Patent Application
20240229119
The present invention relates to nucleic acid amplification method and its device, and nucleic acid detection method and its device, particularly to a method and a device for forming an in-situ environment around a solid-phase carrier and performing nucleic acid amplification within the in-situ environment, and a method and a device for performing nucleic acid detection after completion of in-situ nucleic acid amplification.
Nucleic acid amplification tests (NAAT) have been widely applied in molecular diagnosis, including the diagnosis of infectious pathogens, genetic tests, forensic science, agriculture, and clinical medicine. Even if the analyte only has trace amounts of the target substance, nucleic acid amplification tests exhibit high sensitivity and specificity due to the amplification of the target nucleic acid fragments. Polymerase chain reaction (PCR) is one of the methods used for target nucleic acid amplification testing. It includes three steps for generating new deoxyribonucleic acid (DNA) strands: (a) denaturation of double-strand DNA to single-strand DNA (ssDNA) acting as a template by raising the temperature; (b) annealing of specific primers to the ssDNA template through DNA complementary sequence by lowering the temperature to the range of melting temperature; (c) new DNA strands are synthesized through extension of the thermostable DNA polymerases. The abo-mentioned is the thermal cycling of PCR with 2 to 3 temperature ranges. As the number of thermal cycling increases, newly synthesized amplicons are generated exponentially. However, conventional PCR instruments are bulky, expensive, have high energy consumption (using electric heating modules), and have long turnaround times (usually >1 hour).
Additionally, isothermal nucleic acid amplification technologies (iNAATs) have been developed to replace conventional nucleic acid amplification technologies (NAATs), allowing nucleic acid amplification at a constant and relatively moderate temperature. As compared to conventional NAATs, iNAATs significantly reduce the complexity of temperature management controls (e.g. the setup of thermal cycling, and rapid heating and cooling of the thermal block), its related complex devices, and the overall operating time required for nucleic acid amplification. Due to the absence of complex temperature management controls, iNAATs can be performed by using simple instruments, (i.e. heating plate, oven, or water bath). Among various iNAAT methods/technologies, loop-mediated isothermal amplification (LAMP) is the most widely adopted method, this is mainly because LAMP has good tolerance to DNA polymerase inhibitors (e.g., hemoglobin, IgG, or IgM), moreover, LAMP has high specificity, high sensitivity, and high amplification efficiency. This means that LAMP has the potential to directly amplify target DNA or RNA from partially processed or unprocessed biological samples.
In addition, the literature titled “Loop-mediated isothermal amplification (LAMP)-review and classification of methods for sequence-specific detection” (published in Analytical Methods on Feb. 14, 2020, by Lisa Becherer, Nadine Borst, Mohammed Bakheit, Sieghard Frischmann, Roland Zengerle, and Felix von Stetten) has proposed that LAMP demonstrates increased sensitivity of 10 to 100 times as compared to conventional quantitative PCR (qPCR) or PCR.
Furthermore, the literature titled “Reduced False Positives and Improved Reporting of Loop-Mediated Isothermal Amplification using Quenched Fluorescent Primers” (published in Scientific Reports on May 14, 2019, by Patrick Hardinge and James A. H. Murray) has mentioned that since the use of 4 to 6 target-specific primers are used in LAMP reaction, the specificity of LAMP is expected to be superior to conventional PCR that uses only two primers. Additionally, LAMP can generate more than 109 DNA amplicons within an hour. The presence of target analytes can be determined by directly or indirectly detecting the abundantly amplified DNA amplicons or by-products of the LAMP. However, owing to its high false positive rate and low discrimination, the applications of LAMP-based point-of-care testing are further limited.
Conventional PCR platform mainly uses thermoelectric elements (e.g., Peltier elements) to perform the programmed heating and cooling processes to achieve the required thermal cycling for PCR. This method relies merely on heat conduction to transfer thermal energy between the thermoelectric element and the reaction solution, leading to its relatively slow heating and cooling rates (1.5 to 3° C./second), resulting in a prolonged turnaround time in overall PCR procedures. The literature titled “Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics” (published in Biosensors and Bioelectronics on Jun. 18, 2019, by Sang Hun Lee, Seung-Min Park, Brian N. Kim, Oh Seok Kwon, Won-Yep Rho, Bong-Hyun Jun) proposes that the key factors for achieving ultra-fast PCR, including rapid thermal cycling, low heat capacity materials, high thermal conductivity, and high-speed DNA polymerases. With the development of protein engineering technology on direct molecular evolution, the second-generation DNA polymerase with a high extension rate has been screened out (e.g., KAPA2G, KAPA Biosystems). Its extension rate can reach up to 1 kb/second, significantly reducing the time required for the PCR reaction. In the past, in order to achieve rapid thermal cycling, the PCR reaction volumes were limited to nanoliters to microliters (nL-μL) in capillaries or microfluidic chips, the increased surface area to volume ratios led to improve heat conducting effects between the thermal block and the PCR reactions [e.g., the LightCycler® 2.0 Carousel-Based System (Roche Diagnostics) or microfluidic polymerase chain reactions were employed]. Additionally, materials with low heat capacity are utilized for performing heating and cooling in thermal cycling, [e.g., air was utilized in the LightCycler® 2.0 Carousel-Based System (Roche Diagnostics)]. Although the overall nucleic acid amplification can be completed within several minutes, they are still accompanied by several technical issues, including low amplicon yield, evaporation and bubble generation in the small reaction volumes, platform scalability, and heat control management. Therefore, rapid nucleic acid amplification/detection is not only about the achievement of rapid thermal cycling, but a strategy for nucleic acid amplification that ensures it is not adversely affected while achieving rapid temperature cycling.
In response to the aforementioned issues, many research teams have conducted studies and made improvements. For example, Royal Institution for the Advancement of Learning McGill University has a U.S. patent with the publication number U.S. Pat. No. 10,604,798B2 (titled “HEATING MECHANISM FOR DNA AMPLIFICATION, EXTRACTION, OR STERILIZATION USING PHOTO-THERMAL NANOPARTICLES”). The abstract discloses that the photothermal nanoparticles are used to heat the PCR solution in both contact and non-contact manner under the illuminations of an external light source. Different photothermal converted temperature ranges are achieved by exciting the nanoparticles with external energy of varying intensities, enabling sterilization, and photothermal lysis for the nucleic acid extraction and performing amplification of specific nucleic acid fragments. For example, the Regents of the University of California has a U.S. patent with the publication number U.S. Pat. No. 11,130,993B2 (titled “LED DRIVEN PLASMONIC HEATING APPARATUS FOR NUCLEIC ACIDS AMPLIFICATION”). Its abstract discloses plasmonic heating generated in a 3D miniature reaction chamber with a specific thickness of the gold film by a light-emitting diode (LED) at a specific wavelength, thereby rapid thermal cycling is achieved for PCR under manipulations of LED illuminations. For example, the University of Chicago has a U.S. patent with the publication number U.S. Pat. No. 11,045,874B2 (titled “BIPYRAMID-TEMPLATED SYNTHESIS OF MONODISPERSE NOBLE METAL NANOCRYSTALS”). By using synthesized gold nanoparticles with a bipyramid structure, plasmonic heating is generated under the LED illuminations with a specific wavelength, enabling the rapid thermal cycling required for PCR in the miniature reaction chamber. The above-mentioned methods use nanoparticles with noble metal materials and specific configurations to convert light energy to thermal energy (reaction time >100 picoseconds) through localized surface plasmon resonance (LSPR) effect under the excitation of a specific light source. The photothermal conversion property can be regarded as a fast and immediate response. This concept of plasmonic nanoscale heaters exhibits rapid heating rates and generates a uniform thermal field during PCR thermal cycling. By adjusting the concentration of these nanoparticles, and the intensity of the incident light, the manipulation of photothermal converted temperature can be easily controlled. However, there are still some challenges and issues to be overcome in the above-mentioned methods, which as listed as follows: (1) the liquid evaporation in the miniature reaction chamber, (2) the cooling effect of plasmonic nanoparticles is not obvious during thermal cycling, requiring the use of external devices (e.g., cooling fans) to achieve cooling, (3) the possibility of overlapping wavelength ranges between the incident light source for localized surface plasmon resonance of plasmonic nanoparticles and the spectra of certain organic fluorescent dyes is existed, which may restrict the application in fluorescence-based quantitative PCR.
In addition to the aforementioned photothermal heating method using plasmonic nanoparticles, another method that involves applying an alternating magnetic field to induce heat in magnetic nanoparticles has also been employed in PCR nucleic acid amplification techniques. For example, the team led by Dr. Shich, Dar-Bin at National Cheng Kung University has a U.S. patent with the publication number U.S. Pat. No. 10,913,069B2 (titled “METHOD AND DEVICE FOR POLYMERASE CHAIN REACTION”). Its claims refer to the method for nucleic acid amplification comprising a reaction mixture containing the target nucleic acid and particles containing transitional metal material in a reaction unit under electromagnetic radiations (EMR) with a frequency ranging from approximately 200 kHz to 500 THz. The inducing heating generated by the particles of the transitional metal materials is used for PCR amplification. A similar commercial device known as the Mic qPCR, produced by Bio Molecular Systems (BMS), is the world's first qPCR machine using electromagnetic induction heating as the heat source.
The heating methods described above, such as the use of photothermal nanoparticles or magnetic nanoparticles, or the conventional PCR with electric heating elements to heat the PCR solution, all belong to the category of “volumetric heating” (i.e., increasing the temperature of the entire reaction mixture through heating).
In contrast to this, GNA Biosolutions (GmbH) has developed a unique “Pulse Controlled Amplification (PCA)” to achieve ultra-fast PCR. According to the literature titled “Ultra-fast PCR technologies for point-of-care testing” (published in Journal of Laboratory Medicine on Oct. 12, 2017, by Lars Ullerich, Stephanie Campbell, Frank Krieg-Schneider, Federico Bursgens and Joachim Stehr) and the U.S. patent with publication number U.S. Pat. No. 9,382,583B2 (titled “Method for the amplification of nucleic acids using heat transfer for nanoparticles;” related patent applications: DE102012201475B4, CN107604052A, EP2809806B1, WO2013113910A1), it is disclosed that this pulse-controlled nucleic acid amplification technology is a technique for controlling “localized heating” in a specific region. This technology integrates primer-functionalized nanoparticles with photothermal conversion properties, a laser light source, and a large volume of reaction solution for cooling purposes. By using short-period laser pulses (with time intervals between laser pulses ranging from 10 nanoseconds to 500 milliseconds) to selectively irradiate certain gold nanoparticles, a specific thermal radiation field required for PCR is generated, and the thermal radiation field generated and dissipated rapidly by this method. Furthermore, this approach effectively only heated the microenvironment of the surface region of the gold nanoparticles upon laser irradiations. Once the laser irradiation on the nanoparticles is paused, due to their high surface area-to-volume ratio of plasmonic nanoparticles, the photothermal-induced thermal field around the nanoparticles will dissipate rapidly and cool down to the temperature of the surrounding nucleic acid amplification solution. During the PCA process (i.e., consecutive and repeated thermal cycling), the temperature of the nucleic acid amplification solution surrounding the nanoparticles remains nearly unchanged. As described above, the PCA technique enables PCR to achieve rapid thermal cycling around the microenvironment of nanoparticles. This technique can not only significantly improve the issue of reaction solution evaporation that may arise from the aforementioned “volumetric heating” (which can affect the accuracy of the subsequent detection work), but also reduces the requirement for specialized and precise cooling devices (e.g., using cooling fans and temperature feedback control system) in conventional nucleic acid amplification processes (e.g., PCR). Nevertheless, there are still drawbacks to consider, which will be further discussed in the following paragraphs.
Moreover, regarding the aforementioned phenomenon of achieving “localized heating” through the use of “pulse-controlled amplification,” this technique can not only use “photothermal particles” to achieve the “localized heating,” but also employ other principles or mechanisms to generate the phenomenon of “localized heating.” The GNA Biosolutions has a European patent with publication number EP3733292A1 (titled “METHOD FOR CARRYING OUT A POLYMERASE CHAIN REACTION AND DEVICE FOR CARRYING OUT THE METHOD;” related applications: US2019/0249168A1) and the literature titled “Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions” (published in PLOS NEGLECTED TROPICAL DISEASES on Jan. 29, 2021, by Katharina Müller, Sarah Daßen, Scott Holowachuk, Katrin Zwirglmaier, Joachim Stehr, Federico Buersgens, Lars Ullerich, Kilian Stoecker). It discloses that PCA amplification was achieved by using a rapid energy pulse to heat microcyclers (micro metal heating element directly embedded in nucleic acid amplification reaction vessel). By applying a microsecond-period electric heating, an instantaneously generated thermal radiation field occurs only in the surface area of the micrometal heating element (designed nucleic acid amplification reaction region). Once the electric pulse is ceased, the heat of the metal heating element will dissipate rapidly and cool down by the surrounding large volume of the reaction solution. Through repeated control and generation of the aforementioned temperature field, this design enables the rapid generation of temperature cycles required for nucleic acid amplification reactions (e.g., PCR), thereby achieving the objective of fast nucleic acid amplification.
Based on the aforementioned prior patents and literature, GNA Biosolutions has developed a unique “Pulse-Controlled Amplification (PCA)” that achieves ultra-fast nucleic acid amplification (e.g., PCR), and subsequent detection of nucleic acids. This technique can significantly improve the issues of reaction solution evaporation caused by “volumetric heating” in conventional PCR and reduces the requirement for specialized cooling designs or devices in conventional nucleic acid amplification processes.
However, the PCA technique mainly controls the relative positions of the laser illumination beams within the reaction region of PCR dynamically, allowing high-power NIR laser beams to trigger plasmonic nanoparticles to generate a photothermal effect within the reaction region of PCR with laser illuminations in very short time intervals ranging from 10 nanoseconds to 500 milliseconds. By irradiating a portion of the plasmonic nanoparticles within the reaction region of PCR selectively and specifically, the required temperature fields for performing nucleic acid amplification are generated as mentioned above. Basically, the performing of the PCA technique has certain technical difficulties. In addition, the volume of PCR solution used for cooling purposes in the PCA technology is relatively large (e.g., 100-500 μL). This is because in the PCA technology, the nucleic acid products (e.g., amplicons) are generated in the form of single-strand DNA (ssDNA) within the PCR solution. The detection method of the amplicons, as described in the European patent with publication number EP2481817A1 (titled “Process for detecting nucleic acids”), the European patent with publication number EP3733292A1, and by Müller et al. (2020), is achieved by detecting the polymer (e.g., amplified free single-strand DNA) generated by (primer-functionalized) plasmonic nanoparticles and, based on the spectral difference at a wavelength of 650 nm or the fluorescent signal generated by TaqMan probes. In the aforementioned nucleic acid detection methods, due to the dilution effect [e.g., a large volume of PCR solution (100-500 μL)], the PCA technology will lead to the need for performing more thermal cycles of nucleic acid amplification to accumulate the signal of amplicons. Those drawbacks further affect the total turnaround time required for nucleic acid amplification and detection.
Additionally, according to the prior patents of the GNA Biosolutions, there are still many aspects of PCA nucleic acid amplification methods and devices requiring improvement, summarized as follows:
1. Localized heating: By integrating the monodispersed photothermal nanoparticles within a large volume of PCR solution, PCA technology enables the achievement of “ultra-fast nucleic acid amplification” through “ultra-fast temperature (thermal) cycling,” and during the overall process of ultra-fast nucleic acid amplification, there is no significant change in the temperature of the PCR solution. This technical characteristic can improve the issue of evaporation of the trace volume of PCR solution during the temperature (thermal) cycling of the conventional fast nucleic acid amplification techniques (e.g., the accuracy of detection). However, this technique mainly relies on dynamic control of the relative positions of the laser beam and the illumination regions within the PCR reaction. By allowing high-power NIR laser beams to trigger plasmonic nanoparticles to generate a photothermal effect within the reaction region of PCR with laser illuminations in very short time intervals ranging from 10 nanoseconds to 500 milliseconds. By irradiating a portion of the plasmonic nanoparticles within the reaction region of PCR selectively and specifically, the required temperature fields for performing nucleic acid amplification are generated as mentioned above. Basically, this operation has certain technical difficulties.
2. The prior patents of GNA Biosolutions mention that the PCR used in the PCA nucleic acid amplification reactions, which employs a two-step temperature (thermal) cycling, including a denaturation temperature of 95° C. required for double-strand separations of DNA, and an annealing and extension temperature of 65° C. required for oligo primer/probe annealing and DNA polymerase extension, respectively. Although the PCA technique utilizes a large volume of PCR solution as a cooling medium to cool the localized heating of plasmonic nanoparticles upon laser off (e.g., from 95° C. to 65° C.), because the temperature difference is merely 30° C., the cooling rate may not be as fast as claimed for the “instant cooling” effect by the PCA technology.
3. The PCA technique utilizes a large volume of PCR solution as a cooling medium to cool down the localized heating of plasmonic nanoparticles upon laser off. However, the amplicons of PCA generated in the form of free single-strand DNA (ssDNA) within the PCR solution, due to the dilution effect of a large volume of PCR solution (e.g., 100-500 μL), which will lead to the requirement for more thermal cycles of nucleic acid amplification to accumulate the signal of amplicons. Those drawbacks further affect the total turnaround time required for nucleic acid amplification and detection
4. When encountering complex nucleic acid analytes in a biological sample: the conventional PCR employs a primer pair design to perform nucleic acid amplification. When encountering complex nucleic acid analytes, including partially similar or identical sequences complementary with oligo primer sets, these sequences can undergo partial hybridization during the annealing process, resulting in the generation of non-specific chimeric by-products. This will contribute to imprecise discrimination with point-of-care testing devices.
Therefore, how to implement simple and fast nucleic acid amplification and detection while avoiding the aforementioned technical issues remains a critical unresolved challenge.
In view of the issues with prior art, one of the objectives of the present invention is to provide a nucleic acid amplification method and its device, enabling easy and rapid temperature (thermal) cycling required for nucleic acid amplification, and the nucleic acid amplification involves amplification of nucleic acid molecules partially immobilized on the surface of a solid-phase carrier. Another objective of the present invention is to achieve fast purification, separation, and enrichment of nucleic acids on the surface of the solid-phase carrier, thereby facilitating the subsequent detection and analysis of target nucleic acid molecules with high specificity and sensitivity.
According to one objective of the present invention, a nucleic acid amplification method is provided, comprising: a reaction unit including at least one analyte, a nucleic acid amplification solution, at least one solid-phase carrier within its interior, wherein the reaction unit, and the content within its interior, including the analyte, the nucleic acid amplification solution and the solid-phase carriers are maintained at a cooling temperature in a cooling environment; by modulating the output of an external energy and the timing sequence of on and off of the external energy. Meanwhile, by coordination with the cooling temperature of the cooling environment, thereby generating one or more temperature (thermal) cycles required for nucleic acid amplification happened around solid-phase carriers (i.e., in-situ environment), wherein in each of the temperature (thermal) cycles, all the solid-phase carriers simultaneously form its corresponding in-situ environment triggered upon irradiations of the external energy, when the external energy to the solid-phase carriers is paused, the in-situ environment around solid-phase carriers will be dissipated. By the formation and dissipation of the in-situ environment on solid-phase carriers, which are allowed to perform the nucleic acid amplification under the presence of target analytes, thereby generating amplicons.
Among these, the nucleic acid amplification reactions comprise polymerase chain reaction (PCR), ligase chain reaction (LCR), or isothermal nucleic acid amplification technologies (iNAAT).
Among these, during each turn-on period of external energy irradiation of the thermal cycle, all the solid-phase carriers within the reaction unit are simultaneously triggered, resulting in the formation of the in-situ environment around each of the solid-phase carriers, and further generating the amplicons immobilized on each of the carriers. During each turn-on period of high-power external energy irradiation, the immobilized amplicons will be denatured, and a portion of the amplicons is retained on each of the solid-phase carriers, while another portion of the amplicons is released into the nucleic acid amplification solution as templates; and during each turn-off period of external energy irradiation of the thermal cycle, the excitation of each of the solid-phase carriers is paused, and each of the in-situ environments dissipates through the cooling temperature of the cooling environment.
Among these, the enzyme can be a polymerase, wherein the polymerase comprises DNA polymerase, RNA polymerase, or the enzyme can also be one or a combination of reverse transcriptase (RT), ribonuclease (RNase), helicases, DNA ligase, and can act coordinately with the polymerase.
Among these, the transfer methods of external energy irradiation to each of the solid-phase carriers include contact or non-contact excitation mode.
Among these, the non-contact excitation mode includes photothermal excitation or magnetic excitation. The light source utilized for photothermal excitation is one of either laser or LED arrays, with wavelengths ranging from the visible to near-infrared spectrum, and the wavelength range from 380 nanometers (nm) to 1.4 micrometers (μm).
Among these, the magnetic excitation system uses alternating magnetic fields (AMF) to generate heat (i.e., AMF-induced hyperthermia). 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 temperature conditions required for nucleic acid amplification on the solid-phase carriers. Furthermore, the amplitude of the alternating magnetic field ranges from 0.5 to 550 kA/m, and the frequency of the alternating magnetic field ranges from 3 to 3,500 KHz.
Among these, the contact excitation mode is electric heating, in which electrical energy can be transmitted through electronic circuits or induced magnetic flux for electric energy transmission (wireless charging). The electric heating modes can be Joule heating, thermoelectric heating, or surface acoustic waves (SAWs).
Among these, the ratio of the total volume of the solid-phase carriers to the volume of the nucleic acid amplification solution is from 1:200 to 1:1×109.
Among these, the size of each of the solid-phase carriers ranges from 8 to 2,000,000 nm, among which the preferred size of the solid-phase carrier for photothermal excitation and magnetic field excitation is 8 to 1,000 nanometers; while the preferred size of the solid-phase carrier for electric heating is 1,000 to 2,000,000 nanometers.
Among these, the shape of each of the solid-phase carriers can be in the form of spheres, ellipsoids, disc-shaped, star-shaped, rod-shaped, squares, anisotropic structures, nanoshells, nanocages, bipyramidal structures, microfilament or a combination of two or more of these.
Among these, each of the solid-phase carriers can be either suspended within the reaction unit, or each of the solid-phase carriers can be tethered to the inner wall of the reaction unit, or a combination of both.
Among these, the cooling temperature ranges from −10 to 50° C.
Among these, the reaction unit or the nucleic acid amplification solution can be pre-cooled to the cooling temperature or placed in an external auxiliary cooling unit, wherein the pre-cooled reaction unit or the external auxiliary cooling unit maintains the cooling temperature during one or more thermal cycles.
Among these, the external auxiliary cooling unit comprises ice, a water-swelling macromolecular polymer, chemical endothermic reactants, thermoelectric cooling chips, or a combination of any two of these. The water-swelling macromolecular polymer is carboxymethyl cellulose, among which the chemical endothermic reactants are ammonium nitrate or urea dissolved in water, involving endothermic reactions.
Among these, the analytes to be tested can be a cell, an organelle, a bacterium, a virus, a protozoan, or a combination thereof. The solid-phase carrier comprises a multifunctional body, at least one enrichment ligand, and at least one amplification ligand, among which each of the enrichment ligands is immobilized on the surface of the multifunctional body used for specific capture of the analyte; wherein the amplification ligands are also immobilized to the surface of the multifunctional body and are used for binding a biological substance. Among these, the biological substances are deoxyribonucleic acid or ribonucleic acid released from the analyte, or the amplicons replicated on the amplification ligand and subsequently released into the nucleic acid amplification solution. Among these, the enrichment ligand can be an antibody, nucleic acid aptamer, oligonucleotide, protein, polysaccharide, or a combination thereof. Wherein the amplification ligand can be a nucleic acid aptamer or oligonucleotide, and has a functional group modification for immobilization to the solid-phase carrier, which can be modified with a primary amine, biotin, thiol groups, or a combination thereof. In order to enhance the specificity and stability of oligonucleotide for hybridization capture to biological substances, the modifications can be a locked nucleic acid (LNA), phosphorothioates, morpholino modifications, or a combination thereof.
Among these, the analyte to be tested can be a cell, an organelle, a bacterium, a virus, a protozoan, or a combination thereof. Part of the solid-phase carriers includes an enrichment body and at least one enrichment ligand, each of the enrichment ligands is immobilized to the surface of the enrichment body and is used for specific capture of the analyte; another part of solid-phase carriers includes an amplification body and at least one amplification ligand, each of the amplification ligands are immobilized to the surface of the amplification body and is used for hybridization binding a biological substance released by the analyte. The biological substance is deoxyribonucleic acid or ribonucleic acid released from the analyte, or the amplicons replicated on the amplification ligand and subsequently released into the nucleic acid amplification solution. Among these, the enrichment body corresponded to the multifunctional body functionalized with only the enrichment ligand. Among these, the amplification moiety corresponds to a multifunctional moiety with only the amplification ligand.
Among these, the analyte is a free deoxyribonucleic acid or a free ribonucleic acid, among which each of the solid-phase carriers includes an amplification body and at least one amplification ligand, each of the amplification ligands is immobilized to the surface of the amplification body and are used for hybridization binding of the analyte.
According to one objective of the present invention, a rapid nucleic acid amplification system is provided, comprising: a reaction unit, an auxiliary cooling unit, an external energy excitation unit, an integrated driver; among which, the reaction unit is provided to accommodate a reaction solution, an analyte, a nucleic acid amplification solution, and one or more solid-phase carriers, wherein the auxiliary cooling unit can be a pre-cooled nucleic acid amplification solution at a cooling temperature, or can be an external auxiliary cooling unit arranged around the reaction unit, continuously cooling the reaction unit and maintaining the nucleic acid amplification solution at a cooling temperature; the external energy excitation unit is configured to provide contact and non-contact energy transfer to the solid-phase carriers to generate heat; the integrated driver is configured to control the energy output and timing sequence of on and off of external energy excitation unit, among which the integrated driver is also configured to modulate energy output of the external excitation unit and feedback control of the external auxiliary cooling unit by an external energy calibrator and a temperature detection unit, thereby generating one or more temperature (thermal) cycles required for nucleic acid amplification.
According to another aim of the present invention, a nucleic acid detection method is provided, comprising the following steps for nucleic detection after completing the nucleic acid amplification: an operation unit is used to assist in the purification, separation, and concentration of the amplicons immobilized on the solid-phase carriers; a detection module to detect one or a combination of optical changes, thermal sensing changes, electrochemical changes, magnetic changes, or mass changes (e.g., piezoelectric) occurring on the amplicons bound to the solid-phase carriers. Furthermore, the detection module detects amplification signals by the amplicons bound to the solid-phase carriers.
Among these, the methods for detecting the optical changes on the amplicons immobilized on solid-phase carriers, comprised of detecting the change of light intensity generated directly by the primer labeled with the nucleic acid tags or fluorophore incorporated into amplicons with a spectrophotometer or a fluorometer, or detecting the optical changes or chemiluminescence changes generated by an enzyme-linked immunosorbent assay, or detecting spectral changes resulting from the repulsion on the amplicons immobilized on the solid-phase carriers.
Among these, the method for detecting optical changes on the amplicons immobilized on the solid phase carriers, wherein the detection module comprises nucleic acid lateral flow strips or lateral flow immunoassay strips, which are used to detect the optical changes.
Among these, the sensitivity of the nucleic acid lateral flow strips or the lateral flow immunoassay strips is enhanced by combining the nucleic acid lateral flow strips or the lateral flow immunoassay strips with a thermal sensing detection, surface plasmon resonance spectroscopy, or any combination thereof.
Among these, a method for electrochemical changes occurring on the amplicons immobilized on solid-phase carriers, comprising one or a combination of both electrochemical detections coupled with enzyme-linked immunosorbent assay and electrochemical impedance spectroscopy.
Among these, a method for magnetic changes occurring on the amplicons immobilized on solid-phase carriers, including the detections of frequency-dependent alternating current (AC) susceptibility with the AC susceptometry, and the giant magnetoresistive measurement (GMR), or a combination of thereof.
Among these, a method for mass changes occurring on the amplicons immobilized on solid-phase carriers that is performed using a quartz crystal microbalance (QCM).
According to another aim of the present invention, a rapid nucleic acid detection device is provided, comprising an operation unit and a detection module, wherein the operation unit is configured to assist in purification, separation, and concentration of the solid-phase carriers; the detection module is configured to detect one or a combination of optical changes, thermal sensing changes, electrochemical changes, magnetic changes, or mass changes occurring on the amplicons immobilized to the solid-phase carriers.
In summary, the present invention is different from conventional nucleic acid amplification methods and possesses the following advantages:
(1) By simply controlling the characteristic of the external energy excitation (i.e., the magnitude and frequency of external energy) and the cooling effect in the reaction unit, it is possible to instantly generate the temperature (thermal) cycling required for nucleic acid amplification within the in-situ environment around the surface of a solid-phase carrier, thereby achieving the purpose of fast nucleic acid amplification.
(2) The nucleic acid amplification solution is pre-cooled to the cooling temperature, or using an auxiliary cooling unit to keep the reaction unit in a cooling environment, it is possible to inhibit non-specific primer annealing/extension and non-target nucleic acid amplification, thereby improving the specificity of amplification for the target nucleic acid.
(3) Due to the limited capacity of the amplification ligands on the surface of the solid-phase carrier, the immobilized amplified nucleic acid molecules on the solid-phase carrier surface can reach a saturation state within a relatively short operation time (or a relatively small number of thermal cycles). This will facilitate the subsequent detection of amplified nucleic acid molecules.
(4) If the solid-phase carrier has strong magnetism, the amplicons generated on the solid-phase carrier can be further purified and concentrated through magnetic manipulation by the operation unit. This technological characteristic will facilitate the subsequent detection of the amplicons immobilized on the solid-phase carriers (e.g., enhancing detection performance).
(5) Because the overall nucleic acid amplification solution is maintained at a low temperature, it not only avoids the issues of evaporation and bubble generation in the reaction solution during thermal cycling but also has the advantage of inhibiting or reducing non-specific nucleic acid amplification.
To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following detailed description of the present invention is provided in conjunction with the accompanying drawings and embodiments. It should be noted that the specific embodiments described herein are provided for the purpose of illustrating the present invention and should not be considered as limiting the scope of the present invention.
The present invention is a nucleic acid amplification method, comprised of providing an analyte 20, a nucleic acid amplification solution 30, and at least one solid-phase carrier 40 inside the reaction unit 10. The reaction unit 10 is placed in a cooling environment that achieves the required cooling temperature. The cooling temperature ranges from −10 to 50° C. The preferred cooling temperatures range from 15 to 30° C. and 30 to 50° C., with the optimal cooling temperatures ranging from −10 to 0° C., and from 2 to 15° C. By controlling operating conditions of external energy excitation (e.g., to modulate energy output and frequency), and using the cooling effect achieved by the cooling environment of the reaction unit 10, an in-situ environment 50 is formed around the surface of the solid-phase carrier 40 within the reaction unit 10, and a temperature (thermal) cycling required for nucleic acid amplification is performed within the in-situ environment 50, enabling the generation of amplicons 60 on the solid-phase carrier 40 during each temperature (thermal) cycling within the reaction unit 10. It should be noted that the temperature (thermal) cycling required for nucleic acid amplification within the reaction unit 10 refers to different temperatures needed for nucleic acid denaturation, primer annealing, and DNA polymerase extension, but the actual implementation is not limited thereto. The size of each of the solid-phase carriers ranges from 8 to 2,000,000 nm, wherein the preferred size of the solid-phase carrier for photothermal excitation and magnetic field excitation is 8 to 1,000 nanometers; while the preferred size of the solid-phase carrier for electric heating is 1,000 to 2,000,000 nanometers.
The term “in-situ environment 50” as described in the present invention refers to the configurations shown in
Moreover, the term “in-situ nucleic acid amplification” as described in the present invention refers to that the nucleic acid amplification occurs in the in-situ environment 50 on the surface of the solid-phase carriers 40, and the amplicons 60 are immobilized on the surface of the solid-phase carriers 40. The amplicons 60 includes but is not limited to amplified nucleic acid molecules. A portion of the amplicons 60 generated through the in-situ nucleic acid amplification is retained on each of the solid-phase carriers 40, while another portion of the amplicons 60 is released into the nucleic acid amplification solution 30.
In the present invention, the nucleic acid amplification solution 30 includes primers 301, nucleotides, enzymes, and reaction additives. The enzyme can be a polymerase 302, wherein the polymerase 302 includes DNA polymerase, RNA polymerase, or the enzyme can also be one or a combination of reverse transcriptase (RT), ribonuclease (RNase), helicases, DNA ligase, and can act coordinately with the polymerase.
In the present invention, nucleic acid amplification can include polymerase chain reaction (PCR), ligase chain reaction (LCR), or isothermal nucleic acid amplification technologies (iNAATs). Among the various iNAATs methods, loop-mediated isothermal amplification (LAMP) is preferred. In the polymerase chain reaction, a two-step temperature (thermal) cycling program is used, with denaturation temperatures ranging from 85 to 95° C., and annealing and polymerase extension temperatures ranging from 60 to 65° C.; the temperature for loop-mediated isothermal amplification ranges from 60 to 65° C.
In one embodiment of the present invention, as shown in
In this embodiment, the selected enrichment ligand 70 is capable of capturing specifically the aforementioned analyte 20. The analyte 20 to be tested can be a cell, an organelle, a bacterium, a virus, a protozoan, or a combination thereof. The enrichment ligand 70 specifically binds to the analyte 20, allowing the analyte 20 to be captured onto the enrichment ligand 70 of the solid-phase carrier 40.
In this embodiment, at least one amplification ligand 71 is functionalized on the multifunctional body 41, which is used to bind to the oligonucleotide primers or probes utilized for nucleic acid amplification. Furthermore, the amplification ligand 71 can be designed for hybridization capture to a biological substance 21, which can be the nucleic acid molecules released from the analyte 20 after thermal lysis or the amplicons 60 replicated on the amplification ligand 71 and subsequently released into the nucleic acid amplification solution 30. Additionally, the labeled primer 301 can incorporated into the amplicons in the nucleic acid amplification solution 30. The tag labeling of the labeled primers 301 can be a nucleic acid tag, including but not limited to radioactive isotopes (such as 3H, 14C, and 32P), digoxin (DIG), biotin, fluorophore [e.g., fluorescein isothiocyanate (FITC), Texas Red, Cy2, Cy3, Cy5, Cy7, rhodamine B], and luminescent substances (e.g., 2′,6′-DiMethylcarbonylphenyl-10-sulfopropylacridinium-9-carboxylate 4′-NHS ester), but the actual implementation of the present invention is not limited thereto.
In this embodiment, the surface of the solid-phase carrier 40 is subjected to one or more surface modification procedures, resulting in the formation of dual-functionalization of enrichment ligands 70 and amplification ligands 71 simultaneously. These ligands are used for the specific analysis of trace amounts of the analyte 20.
In another embodiment of the present invention, the analyte 20 to be tested can be a cell, an organelle, a bacterium, a virus, a protozoan, or a combination thereof. A portion of the solid-phase carriers 40 are enrichment bodies with at least one enrichment ligand 70, while another portion of the solid-phase carriers 40 are amplification bodies 42 with at least one amplification ligand 71. The enrichment ligand 70 is immobilized to the enrichment body, and the enrichment ligand 70 is used to specifically capture the analyte 20. The amplification ligand 71 is immobilized to the amplification body 42, and the amplification ligand 71 is used to bind to the biological substance 21 released by the analyte 20, or the amplicons 60 replicated on the amplification ligand 71 and subsequently released into the nucleic acid amplification solution 30.
In another embodiment of the present invention, the analyte 20 can be free deoxyribonucleic acid or a free ribonucleic acid. Moreover, free nucleic acid includes cell-free nucleic acid, free tumor nucleic acid, or a combination thereof. Each solid-phase carrier 40 includes the amplification body 42 and at least one amplification ligand 71, amplification ligand 71 is immobilized to the amplification body 42, and the amplification ligand 71 is used to bind to analyte 20, or the amplicons 60 replicated on the amplification ligand 71 and subsequently released into the nucleic acid amplification solution 30.
In each embodiment of the present invention, to allow the solid-phase carriers 40 to collaborate with different external energy excitation modes, various external excitation methods for the corresponding solid-phase carriers 40 are employed, including instant heating through photothermal conversion or magnetic hyperthermia under irradiations with the specific external energy. The solid-phase carrier 40 composed of specific material deposition or structure can be heated by using plasmonic heating under the light irradiations with a specific spectrum, or the solid-phase carrier 40 decorated with an organic photothermal conversion coating on the surface can be heated; alternatively, the solid-phase carrier 40 can be a magnetic carrier, enabling to produce the magnetically induced heating by magnetic solid carrier-mediated conversion of the AMF (alternating magnetic field). By placing the magnetic solid-phase carrier 40 in an externally applied magnetic field with an amplitude ranging from 0.5 to 550 kA/m and a frequency ranging from 3 to 3,500 kHz, the required localized temperature range on solid-phase carriers 40 for performing the nucleic acid denaturation, primer annealing, and polymerase extension can be achieved.
Furthermore, the multifunctional body 41, enrichment body, and amplification body 42 in the solid-phase carrier 40 can be of different configurations. The solid-phase carrier 40 can be implemented in the form of a spherical structure that includes a magnetic core 401 and an intermediate layer 402, which ensures the stability and maintenance of the outer photothermal conversion layer and can be made from metal or composite materials. The intermediate layer 402 can be a silicon shell, and its overall configuration can be spheres, ellipsoids, discs, stars, rods, squares, anisotropic spike structures (i.e., nanostar), nanoshells, nanocages, bipyramidal structures, microfilaments or a combination of two or more of these, but is not limited thereto; the magnetic core 401 includes transitional metals and their oxides, such as FeO, Fe2O3, Fe3O4, FeO(OH), Fe(OH)2, Fc(OH)3, COO, CoO(OH), Co3O4 and their derivatives or their mixture, but the present invention is not limited thereto. The surface of the solid-phase carrier 40 is composed of a noble metal layer localized surface plasmon resonance (LSPR) effects, such as gold, silver, palladium, platinum, or a combination thereof, or an organic layer with efficient photothermal conversion, which may also incorporate materials that exhibit near-infrared spectroscopy (NIR) absorption spectra, such as cyanine, polypyrrole, or graphene.
Please refer to
1. The nucleic acid amplification solution 30 (with a volume of at least 100 μL) serves as the auxiliary cooling unit. The nucleic acid amplification solution 30 is pre-cooled to the cooling temperature by using external auxiliary cooling unit 13 followed by being introduced into reaction unit 10, thus the nucleic acid amplification solution 30 in the reaction unit 10 can serve as the auxiliary cooling unit. In this formation, after the external energy is provided, the temperature of the nucleic acid amplification solution 30 will gradually increase but still remain at the cooling temperature during the reaction. Therefore, the efficiency of the nucleic acid amplification is worse than that of the auxiliary cooling unit described below, but the overall operation time is still shorter than the nucleic acid amplification of the conventional polymerase chain reaction.
2. The nucleic acid amplification solution 30 and the external auxiliary cooling unit 13 collaborate with each other to form an auxiliary cooling unit, the external auxiliary cooling unit 13 is arranged around the reaction unit 10 (as shown in
Moreover, the external auxiliary cooling unit 13 can be selected to use ice or the water-swelling macromolecular polymer with high specific heat capacity (e.g., carboxymethyl cellulose) or thermoelectric cooling chips (e.g., Peltier Cooler). The external auxiliary cooling unit 13 can also be water (i.e., 0.5 mL to 1 mL) or can be selected to contain the chemical endothermic reactants, the chemical endothermic reaction caused by its crystal dissolving in water, [e.g., ammonium nitrate molar heat of solution (ΔHsol)=26.2 KJ, urea (ΔHsol=15 KJ].
Please refer to
In the present invention, the contact excitation mode is electric heating. The electric energy transmission can be achieved through electric circuits or electromagnetic induction (wireless charging). The electric heating module can be Joule heating, thermoelectric heating, and surface acoustic waves (SAWs) to generate heat energy.
In the present invention, the preferred mode for the non-contact excitation to the solid-phase carrier 40 is plasmonic heating. As shown in
Furthermore, the energy excitation unit 12 can trigger the solid-phase carrier 40 in a non-contact manner and it can be selected as photothermal irradiation mode. As shown in
In the present invention, the rapid nucleic acid amplification system further includes a temperature detection unit 14 (e.g., K-type thermocouple) to detect the temperature feedback of the external auxiliary cooling unit 13; under the collaborated control of the nucleic acid amplification reaction in the auxiliary cooling unit and the heating provided by the energy excitation unit 12 to the solid-phase carrier 40, the required localized temperature (thermal) cycling for performing PCR is rapidly achieved, enabling the rapid completion of nucleic acid amplification.
In the present invention, the rapid nucleic acid amplification system further includes an operation unit 15, which can be a permanent magnet, an electromagnet, or a combination thereof, but the present invention is not limited thereto; the operation unit 15 allows the solid-phase carrier 40 in the reaction unit 10 for performing the procedures of purification, separation, and concentration/enrichment via a simple magnetic manipulation, which will facilitate the subsequent detection and analysis of the amplicons 60 immobilized on the solid-phase carrier 40 (i.e., enhancing the signal readouts of the amplicons 60 in detection), the present invention is not limited thereto. As shown in
It should be specifically noted that this invention can simplify temperature control devices and achieve localized temperature control with only a single energy excitation unit 12 and an external energy calibrator 16. The external energy calibrator 16 can be a light intensity meter calibrating the energy output of the energy excitation unit 12. By controlling the energy output and excitation timing sequence of the energy excitation unit 12 to achieve localized temperature (thermal) cycling control around the solid-phase carrier 40, forming an in-situ environment 50 around all the solid-phase carriers 40. This invention eliminates the need of the two-dimensional laser scanning with a two-dimensional mirror scanner, as disclosed in GNA's patent with the publication number U.S. Pat. No. 9,382,583B2 (titled “Method for the amplification of nucleic acids using heat transfer for nanoparticles”), in which laser beams are selectively and specifically focused on a portion of nanoparticles in a PCR sample for 10 nanoseconds to 500 milliseconds.
In one embodiment of the present invention, the analyte 20 is a free deoxyribonucleic acid, ribonucleic acid, or a biological substance 21 released from the analyte 20, and the solid-phase carrier 40 acts as an amplification body 42. Please refer to
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In one embodiment of the present invention, regarding the “photothermal” excitation of the solid-phase carrier 40, the surface of the solid-phase carrier 40 is preferably coated with a gold nanoshell 403. The gold nanoshell 403 includes a magnetic core 401 and an intermediate layer 402 between the gold nanoshell 403 and the magnetic core 401, collectively referred to as “magnetic gold nanoshell, MGN.” The photothermal excitation can be achieved using a laser, an LED array, or a combination of both, the preferred external energy source is a laser, and the laser wavelength mainly ranges from visible spectrum to near-infrared spectrum (380 nm to 1.4 μm). Besides, the monodispersed suspension of magnetic gold nanoshells (MGNs) exhibits the characteristic of near-infrared absorbance (750 nm to 1.4 μm), particularly, the LSPR (localized surface plasmon resonance) absorbance peak is closed to the wavelength at 808 nm. Under irradiations with an 808 nm laser, the MGNs can generate the photothermal conversion highly efficiently (i.e., plasmonic heating).
In another embodiment of the present invention, regarding the “alternating magnetic field (AMF)-induced heating of the solid-phase carrier 40”, the solid-phase carrier 40 comprises a magnetic core 401. Under an applied alternating magnetic field, the solid-phase carrier 40 exhibits an induced heating effect that is mainly attributed to the magnetization reversal of the nanoscale structure, including the Néel relaxation mechanism (reorientation of the magnetization of the solid-phase carrier 40) and the Brown relaxation mechanism (physical rotation of the solid-phase carrier 40 in aqueous medium). This heating capability depends on the characteristics of nanoscale structures, (e.g., average size, configuration, magnetization intensity, magnetic anisotropy), and the amplitude (Hac) and frequency (f) of the applied alternating magnetic field. The alternating magnetic field is generated by an alternating magnetic field generator, and the amplitude and frequency of the alternating magnetic field are set based on the field conditions required for the temperature needed for forming in-situ environment 50 by each solid-phase carrier 40. Moreover, the amplitude of the alternating magnetic field ranges from 0.5 to 550 kiloamps per meter (kA/m), and the frequency of the alternating magnetic field ranges from 3 to 3,500 kilohertz (kHz).
Furthermore, the solid-phase carrier 40 can be suspended with the nucleic acid amplification solution 30 and be a stable colloidal monodispersed suspension, in which the monodispersed status refers to a single dispersed suspension, and if the size of the solid-phase carrier 40 reaches the nanoscale, due to the surface potential of solid-phase carriers 40 it forms a stable colloidal monodispersed solution; the solid-phase carrier 40 can be a strong magnetic or superparamagnetic iron oxide solid-phase carrier (i.e., Superparamagnetic Iron Oxide Nanoparticles; SPIONs), especially Fe3O4 (magnetite), γ-Fe2O3, (maghemite) or spinel ferrite nanomaterials including MIIFe2O4 spinel ferrites (in which MII=Co2+, Ni2+, Zn2+, Mn2+). The addition of transition metal elements such as Ni, Co, etc mixed into the ferrite nanomaterials, can obtain a larger saturation magnetization, stable effective magnetic anisotropy (Keff), and stronger magnetization loss under the external alternating magnetic field.
The size of solid-phase carriers 40 for alternating magnetic field (AMF)-induced heating ranges from 8 to 1,000 nm. In addition to the common spheres, its shape type includes nanocubes, nanoctahedra, rods, disks, hollow spheres, stars, tetrapods, and so on, but not limited thereto. The amplitude (Hac) and frequency (f) of the applied alternating magnetic field can be set to a wide range with wide frequencies (ranging from 3 to 3,500 kHz) and field amplitudes (ranging from 0.5 to 550 kA/m), to generate the field conditions required for the temperature needed for forming in-situ environment 50 by each solid-phase carrier 40.
In the above embodiments, the surface modification of the multifunctional body 41, enrichment body, or amplification body 42 of the solid-phase carrier 40 further includes a surface-filling substance. The surface-filling substance is used to prevent aggregation of the solid-phase carriers 40, improve the hydrophilicity and confer the water-solubility of solid-phase carriers 40, reduce the adsorption inhibition of polymerase 302, and prevent non-specific adsorptions of proteins and primer 301 on the solid-phase carrier 40. The surface-filling substances can be polyethylene glycol (PEG) 72 with a molecular weight of 5 to 10 kDa and bovine serum albumin (BSA) 73. The enrichment ligand 70 or amplification ligand 71 can form stable covalent bonds with the functional groups provided by the surface-filling substances on the solid-phase carriers 40, the functional groups provided by surface-filling substance are preferred for carboxyl, amine, or thiol groups for perming chemical crosslinking in this embodiment.
As shown in
Step S10: Mixing a sample with a plurality of solid-phase carriers 40, the sample includes at least one analyte 20, and the plurality of solid-phase carriers 40 binds to at least one target analyte 20, then proceed to step S20.
Step S20: In the reaction unit 10, use the operation unit 15 to wash away impurities, and concentrate (enrich) the target analyte 20. If the at least one analyte 20 is a free deoxyribonucleic acid or ribonucleic acid, proceed directly to step S51; otherwise, proceed to step S30.
Step S30: At least one energy excitation unit 12 provides external energy to the plurality of solid-phase carriers 40, raising the thermal fields of the plurality of solid-phase carriers 40 to the denaturation temperature (e.g., 80 to 95° C.) of the analyte 20, then proceed to step S40.
Step S40: The at least one analyte 20 undergoes lysis, releasing a biological substance 21 (e.g., deoxyribonucleic acid or ribonucleic acid), then proceed to step S50.
Step S50: Controlling the energy excitation unit 12 to provide external energy to the plurality of solid-phase carriers 40 until reaching a nucleic acid hybridization temperature, causing the plurality of solid-phase carriers 40 to bind with at least one biological substance 21, and using the operation unit 15 to purify, separate, and enrich the multiple solid-phase carriers 40, then proceed to step S60.
Step S51: Controlling the energy excitation unit 12 to provide an external energy to the plurality of solid-phase carriers 40 until reaching a nucleic acid hybridization temperature, causing the plurality of solid-phase carriers 40 to bind with at least one analyte 20, and using the operation unit 15 to purify, separate, and enrich the multiple solid-phase carriers 40, then proceed to step S60.
Step S60: Adding pre-cooled nucleic acid amplification solution 30 (i.e., with a temperature of the nucleic acid amplification solution 30 ranging from −10 to 4° C.) to the reaction unit 10 and combining it with the external auxiliary cooling unit 13. If performing water cooling on-bead polymerase chain reaction, then proceed directly to step S70. If performing water cooling water cooling on-bead loop-mediated isothermal amplification, then proceed directly to step S71.
Step S70: Controlling the energy excitation unit 12 to provide external energy to the plurality of solid-phase carriers 40, allowing the in-situ environment 50 of the solid-phase carriers 40 to reach the temperature required for nucleic acid denaturation (i.e., 90 to 95° C.) and primer annealing/polymerase extension (i.e., 60 to 65° C.) in the polymerase chain reaction, and at least one enzyme will use for performing in-situ nucleic acid amplification on the plurality of solid-phase carriers 40. If performing the detection procedure, then proceed to step S80.
Step S71: Controlling the energy excitation unit 12 to provide an external energy to the plurality of solid-phase carriers 40, allowing the in-situ environment 50 surrounding their surfaces to reach the temperature required for performing loop-mediated isothermal amplification (i.e., 60 to 65° C.), and at least one enzyme will use for performing in-situ nucleic acid amplification on the plurality of solid-phase carriers 40. If performing the detection procedure, then proceed to step S80.
Step S80: Using the operation unit 15 to separate the amplification bodies 42 from the supernatant.
Step S90: Use a detection module 17 to detect the changes of signal readouts on the amplicons 60 immobilized on the amplification bodies 42, by detecting colorimetric changes, luminescence changes, or a combination thereof generated by recognizing the nucleic acid tags on the amplicons 60 with antibodies conjugated with enzymes (i.e., horseradish peroxidase, alkaline phosphatase).
In one embodiment, the water cooling on-bead loop-mediated isothermal amplification (LAMP) of the present invention is performed. As shown in
Furthermore, according to the disclosure of U.S. patent with publication number U.S. Pat. No. 9,382,583 B2 by GNA Biosolutions, the occurrence of “localized heating” on the photothermal solid-phase carrier 40 is based on the following conditions. When the interval of energy excitation is shorter or equal to the critical excitation interval t1, the critical excitation interval t1 can be represented by the following equation:
According to the conditions disclosed in the aforementioned patent, it is required to use the two-dimensional laser scanning controlled with a complex and sophisticated two-dimensional mirror scanner, in which laser beams are selectively and specifically focused on a portion of plasmonic nanoparticles within the nucleic acid solution 30 for 10 nanoseconds to 500 milliseconds. Therefore, in the overall implementation of the aforementioned patent, it is necessary to precisely control the actions as mentioned above to control the time and relative positions of light irradiations of the plasmonic particles within the nucleic acid solution 30, which are critical factors for the successful implementation of the PCA technology.
In contrast, the present invention provides a relatively easy strategy to control the excitation of the solid-phase carrier 40 by external energy, resulting in the generation of localized thermal radiation surrounding the solid-phase carrier 40. Firstly, the present invention uses solid-phase carriers 40 suspended in the nucleic acid amplification solution 30, with a ratio of the total volume of solid-phase carriers 40 to the volume of the nucleic acid solution 30, ranging from 1:200 to 1:1×109. The preferred volume ratio is 1:1×104 to 1:1×108, meaning that the distance |X| between the solid-phase carriers 40 and the distance of heat front of the solid-phase carrier 40 spreading during the critical excitation interval t1 excitation (i.e., the s1; the scaling factor) are significantly increased. Therefore, the longer critical excitation intervals t1 can be used to modulate the laser irradiations on the solid-phase carriers 40 to generate localized thermal radiation surrounding the solid-phase carriers 40.
Moreover, the nucleic acid amplification solution 30 is placed within an external auxiliary cooling unit 13, which provides a cooling environment to significantly constrict the spreading of localized thermal field (i.e., in-situ environment 50) generated by the solid-phase carriers 40 upon laser irradiations, ensuring that the localized thermal fields generated by different solid-phase carriers 40 do not overlap each other and remain in an independent state. In view of this, the present invention does not require controlling for the relative movement between the energy excitation unit 12 and the reaction unit 10 (i.e., To control the irradiation direction of laser beams on a portion of plasmonic nanoparticles within the nucleic acid solution 30 with very short intervals), and can use a relatively long excitation intervals (ranging from one second to several tens of seconds) to excite “all” suspended solid-phase carriers 40 and the solid-phase carriers 40 are tethered or the solid-phase carriers 40 are embedded in the inner wall of the reaction unit 10. Additionally, during each activation of the energy excitation unit 12, there is no implementation of controlling relative movement between the energy excitation unit 12 and the reaction unit 10. The above-mentioned characteristics have a distinct difference from those disclosed in the U.S. patent (with publication number U.S. Pat. No. 9,382,583 B2) of the GNA Biosolutions.
Due to the high surface area to volume ratio of the solid-phase carriers 40, the solid-phase carriers 40 not only serve as efficient platforms for separating/concentrating the amplicons 60 of the analyte 20 but also can be used to remove potential inhibitory substances originating from the analyte 20. In addition, the in-situ nucleic acid amplification is performed in the in-situ environments 50 formed on the surrounding space of the solid-phase carriers 40. Since the limited capacity of immobilized amplicons 60 on the surface of the solid-phase carriers 40, which enables to achieve saturation status of the capacity of the solid-phase carriers 40 in relatively short operation times (or a relatively small number of nucleic acid amplification cycles). This will facilitate the subsequent detection of amplicons 60.
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In the process of “capture and concentration of the target analyte 20,” 5 μL of the suspension of the enrichment bodies, 15 μL of the suspension of the amplification bodies 42, and 1 μL of the analyte 20, were added into 19 μL of TBST buffer including 1% bovine serum albumin, and then incubated at 25° C. with shaking for 20 minutes. This allows the amplification ligands 71 functionalized on the amplification bodies 42 to capture the cells of E. coli, subsequently, the magnetic separation is used to concentrate (enrich) the enrichment bodies and amplification bodies 42 with the operation unit 15, and remove the supernatant and wash away impurities with the TBST buffer. This process can be completed within 25 minutes.
In the process of “photothermal lysis of the analyte and capture of released biological substance 21”. After the process of “capture and concentration of the target analyte 20,” the mixed enrichment bodies and the amplification bodies 42 were resuspended in 20 μL of TBST buffer, and then those were subjected to continuous 808 nm laser irradiations at 400 mW for 5 minutes to perform photothermal lysis, and then the biological substances 21 are released from the lysed analyte 20. The released biological substances 21 are bound by the amplification ligands 71 on the amplification bodies 42 through the hybridization capture. The magnetic operation unit 15 is used to concentrate the enrichment bodies and amplification bodies 42, and remove the putative PCR inhibitory substances originating from target analytes 20 in the supernatant. Subsequently, add pre-cooled 100 μL of nucleic acid amplification solution 30 to resuspend the mixed enrichment bodies and amplification bodies 42.
In the process of “water cooling on-bead polymerase chain reaction using laser excitation”. After the process of “photothermal lysis of the analyte and capture of released biological substance 21”, the mixed enrichment bodies and the amplification bodies 42 are resuspended in the pre-cooled nucleic acid amplification solution 30 or placed in an external auxiliary cooling unit 13, wherein the pre-cooled reaction unit 10 or the external auxiliary cooling unit 13 maintains the cooling temperature, and then those are excited under the NIR laser irradiations with 40 photonic cycles (i.e., each photonic cycle is modulated by the integrated driver at 400 mW/0.16 cm2 for 1.25 seconds, followed by 0.5 seconds of laser off, and then at 150 mW/0.16 cm2 for 7.5 seconds of irradiation). After the water cooling on-bead polymerase chain reaction. The magnetic operation unit 15 is then used to concentrate the enrichment bodies and amplification bodies 42 and remove the supernatant, subsequently, add 20 μL of TBST buffer to resuspend the mixed enrichment bodies and the amplification bodies 42. This process can be completed within 6 minutes.
In the process of “detection using lateral flow immunoassay strip 171”. After the process of “the water cooling on-bead polymerase chain reaction using laser excitation”, the mixed enrichment bodies and the amplification bodies 42 are resuspended in TBST buffer, those are subjected to detection using the lateral flow immunoassay strip 171. The signal readouts on the test line 1711 of the lateral flow immunoassay strip 171 can be directly observed with the visual inspection or those can be detected using plasmonic thermal sensing with the NIR laser-coupled infrared thermal sensor 172. This process can be completed within 3 to 5 minutes. The overall process of the integrated platform can be completed within 45 minutes.
As shown in
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To understand the effect of the cooling temperature of the nucleic acid amplification solution 30 on the in-situ amplification performance of the water-cooling on-bead PCR, the issues about the influence of rapid heat dissipation under laser excitation were further investigated by testing the presence or absence of the external auxiliary cooling units 13 (e.g., an ice pack or an enlarged ice pack), the specific heat capacity of endothermic substances (e.g., the size of the ice pack, and the volume of the nucleic acid amplification solution 30). The overall temperature difference (ΔT; ° C.) of the nucleic acid amplification solution 30 was monitored under various above-mentioned conditions during the performing of the water cooling on-bead PCR. All experiment groups had been classified depicted as the “O group” (i.e., with an ice pack as an external axillary cooling unit 13, 100 μL nucleic acid amplification solution 30), the “Non group” (i.e., without an ice pack, 100 μL nucleic acid amplification solution 30), the “Non (2×) group” (i.e., without an ice pack, 200 μL nucleic acid amplification solution 30), and the “E group” (i.e., with an enlarged ice pack, 100 μL nucleic acid amplification solution 30). Each experiment group contains two duplicated experiments and each experiment has equal amounts of MGNs at 2.8×108 particles. The ice pack was prepared by freezing 550 μL of water, while the enlarged ice pack was prepared by freezing 2,200 μL of water. Please refer to
In the embodiment of the present invention, the water cooling on-bead PCR was performed under the photonic cycles with the NIR laser irradiations, in which the thermal cycling generated around the solid-phase carriers 40 is controlled by the photonic cycles, including power output and timing sequence of on and off of the NIR laser irradiations modulated by the integrated driver 11. Each photonic cycle triggered the thermal cycling generated around the solid-phase carriers 40 including the sequential processes of the nucleic acid denaturation, primer annealing, and polymerase extension. The interval of the photonic cycle for performing the nucleic acid denaturation ranges from 0.5 to 5 seconds, preferably 1 to 2.5 seconds, the power of the NIR laser ranges from 400 to 800 mW/0.16 cm2, and the temperature generated around the solid-phase carriers 40 for performing the nucleic acid denaturation ranges from 85 to 95 C, preferably 95° C. The interval of the photonic cycle for performing the primer annealing and polymerase extension ranges from 2 to 15 seconds, preferably 5 to 15 seconds, with laser power ranges from 100 to 400 mW/0.16 cm2, and the temperature generated around the solid-phase carriers 40 for performing the primer annealing and polymerase extension ranges from 55 to 65° C., preferably 60° C. Furthermore, the total turnaround time of 50 photonic cycles for performing the water cooling on-bead PCR time ranges from 5 to 15 minutes.
The present invention is a nucleic acid detection method, wherein the aforementioned solid-phase carriers 40 exhibit magnetism, after the procedure of the water cooling on-bead PCR, the target amplicons 60 are generated and immobilized on the solid-phase carriers 40, and the detection of target amplicons 60 immobilized on the solid-phase carriers 40 is performed as the following steps:
In the present invention, the methods for detecting the optical changes on the amplicons 60 immobilized on solid-phase carriers 40, comprised of detecting the change of light intensity generated directly by the primer 301 labelled with the nucleic acid tags or fluorophore 3011 incorporated into amplicons 60 with a spectrophotometer or a fluorometer, or detecting the optical changes or chemiluminescence changes generated by an enzyme-linked immunosorbent assay, or detecting spectral changes resulting from the repulsion on the amplicons 60 immobilized on the solid-phase carriers 40.
In the present invention, the method for detecting optical changes of the amplicons 60 immobilized on the solid-phase carriers 40, wherein the detection module comprises the nucleic acid lateral flow strips or the lateral flow immunoassay strips 171, further combined with thermal sensing, surface-enhanced Raman spectroscopy (SERS) or a combination thereof, thus enhancing the sensitivity of the nucleic acid lateral flow strip detection or lateral flow immunoassay strip 171.
In the present invention, the method for magnetic changes occurring on the amplicons 60 immobilized on the solid-phase carriers 40, including the detections of frequency-dependent AC susceptibility with the AC susceptometry, and the giant magnetoresistive measurement (GMR), or a combination of both.
In the present invention, the method for electrochemical changes occurring on the amplicons 60 immobilized on the solid-phase carriers 40, including one or a combination of both electrochemical detections coupled with enzyme-linked immunosorbent assay and electrochemical impedance spectroscopy (EIS).
In the present invention, a method for mass changes occurring on the amplicons 60 immobilized on the solid-phase carriers 40 is performed using a quartz crystal microbalance (QCM).
As illustrated above, the present invention has completed various experimental verifications for performing the water cooling on-bead PCR. Firstly, the linear calibration curve of the applied NIR laser powers and the micro-scale photothermal converted temperature of the solid-phased carriers 40 suspensions has been established to estimate the suitable temperature ranges for performing the in-situ amplification on the solid-phase carriers 40. In addition to the optimal conditions in photonic cycles of the external energy excitation (e.g., the output powers and irradiation frequency) and the cooling temperature of the nucleic acid amplification solution 30 on the in-situ amplification performance have also been discussed. By the coordination of the localized heating of the solid-phase carriers 40 induced by external energy and cooling temperature provided by the PCR reaction solutions 30, rapid thermal cycling generated around the solid-phase carriers 40 could be achieved. Furthermore, the subsequent modes for performing the detections of the amplicons 60 generated on the solid-phase carriers 40 and the operation examples for pretreatment of the analyte 20 have also been discussed. Taken together, our results demonstrated that the integrated platform of the present invention could achieve the target analytes enrichment, rapid nucleic acid amplification and detection in the sample-in and result-out manner. Therefore, the present invention can be applied to nucleic acid-based point-of-care testings (POCTs). In conclusion, the present invention confers the following advantages:
1. By using solid-phase carriers 40 with the high surface area to volume ratio, the combination of ligands on their surfaces can be flexibly functionalized according to the intended application (e.g., antibodies, nucleic acid aptamers, oligonucleotides, proteins, polysaccharides, or a combination thereof).
2. By controlling the coordination of the external energy excitation and the cooling temperature provided by the nucleic acid amplification solution 30 or the auxiliary cooling unit, the present invention can rapidly achieve the in-situ lysis of cells, the hybridization capture of target nucleic acids released from the analytes 20, and the localized temperature conditions required for performing in-situ nucleic acid amplification on the surface of the solid-phase carriers 40 (i.e., in-situ environment 50).
3. Since the above-mentioned in-situ environment 50 occurred on the surface of the solid-phase carriers 40, the in-situ nucleic acid amplification is constricted to the space adjacent to the surface of the solid-phase carriers 40. The pre-cooled nucleic acid amplification solutions 30 surrounding the solid-phase carriers 40 not only rapidly dissipate the heat from the solid-phase carriers 40 but also further constrict the spreading of heat front of the solid-phase carrier 40 upon the external excitation. This effect of localized heating contributes to the space adjacent to the surface of the solid-phase carriers 40 form the independent in-situ environment 50. However, in non in-situ environment 50, the cooling temperature of the pre-cooled nucleic acid amplification solution 30 can inhibit the polymerase activity, resulting in a reduction of the occurrence of the non-specific amplification. Furthermore, the technical feature of the water cooling on-bead PCR leads to create a thermal filed partitioning for performing the in-situ nucleic acid amplification on the solid-phased carriers 40, which is similar to the concept of droplet digital PCR (ddPCR) or emulsion PCR, the PCR reactions are further partitioned into tens of thousands of nano-liter sized droplets, where an independent PCR reaction takes place in physically isolated chambers. However, unlike the physical partitioning of PCR reactions that occurred in the ddPCR or emulsion PCR, the technical feature of the “virtual emulsion PCR or virtual droplet digital PCR” of the disclosed invention, which means that it does not need the additional the droplet generator and the assistance of the mineral oil and surfactants to partition test samples into the physically isolated droplets. The effect of virtual partitioning in the water cooling on-bead PCR can reduce the influences originating from the complexity of the analytes 20 on the nucleic acid amplification and subsequent detection reaction.
4. The low temperature of the nucleic acid amplification solution 30 significantly improves technical issues, such as evaporation and bubble generation that occur in conventional miniaturized nucleic acid amplification devices.
5. The present invention can achieve the ultra-fast thermal cycling required for performing in-situ nucleic acid amplification in the in-situ environment 50 on the solid-phase carriers 40, and subsequent purification, separation, and concentration of the amplicons 60 immobilized on the solid-phase carriers 40 under assistance with the magnetic operation unit 15. These advancements significantly reduce the overall turnaround time required for nucleic acid amplification and detection.
6. The present invention greatly simplifies thermal management in performing the nucleic acid amplification.
7. Simplified pretreatment of the analytes 20 and detection of the amplicons 60: The magnetic solid-phase carriers 40 can be applied for purification and concentration (enrichment) of the analytes 20, the biological substances 21, and the amplicons 60. Furthermore, the solid-phase carriers 40 can act as the signal substances on the test line 1711 of the lateral flow immunoassay strips 171, (e.g., to form the visually observable bandings on the test line and those of the thermal images detected through plasmonic thermal sensing). Altogether, by using the unique properties of the solid-phase carriers 40 (e.g., magnetism, the enrichment and amplification ligands functionalized, LSPR, and plasmonic heating). These characteristics facilitate the pretreatment of the analyte 20 and detection of the analyte 20 and the amplicons 60.
The above brief description of the present invention is intended to provide a basic explanation of several aspects and technical features of the present invention. A brief description of the invention is not a detailed representation of the present invention. Therefore, its purpose is not to specifically enumerate the critical or the essential elements of the invention, nor to define the scope of the invention. It is merely to present several concepts of the invention concisely.
This application is a continuation application of International Application No. PCT/CN2021/142714, filed Dec. 29, 2021, and the disclosure of which is hereby incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/142714 | Dec 2021 | WO |
| Child | 18617874 | US |
