Nucleic acid amplification system and method thereof
A nucleic acid amplification system and a method thereof. An external energy source is used to drive nanoparticles with a regional thermal transfer to generate an energy response, so that the target nucleic acids on the nanoparticles are subjected to a polymerase chain reaction (PCR) or a nucleic acid isothermal amplification reaction, and the liquid in the reaction space is used for refrigeration to achieve rapid temperature regulation, such that the target nucleic acids accumulated on the surface of the nanoparticles are amplified. The concentration, capture and collection of the nanoparticles can be carried out by means of centrifugation, magnetic capture, lateral flow chromatography, etc. Subsequently, detection can be carried out by means of analyzing the fluorescence intensity and the color change of the layer after amplification.
1 . An in-situ nucleic acid amplification system, comprising:
a reaction space;
a sample placed in the reaction space and including at least one analyte, wherein the at least one analyte comprises an individual microbe or a cell;
a plurality of particles mixed with the sample in the reaction space, wherein the plurality of particles are magnetic nanoparticles, each of the plurality of particles including:
a body configured to have at least one property of photothermal conversion or thermomagnetic conversion, and configured to generate heat via external energy excitation;
at least one first ligand functionalized on the body and configured to match the at least one analyte, wherein the at least one first ligand comprises an antibody, an aptamer, an oligonucleotide, or a combination thereof; and
at least one second ligand functionalized on the body through a stabilizing bond, wherein the at least one second ligand is a single-strand nucleic acid sequence having a complementary sequence to a corresponding target gene of at least one biological substance released from the analyte or a cell-free nucleic acid, wherein the at least one second ligand is configured to bind the at least one biological substance or the cell-free nucleic acid on the body;
at least one enzyme mixed with the sample, wherein the at least one enzyme is a polymerase;
at least one tag mixed with the sample, wherein the at least one tag is a fluorescent or a nucleic acid tag;
at least one energy supply module configured to provide external energy to the plurality of particles, wherein the external energy comprises light energy or an alternating magnetic field;
at least one temperature equilibrium substance placed in the reaction space, wherein the temperature equilibrium substance is a cooling substance, and comprises a nucleic acid amplification solution or an ice pack externally in contact with the reaction space to cool down the nucleic acid amplification reaction on the plurality of particles; and
an operating module configured to control the plurality of particles in the reaction space, wherein the operating module is a magnetic component configured to concentrate and focus the analyte and the free nucleic acid substance and amplicons incorporated with the at least one tag on the plurality of particles;
wherein the at least one energy supply module is configured to excite the plurality of particles to generate a reaction temperature range for performing photothermal lysis, DNA hybridization capture and nucleic acid amplification, and the at least one temperature equilibrium substance is configured to maintain a constant temperature range for isothermal amplification or to achieve rapid temperature cycling for PCR by controlling the energy supply module with assistance of the at least one temperature equilibrium substance; and
wherein the at least one enzyme is configured to amplify, on the body, the at least one biological substance or the cell-free nucleic acid captured by the at least one second ligand, such that amplicons generated from the at least one biological substance or the cell-free nucleic acid are captured by the plurality of particles.
2 . The nucleic acid amplification system of claim 1 , wherein the at least one temperature equilibrium substance is a cooling substance.
3 . 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 cell-free nucleic acid or a combination thereof.
4 . The nucleic acid amplification system of claim 3 , wherein the at least one cell-free nucleic acid comprises cell-free nucleic acid derived from body fluid, tumor or a combination thereof.
5 . The nucleic acid amplification system of claim 1 , wherein each of the at least one first ligand and the at least one second ligand is functionalized on the body through a stabilizing structure, and the stabilizing structure comprises a thermostable bond, a contact inhibition coating or a combination thereof.
6 . The nucleic acid amplification system of claim 1 , wherein the at least one enzyme is a polymerase.
7 . The nucleic acid amplification system of claim 6 , wherein the polymerase further comprises DNA polymerase, RNA polymerase or a combination thereof.
8 . The nucleic acid amplification system of claim 6 , wherein the polymerase further comprises a reverse transcriptase (RT), a ribonuclease (RNase), a helicase, a DNA ligase or a combination thereof.
9 . The nucleic acid amplification system of claim 1 , wherein the energy supply module comprises a laser transmitter, a light-emitting diode or a magnetic field generator.
10 . The nucleic acid amplification system of claim 1 , wherein the operating module comprises a magnetic component, a centrifuge or a combination thereof.
11 . The nucleic acid amplification system of claim 10 , wherein the magnetic component serves as the operating module, and the plurality of particles comprises a plurality of magnetic particles.
12 . The nucleic acid amplification system of claim 1 , further comprising a detection module that detects the specific tags in each of the at least one second ligand of the plurality of particles concentrated by the operating module.
13 . A method for operating the nucleic acid amplification system of claim 1 , comprising the steps of:
(a) mixing the sample and the plurality of particles so that the at least one first ligand of the plurality of particles matches the at least one analyte and captures the at least one analyte, wherein the at least one first ligand comprises an antibody, an aptamer, an oligonucleotide or a combination thereof;
(b) using the operating module to remove an impurity and concentrate the sample in the reaction space, wherein the sample includes the at least one analyte, and wherein step (e) immediately follows the step (b) if the at least one analyte is a cell-free nucleic acid, otherwise step (c) is performed;
(c) in which the at least one energy supply module provides the external energy to the plurality of particles so that a temperature of the plurality of particles rises to a thermolysis temperature;
(d) lysing the at least one analyte, thereby releasing at least one biological substance;
(e) in which the at least one energy supply module provides the external energy to the plurality of particles up to a hybridization capture temperature, so that the at least one second ligand captures the at least one biological substance released from the at least one analyte; and
(f) providing the external energy to the plurality of particles up to an amplification temperature with the at least one energy supply module, and then using the enzyme to amplify the at least one biological substance on the plurality of particles, so that the at least one biological substance that is amplified is tethered on the plurality of particles.
14 . The method of claim 13 , after step (f), further comprising step (g), wherein the step (g) follows the step (f), and wherein a detection module is used in the step (g) to detect a colorimetric change, a luminescence or a combination thereof caused by the specific tags on the at least one biological substance or the cell-free nucleic acid that is amplified so as to detect the at least one biological substance or the cell-free nucleic acid, which is amplified, on the plurality of particles that is concentrated.
15 . The method of claim 13 , wherein the at least one enzyme is at least one polymerase, and the at least one polymerase is selected corresponding to the type of the at least one biological substance or the cell-free nucleic acid, and wherein the at least one polymerase comprises deoxyribonucleic acid polymerase (DNA polymerase) or ribonucleic acid polymerase (RNA polymerase).
16 . The method of claim 13 , wherein the operating module comprises a magnetic component, a centrifuge or a combination thereof.
17 . The method of claim 13 , wherein in the step (f), the method for amplifying the at least one biological substance or the cell-free nucleic acid comprises polymerase chain reaction (PCR) or an isothermal amplification reaction.
18 . The method of claim 13 , wherein in the step (f), the at least one temperature equilibrium substance rapidly cools the plurality of particles, thereby achieving rapid thermal cycling.