Furthermore, according to the disclosure of U.S. Patent No. US9,382,583 B2 of GNA Biosolutions, "local heating" of the solid support 40 occurs under the following condition: when the energy excitation interval is shorter, or when the excitation interval is t1, the critical excitation interval t1 can be expressed by the following formula: t1=(s1×|x|)^2 / D t1 represents the time required for heat to diffuse from one nanoparticle to the next at the average nanoparticle distance. s1 represents a scaling factor, which is a measure of the diffusion length of the heat flow generated by the thermal force field when the solid support 40 is irradiated with the critical excitation interval t1, and s1 is less than or equal to 1 when the solid support 40 is locally heated. |X| represents the average distance between the solid supports 40 . D represents the thermal diffusion coefficient of the medium between the solid supports 40.

[0100] According to the conditions disclosed in the aforementioned patent, the two-dimensional mirror scanner must be operated to scan light through the nucleic acid amplification reaction solution 30 containing photothermal particles at a frequency of 10 nanoseconds (ns) to 500 milliseconds (ms). Therefore, when implementing the aforementioned patent, the above operation must be precisely controlled to precisely control the exposure time of the photothermal particles, which is a key factor for the successful realization of this technology.

[0101] In contrast, 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 localized thermal radiation field phenomenon. In particular, the present invention uses a nucleic acid amplification reaction solution 30 in which the solid-phase carrier 40 is suspended. 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 1:1×109. In one embodiment, the preferred volume ratio is 1:1×104 to 1:1×108. This significantly improves the distance |X| between the solid-phase carriers 40 and the heat flow diffusion distance s1 of the thermal force field. Therefore, a relatively long critical excitation interval t1 can be used to irradiate and excite the solid-phase carrier 40 to generate a localized heating phenomenon.

[0102] Furthermore, the nucleic acid amplification reaction solution 30 is disposed in the external auxiliary cooling unit 13, which provides a cooling temperature environment that largely confines the in situ environment 50 generated by the solid phase carrier 40, so that the ideal state does not overlap with the in situ environment 50 formed by other solid phase carriers 40. In this way, the present invention does not need to control the relative motion relationship between the energy excitation unit 12 and the reaction unit 10, and can use a relatively long excitation time (between 1 second and tens of seconds) to excite all the suspended or immobilized solid phase carriers 40 in the reaction unit 10. Furthermore, the relative position between the energy excitation unit 12 and the reaction unit 10 does not move every time the energy excitation unit 12 is driven. This characteristic also causes an obvious difference between the present invention and GNA Biosolutions' U.S. invention patent (U.S. 9,382,583 B2 Publication No.).

[0103] Furthermore, since the solid phase carrier 40 of the present invention has a high surface area ratio (as described above), it can be used not only as a stage for efficiently separating the amplification reaction product 60 in the test substance 20, but also to remove inhibitors of the nucleic acid amplification reaction in the test substance 20. Furthermore, when an in situ nucleic acid amplification reaction is performed in the in situ environment 50 formed on the surface of the solid phase carrier 40, the surface of the solid phase carrier 40 has a limited space available for supporting the immobilized amplification reaction product 60, and therefore, the amplification reaction product 60 on the surface of the solid phase carrier 40 can reach a saturated state in a relatively short operation time (or a relatively small number of nucleic acid amplification cycles), thereby facilitating subsequent detection of the amplification reaction product 60.

[0104] In one embodiment of the present invention, as shown in Figures 10(a) and 10(b), the magnetic gold nanoshell of the solid support 40 has good photothermal conversion properties and stability, and the light energy absorption effect is better when the wavelength of the laser light is between 780 and 1,000 nm, preferably between 800 and 950 nm.

[0105] In this embodiment, magnetic gold nanoshells are added to a small amount of nucleic acid amplification reaction solution 30 (20 μL) to form a small amount of single-phase magnetic gold nanoshell suspension. The concentration of magnetic gold nanoshells is 2.9 × 1010 particles / ml, and the photothermal conversion and stability experiments were carried out using an 808 nm laser. As shown in Figure 10(a), the integrated driver 11 drives the 808 nm fiber-coupled laser to excite this small amount of single-phase magnetic gold nanoshell suspension with 10 consecutive laser cycles (laser power is 400 milliwatts (mW) / 0.16 square centimeters (cm2), first turned on for 60 seconds and then turned off for 120 seconds), so that the maximum temperature of the photothermal conversion of this small amount of single-phase magnetic gold nanoshell suspension can reach 62.29 ± 1.06 °C, and its coefficient of variation is 1.70%. This result indicates that the magnetic gold nanoshells have stable photothermal conversion properties and the shape of the solid support 40 does not change due to high-power laser irradiation. As shown in Fig. 10(b), the trace single-phase magnetic gold nanoshell suspension in Fig. 10(a) was excited by 808 nm laser with different powers. In the embodiment of the present invention, the different powers used include 200 mW / 0.16 cm2, 250 mW / 0.16 cm2, 300 mW / 0.16 cm2, 400 mW / 0.16 cm2, 500 mW / 0.16 cm2, 600 mW / 0.16 cm2 and 700 mW / 0.16 cm2. In addition, in the control group, a blank sample solution without magnetic gold nanoshells was excited by 1 W / 0.16 cm2 laser, and the trace single-phase magnetic gold nanoshell suspension was locally photothermally heated to a steady-state temperature and its temperature was measured in microscale, and the results show that the magnetic gold nanoshells have good photothermal conversion properties and the temperature at which the photothermal conversion reaches a steady state is directly proportional to the input laser power.

[0106] In one embodiment of the present invention, the temperature of the magnetic gold nanoshell suspension (20 μL) photothermally heated to a steady state is measured in a microscale to establish the relationship between the laser power intensity and the microscale environment temperature around the magnetic gold nanoshells, as shown in FIG. 11(a) and FIG. 11(b). The relationship between the temperature field of the in-situ environment 50 of the magnetic gold nanoshells and the wattage of the laser is estimated through the microscale measurement. As shown in FIG. 11(a), the test object is a 20 μL magnetic gold nanoshell suspension with a concentration of 2.9×1010 (particles / ml), and under various laser powers shown in FIG. 10(b), the test object is to directly measure the photothermal conversion temperature in a microscale to establish the photothermal conversion temperature rise curve of the magnetic gold nanoshells in a steady state (its linear equation is y=0.0778x+44.126; R2=0.9104). The temperature region boxed in the upper box in Figure 11(a) is the temperature range (85-95 °C) required for cell lysis and DNA denaturation. The temperature region boxed in the lower box in Figure 11(a) is the temperature range (55-65 °C) required for the primer binding and polymerase extension operations of loop-mediated isothermal amplification reaction (LAMP). Figure 11(b) shows infrared thermography images of reaction unit 10 of magnetic gold nanoshell suspension at steady state under different 808 nm laser powers (200 mW / 0.16 cm2, 250 mW / 0.16 cm2, 400 mW / 0.16 cm2). The top part of Figure 11(b) shows the results of irradiating the blank sample solution with 808 nm laser power (200 mW / 0.16 cm2, 250 mW / 0.16 cm2, 400 mW / 0.16 cm2) and the temperature is 27.9-29 °C. In one embodiment of the invention, the magnetic gold nanoshell suspension is excited to a steady state with a 200 mW / 0.16 cm2 laser and the core temperature is 51.6 °C. In yet another embodiment of the invention, the magnetic gold nanoshell suspension is excited to a steady state with a 250 mW / 0.16 cm2 laser and the core temperature is 63.9 °C. In yet another embodiment of the invention, the magnetic gold nanoshell suspension is excited to a steady state with a 400 mW / 0.16 cm2 laser and the core temperature is 79.1 °C.

[0107] As shown in Figures 12(a) and 12(b), the auxiliary cooling unit of one embodiment of the present invention performs 100 laser excitation cycles (one cycle includes first irradiating with 400 mW / 0.16 cm2 for 1.25 seconds, then turning off for 0.5 seconds and irradiating with 150 mW / 0.16 cm2 for 7.5 seconds) on the nucleic acid amplification reaction solution 30 containing the single-phase magnetic gold nanoshells, and observes the temperature change of the nucleic acid amplification reaction solution 30 containing the suspended single-phase magnetic gold nanoshells. Figure 12(a) shows the structural configuration of the auxiliary cooling part of this embodiment, which includes a large volume (100 μL) of nucleic acid amplification reaction solution 30 and its external auxiliary cooling unit 13. In this embodiment, the external auxiliary cooling unit 13 is ice with a large specific heat capacity (ice made by freezing water with a volume of 550 μL), and is used to surround the reaction unit 10 containing the nucleic acid amplification reaction solution 30 and maintain the temperature of the nucleic acid amplification reaction solution 30 in the reaction unit 10 at a cooling temperature (2 to 10 °C). As shown in FIG. 12(b), the number of magnetic gold nanoshell particles (5.8 × 108 particle number) is fixed, and magnetic gold nanoshell suspensions are prepared using different volumes of nucleic acid amplification reaction solution 30 (20 μL and 100 μL), and some groups use the external auxiliary cooling unit 13 as shown in FIG. 12(a). In addition, a blank sample solution containing only the nucleic acid amplification reaction solution is used as a control group. Next, 100 external laser excitation cycles (one cycle includes first irradiating with 400 mW / 0.16 cm2 for 1.25 seconds, then turning off for 0.5 seconds and irradiating with 150 mW / 0.16 cm2 for 7.5 seconds) are performed to observe the temperature change of the nucleic acid amplification reaction solution 30 of each group. At the same time, it is also evaluated how many external laser excitation cycles the temperature of the nucleic acid amplification reaction solution 30 can be maintained at the cooling temperature (2 to 10°C) by combining the above operating conditions, or whether the temperature change does not change significantly even if the number of external laser excitation cycles changes.In this example, some groups use 100 μL of magnetic gold nanoshell suspension and an external auxiliary cooling unit, some groups use 100 μL of blank sample solution and an external auxiliary cooling unit, 13 and some groups use 100 μL of magnetic gold nanoshell suspension but no external auxiliary cooling unit, and some groups use 20 μL of magnetic gold nanoshell suspension but no external auxiliary cooling unit. The laser cycle period corresponds to the Y-axis on the right, and the Y-axis on the right represents the laser power (mW). The results of this embodiment are shown (Figure 12(b)). Specifically, when 100 μL of the nucleic acid amplification reaction solution 30 is used to disperse the suspended single-phase magnetic gold nanoshells and an external auxiliary cooling unit 13 (ice volume: 550 μL) is used, after 65 external laser excitation cycles (one cycle includes first irradiating at 400 mW / 0.16 cm2 for 1.25 seconds, then turning off for 0.5 seconds and irradiating at 150 mW / 0.16 cm2 for 7.5 seconds), the temperature (℃) of the nucleic acid amplification reaction solution 30 on the left Y-axis is maintained at the cooling temperature (2-10℃). This result also indicates that under this design and operating conditions, the heating method of the magnetic gold nanoshells when excited by the laser belongs to the "local heating" mode, that is, the heating is limited only to the in situ environment 50. The average temperature of the entire nucleic acid amplification reaction solution 30 is maintained at the cooling temperature (2-10℃).

[0108] 13(a) and 13(b) show an embodiment of the present invention, in which an immune lateral flow test strip 171 is used to detect the amplification reaction product 60 generated by the water-cooled on-bead polymerase chain reaction. As shown in FIG. 13(a), after performing the water-cooled on-bead polymerase chain reaction using laser excitation, an operation unit 15 equipped with a magnetic force is used to purify and concentrate (concentrate) the magnetic gold nanoshells and remove the nucleic acid molecule products generated by the non-specific nucleic acid amplification reaction and other substances (e.g., primers 301, polymerase 302, etc.) in the nucleic acid amplification reaction solution 30. At the same time, the amplification reaction product 60 generated by the specific in situ nucleic acid amplification reaction is concentrated (concentrated). The magnetic gold nanoshells bind to the amplification reaction product 60 generated by the in situ nucleic acid amplification reaction. As shown in FIG. 13(a) and 13(b), the purified and concentrated magnetic gold nanoshells are resuspended in TBST buffer, and then rapid detection is performed using the immune lateral flow test strip 171. Here, the magnetic gold nanoshells bind to the amplification reaction products 60, which in turn bind to the nucleic acid label. Anti-fluorescein isothiocyanate antibodies are then attached to the test line 1711 of the lateral flow test strip to identify and capture the magnetic gold nanoshells that are attached to the amplification reaction products 60, which are nucleic acid labels, and form a visually detectable mark (e.g., form a row at the test line 1711). This reaction can be completed within 3-5 minutes, and the control line is used for quality control of conventional immune lateral flow test strips 171. The nucleic acid label in this embodiment is a primer 301 labeled with fluorescein isothiocyanate (FITC).

[0109] As shown in FIG. 14(a) and FIG. 14(b), another embodiment of the present invention uses plasma thermal sensing technology to interpret the test results of the immune lateral flow test strip 171 (FIG. 13(b)) and improve the detection sensitivity of the conventional immune lateral flow test strip 171. As shown in FIG. 14(a), the amplification reaction product 60 on the magnetic gold nanoshell is bound with the fluorescein isothiocyanate (FITC)-labeled primer 301. Then, the anti-fluorescein isothiocyanate antibody bound to the test line 1711 of the lateral flow test strip identifies and captures the magnetic gold nanoshell bound to the amplification reaction product 60 that is the nucleic acid label of the primer 301. Here, the magnetic gold nanoshell is excited by the laser-combined infrared thermal sensor 172 to generate heat, which is further thermally imaged. This plasma thermal sensing technology can increase the sensitivity of the interpretation of the test results of the immune lateral flow test strip 171. As shown in FIG. 14(b), the immunological lateral flow test strip 171 of FIG. 13(b) is analyzed by plasma thermal sensing to detect nucleic acid amplification, but there is no capture of the solid support 40 at the test line 1711 (i.e., a negative reaction).

[0110] As shown in FIG. 15(a) and FIG. 15(b), in one embodiment of the present invention, an 808 nm laser processes a malB gene fragment of E. coli (ATCC: 35218) in the following laser cycles (one cycle includes irradiation at 400 mW / 0.16 cm2 for 1.25 seconds and then irradiation at 150 mW / 0.16 cm2 for 7.5 seconds). In this example, the malB gene fragment is a detection target of conventional polymerase chain reaction for E. coli detection, and the water-cooled bead polymerase chain reaction is performed on a magnetic gold nanoshell with different laser cycle times (10 to 50 cycles). After the in-situ nucleic acid amplification reaction is completed, the results of the nucleic acid amplification performed by the water-cooled bead polymerase chain reaction on the solid phase carrier 40 and the nucleic acid amplification reaction solution 30 are sequentially analyzed using an immune lateral flow test strip 171 and urea gel electrophoresis. As shown in FIG. 15(a), the immune lateral flow test strip was used for detection at different laser cycle periods (10 to 50 cycles). It was found that the in situ nucleic acid amplification reaction on the magnetic gold nanoshell increased over time. As the number of laser cycles increased, the signal intensity of the test line 1711 on the immune lateral flow test strip 171 also gradually increased. This result indicates that the super-amplification formed by the short laser cycle can compensate for the loss of a small amount of nucleic acid amplification in the nucleic acid amplification reaction cycle by increasing the number of laser cycles. As shown in FIG. 15(b), the supernatant remaining after separating the magnetic gold nanoshell that completed the in situ nucleic acid amplification reaction by the operation unit 15 was analyzed by electrophoresis using a denatured agar colloid made of urea agar gum. After recovering the DNA contained in the supernatant by DNA precipitation, the DNA was denatured by heating with high concentration urea, and then analyzed by urea gel electrophoresis to observe whether the amplification reaction product 60 remains in the supernatant. The purified malB-FITC fragment is an amplification product of a malB gene fragment labeled with FITC and is used as a positive control group of amplification reaction products 60.The B3-FITC calibration primer is the B3 primer labeled with FITC and is used as a positive control group of primers. As a result, it was found that only free primer 301 was detected in the supernatant separated after different laser cycles (10 to 30 cycles), and no amplification reaction product 60 was present. This result also indirectly proves that the nucleic acid amplification reaction occurs on the magnetic gold nanoshell. In Figure 15(b), a DNA molecular weight standard (DNA ladder) is added as a standard for comparing DNA molecular weights. All DNA molecular weight standards are represented by "L". In Figure 15(b), non-thermally denatured ones are represented by "N" and heat-denatured ones are represented by "D".

[0111] As shown in Figure 16(a) and Figure 16(b), in one embodiment of the present invention, under the condition of a simple sample, the difference in nucleic acid amplification and detection efficiency between the conventional polymerase chain reaction and the water-cooled bead polymerase chain reaction using 808 nm laser excitation is compared. As shown in the top of Figure 16(a), the sensitivity of the conventional polymerase chain reaction is measured by performing the same 40 thermal cycles (one thermal cycle is first 95 ° C, 15 seconds, then 60 ° C, 30 seconds) operation using E. coli malB gene fragments with copy numbers 0, 10, 102, 103, 104, 105 and 106, and then performing a sensitivity test using agaricus gel electrophoresis. The results show that the sensitivity of the conventional detection polymerase chain reaction for detecting a simple sample is 10 to 102 copies of the malB gene fragment. Moreover, the bottom panel of Figure 16(a) confirms the specificity of the conventional polymerase chain reaction, which mainly uses a control group and the genomic DNA of a microbial strain described below as individual samples. The leftmost sample is a DNA molecular weight standard, and the others are simple samples. The naïve samples, from left to right, were: no template control (NTC), Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, Staphylococcus aureus (ATCC: BAA977), Streptococcus agalactiae, Enterococcus faecalis, Mycobacterium abscessus, Candida albicans, Aspergillus niger, and Streptococcus niger. niger and Escherichia coli (ATCC: 35218). To clearly indicate the simplified samples at the bottom of Figure 16(a), numbers 1 to 12 are assigned from left to right.Each simplified sample is targeted for detection of E. coli malB gene fragments. Sensitivity and specificity tests are performed using agar gel electrophoresis. The results show that only E. coli samples showed detection signals. Note that the upper and lower parts of Figure 16(a) have added DNA molecular weight standards as a reference for DNA molecular weight comparison, and all DNA molecular weight standards are represented by "L". Similarly, the same E. coli malB gene fragments are used for detection in water-cooled bead polymerase chain reaction, as shown in Figure 16(b). This means that the E. coli malB gene fragments with copy numbers of 0, 1, 10, 102, 103, and 104 from left to right correspond to 0, 0.00675, 0.0675, 0.675, 6.75, and 67.5 picograms (pg) of E. coli malB gene fragments, respectively. The fluorescein isothiocyanate labeled primer 301 bound to the amplification reaction product 60 is detected by binding an anti-fluorescein isothiocyanate antibody to the test line 1711 of the immune lateral flow test strip 171. The results show that the detection sensitivity is also in the range of 10 to 102 copy numbers. The amplification reaction product 60 is an amplification reaction product of the malB gene fragment bound to the magnetic gold nanoshell.

[0112] As shown in Figures 17(a) and 17(b), one embodiment of the present invention compares the difference in nucleic acid amplification and detection efficiency between conventional polymerase chain reaction and water-cooled bead-polymerase chain reaction using 808 nm laser excitation under complex sample conditions. The complex sample includes a mixture of genomic DNA from Escherichia coli (ATCC: 35218), the aforementioned simple sample, and genomic DNA from five other common human pathogenic microbial strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus (ATCC: BAA977), Streptococcus agalactiae, and Enterococcus faecalis. First, 0, 1, 10, 102, 103, 104, 105 and 106 copies of E. coli genomic DNA were grouped, and 106 copies of the genomic DNA mixture of the above five common human pathogenic microbial strains were added to each group, which is equivalent to 6.75 nanograms (ng) of genomic DNA of the above five common human pathogenic microbial strains. As shown in Figure 17(a), in the conventional polymerase chain reaction detection, a complex sample is used to perform the same 40 thermal cycles (one thermal cycle is first 95°C, 15 seconds, then 60°C, 30 seconds) operation, and then perform nucleic acid amplification and detection sensitivity tests on the malB gene. The results show that the sensitivity of conventional polymerase chain reaction detection is reduced to 105 copies to achieve specific identification, and the detection sensitivity is greatly reduced. Also, in another embodiment of the present invention, as shown in FIG. 17(b), laser excitation was used to perform water-cooled on-bead polymerase chain reaction, and E. coli genomic DNA with copy numbers of 10, 102, 103, and 104 were added as groups, which correspond to 0.0675, 0.675, 6.75, and 67.5 picograms of E. coli genomic DNA, respectively.In addition, 106 copies of the mixture of genomic DNA of the above five common human pathogenic microbial strains are added to each group, which corresponds to 6.75 nanograms of the mixture of genomic DNA of the above five common human pathogenic microbial strains. Then, the immune lateral flow test strip 171 is used to detect the amplified nucleic acid molecules on the magnetic gold nanoshell, and even under the conditions of complex samples as mentioned above, the detection sensitivity is still maintained at 10-102 copy numbers. The test results show that the sensitivity of nucleic acid amplification using laser excitation and detection of water-cooled bead polymerase chain reaction is not affected by the complex nucleic acid analyte 20.

[0113] As shown in FIG. 18(a) and FIG. 18(b), in one embodiment of the present invention, an experiment is carried out using a suspension of mixed enrichment bodies and amplification bodies 42, where both enrichment bodies and amplification bodies 42 are magnetic gold nanoshells. The enrichment bodies are used to capture E. coli, which are purified and separated by a magnetic operation unit 15, and the analyte 20 captured on the enrichment bodies is photothermolyzed. The analyte 20 is a mixture of E. coli (Escherichia coli, ATCC: 35218), which causes E. coli to release biological material 21. The amplification bodies 42 are used to capture the biological material 21 released by E. coli. In another embodiment, a suspension containing only the amplification bodies 42 and E. coli are used in the reaction. Specifically, the concentrate suspension and the amplification body 42 are mixed, the concentrate has an antibody capable of specifically recognizing the O antigen and K antigen of E. coli as an enrichment ligand, and the amplification body 42 has an F3 primer 301 (malB F3 primer) capable of specifically hybridizing with a malB gene fragment specific to E. coli as an amplification ligand 71. In one embodiment of the present invention, 20 μL of the mixed suspension of the concentrate and the amplification body 42 is mixed with 1 μL of the E. coli mixture and 19 μL of TBST buffer containing 1% BSA. The 1 μL E. coli mixture contains 8×103 colony forming units (CFU), and after mixing and reacting for 20 minutes at room temperature (25° C.), the concentrate and the amplification body 42 are purified and concentrated by the magnetic operation unit 15, and then washed with the TBST buffer. After the washing is completed, the TBST buffer used for washing is concentrated and removed using the operation unit 15. Next, the washed enrichment and amplification body 42 are resuspended in 20 μL of TBST buffer to achieve low background and high specificity for capturing E. coli. In another embodiment of the present invention, the suspension in which the enrichment and amplification body 42 are mixed in the above step is replaced with a suspension containing only the amplification body 42, and the same steps are performed to confirm the capture efficiency of E. coli when the enrichment is not included. As shown in the upper part of Figure 18(a), the petri dish number 1 shows a schematic diagram of the result of smearing and culturing 1 microliter of E. coli (OD600 = 0.01) on the petri dish.Number 2 shows a schematic diagram of the culture result when 0.1 microliters of E. coli (OD600=0.01) was smeared on a petri dish, and a total of 238 E. coli colonies were cultured. Number 3 shows a schematic diagram of the culture result when 1 microliter of a suspension containing only the amplification body 42 was taken and smeared on a petri dish, and no E. coli colonies were cultured. Number 4 shows a schematic diagram of the culture result when 19 microliters of a suspension containing only the amplification body 42 was smeared on a petri dish, and one E. coli colony was cultured. Number 5 shows a schematic diagram of the culture result when 19 μL of a suspension of the mixed concentrate and the amplification body 42 was smeared on a petri dish, and three E. coli colonies were cultured. Number 6 shows a schematic diagram of the culture result when 19 μL of a suspension of the mixed concentrate and the amplification body 42 was taken and smeared on a petri dish, and 83 E. coli colonies were cultured. From the results in FIG. 18(a), it can be seen that when the above steps of this embodiment are performed using a suspension containing only the amplification body, E. coli cannot be effectively captured, and when the steps are performed using a suspension containing a mixture of the enrichment body and the amplification body, E. coli can be effectively captured. As shown in the lower part of FIG. 18(a), the suspension containing the enrichment of the captured E. coli is excited with an 808 nm laser, and the excitation is continued for 5 minutes with different laser powers, including 0 mW / 0.16 cm2, 300 mW / 0.16 cm2, 400 mW / 0.16 cm2 and 500 mW / 0.16 cm2. Each group takes 1 μL of the suspension containing a certain enrichment of the captured E. coli and applies it to the LB solid medium, and the photothermal lysis efficiency of E. coli is judged by the formation of bacterial cells. If new cells cannot be generated and the E. coli is dissolved, the results show that continuous stimulation at 400 mW / 0.16 cm2 for 5 minutes can lyse the E. coli. As shown in FIG. 18(b), malB gene fragments in biological material 21 decomposed by photothermolysis of E. coli (ATCC: 35218) are captured via amplification ligand 71, and water-cooled on-bead polymerase chain reaction is carried out by laser excitation, and then evaluated using immune lateral flow test strip 171.The results show that under photothermolysis at 400 mW / 0.16 cm2 for 5 minutes, E. coli is successfully photothermolyzed and releases biological material 21, the malB gene fragment contained in the biological material 21 is captured by the amplification body 42, and then the water-cooled bead polymerase chain reaction is carried out by laser excitation, and the signal can be detected on the test line 1711 on the immune lateral flow test strip 171.

[0114] As shown in FIG. 19(a) and FIG. 19(b), in one embodiment of the present invention, an experiment is carried out using a mixed suspension of enrichment body and amplification body 42. Here, the enrichment body is a magnetic gold nanoshell, the enrichment ligand is an antibody that can specifically recognize O antigen and K antigen of E. coli, and the amplification body 42 is a magnetic gold nanoshell. In addition, the amplification ligand 71 is an F3 primer 301 (malB F3 primer) that can recognize malB gene fragment, and E. coli (Escherichia coli, ATCC: 35218) with different colony forming units is used as the test substance 20. In an embodiment of the present invention, different colony forming units, including 0 CFU, 8 CFU, 8×10 CFU, 8×102 CFU and 8×103 CFU, are measured and grouped by absorbance at a wavelength of 600 nm, and a water-cooled bead polymerase chain reaction is carried out using laser excitation, and then the sensitivity of the immune lateral flow test strip 171 is tested and the time required for the whole process is evaluated. As shown in Figure 19(a), the overall operation process of this integrated platform includes four major steps: capture and concentration of analyte 20, photothermal dissociation of analyte and capture of released biological material 21, water-cooled bead polymerase chain reaction using laser excitation, and testing of immune lateral flow test strip 171.

[0115] In the step of "capturing and concentrating the test substance 20", 5 μL of the suspension of the concentrate, 15 μL of the suspension of the amplification body 42, 1 μL of the test substance 20, and 19 μL of TBST buffer containing 1% bovine serum albumin are mixed and reacted. Furthermore, the amplification body 42 is shaken and mixed at 25° C. for 20 minutes to allow the amplification ligand 71 on the amplification body 42 to capture E. coli, and then the operation unit 15 concentrates the concentrate and the amplification body 42 to remove the supernatant, washes with TBST buffer to remove impurities, and then removes the TBST buffer. This is completed within 25 minutes.

[0116] In the step of "photothermal dissociation of the test substance and capture of the released biological substance 21", the concentrate and amplification body 42 washed with a buffer solution after the step of "capturing and concentrating the test substance 20" is resuspended in 20 μL of TBST buffer and photothermally dissociated by irradiation with 400 mW for 5 minutes using an 808 nm laser, whereby the biological substance 21 released from the test substance 20 is captured by the amplification ligand 71 on the amplification body. Next, the concentrate and amplification body 42 are concentrated using the magnetic operation unit 15, and after removing the supernatant, 100 μL of the nucleic acid amplification reaction solution 30 is added and resuspended.

[0117] In the step of "water-cooled bead polymerase chain reaction using laser excitation", the concentrate and amplified bodies 42 resuspended in the nucleic acid amplification reaction solution 30 in the step of "photothermal dissociation of the test substance and capture of the released biological substance 21" are subjected to water-cooled on-bead polymerase chain reaction under 40 laser cycles (each cycle includes irradiation at 400 mW / 0.16 cm2 for 1.25 seconds and irradiation at 150 mW / 0.16 cm2 for 7.5 seconds) using an 808 nm laser. Next, the concentrate and amplified bodies 42 are concentrated using the magnetic operation unit 15, and after removing the supernatant, 20 μL of TBST buffer is added and resuspended. This is completed within 6 minutes.

[0118] In the step of "testing the immune lateral flow test strip 171", when the enrichment and amplification bodies 42 resuspended in TBST buffer in the step of "water-cooled bead polymerase chain reaction using laser excitation" are detected by the immune lateral flow test strip 171, the signal reaction of the test line 1711 on the immune lateral flow test strip 171 can be directly observed by the naked eye, or can be completed in 3 to 5 minutes when detected using the laser combined infrared thermal sensor 172. The whole process can be completed within 45 minutes.

[0119] Figure 19(b) is a schematic diagram to evaluate the detection result of the sensitivity of laser-induced water-cooled on-bead PCR using the immune lateral flow test strip when completing the procedure of Figure 19(a). The result shows that the signal response can be visually observed on the immune lateral flow test strip 171 in the group of 8 CFU. This means that the detection sensitivity falls within the range of each reaction containing 8 CFU of E. coli.

[0120] As shown in FIG. 20(a), FIG. 20(b), and FIG. 20(c), in another embodiment of the present invention, the detection sensitivity of the immune lateral flow test strip 171 combined with plasmonic thermal sensing is compared with that of a general immune lateral flow test strip 171. Using image analysis software (Image J), ​​pixel intensity analysis is performed on the image of the test line 1711 in the immune lateral flow test strip 171 shown in FIG. 19(b). The image quantification result is shown in FIG. 20(a), and the tested E. coli colony forming units are 0 CFU, 8 CFU, 8×10 CFU, 8×102 CFU, and 8×103 CFU, respectively. Although the detection sensitivity of the image analysis falls within the range of each reaction containing 8 CFU of E. coli, the detection of E. coli containing 8 CFU to 8×10 CFU in the reaction is difficult to visually identify and judge. In the embodiment of the present invention, the identification rate can be significantly improved by integrating the immune lateral flow test strip 171 with plasmonic thermal sensing. As shown in FIG. 20(b), the immune lateral flow test strip 171 is excited with 808 nm laser energy of 24 mW and 140 mW, respectively, and the laser combined infrared thermal sensor 172 focuses on the test line 1711, and the thermal imaging image displayed after detection. FIG. 20(c) is a graph quantifying the temperature measured by thermal imaging after FIG. 20(b) is excited with 808 nm laser energy of 24 mW and 140 mW. This temperature is the environmental temperature rise difference (ΔT(℃)) obtained after subtracting the background temperature in the environment (18.2℃). In particular, the average value of the rise temperature difference measured in the 0 CFU group plus three standard deviations is set as the boundary of the detection limit, and if the temperature difference is larger than this, it is judged as positive. As a result, even when excited with 808 nm laser power of 24 mW or 140 mW, the rise temperature difference of thermal imaging clearly shows that the detection sensitivity is within the range of 8 CFU of E. coli per reaction.

[0121] To further understand the effect of laser excitation on the performance of water-cooled bead-PCR when performing PCR at different cooling temperature ranges, in an embodiment of the present invention, the nucleic acid amplification reaction solution 30 is grouped according to the presence or absence of ice pack, the presence or absence of ice pack expansion, and the volume of the reaction solution, and the temperature rise difference (ΔT (°C)) of the reaction solution 30 is tested under different conditions. The number of magnetic gold nanoshells in each group is 2.8×108 particles. Groups include, for example, O1 (with ice pack, 100 μL of nucleic acid amplification reaction solution 30), O2 (with ice pack, 100 μL of nucleic acid amplification reaction solution 30), Non-1 (without ice pack, 100 μL of nucleic acid amplification reaction solution 30), Non-2 (without ice pack, 100 μL of nucleic acid amplification reaction solution 30), Non(2x)-1 (without ice pack, 200 μL of nucleic acid amplification reaction solution 30), Non(2x)-2 (without ice pack, 200 μL of nucleic acid amplification reaction solution 30), E1 (with spare ice pack, 100 μL of nucleic acid amplification reaction solution 30), E2 (with spare ice pack, 100 μL of nucleic acid amplification reaction solution 30). The ice pack contains 550 μL of ice, and the large ice pack contains 2,200 μL of ice. Each group performs water-cooled on-bead polymerase chain reaction using laser excitation, which is quantified by bead-based ELISA to evaluate the impact of each group of nucleic acid amplification reaction solution 30 on the performance of water-cooled on-bead polymerase chain reaction using laser excitation. As shown in Figure 21(a), 190 nm magnetic gold nanoshell suspensions are subjected to 50 laser cycles (first irradiated at 690 mW for 1.25 seconds, then irradiated at 360 mW for 7.5 seconds) at different starting temperatures of the above groups, and the temperature change of the nucleic acid amplification reaction solution 30 is detected in real time using a thermocouple. As shown in Figure 21(b), the laser excitation efficiency of water-cooled bead-polymerase chain reaction is evaluated for each group of nucleic acid amplification reaction solution 30 through bead-based ELISA quantification. The left Y axis shown in Figure 21(b) corresponds to the scatter line, which is a scatter plot of the elevated temperature change of the nucleic acid amplification reaction solution 30 after water-cooled on-bead polymerase chain reaction by laser excitation. Of these, the temperature difference during the rise of O1 was 11.2°C (from 3.8°C to 15°C), the temperature difference during the rise of O2 was 10.1°C (from 2.1°C to 12.2°C), the temperature difference during the rise of Non-1 was 16.8°C (from 13.5°C to 30.3°C), the temperature difference during the rise of Non-2 was 20.1°C (from 12.6°C to 32.7°C), the temperature difference during the rise of Non(2x)-1 was 14.2°C (from 17.2°C to 31.4°C), the temperature difference during the rise of Non(2x)-2 was 14.8°C (from 17.5°C to 32.3°C), the temperature difference during the rise of E1 was 9.8°C (from 1.1°C to 10.9°C), and the temperature difference during the rise of E2 was 6.7°C (from -8.7°C to -2°C). The Y-axis on the right corresponds to a histogram, which represents the absorbance values ​​at 450 nm using a bead-based enzyme-linked immunosorbent assay to quantify and evaluate the performance of water-cooled bead-polymerase chain reaction excited by an 808 nm laser. Among them, the absorbance value of O1 is 0.147, the absorbance value of O2 rise is 0.168, the absorbance value of Non-1 is 0.132, the absorbance value of Non-2 is 0.120, the absorbance value of Non(2x)-1 is 0.128, the absorbance value of Non(2x)-2 is 0.138, the absorbance value of E1 is 0.148, and the absorbance value of E2 is 0.195. The higher the absorbance value, the higher the overall nucleic acid amplification efficiency.

[0122] In one embodiment of the present invention, the water-cooled on-bead polymerase chain reaction is stimulated by a laser. A laser cycle includes multiple cycles of reaction periods and controls the temperature cycle process around the solid support 40. Each cycle reaction period includes a nucleic acid molecule denaturation reaction period, a primer attachment period, and a polymerase extension period. The reaction time for denaturing the nucleic acid molecule is 0.5 to 5 seconds, preferably 1 to 2.5 seconds. The laser output is 400 to 800 mW. The temperature during the denaturation reaction of the nucleic acid molecule is 85 to 95°C, preferably 95°C. The primer attachment time and polymerase extension time are 2 to 15 seconds, preferably 5 to 15 seconds. The laser output is 100 to 400 mW, and the temperature during the primer attachment period and the polymerase extension period is 55 to 65°C, preferably 60°C. In addition, the laser irradiation cycle time during each cycle reaction period is 5 to 15 minutes.

[0123] In the present invention, each solid phase carrier 40 has magnetic properties, and after the in situ nucleic acid amplification is completed on each solid phase carrier 40 and the amplification reaction product 60 is generated, nucleic acid detection is performed in the following manner. The amplification reaction product 60 bound to the solid phase carrier 40 is purified, separated, and concentrated by the operation unit 15. The detection module 17 is used to detect one or any combination of two or more of optical changes, thermosensing changes, electrochemical changes, magnetic changes, and mass changes caused by the amplification reaction product 60 bound to the solid phase carrier 40.

[0124] In an embodiment of the present invention, the method of generating an optical change in each amplification reaction product 60 is to use a primer 301 having a nucleic acid label to bind to the amplification reaction product 60 and detect the light intensity using a luminance meter. The nucleic acid label may be a fluorescent label 3011, and the fluorescence intensity generated thereby may be detected by a fluorometer. In another embodiment of the present invention, an enzyme-linked immunosorbent assay may be used to detect the optical or chemiluminescent change generated by the amplification reaction product 60. In another embodiment of the present invention, an enzyme-linked immunosorbent assay may be used to detect the spectral change caused by the combination of the solid phase support 40 and the amplification reaction product 60.

[0125] In an embodiment of the present invention, the method of generating an optical change in the amplification reaction product 60 utilizes the nucleic acid lateral flow strip or the lateral flow immunoassay strip 171 to detect the optical change, and further, in addition, uses thermal sensor detection, surface plasmon resonance spectroscopy, or any combination thereof to increase the sensitivity of detection of the nucleic acid lateral flow strip or the lateral flow immunoassay strip 171.

[0126] In an embodiment of the present invention, the method of producing a magnetic change in the amplification reaction product 60 is one or a combination of AC magnetic induction detection (i.e., detection of frequency-dependent AC magnetic susceptibility by AC magnetometry) and giant magnetoresistance measurement.

[0127] In an embodiment of the present invention, the method for electrochemically altering the amplification reaction product 60 is one of electrochemical enzyme-linked immunosorbent assay, electrical impedance spectroscopy (EIS) detection, or a combination thereof.

[0128] In an embodiment of the present invention, the method of generating a change in the electrical mass of the amplification reaction product 60 is carried out using a Quartz Crystal Microbalance (QCM).

[0129] According to the above, the contents included in the embodiments of the present invention include exciting external energy to the solid phase carrier 40 to generate a rapid hot / cold temperature cycle process, and then detecting the amplification reaction product 60 generated by the in-situ nucleic acid amplification reaction on the solid phase carrier 40. In addition, the contents included in the embodiments of the present invention also include the temperature range required for nucleic acid amplification by external energy excitation on the solid phase carrier 40, and the external energy excitation parameters (energy output and irradiation frequency period). All these contents prove that this case can complete the purpose of nucleic acid amplification and back-end detection in a rapid and stable state. In addition, a workable embodiment is also provided for the sample pretreatment of the test substance 20, which can be connected with the back-end rapid nucleic acid amplification and detection to achieve the purpose of rapid nucleic acid detection. Therefore, the present invention can be applied to point-of-care testing (Point of Care Testing, POCT) based on nucleic acid detection. Overall, the present invention has the following advantages:

[0130] First, because a solid phase carrier 40 with a high surface area to volume ratio is used, the surface can be flexibly modified with a combination of ligands (e.g., antibodies, nucleic acid aptamers, oligonucleotides, proteins, polysaccharides, or combinations thereof) depending on the application.

[0131] Second, by controlling the relationship between the conditions of external energy excitation and the low-temperature reaction solution, the present invention can rapidly create the temperature conditions required for in situ lysis (or dissociation) of cells, capture of target nucleic acid, and in situ nucleic acid amplification reaction in the in situ environment 50 on the surface of the solid support 40.

[0132] Third, due to the 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 nucleic acid amplification reaction solution 30 having a cooling temperature around the solid-phase carrier 40 can not only rapidly cool the solid-phase carrier 40, but also suppress the amplification reaction of unintended nucleic acids, which is advantageous for the amplification and detection of nucleic acids (i.e., detection of nucleic acid molecules). In addition, this technical feature can also be a physical (e.g., temperature field, etc.) division for the amplification and detection of nucleic acids, forming a form of nucleic acid amplification and detection similar to virtual emulsion polymerase chain reaction or droplet digital polymerase chain reaction (Droplet Digital PCR, ddPCR). This makes the amplification and detection reaction of nucleic acids less affected by the complexity of the nucleic acids of the test substance 20.

[0133] Fourth, the overall low-temperature nucleic acid amplification reaction solution 30 can greatly improve the technical problems of evaporation of the nucleic acid amplification reaction solution 30 and generation of bubbles in the conventional small-sized nucleic acid amplification device.

[0134] Fifth, the temperature (heat) cycle required for nucleic acid amplification reactions, as well as the in situ solid-phase nucleic acid amplification reaction and the subsequent purification, separation, and concentration of the amplified nucleic acid molecules can be realized at ultra-high speeds, significantly shortening the overall nucleic acid amplification and detection time.

[0135] Sixth, the present invention greatly simplifies temperature control devices and equipment.

[0136] Seventh, it simplifies the pretreatment of the test substance 20 and the detection of the amplification reaction product 60. The solid phase carrier 40 can be applied to the purification and concentration (enrichment) of the test substance 20, the biological substance 21, the amplification reaction product 60, etc. Furthermore, according to the present invention, the solid phase carrier 40 can be used in the immune lateral flow test strip 171 to amplify a signal substance for nucleic acid detection (e.g., a visual image or a thermal image detected through plasma thermal sensing), which facilitates the pretreatment of the test substance 20 and the collection and detection of the amplification reaction product 60.

[0137] It will be apparent to those skilled in the art that various improvements and modifications can be made to the present invention without departing from the spirit and scope of the present invention. It is not intended to limit the present invention to the particular form(s) disclosed, but on the contrary, the intention is to embrace all modifications, alternative constructions, and equivalents falling within the spirit and scope of the present invention as defined by the appended claims. It is therefore intended that the present invention cover modifications and modifications of this invention provided they come within the scope of the appended claims and their equivalents. [Explanation of symbols]

[0138] 10 reaction units, 11 Unified driver, 12 Energy Excitation Unit, 121 Laser collimator, 122 Laser Launcher, 13 External auxiliary cooling unit, 14 Temperature detection unit, 15 Operation Unit, 16 External energy calibrator, 17 Detection module, 171 Immuno-lateral flow test strips; 1711 Test Line, 1712 control line, 172 Laser combined infrared thermal sensor, 20 test substances, 21 biological substances; 30 Nucleic acid amplification reaction solution, 301 Primer, 3011 Fluorescent labeling, 302 polymerase, 40 Solid phase support, 401 magnetic core, 402 Middle class, 403 Gold nanoshells, 41 Multifunctional body, 42 Amplification body, 43 Concentrate, 50 In situ environment, 60 amplification reaction products, 70 enriched ligands, 71 Amplification ligand; 72 Polyethylene glycol, 73 Bovine serum albumin, Steps S10~S90, Steps S100~S400

Claims

1. disposing at least one test substance, at least one nucleic acid amplification reaction solution, and at least one solid phase carrier in a reaction unit; forming a cooling environment in the reaction unit and its interior, and maintaining a predetermined cooling temperature; Controlling the output of external energy and its opening and closing timing, while forming one or more reaction temperature cycles required for nucleic acid amplification reaction to occur through the predetermined cooling temperature of the cooling environment, in each reaction temperature cycle, all the solid phase carriers are excited by the external energy simultaneously to form an in-situ environment, or each solid phase carrier stops receiving the external energy, and the in-situ environment gradually dissipates; The solid phase carrier, the test substance, and the nucleic acid amplification reaction solution carry out a nucleic acid amplification reaction in the formation and disappearance of an in situ environment to generate an amplification reaction product.

2. The nucleic acid amplification method of claim 1, characterized in that during the start of each reaction temperature cycle, all the solid phase carriers in the reaction unit are excited simultaneously, a field environment is formed around each solid phase carrier, the amplification reaction products are generated on each solid phase carrier, a portion of the amplification reaction products is retained on each solid phase carrier, while another portion of the amplification reaction products is released into the nucleic acid amplification reaction solution, and further, during the shutdown period of each reaction temperature cycle, the excitation of each solid phase carrier is stopped, and each field environment is gradually dissipated by the cooling temperature of the cooling environment.

3. 2. The method for amplifying nucleic acid according to claim 1, wherein the ratio of the total volume of all the solid phase carriers to the volume of the nucleic acid amplification reaction solution is 1:200 to 1:1 x 10 9 .

4. 2. The method for amplifying nucleic acid according to claim 1, wherein the size of the solid phase carrier is 8 to 2,000,000 nm.

5. 2. The nucleic acid amplification method according to claim 1, wherein the cooling temperature is -10 to 50°C.

6. The nucleic acid amplification method according to claim 1, characterized in that the reaction unit or the nucleic acid amplification reaction solution is pre-cooled to the cooling temperature or contained in an external auxiliary cooling unit and can be maintained at the cooling temperature by the pre-cooled reaction unit or the external auxiliary cooling unit, and the reaction temperature cycle is performed one or more times.

7. 2. The nucleic acid amplification method according to claim 1, wherein the test substance is a cell, a cell organelle, a bacterium, a virus, a protozoan, or a combination thereof.

8. the solid support comprises a multifunctional body, at least one enrichment ligand, and at least one amplification ligand; the enrichment ligands are bound to a surface of the multifunctional body, the enrichment ligands being used to bind the analyte; 8. The method of claim 7, further comprising: at least one amplification ligand attached to a surface of the multifunctional body, the amplification ligand being used to bind a biological substance.

9. The nucleic acid amplification method according to claim 8, characterized in that the biological substance is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) released from the test substance, or an amplification reaction product released into the nucleic acid amplification reaction solution after the amplification ligand is replicated.

10. the portion of the solid support comprises an enrichment body and at least one enrichment ligand, the enrichment ligand being bound to a surface of the enrichment body, the enrichment ligand being used to bind the analyte; The nucleic acid amplification method of claim 7, further comprising: another portion of the solid phase support, an amplification body and at least one amplification ligand, the amplification ligand being bound to a surface of the amplification body, the amplification ligand being used to bind biological material released from the test substance.

11. The nucleic acid amplification method according to claim 10, characterized in that the biological substance is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) released from the test substance, or an amplification reaction product released into the nucleic acid amplification reaction solution after the amplification ligand is replicated.

12. 2. The method for amplifying nucleic acid according to claim 1, wherein the test substance is a free DNA or RNA substance.

13. The nucleic acid amplification method of claim 12, wherein each solid support comprises an amplification body and at least one amplification ligand, each amplification ligand being bound to the surface of the amplification body, and each amplification ligand being used to bind the test substance.

14. The present invention comprises a reaction unit, an auxiliary cooling unit, an external energy excitation unit, and an integrated driver; The reaction unit contains a reaction solution, a test substance, a nucleic acid amplification reaction solution, and at least one solid phase carrier; The auxiliary cooling unit is an external auxiliary cooling unit arranged around the nucleic acid amplification reaction solution that has been cooled to a cooling temperature in advance or the reaction unit, and the external auxiliary cooling unit continues to cool the reaction unit so that the nucleic acid amplification reaction solution maintains the pre-cooled temperature; The external energy excitation unit excites the solid support to generate heat; The integrated driver controls the energy output and opening / closing timing of the external energy excitation unit, performs energy output based on the external energy calibrator and the temperature detection unit, feedback controls the external auxiliary cooling unit, and forms one or more reaction temperature cycles required for the nucleic acid amplification reaction, in a rapid nucleic acid amplification system.

15. A nucleic acid detection method for detecting a nucleic acid by the following procedure after the nucleic acid amplification method according to any one of claims 1 to 13 is completed: purifying, separating and concentrating the amplification reaction products bound to the solid support by an operational unit; A nucleic acid detection method comprising: using a detection module to detect one or any combination of two or more of an optical change, a thermosensing change, an electrochemical change, a magnetic change, or a mass change caused by the amplification reaction product bound to the solid phase support.

16. The method for causing an optical change in the amplification reaction product includes: mixing a nucleic acid label with said amplification reaction product and detecting light intensity using a photometer; Detecting an optical or chemiluminescent change in the amplification reaction product using an enzyme-linked immunosorbent assay; or 16. The method for detecting nucleic acids according to claim 15, characterized in that it comprises one or more of the following: a spectral change caused by binding of the solid phase carrier to the amplification reaction product.

17. The method for generating an optical change in the amplification reaction product according to claim 16, characterized in that the detection module is a nucleic acid lateral flow strip or a lateral flow immunoassay strip used to detect the generated optical change.

18. The nucleic acid detection method of claim 17, further comprising using thermal sensor detection, surface plasmon resonance spectroscopy, or any combination thereof in addition to the nucleic acid lateral flow strip or the lateral flow immunoassay strip to enhance the sensitivity of detection of the nucleic acid lateral flow strip or the lateral flow immunoassay strip.

19. The nucleic acid detection method according to claim 15, wherein the method for causing an electrochemical change in the amplification reaction product is one or a combination of electrochemical detection, enzyme-linked immunosorbent assay, and electrical impedance spectroscopy (EIS) detection.

20. The nucleic acid detection method of claim 15, wherein the method for inducing a magnetic change in the amplification reaction product is one or a combination of detection of frequency-dependent AC magnetic susceptibility by AC magnetometry and giant magnetoresistance measurement.

21. 16. The method for detecting nucleic acids according to claim 15, wherein the method for generating a change in the electrical mass of the amplification reaction product is carried out using a quartz crystal microbalance.

22. A rapid nucleic acid detection device including an operation unit and a detection module, The operational unit purifies, separates and concentrates the amplification reaction products produced by the method according to claims 1 to 13; The detection module detects one or any combination of two or more of optical changes, thermosensing changes, electrochemical changes, magnetic changes, or mass changes occurring on the amplification reaction products.

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