TWI765431B - Nucleic acid amplification system and method thereof - Google Patents

Abstract

The invention discloses a nucleic acid-based amplification system and its operating method, using external energy to drive nanoparticles to generate localized thermal conversion, and use the reaction solution for cooling to achieve rapid temperature modulation, resulting in nucleic acid amplification reactions, including in-situ polymerase chain reaction (in-situ PCR) or in situ isothermal DNA amplification reactions to be achieved. The increased target nucleic acid product accumulates on the surface of the nanoparticles. And through centrifugation, magnetic capture or lateral flow test strips the amplicons on the nanoparticles can be detected by fluorescence intensity analysis or colorimetric methods generated by the coupling method of enzymes with specific DNA fragment and antibody specific against fluorophores.

Description

Nucleic acid amplification system and method thereof

The present invention relates to a nucleic acid amplification system, which uses magnetic or non-magnetic nanoparticles capable of converting external energy into regional thermal energy by integrating photothermal heating, and performs polymerase chain reaction or isothermal amplification on the surface of the nanoparticles Reactions for rapid amplification of specific nucleic acid fragments. In addition, using single-molecule concentration procedures such as magnetic capture, centrifugation, lateral flow chromatography, etc., the proliferating nucleic acid fragments are identified by nucleic acid markers or antibodies and coupled with enzyme reactions to achieve rapid detection by colorimetry.

For molecular biological detection of trace biological samples, rapid nucleic acid amplification and detection has always faced difficulties. In the past ten years, rapid PCR has been validated through rapid thermal cycling and the development of second-generation DNA polymerases, enabling rapid nucleic acid amplification reactions, which can be completed in minutes as a whole, but it is also accompanied by many problems, such as low Amplicon yield, platform scalability, and platform thermal management. Rapid nucleic acid amplification technology is the core technology of point of care (POC). Compared with traditional polymerase or other isothermal nucleic acid amplification methods, microelectromechanical technology realizes miniaturized PCR to achieve rapid heating and cooling operations and reduce the amount of reagents and samples required for the reaction. It is a portable analysis device, but such systems require precise temperature control and fluid operation, which relatively increases the difficulty of design, process and operation, and is not suitable for the development and application of simple point-of-care diagnosis. The back-end nucleic acid molecule detection technologies mostly rely on physical or chemical expressions, including absorbance, fluorescence, concentration, electrical impedance, and even fluid viscosity.

In the identification of cells, bacteria or viruses, and even in the screening of cancer cells, such as the presence of very small circulating tumor cells in the blood circulation system, the identification of cell markers by specific antigens on the cell surface also plays a very important role. . However, taking circulating tumor cells as an example, cell markers such as EpCAM or CKs do not appear on all circulating tumor cells. In addition, in the identification of some RNA viruses, due to the high mutation of structural proteins, traditional antibody detection, It takes a lot of time just to screen out the monoclonal antibody of the target marker. Therefore, the design of nucleic acid detection is easier than that of antibody detection. In addition, even if a subsequent detection reaction is required after screening, nucleic acid detection can improve whether the concentration of the sample is sufficient for biological detection by multiplying and amplifying specific nucleic acid fragments.

For example, if you want to screen specific cancer cells, bacteria or viruses, most of the existing technologies need to release various targets or nucleic acids that may contain antigens through complex methods; and even after the nucleic acids or antigens are released, It is more likely that because the sample concentration is too low, it takes a long time to react and scale up, so that devices such as biochips can perform their functions.

Therefore, related biological testing often requires multiple steps such as screening, culturing, controlling the number of sample concentrations, selecting appropriate testing instruments, and finally performing data analysis, resulting in a difficult situation. There is currently a need for a system and method that can quickly solve the above problems without losing detection accuracy.

In order to solve the problems mentioned in the prior art, the present invention provides a nucleic acid amplification system and method thereof. Among them, the thermal cycling nucleic acid proliferation reaction does not use a metal heating vehicle or a cooling fan to conduct the thermal cycling reaction by thermal conduction. The nucleic acid amplification system mainly includes a reaction space, a sample, a plurality of particles, an enzyme, and at least one energy supply. a module, at least one temperature balancing substance and a control module.

Wherein, the sample contains at least one analyte, and the sample is placed in the reaction space. The plurality of particles are mixed with the sample, and each of the particles includes a body, at least one first ligand and at least one second ligand. The at least one first ligand is disposed on the body, and the at least one first ligand matches the at least one analyte. The at least one second ligand is disposed on the body, and each of the second ligands includes a specific mark.

As for the polymerase also mixed with the sample, the at least one energy supply module provides an external energy to the plurality of particles. The manipulation module is used to manipulate the plurality of particles in the reaction space. The reaction space further includes at least one temperature balancing substance for balancing the temperature of the plurality of particles.

Further, the method of the nucleic acid amplification system of the present invention is mainly composed of seven steps, including step (a), mixing the sample with a plurality of nanoparticles, so that the plurality of nanoparticles capture the at least one Analyte. Following step (b), a control module is used to concentrate a sample in a reaction space, and the sample contains the at least one analyte. If the at least one analyte is a free nucleic acid substance, then proceed to step (e), If not, go to step (c).

In step (c), at least one energy supply module provides an external energy to the plurality of particles, so that the micro-environment around the plurality of nanoparticles rises to a burst temperature. Then step (d) is performed, the at least one object to be tested bursts to release at least one biological substance; followed by step (e), the at least one energy supply module provides an external energy to the plurality of particles until a combination The temperature causes the plurality of nanoparticles to combine with the at least one biological substance or the free nucleic acid substance, and concentrate the plurality of particles through the manipulation module. In step (f), the at least one energy supply module provides an external energy source to a plurality of particles until an amplification temperature is reached, and an enzyme is used to in situ amplify the at least one biological substance or the free nucleic acid substance on the plurality of particles , contacting the amplified at least one biological substance or the free nucleic acid substance with the plurality of particles. In this reaction, at least one temperature balancing substance rapidly cools the plurality of particles, thereby achieving rapid temperature cycling. Finally, in step (g), a detection module is used to identify the color change, luminescence or combination of specific markers on the amplified biological material or free nucleic acid material, so as to detect the amplified biological material on the plurality of particles after concentration. substance or free nucleic acid substance. Fluorescence or spectrophotometric detection devices, including fluorometers or fluorescence scanners to analyze differences in fluorescence intensity, or to identify specific polymerase-amplified biological substances by binding specific DNA fragments or antibody-coupled enzyme reactions logo.

The purpose of the above brief description of the present invention is to provide a basic description of several aspects and technical features of the present invention. The Brief Description of the Invention is not a detailed description of the invention, and therefore its purpose is not to specifically list key or important elements of the invention, nor to delineate the scope of the invention, but merely to present several concepts of the invention in a concise manner.

In order to understand the technical features and practical effects of the present invention, and to implement according to the contents of the description, hereby further take the preferred embodiment as shown in the drawings, and the detailed description is as follows:

First, please refer to FIG. 1 . FIG. 1 is a schematic diagram of the structure of a nucleic acid amplification system 10 according to an embodiment of the present invention. As shown in FIG. 1 , this embodiment provides a nucleic acid amplification system 10 . The nucleic acid amplification system 10 mainly includes a reaction space 100, a sample 200, a plurality of particles, enzymes, an energy supply module 500, and a control module. The control module in this embodiment is a magnetic element 600 , the plurality of particles are selected from a plurality of magnetic nanoparticles 300 , and the enzyme is a polymerase 400 in the embodiment of the nucleic acid amplification system method.

The sample 200 , the plurality of particles, and the polymerase 400 in this embodiment are all placed in the reaction space 100 , and the sample 200 in the reaction space 100 further includes a temperature balancing substance 203 . The temperature balancing substance 203 can be a large volume (greater than 100ul) nucleic acid proliferation reaction solution or an external cooling ice pack in the reaction space 100, and the content can be ice or a high molecular polymer water-absorbent resin, such as carboxymethyl cellulose ( Carboxymethyl Cellulose). With the large specific heat properties of the above-mentioned substances, the magnetic nanoparticles 300 can be rapidly cooled by photothermal conversion to rapidly heat the temperature of the surrounding microenvironment, so as to achieve rapid thermal cycling of nucleic acid amplification reactions. Further, the energy supply module 500 rapidly heats the plurality of nanoparticles and the microenvironment in which they are located by the photothermal effect, so that the nucleic acid amplification reaction on the plurality of nanoparticles can be rapidly heated; The temperature balancing substance 203 is used to rapidly cool down the nucleic acid amplification reaction on the nanoparticle to form an efficient temperature cycle for the in situ nucleic acid amplification reaction.

The sample 200 in this embodiment includes at least one object 201 to be tested, and the sample 200 is placed in the reaction space 100 . Specifically, the sample 200 referred to in this embodiment may be any biological fluid or its supernatant that requires biological detection, such as serum, plasma, urine, lymph fluid, throat swab, nasopharyngeal swab, bronchoalveolar lavage Biological specimens such as fluid, sputum, feces, cerebrospinal fluid, skin vesicle fluid, crusts and other biological samples are even their combinations, and the primary samples of food or processed agricultural products are even their combinations. Further, in the present invention, the used sample 200 may be a purified sample 200 or a sample 200 without preliminary purification and pretreatment. No matter what conditions the sample 200 is, the nucleic acid amplification system 10 shown in this embodiment can be used for subsequent regional thermal blasting of the test object 201 . The analyte 201 can be any microbial individual, tissue or cell, including at least one cell, at least one bacterium, at least one algae, at least one protozoa or protozoa, at least one fungus, at least one virus or phage, at least one A protist, at least one free nucleic acid species, or a combination thereof. Wherein, the at least one free nucleic acid substance further comprises at least one body fluid free nucleic acid substance, at least one tumor free nucleic acid substance or a combination thereof, which is not limited in the present invention.

In this embodiment, a plurality of magnetic nanoparticles 300 are mixed with the sample 200 . Please further refer to FIG. 2 , which is a schematic structural diagram of a magnetic nanoparticle 300 according to an embodiment of the present invention. In this embodiment, the plurality of magnetic nanoparticles 300 can be selected from magnetic gold nanoshells.

As shown in FIG. 2 , each magnetic nanoparticle 300 in this embodiment includes a body 301 , at least one first ligand 302 or a specific aptamer 305 , and at least one second ligand 303 . Under the concept of the present invention, other possible forms of the body 301 of the magnetic nanoparticles 300 may be spherical, short rods, anisotropic protrusions (such as nanometers) formed of metal or composite materials. star), nanoshells, nanocages, double triangular pyramids, but not limited to this form. The material system of the body 301 may include at least one precious metal, such as gold, silver, palladium (alladium), platinum or a combination thereof. The structure of the body 301 for at least one precious metal may be the aforementioned precious metal types such as gold, silver, palladium, platinum, etc. coated on the surface or configured with at least one layer. What's more, the body 301 of this embodiment can incorporate materials such as cyanine, polypyrrole, or graphene, which have a near-infrared (NIR) absorption spectrum. Transition metals such as iron, cobalt, etc. may also be included. Of course, the aforementioned transition metals must also include their oxides, such as ferrous oxide (FeO), iron oxide (Fe ₂ O ₃ ), ferric oxide (Fe ₃ O ₄ ), iron oxide (hydroxide) (FeO ( OH)), ferrous hydroxide (Fe(OH) ₂ ), ferric hydroxide (Fe(OH) ₃ ), cobalt monoxide (CoO), cobalt oxy (hydroxide) (CoO(OH)), cobalt tetroxide ( Co ₃ O ₄ ) and its derivatives and mixtures are not limited in the present invention, as long as the body 301 can contain photothermal or magnetothermal conversion properties or further magnetic properties according to requirements and its related technical means, that is, the conditions of the embodiments of the present invention are met.

Referring to FIG. 2 , regarding at least one first ligand 302 disposed on the body 301 , the first ligand may be an antibody, an aptamer 305 , an oligonucleotide or a combination thereof, which is not limited thereto. The at least one first ligand 302 selected in this embodiment can be matched with the at least one analyte 201 mentioned above. The first ligand 302 specifically binds the analyte 201 to adsorb the surface of the sample 200 on the body 301 . In this embodiment, at least one second ligand 303 is disposed on the body 301, and the second ligand 303 may be an antibody, an aptamer, an oligonucleotide or a combination thereof, which is not limited thereto. And each second ligand 303 contains a specific mark, and the specific mark can be a fluorescent group 3061 or a nucleic acid mark 3062. Furthermore, the second ligand 303 can be a single-stranded nucleic acid sequence, and the biological substance (such as a nucleic acid sample) of the test object 201 released at high temperature can be functionalized on the surface of the nanoparticle body 301 by the second ligand. Ligand 303 grabs to capture specific genetic material for amplification reaction. Therefore, the surface of each magnetic nanoparticle 300 in this embodiment is subjected to surface processing by at least two procedures, so that it has extremely high specificity. The functionalized and specific first ligand 302 and the second ligand 303 in this embodiment are specialized to be able to analyze a small amount of the sample 200 accordingly.

Each of the first ligand 302 and the second ligand 303 in this embodiment is connected to the body 301 through the stabilization structure 304 . Wherein, the stabilizing structure 304 optionally includes a thermally stable bond, a contact suppression coating layer or a combination thereof. Further, the thermostable bond further includes the connection of biotin and streptavidin or a cross linker, or other covalent bonds with thermostable properties. Since the combination of streptavidin and biotin has extremely high thermal stability (> 105 degrees) in its saturated state, and the preparation method is simple, streptavidin is used to combine with biotin. The high-strength and high-affinity binding between biotins (Biotin-streptavidin interaction) creates a stable reaction environment in the high temperature environment of polymerase chain reaction (PCR). Compared with the prior art, most of the disulfide bonds formed by thiol (thiol) are covalently bonded, resulting in the presence of dithiothreitol (DTT) or other reducing agents in the PCR reaction solution. In the presence of the aforementioned substances, it is easy to reduce the covalent bond of the originally formed disulfide bond to a thiol (-S-H bond), causing the primer in the polymerase chain reaction to be detached from the surface of the nanoparticle. Avidin proteins can form a protein coating on the nanoparticle surface to reduce nonspecific adsorption of nanoparticles.

The polymerase 400 is also mixed with the sample 200. The polymerase 400 used in this embodiment may be DNA polymerase (DNA Polymerase), ribonucleic acid polymerase (RNA Polymerase) or a combination thereof, which can only be replaced according to the type of nucleic acid to be detected or the type of the second ligand 303 , the present invention is not limited. In addition, in this embodiment, the enzyme further comprises reverse transcriptase (RT), ribonuclease (RNase), helicases (helicases), DNA ligase or a combination thereof, according to the 200 kinds of samples or nucleic acids The type of amplification reaction varies depending on the situation.

In addition, in order to reduce the contact inhibition between the surface of the nanoparticle body 301 and deoxyribose nucleic acid polymerase (DNA Polymerase) or ribose nucleic acid polymerase (RNA Polymerase), this embodiment optionally coats the surface of the body 301 The contact suppressing cladding. The contact inhibition coating layer may include a silica coating, polyethylene glycol (PEG) or a combination thereof, which is used as a filling molecule on the surface of the nanoparticle body 301 to reduce nanoparticle size. Aggregate between particles improves the stability of the nanoparticle surface.

The at least one energy supply module 500 in this embodiment can select a laser emitter, an LED diode light source or a magnetic field generator, thereby providing an external energy source B to a plurality of magnetic nanoparticles 300. The external energy source B can be light energy or an alternating magnetic field. Of course, the external energy B referred to in this embodiment (refer to FIG. 7 first) is laser light energy. The control module is used to control the plurality of nanoparticles in the reaction space 100 . Among them, although the control module in this embodiment is only realized by using the magnetic element 600, in fact, the control module can further select the magnetic element 600, centrifugal force device or a combination thereof. When the manipulation module is the magnetic element 600 , the nanoparticle should be designed as the magnetic nanoparticle 300 with magnetic force, so that the magnetic element 600 can operate the plurality of magnetic nanoparticles 300 in the reaction space 100 . Further, the magnetic element 600 may be a permanent magnet, an electromagnet or a combination thereof, which is not limited in the present invention.

The embodiment of the nucleic acid amplification system 10 of the present invention further includes a detection module for detecting the fluorescent light F released by the fluorescent group 3031 on the second ligand 303 concentrated and aggregated by the manipulation module , to quantify the amplified biological substance 202 of the analyte 201 on the surface of the nanoparticle. In this embodiment, the biological substance 202 may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and is not limited thereto. If the biological material 202 is a nucleic acid, it is subjected to in-situ PCR, reverse transcription or isothermal amplification, wherein the isothermal amplification refers to a technology with an operating temperature greater than 37 degrees. , including isothermal circular amplification (LAMP), strand displacement amplification (SDA), helicase strand displacement amplification (HDA), nucleic acid delisting-based amplification (NASBA) and transcription-mediated amplification ( TMA), Nicking Enzyme Amplification Reaction (NEAR), isothermal recombinant polymerase nucleic acid amplification reaction (Recombinase polymerase amplification, RPA).

The amplified nucleic acid sequence fragment, that is, the biological substance 202 released by the thermal explosion of the analyte 201 after being amplified by the nucleic acid amplification reaction, is called an amplicon.

Please refer to FIG. 3 , which is a flowchart of a method of the nucleic acid amplification system 10 according to an embodiment of the present invention. As shown in FIG. 3 , the method of the nucleic acid amplification system 10 described in this embodiment mainly consists of seven steps, including step (a), mixing a sample 200 with a plurality of magnetic nanoparticles 300 to make the plurality of magnetic nanoparticles The nanoparticle 300 captures the at least one analyte 201 . Following step (b), a control module is used in a reaction space 100 to condense a sample 200 , and the sample 200 includes the at least one analyte 201 . If the at least one analyte 201 is a free nucleic acid substance, proceed directly to step (e), otherwise proceed to step (c).

In step (c), at least one energy supply module 500 provides an external energy source B to the plurality of magnetic nanoparticles 300 to raise the plurality of magnetic nanoparticles 300 to a burst temperature. Then step (d) is performed, the at least one object to be tested 201 bursts to release at least one biological substance 202 ; followed by step (e), the at least one energy supply module 500 provides an external energy B to the plurality of When the particles reach a binding temperature, the plurality of magnetic nanoparticles 300 are combined with the at least one biological substance 202 or the free nucleic acid substance, and the plurality of magnetic nanoparticles 300 are concentrated through the control module. Finally, in step (f), the at least one energy supply module 500 provides an external energy source B to the plurality of magnetic nanoparticles 300 until an amplification temperature is reached, and the plurality of magnetic nanoparticles 300 are amplified by a polymerase 400 The at least one biological substance 202 or the free nucleic acid substance. Finally, in step (g), a detection module is used to identify the color change, luminescence or combination of the specific mark on the amplified biological substance or the free nucleic acid substance, so as to detect the plurality of particles after concentration The at least one biological substance or the free nucleic acid substance amplified above.

For a clearer understanding of the method of the nucleic acid amplification system 10 described in this embodiment, please refer to FIGS. 3-5 at the same time. FIG. 4 is a schematic diagram of the sample 200 according to the embodiment of the present invention before concentration; FIG. 5 is the sample 200 according to the embodiment of the present invention. Schematic diagram after concentration.

In step (a) of FIG. 3 , the sample 200 is mixed with a plurality of magnetic nanoparticles 300 , so that the plurality of magnetic nanoparticles 300 capture at least one test object 201 . The analyte 201 of this embodiment includes at least one cell, at least one bacteria, at least one algae, at least one protozoa or protobacteria, at least one fungus, at least one virus or bacteriophage, at least one protist, or a combination thereof. Basically, the analyte 201 must have an antigen that can be recognized by a specific antibody or a specially designed antibody, and carry genetically related substances such as nucleic acid, so that further detection can be carried out in this embodiment. As shown in FIG. 2 , a specific antibody 302 or an aptamer 305 capable of recognizing the antigen of the analyte 201 is disposed on the nanoparticle with surface functionalization, for the subsequent thermal detection of the analyte 201 through nanoparticle identification. Blast and nucleic acid material amplification.

Please refer to FIGS. 3-5 at the same time. FIG. 4 is a schematic diagram of a sample 200 according to an embodiment of the present invention before concentration. In order to speed up the detection of this embodiment, a control module is used in the reaction space 100 in step (b) of this embodiment. The sample 200 is concentrated and washed to remove an impurity, so that it assumes the state of FIG. 5 . The concentrated sample 200 in FIG. 5 includes at least one analyte 201 . The manipulation module includes centrifugal equipment, magnetic element 600 or a combination thereof. The concentration method in this embodiment may include centrifugation and then extracting the supernatant, using a magnetic element 600 to concentrate a plurality of magnetic nanoparticles 300 or a combination thereof, which is not limited in the present invention. In this embodiment, the impurity is a substance other than the analyte. The analyte 201 is captured by the plurality of magnetic nanoparticles 300 through the combination with the first ligand 302, and then the plurality of magnetic nanoparticles are concentrated through the control module. After particle 300, the analyte 201 is separated from the impurity and removed.

Next, please refer to FIG. 3 and FIGS. 6-7 at the same time. FIG. 6 is a schematic diagram of adding polymerase 400 according to an embodiment of the present invention; FIG. 7 is a schematic diagram of blasting a test object 201 according to an embodiment of the present invention. Step (c) of FIG. 3 corresponds to those shown in FIGS. 6-7 . As shown in FIG. 6 , step (c) of this embodiment may further add polymerase 400 . Of course, in other possible implementations, if the blasting temperature of step (c) may affect the polymerase 400 and cause the risk of denaturation, the polymerase 400 can also be added in step (f) instead. Inventions are not limited.

Next, as shown in FIG. 7 , through at least one first ligand 302 or one aptamer 305 on the magnetic nanoparticle 300 (refer to FIG. 2 ), the magnetic nanoparticle 300 can smoothly use the antigen-recognition region to rapidly enter the The analyte 201 is matched and captured in the sample 200 . In this embodiment, the analyte 201 may be a microorganism or a specific cell. Thereby, when step (c) is performed, the energy supply module 500 provides the external energy B (the gray ripples emitted by the energy supply module 500 in FIG. 7 ) to a plurality of magnetic nanoparticles 300 , so that the plurality of magnetic nanoparticles The temperature of the particles 300 rises to the burst temperature.

As mentioned above, the energy supply module 500 of this embodiment is a laser transmitter, and the external energy B is of course laser light energy. However, in fact, the type of the energy supply module 500 must be selected according to the material of the body 301 of the magnetic nanoparticle 300, mainly the energy supply module 500 that can freely heat the body 301, and the laser wavelength ranges from the visible spectrum to the near-infrared spectrum. (380nm-1.4μm) is the main range, preferentially in the near-infrared spectral region (750nm-1.4μm). Nanoparticles that can convert external energy into thermal energy, and the external energy can be energy such as light energy, alternating magnetic field, etc., which is not limited in the present invention. In this embodiment, the energy supply module 500 mainly heats the nanoparticle itself and the microenvironment of the nearby area (the distance is in micrometers (μm)), so it is more suitable for in-situ polymerase chain reaction ( in-situ PCR) .

Please refer to FIG. 3 and FIGS. 8-9 at the same time. FIG. 8 is a schematic diagram of the biological substance 202 released by the test object 201 according to an embodiment of the present invention; FIG. FIG. 8 corresponds to step (d) of FIG. 3 . In this embodiment, since a plurality of magnetic nanoparticles 300 have already passed through the at least one first ligand 302 to grasp the test object 201 in step (c), therefore The distances between the plurality of magnetic nanoparticles 300 and the object to be tested 201 are very close. The plurality of magnetic nanoparticles 300 use the photothermal effect property of the body 301 to absorb the external energy B provided by the energy supply module 500 and convert it into heat, and when the plurality of magnetic nanoparticles 300 have risen to the explosion temperature On the premise, the test object 201 will be degraded and ruptured due to the physical action of high heat, such as cell membrane, cell wall, etc., to achieve the purpose of blasting the test object 201 . Accordingly, the at least one test object 201 in step (d) is then burst to release at least one biological substance 202 .

In this embodiment, the aforementioned biological material 202 is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Referring to FIG. 2 at the same time, since the analyte 201 bursts in a state where a plurality of magnetic nanoparticles 300 are very close together, the biological substance 202 released by the analyte 201 will also be in a high concentration manner in the magnetic nanoparticles 300 surrounding release. Thereby, as described in step (e) of FIG. 3 , a plurality of magnetic nanoparticles 300 are combined with at least one biological substance 202 (refer to FIG. 9 ). If the analyte is a free nucleic acid substance, the step (b) is executed and the step (e) is directly entered. At this time, the at least one energy supply module provides an external energy to the plurality of particles until a binding temperature is reached, so that the A plurality of magnetic nanoparticles 300 are combined with the free nucleic acid substance. Furthermore, the reason why the plurality of magnetic nanoparticles 300 can be combined with at least one biological substance 202 or the free nucleic acid substance is that the magnetic nanoparticles 300 have the second ligand 303 (refer to FIG. 2 ).

In this embodiment, since the biological substance 202 is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the second ligand 303 also selects deoxyribonucleic acid (DNA) or ribose sugar that is complementary to the biological substance 202 accordingly. Nucleic acid (RNA) short chains. Accordingly, the goal of being able to combine with at least one biological substance 202 is achieved.

Next, please refer to FIG. 3 and FIG. 10 at the same time. FIG. 10 is a schematic diagram of detecting the fluorescent F reaction according to an embodiment of the present invention. As shown in FIG. 10 , corresponding to steps (e)-(f) of FIG. 3 , after step (e) as shown in FIG. 9 , a plurality of magnetic nanoparticles 300 are combined with at least one biological substance 202 , as shown in FIG. 10 . As shown in general, a plurality of magnetic nanoparticles 300 are concentrated through the operation of the magnetic element 600 .

In this way, the polymerase 400 will assist the biological substances 202 captured on the plurality of magnetic nanoparticles 300 to perform step (f), and the energy supply module 500 provides the external energy B to the plurality of magnetic nanoparticles in the reaction space 100 . The rice particles 300 reach the constant temperature amplification temperature, and the plurality of magnetic nanoparticles 300 amplify at least one biological substance 202 through the polymerase 400, and at the same time generate the color change of the fluorescent F or the nucleic acid marker 3062 coupled enzyme. The temperature balancing substance 203 in this embodiment can be a large-volume (greater than or equal to 100 microliters) PCR proliferation reaction solution, providing heat capacity to rapidly cool a plurality of magnetic nanoparticles 300 with a large specific heat, so as to achieve rapid temperature cycling for nucleic acid amplification reactions.

As mentioned above, if the polymerase 400 has been added in step (b), the polymerase 400 in this embodiment should be selected as the type of at least one biological substance 202 as DNA polymerase, ribonucleic acid Polymerase (RNA Polymerase) or reverse transcriptase (reverse transcriptase), in order to carry out the amplification reaction in the subsequent step (f). Of course, the polymerase 400 can also be added between the steps (e)-(f), and the present invention does not limit the time for adding the polymerase 400.

Compared with the prior art, since the nucleic acid amplification product is not in the reaction space 100 but only on the second ligand 303 on the surface of the nanoparticle, in-situ polymerase chain reaction ( in-situ polymerase chain reaction) is performed on the surface of the nanoparticle. PCR), the overall in situ proliferation amplification load is much smaller than when the polymerase chain reaction is in the entire reaction space 100, so as long as the nucleic acid load on the nanoparticles is saturated, this also represents a relatively short Saturation can be achieved with time or a relatively small number of nucleic acid amplification cycles.

Finally, step (g) is performed, through a detection device, in this embodiment, a fluorometer or a fluorescence scanner can be used to analyze the difference in fluorescence intensity, or a spectrophotometric detection device can be used to identify the Amplified biological material 202 or nucleic acid marker 3062 on free nucleic acid or color change produced by antibody-coupled enzyme reaction. The nucleic acid marker 3062 can be a DNA marker that can be recognized by an antibody, and the color change generated by the coupled enzyme reaction after the antibody recognizes the DNA fragment can be analyzed by colorimetry, thereby detecting the biological substance 202 amplified by the polymerase.

As for the cause of the color change caused by fluorescent F or nucleic acid marker 3062, reference can be made to FIG. 2 . The free nucleic acid primer 306 in this embodiment further has a fluorescent group 3061 or nucleic acid marker 3062. In other words, when the at least one analyte 201 in the sample 200 can release the correct at least one biological substance 202 or the sample 200 contains free nucleic acid substances, the second ligand 303 can interact with the biological substances 202 or free nucleic acid substances. The sequences of the nucleic acid substances are complementary and use the free nucleic acid primer 306 as a template to amplify and react with the polymerase 400, so that the fluorescent group 3061 or the nucleic acid marker 3062 is inserted into the amplified product; When the analyte 201 cannot release at least one correct biological substance 202 or there is no free nucleic acid substance in the sample 200, then the fluorescent group 3061 or the nucleic acid marker 3062 on the second ligand 306, because the polymerase 400 does not work Amplification proceeds without fluorescence F or subsequent colorimetric changes.

As mentioned above, there are several embodiments for signal detection of fluorescence F generated by amplicons on nanoparticles, and are not limited here. In one of the preferred embodiments, a fluorescent method can be used, and a primer labeled with a fluorescent group 3061 can be used. In this embodiment, the fluorescent group 3061 can be selected from Fluorescein isothiocyanate (FITC). ). Therefore, under the premise that Fluorescein isothiocyanate (FITC) is selected for the fluorescent group 3061, the present embodiment is designed to specifically identify the primer pair (F3 & B3) for the E.coli malB gene, Among them, F3 is bound to nanoparticles by means of streptavidin and biotin or by covalent bonding of carboxyl and amine cross-linking. In addition, B3 primer is labeled with fluorescein isothiocyanate and its 5 end to form FITC- B3 primer. However, in fact, the selection of the fluorescent group 3061 and the primer can be arbitrarily replaced according to different detection requirements, which is not limited in the present invention. To sum up, during the above-mentioned in situ nucleic acid amplification in this embodiment, an amplicon with a fluorescent group 3061 is formed on the surface of the magnetic nanoparticle 300. As for the free form of isothiocyanate The acid luciferin-B3-primer (FITC-B3 primer) can be removed by the process of magnetically grabbing the magnetic nanoparticles 300 and cleaning. The fluorescent F signal on the surface of the magnetic nanoparticle 300 can be concentrated and concentrated by magnetic force (or a combination of centrifugal means) later. A suitable spacer is formed on the surface of the rice particle 300, so that the magnetic nanoparticle 300 and the fluorescent group 3031 can enhance the fluorescence F intensity through the metal enhanced fluorescence effect (MEF), and analyze the fluorescence intensity image (fluorescence scanner) or by fluorescence spectroscopy (fluorescence spectrometry) for analysis.

Another example of detecting colorimetric signals from amplicons on nanoparticles is to use a Lateral flow dipstick. This method is slightly different from the traditional lateral flow immunogold method. The traditional immunogold will bind to the antibody to be tested and calibrated; however, the detection process in this example will not add colloidal gold particles bound to the antibody, and will only The antibody that recognizes the fluorescent group 3031 is immobilized on the detection line of the lateral flow. When the liquid containing the target amplicon (amplicon) flows through, the target amplicon (amplicon) will be immobilized on the detection line to obtain the detection signal. ; On the control line, only the first ligand 302 for identifying the magnetic nanoparticle 300 itself is fixed.

In addition to the above-mentioned embodiments for detecting the fluorescent F signal, there are also methods such as bead-based ELISA, Litmus test, etc., which are not limited to the above. The purpose of Bead-based ELISA is to increase the sensitivity of the fluorometric method, or to convert the fluorescent F signal into a colorimetric method. In this method, an antibody that can specifically recognize the fluorescent group 3031 (FITC) is coupled to horseradish peroxidase (HRP), and tetramethyl tetramethylmethane is converted by horseradish peroxidase (HRP). After the conversion of the TMB substrate to a blue product, acidification terminates the reaction of horseradish peroxidase (HRP), and the magnetic nanoparticles 300 are detected at a wavelength of 450 nanometers (nm). In situ amplified nucleic acid product on the surface.

The Litmus test has the same purpose as the Bead-based ELISA described above, but the difference is that it is not identified by specific antibodies, but in situ polymerization The reverse primer on the enzyme chain reaction ( in situ PCR) is designed and reserved with a tag (conjugated urease-DNA (UrD) binding tag) that can bind to the DNA fragment labeled with urease. Among them, the DNA (conjugated urease-DNA, UrD) marked with urease is a single-stranded nucleotide coupled to urease, which decomposes urea to produce ammonia (NH ₃ ). ), and increase the pH value in the reaction solution, which can be used to measure the amount of amplicons on the magnetic nanoparticle 300 by using phenol red or cresol red as an acid-base indicator. Converts to differences in colorimetric methods.

To sum up, the embodiment of the present invention can simultaneously overcome multiple limitations such as concentration and processing and screening of samples 200 in a single reaction space 100 , and can, through the specific design, photothermal properties and magnetic properties of the precise magnetic nanoparticles 300 , simultaneously It achieves the functions of a heater and a single carrier, so that it is possible to quickly identify whether the analyte 201 exists in the sample 200 with the naked eye, and the temperature balancing substance 203 in the sample 200 can further accelerate the polymerase chain reaction The temperature cycle has greatly shortened the detection process that was protracted in the past.

For a clearer understanding of the actual operation of the present invention, please refer to the experimental data in FIGS. 11 to 23 .

In one of the experiments of applying the embodiments of FIGS. 1-10 of the present invention, the photothermal temperature change experiment is carried out when the magnetic nanoparticles 300 are magnetic gold nanoparticles (Magnetic Gold Nanoshells, MGNs). In this embodiment, the concentration of the magnetic nanoparticles 300 in water is 2.9× ^{10 10} particles/ml, and the energy supply module 500 uses laser light with a wavelength of 808 nm as the external energy source B to conduct energy with different wattages (200mw-700mw) heating the magnetic nanoparticles 300 to produce photothermal reaction changes.

Please refer to FIG. 11 . FIG. 11 is a temperature change diagram of the ambient temperature to a steady state caused by photothermal changes generated by the energy supply module 500 applying laser light energy of different intensities (200mw-700mw) to the magnetic nanoparticles 300 . 12 is a graph showing the relationship between the laser intensity applied to the magnetic nanoparticles 300 and the ambient temperature around the particles when using Magnetic Gold Nanoshells (MGNs) as the magnetic nanoparticles 300. In this embodiment, the wavelength 808 is used first. After being heated by laser light for 5 minutes, the temperature of the Magnetic Gold Nanoshells (MGNs) rose to a steady state, and the entire heating process was detected using a k-type thermocouple. Referring to FIG. 12 , it can be known that the stable temperature of the magnetic nanoparticle 300 selected from Magnetic Gold Nanoshells (MGNs) is linearly related to the laser intensity of the external energy source, and the shallow temperature in FIG. 12 The gray band and the dark gray band respectively indicate the optimum temperature range of the magnetic nanoparticles 300 for photothermal cracking and isothermal loop amplification reaction (LAMP). FIG. 13 is a temperature change diagram when the magnetic nanoparticle 300 is heated by a laser, a photothermal reaction is performed in water, and a constant temperature loop amplification reaction is performed. In FIG. 13 , the magnetic nanoparticle 300 was firstly subjected to photothermal reaction cracking at a power of 300mw-700mw for 5 minutes using a laser light with a wavelength of 808 nm, and then the magnetic nanoparticles were decomposed with an application power of 10mw-50mw. 300 was slowly cooled to capture the DNA on the particles, and finally a constant temperature circular amplification reaction on the magnetic nanoparticles 300 was performed with a laser with an applied power of 200mw-250mw, and the maintenance time was 30 minutes.

In another embodiment of the present invention, the magnetic nanoparticles 300 use polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) to perform photothermal temperature change experiments. In this embodiment, the concentration of polypyrrole-capped iron oxide particles (PPy-capped Fe ₃ O ₄ particles) in water as the magnetic nanoparticles 300 is 1000 ppm, and laser light with a wavelength of 808 nm is also used for different The wattage energy (200mw-1000mw) is heated to make the polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) as the magnetic nanoparticles 300 to produce photothermal reaction changes.

Please refer to FIG. 14 . FIG. 14 shows the photothermal changes caused by the use of laser light energy of different intensities (200mw-1000mw) applied to the magnetic nanoparticles 300 when polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) are selected. Graph of temperature change from ambient temperature to steady state. Figure 15 is a graph of the temperature change of the surrounding environment from the steady state caused by the photothermal change generated by the application of laser light energy of different intensities (200mw-1000mw) to the iron oxide particles (Fe ₃ O ₄ particles) without polypyrrole outer layer. 16 is a graph showing the relationship between the laser intensity applied to the magnetic nanoparticles 300 and the ambient temperature around the particles when PPy-enveloped Fe ₃ O ₄ particles are used as the magnetic nanoparticles 300 . In this embodiment, the temperature of the PPy-enveloped Fe 3 O 4 particles (PPy-enveloped Fe ₃ O ₄ particles) used as the magnetic nanoparticles 300 is raised to a temperature of 5 minutes after heating with a laser light with a wavelength of 808 nm. Steady state, and use a K-type thermocouple (k-type thermocouple) to detect the entire heating process. Referring to FIG. 16 , it can be known that the stable temperature of the magnetic nanoparticle 300 when polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) are selected has a linear relationship with the laser intensity of the external energy source, and The light gray band and the dark gray band in Fig. 16 respectively indicate that the magnetic nanoparticles 300 are subjected to photothermal cracking and isothermal cyclic amplification reaction (LAMP) when polypyrrole-enveloped iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) are selected. optimum temperature range. FIG. 17 is a graph showing the temperature change when PPy-enveloped Fe 3 O 4 particles (PPy-enveloped Fe ₃ O ₄ particles) as the magnetic nanoparticles 300 are heated by a laser and subjected to a photothermal reaction in water to perform a constant temperature loop amplification reaction. In Fig. 17, a laser light with a wavelength of 808 nanometers was first used to perform photothermal treatment for 5 minutes on polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) as magnetic nanoparticles 300 with an applied power of 500mw-1000mw. Reaction cleavage. Then, the applied power of 20mw-80mw is used to slowly cool down the PPy-enveloped Fe ₃ O ₄ particles as the magnetic nanoparticles 300 to capture the DNA on the magnetic nanoparticles 300 . A laser with a power of 300mw-450mw was used as a magnetic nanoparticle 300 to perform a constant temperature circular amplification reaction on PPy-enveloped Fe 3 O 4 particles (PPy-enveloped Fe ₃ O ₄ particles) for 30 minutes.

For further details of the aforementioned embodiment, please refer to FIG. 18 . FIG. 18 is a graph showing the relationship between the survival rate of the bacteria and the ambient temperature when using polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) as the magnetic nanoparticles 300 to perform photothermal reaction to decompose Escherichia coli. It can be seen from Figure 18 that the optimal power used for photothermal cracking of the test object is 700mw-800mw, and the operating conditions are to provide 1000ppm concentration of polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) as magnetic nanometers. particle 300, and the optimal effect of this optimal blast temperature range is not exactly the highest temperature.

In the aforementioned embodiment, please refer to FIG. 19 for further details. Figure 19 shows the effects of photothermal lysis of bacteria and quantitative real-time polymerase chain reaction (Q-PCR) quantification of the release effects of the genomic DNA and plastid DNA, respectively. The experimental data confirmed that when using PPy-enveloped Fe ₃ O ₄ particles as the magnetic nanoparticles 300 , the effect of near-infrared laser photothermal reaction cracking is better than the traditional thermal cracking method.

In the aforementioned embodiment, please refer to FIG. 20 further. Figure 20 shows the sensitivity performance of photothermal in situ isothermal loop amplification reaction (photonic in situ LAMP) by copy number and fluorescence intensity (FI). It can be seen from Figure 20 that when the fluorescence intensity reaches the threshold value (the fluorescence intensity of the control group without template plus three standard deviations) at 30 minutes of reaction, the gene body DNA copy number (copy number) of the lowest concentration of E.coli Between 10 ² and 10 ³ . Further, the same experiment was performed using a conventional isothermal loop amplification reaction, see Figure 21. Figure 21 shows the sensitivity performance of conventional isothermal loop amplification reaction (LAMP) at different time points by copy number (copy number) and fluorescence intensity (FI). The comparison between Figure 20 and Figure 21 shows that the sensitivity performance of the photonic in situ isothermal loop amplification reaction (photonic in situ LAMP) is better than that of the traditional isothermal loop amplification reaction (LAMP).

In this embodiment, please refer to FIG. 22 . Figure 22 shows the performance of photonic in situ LAMP using a particle-bound bead-based ELISA. Similarly, when the laser energy of 700-800mw (milliwatts) is applied, there is a better performance of the analyte protein signal.

In another embodiment, please refer to FIG. 23 . In this example, the aforementioned Magnetic Gold Nanoshells (MGNs) were selected as the magnetic nanoparticles 300 to conduct the photothermal reaction experiment, and a two-step photothermal in situ polymerase chain reaction (photonic in situ PCR) was used to obtain the magnetic nanoparticles 300 . The relationship between cycles of amplification and normalized fluorescence intensity was further evaluated. In this embodiment, a two-stage polymerase chain reaction is used, and different intensities of laser light energy are input in different stages. In the first embodiment, 457mw is used for 10 seconds, and then 240mw is used for 60 seconds; and the second embodiment is 400mw for 10 seconds. After 145mw for 60 seconds. It can be seen from FIG. 23 that in the first embodiment, when the number of amplification cycles reaches 20 to 30 cycles, the bonding intensity reaches saturation, so the fluorescence intensity detected when reaching 30 cycles varies with the decline. Further improvement in the second embodiment, the fluorescence intensity increases when the number of cycles reaches 30. In other words, the second embodiment uses lower energy application, which can effectively improve the bonding strength of the amplified nucleic acid. It can be seen that the positive correlation between the number of amplification cycles and the detected fluorescence intensity is limited by the strength of nucleic acid binding and the degree of saturation of the amplified nucleic acid.

However, the above are only preferred embodiments of the present invention, and should not limit the scope of implementation of the present invention, that is, the simple changes and modifications made according to the scope of the patent application of the present invention and the description content still belong to the present invention. within the scope of coverage.

10: Nucleic acid amplification system 100: React Space 200: Sample 201: Object to be tested 202: Biomass 203: Temperature Equilibrium Substances 300: Magnetic Nanoparticles 301: Ontology 302: first ligand 303: second ligand 304: Stable Structure 305: aptamer 306: free nucleic acid primer 3061: Fluorescent group 3062: Nucleic Acid Markers 400: polymerase 500: Energy Supply Module 600: Magnetic element B: External energy F: Fluorescent (a)~(g): Steps

FIG. 1 is a schematic diagram of the structure of a nucleic acid amplification system according to an embodiment of the present invention. FIG. 2 is a schematic structural diagram of a nanoparticle according to an embodiment of the present invention. FIG. 3 is a flow chart of the method of the nucleic acid amplification system according to the embodiment of the present invention. FIG. 4 is a schematic diagram of a sample of an embodiment of the present invention before concentration. FIG. 5 is a schematic diagram of a sample of an embodiment of the present invention after concentration. FIG. 6 is a schematic diagram of adding a polymerase in an embodiment of the present invention. FIG. 7 is a schematic diagram of blasting a test object according to an embodiment of the present invention. FIG. 8 is a schematic diagram of the biological substance released from the analyte according to the embodiment of the present invention. FIG. 9 is an enlarged schematic diagram of the capture of biological substances according to an embodiment of the present invention. FIG. 10 is a schematic diagram of the detection reaction according to the embodiment of the present invention. FIG. 11 is an energy supply module of one embodiment of the present invention that uses laser light energy of different intensities (200mw-700mw) to apply the photothermal changes to Magnetic Gold Nanoshells (MGNs), resulting in a stable ambient temperature. The temperature change diagram of the state. 12 is a graph showing the relationship between the intensity of the laser applied to the magnetic nanoparticles (Magnetic Gold Nanoshells, MGNs) and the ambient temperature around the particles when magnetic nanoparticles according to one embodiment of the present invention are selected. 13 is a temperature change diagram of an energy supply module according to one embodiment of the present invention using a laser to heat magnetic gold nanoparticles (Magnetic Gold Nanoshells, MGNs) in water for photothermal reaction and constant temperature circular amplification reaction. Fig. 14 is an energy supply module of one embodiment of the present invention when laser light energy of different intensities (200mw-1000mw) is applied to the magnetic nanoparticles when polypyrrole-enveloped iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) are selected The resulting photothermal change causes the temperature change diagram of the ambient temperature to a steady state. FIG. 15 shows the energy supply module of another embodiment of the present invention using different intensities (200mw-1000mw) of laser light energy to be applied to the magnetic nanoparticles when iron oxide particles (Fe ₃ O ₄ particles) without polypyrrole outer layer are selected A graph of the temperature change from the ambient temperature to a steady state caused by photothermal changes. 16 is a graph showing the relationship between the intensity of the laser applied to the magnetic nanoparticles of one embodiment of the present invention and the ambient temperature around the particles when polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) are selected. 17 shows the temperature of the energy supply module according to one embodiment of the present invention using laser to heat polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) in water for photothermal reaction and constant temperature circular amplification reaction Change graph. Figure 18 is a graph showing the relationship between the survival rate of the bacteria and the ambient temperature when the magnetic nanoparticles according to one embodiment of the present invention use polypyrrole-encapsulated iron oxide particles (PPy-enveloped Fe ₃ O ₄ particles) to decompose Escherichia coli by photothermal reaction. Figure 19 shows the use of photothermal reaction to lyse bacteria for DNA extraction and quantitative real time polymerase chain reaction (Q-PCR) to quantify the effect of the release of genomic DNA and plastid DNA, respectively. Figure 20 evaluates the sensitivity performance of photonic in situ LAMP by copy number (copy number) and fluorescence intensity (FI). Figure 21 evaluates the sensitivity performance of conventional isothermal loop amplification reaction (LAMP) at different time points by copy number (copy number) and fluorescence intensity (FI). Figure 22 evaluates the performance of photonic in situ LAMP using a particle-bound bead-based ELISA. Figure 23 shows the magnetic nanoparticles of one embodiment of the present invention. The aforementioned Magnetic Gold Nanoshells (MGNs) are used to perform a two-step photonic in situ PCR (photonic in situ PCR) for further evaluation of amplification. The relationship between the number of cycles and the normalized fluorescence intensity detected.

(a)~(g): Steps

Claims (24)

A nucleic acid amplification system comprising: a reaction space; a sample, including at least one analyte, the sample is placed in the reaction space; a plurality of particles, mixed with the sample, each of the particles containing: a body; at least one first ligand, disposed on the body, the at least one first ligand matches the at least one analyte; at least one second ligand, disposed on the body, each of the at least one second ligand includes a specific mark; at least one enzyme, mixed with the sample; at least one energy supply module for providing an external energy to the plurality of particles; At least one temperature-equilibrating substance is placed in the reaction space; and a manipulation module for manipulating the plurality of particles in the reaction space. The nucleic acid amplification system of claim 1, wherein the plurality of particles are nanoparticles. The nucleic acid amplification system of claim 1, wherein the temperature balancing substance is a cooling substance. The nucleic acid amplification system of claim 1, wherein the at least one analyte comprises at least one cell, at least one organelle, at least one bacterium, at least one virus, at least one protist, at least one free nucleic acid substance or a combination thereof . The nucleic acid amplification system according to claim 4, wherein the at least one free nucleic acid substance comprises at least one body fluid free nucleic acid substance, at least one tumor free nucleic acid substance or a combination thereof. The nucleic acid amplification system of claim 1, wherein the at least one first ligand or the at least one second ligand comprises an antibody, an aptamer, an oligonucleotide or a combination thereof. The nucleic acid amplification system of claim 1, wherein each of the first ligand and the second ligand is connected to the body through a stable structure, the stable structure comprising a thermally stable bond, a contact inhibitory coating or its combination. The nucleic acid amplification system of claim 1, wherein the specific marker comprises a fluorescent group or a nucleic acid marker. The nucleic acid amplification system of claim 1, wherein the enzyme is a polymerase. The nucleic acid amplification system according to claim 9, wherein the polymerase further comprises deoxyribose nucleic acid polymerase (DNA Polymerase), ribose nucleic acid polymerase (RNA Polymerase) or a combination thereof. The nucleic acid amplification system of claim 1, wherein the enzyme further comprises reverse transcriptase (RT), ribonuclease (RNase), helicases, DNA ligase or a combination thereof. The nucleic acid amplification system according to claim 1, wherein the energy supply module is a laser emitter, a light-emitting diode or a magnetic field generator. The nucleic acid amplification system of claim 1, wherein the external energy source comprises light energy or an alternating magnetic field. The nucleic acid amplification system of claim 1, wherein the manipulation module comprises a magnetic element, a centrifugal device or a combination thereof. The nucleic acid amplification system of claim 14, wherein when the manipulation module includes the magnetic element, the plurality of particles include a plurality of magnetic particles. The nucleic acid amplification system of claim 1, further comprising a detection module for detecting the specific marker contained in each of the at least one second ligand of the plurality of particles concentrated and aggregated by the manipulation module . The method for the nucleic acid amplification system as claimed in claim 1, comprising: (a) mixing a sample with a plurality of particles, so that the plurality of particles capture the at least one analyte; (b) using a control module in a reaction space to clean the sample to remove impurities and to concentrate the sample to remove impurities For the at least one analyte, if the at least one analyte is a free nucleic acid substance, proceed directly to step (e), otherwise proceed to step (c); (c) at least one energy supply module provides an external energy source to the plurality of particles to raise the plurality of particles to a blasting temperature; (d) the at least one analyte bursts, releasing at least one biological substance; (e) The at least one energy supply module provides an external energy to the plurality of particles until a binding temperature, so that the plurality of particles are combined with the at least one biological substance or the free nucleic acid substance, and through the manipulation module concentrate the plurality of particles; and (f) the at least one energy supply module provides the external energy to the plurality of particles up to an amplification temperature, and in situ amplifies the at least one biological substance or the free nucleic acid substance on the plurality of particles through an enzyme, The amplified at least one biological material or the free nucleic acid material is contacted with the plurality of particles. The method for a nucleic acid amplification system according to claim 17, wherein step (g) after step (f) is followed: identifying the specific biological substance or the free nucleic acid substance on the amplified at least one biological substance through a detection module The color change, luminescence or combination thereof produced by the marker is used to detect the at least one biological substance or the free nucleic acid substance amplified on the plurality of particles after concentration. The method for a nucleic acid amplification system according to claim 17, wherein the enzyme selects at least one polymerase corresponding to the at least one biological substance or the type of the free nucleic acid substance, and the at least one polymerase comprises a DNA polymerase ( DNA Polymerase) or RNA Polymerase. The method of the nucleic acid detection system according to claim 17, wherein the control module comprises a magnetic element, a centrifugal device or a combination thereof. The method of the nucleic acid amplification system of claim 17, wherein the plurality of particles in step (a) are matched with the at least one analyte with the at least one first ligand and capture the at least one analyte. The method for a nucleic acid amplification system according to claim 17, wherein the plurality of particles in step (e) are linked to the at least one biological substance or free nucleic acid substance with at least one second ligand. The method for a nucleic acid amplification system according to claim 17, wherein the method for amplifying the at least one biological substance by an enzyme in step (f) comprises polymerase chain reaction (PCR) or nucleic acid isothermal amplification reaction. The method for a nucleic acid amplification system according to claim 17, wherein in step (f), at least one temperature balancing substance rapidly cools the plurality of particles, thereby achieving rapid temperature cycling.

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