Methods and photothermal nanoparticles for rapid nucleic acid amplification

Abstract

To provide a rapid nucleic acid amplification method. [Solution] A method for using photothermal nanoparticles in rapid nucleic acid amplification and photothermal nanoparticles comprises forming a reaction mixture comprising a nucleic acid template, a polymerase enzyme, and a heating medium. The heating medium comprises a plurality of photothermal nanoparticles suspended in a solution, fabricated on a microchip, or fabricated on the surface of a well of a multiwell plate. Photothermal nanoparticles are nanoparticles having a specific geometric shape and may be single-component or multi-component. The plurality of photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonance wavelengths for converting energy absorbed from a light source to sufficiently heat the volume of the composition for the transfer of a new nucleic acid chain. The specific geometric shapes of the photothermal nanoparticles are selected from the group consisting of polyhedra, ellipsoids, rings, and hollow shapes.

Description

Technical Field

[0001] Incorporation by Reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 851,403, filed May 22, 2019, entitled "Methods and Photothermal Nanoparticles for Use in Rapid Nucleic Acid Amplification," which is hereby incorporated by reference in its entirety.

Background Art

[0002] This specification describes a method of using nanoparticles as a heating medium in a polymerase chain reaction (PCR) mixture, which can be heated at a rate of up to 75 °C / second to complete an amplification thermal cycle in less than 15 minutes. Typical PCR can take up to several hours to complete, depending on the quality and concentration of the target nucleic acid. This slow cycle time cannot meet the requirements of many current gene amplification applications. Therefore, various aspects of the embodiments herein are directed to rapid nucleic acid amplification using nanoparticles as a heating medium to accelerate PCR cycling. Further, this specification describes plasmonic nanoparticles that absorb energy from light (e.g., from a light-emitting diode or a laser) and convert this into heat, thereby becoming photothermal nanoparticles. The photothermal nanoparticles described in the present invention have various geometric shapes derived to satisfy an extinction peak wavelength in the range of about 300 nanometers (nm) to 1500 nm. These photothermal nanoparticles can be used as a heating medium in a PCR mixture to provide faster thermal cycles, thereby providing a faster PCR cycle time (e.g., less than 15 minutes for 30 cycles or more), which will dramatically improve the efficiency of the workflow of PCR-based technologies. Various exemplary embodiments can be more fully understood in view of the following detailed description in connection with the accompanying drawings.

Brief Description of the Drawings

[0003] [Figure 1A-1B]The present disclosure shows a diagram of a photothermal testing system for evaluating the conversion of nanoparticles from light to heat for rapid nucleic acid amplification. [Figure 2A] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2B] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2C] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2D] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2E] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2F] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2G] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2H] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2I] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2J] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2K] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2L] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2M] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2N] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2O] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2P] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2Q] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2R] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2S] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2T] Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 2U]Exemplary photothermal nanoparticles are shown in this disclosure. [Figure 3A] This disclosure illustrates an exemplary photothermal cycle of silver nanoprismatics coated with polyvinylpyrrolidone. [Figure 3B] The following are exemplary transmission electron microscope (TEM) images of silver nanoprismatics according to this disclosure. [Figure 3C] This disclosure illustrates an exemplary photothermal cycle of a polyethylene glycol-coated gold nanostar. [Figure 3D] The following are exemplary TEM images of gold nanostars according to this disclosure. [Figure 3E] This disclosure illustrates an exemplary photothermal cycle of a polyethylene glycol-coated gold nanostar. [Figure 3F] The following are exemplary TEM images of gold nanostars according to this disclosure. [Figure 3G] This disclosure shows exemplary ultraviolet-visible spectra illustrating the maximum light absorption peaks of gold nanostars and silver nanoprisms. [Figure 4A] This disclosure shows the results of gel electrophoresis following rapid nucleic acid amplification using photothermal gold nanocages. [Figure 4B] This disclosure shows the results of gel electrophoresis following rapid nucleic acid amplification using photothermal gold nanocages. [Figure 5] This disclosure shows the results of gel electrophoresis following rapid nucleic acid amplification using photothermal nanocages. [Modes for carrying out the invention]

[0004] The various embodiments discussed in this specification are capable of variations and alternative forms, and those aspects are shown by way of example in the drawings and described in detail. However, it should be understood that it is not intended to limit the present disclosure to the specific embodiments described. Rather, it is intended to cover all modifications, equivalents, and alternatives falling within the scope of the present disclosure, including the aspects defined in the claims. Further, the term "example" used throughout this application is merely illustrative and not limiting.

[0005] Aspects of the present disclosure are contemplated to be applicable to a variety of different types of devices, systems, and methods involving rapid nucleic acid amplification using nanoparticles. Nanoparticles having specific geometries can be selected with respect to their photothermal profiles such that nucleic acid amplification can be carried out rapidly. At a particular wavelength of light, the collective oscillation of electrons on the nanoparticle surface causes a phenomenon called surface plasmon resonance. The particular wavelength of light at which this occurs depends on the size, shape, and composition of the nanoparticle. For example, nanoparticles having a particular geometry (e.g., shape and / or size), and / or having a particular composition and / or surface coating can be selected to have a maximum peak extinction wavelength in the range of 300 nm to 1500 nm that enables the conversion of photons to heat for polymerase chain reaction (PCR). In certain implementations, aspects of the present disclosure have been shown to be beneficial in situations where nucleic acids are amplified using PCR with plasmonic photothermal nanoparticles used as a heating medium to increase the heating cycle of the PCR reaction. Although not necessarily limited thereto, various aspects can be understood by discussing non-limiting examples using illustrative situations below.

[0006] PCR is a method widely used in molecular biology to create many copies of a nucleic acid segment. Using PCR, one (or more) copy of a nucleic acid sequence is exponentially amplified, generating thousands to millions or more copies of that particular nucleic acid segment. Most of the PCR method relies on thermal cycling. In thermal cycling, the reactants are exposed to repeated cycles of heating and cooling, resulting in different temperature-dependent reactions.

[0007] A typical PCR reaction can take up to several hours to complete, depending on the quality and concentration of the target nucleic acid. This cycle time cannot meet the requirements of many current gene amplification applications. Faster PCR cycle times (e.g., less than 15 minutes for 30 cycles) can improve the efficiency of the workflow of PCR-based technologies and thus downstream applications such as next-generation sequencing (NGS). Consistent with various examples of the present disclosure, plasmonic photothermal nanoparticles are used as a heating medium to accelerate the heating cycle of PCR.

[0008] Many embodiments of this disclosure relate to plasmon photothermal nanoparticles of various different shapes and / or combinations of different shapes for use as a heating medium for PCR-based reaction mixtures. These nanoparticles absorb energy from a light source such as a laser or light-emitting diode (LED) having a wavelength of about 300 nm to 1500 nm, converting the light into heat to rapidly heat the PCR mixture at a rate of up to 75°C / second. In some embodiments, the nanoparticles rapidly heat the PCR mixture at a rate of up to 40°C / second. In some embodiments, the nanoparticles rapidly heat the PCR mixture at a rate of up to 20°C / second. In some embodiments, the nanoparticles rapidly heat the PCR mixture at a rate of up to 10°C / second. These nanoparticles are stable under light irradiation and have high efficiency in converting light to heat. The photothermal nanoparticles described herein may consist of metals or alloys with or without inorganic or organic surface coatings. Nanoparticles may consist of metals, including but not limited to gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), copper (Cu), aluminum (Al), zinc (Zn), and combinations thereof, with or without the presence of alloys.

[0009] The plasmon photothermal nanoparticles described herein have specific geometric structures and / or shapes that are non-spherical and / or non-rod-shaped. For example, nanoparticles may have shapes (e.g., geometric structures) including, but not limited to, nano-stars, nano-triangular / prisms, nano-hexapods, hexagonal rings, nano-cubes, nano-rings, nano-cages, nano-quadrilateral plates, trioctahedrons, rhombododecahedrons, truncated tetrahedrons, tetrahexahedrons, octahedrons, decahedrons, icosahedrons, nanobars, hexagonal plates, grain-like shapes, disks, and combinations thereof. The photothermal nanoparticles described herein may also be coated with organic or inorganic materials to prevent interference by PCR-based reaction components due to the bare particle surface and / or to enhance the stability of the nanoparticles under irradiation and during storage. These coating materials include, but are not limited to, silica, synthetic polymers (e.g., polyvinylpyrrolidone (PVP), thiol-terminated polystyrene, polyacrylamide), oligo- or polyethylene glycol, peptides, polysaccharides, and non-polymer coatings. As an example, photothermal nanoparticles may be coated with gold and / or gold-silver alloys. Such photothermal nanoparticles may have specific geometric structures and chemical coatings sufficient to maintain stability under irradiation and maintain a high efficiency of light-to-heat conversion. To ensure the photothermal properties of the nanoparticles, the light source may be a laser or LED, and the input power is required to be sufficient for a rapid heating gradient at a rate of up to 75°C / second. The described geometric structures, shapes, and / or surface coatings of the nanoparticles provide higher efficiency of light-to-heat conversion and higher morphological stability during irradiation, thereby preventing the melting of the nanoparticles during irradiation by the light source.

[0010] Photothermal nanoparticles absorb energy from light sources such as light-emitting diodes (LEDs) and convert it into heat. According to various embodiments of this disclosure, by adjusting the concentration of photothermal nanoparticles, a PCR mixture containing the nanoparticles can be heated at a rate of up to 75°C per second, completing 30 or more cycles in 15 minutes or less. In some embodiments, the nanoparticles heat the PCR mixture at a rate of up to 40°C / second. In some embodiments, the nanoparticles rapidly heat the PCR mixture at a rate of up to 20°C / second. In some embodiments, the nanoparticles rapidly heat the PCR mixture at a rate of up to 10°C / second. Furthermore, these photothermal nanoparticles can be tuned to have various peak wavelengths in the range of 300 nm to 1500 nm by adjusting the composition and shape of the nanoparticles. This enables not only endpoint PCR-based applications but also real-time PCR-based applications where a fluorescent dye is present in the reaction mixture.

[0011] PCR is a temperature-mediated process that requires cycling between set temperatures. Single-stranded nucleic acid is required for two primer sequences to bind upstream and downstream of the region to be amplified. The first step, to allow amplification to occur, is the denaturation or separation of the two strands at approximately 94–98°C. Primer annealing occurs at approximately 45–55°C, allowing a heat-stable polymerase to bind to a defined region of double-stranded deoxyribonucleic acid (DNA). The next step is the extension of the double-stranded copy, with the temperature rising to the optimal temperature (approximately 72°C) for enzyme catalysis to proceed. Finally, the temperature is returned to 94°C for denaturation to single-stranded DNA, thereby repeating the cycle. As can be understood by those skilled in the art, the PCR process may further include a cooling step.

[0012] The efficiency of the PCR process can be improved by increasing the rate. Thermocycling of the PCR process can depend on several factors, including the experimenter's requirements. The additional time to complete PCR is a function of the time it takes to reach the desired temperature for the temperature-mediated processes (e.g., denaturation, primer annealing, and extension / synthesis). Shortening the gradient and cooling times will result in faster transitions and shorter cycling times.

[0013] PCR is performed in a reaction volume. The reaction volume / mixture contains one type of nucleic acid to be amplified, called the “original” or “sense” strand. In the reaction volume, the sense strand can be in a double-stranded form with its complementary strand, called the “complement” or “antisense” strand. If the sense and antisense strands exist as a double-stranded DNA molecule, this double-stranded DNA molecule is denatured in the first step of PCR, i.e., the double-stranded DNA molecule is split into two single strands, e.g., a sense strand and an antisense strand. In the first step of PCR, the two strands of the double-stranded molecule (e.g., DNA or RNA) are physically separated at a high temperature in a process called denaturation or melting. Denaturation occurs at a temperature called the denaturation temperature. The reaction volume / mixture further contains at least two types of primers. A “primer” is related to, or contains, short single-stranded nucleic acid segments, also known as oligonucleotides, which are complementary sequences to the target nucleic acid sequence. One of the primers is called the forward primer and the other is called the reverse primer. The forward primer is complementary to the 3' end of the sense strand. The reverse primer is complementary to the 3' end of the antisense strand.

[0014] In the second step of PCR, the temperature is lowered, and the primers hybridize / bind to their complementary sequences on the nucleic acid sequence. The two resulting double-stranded nucleic acid strands then serve as a template for the enzymatic reaction using polymerase, which transcribes new nucleic acid strands from free nucleotides also present in the reaction volume / mixture. The forward primer hybridizes to the sense strand sequence, and the reverse primer hybridizes to the antisense strand sequence. The hybridization of primers with complementary sequences to the sense or antisense strand is called annealing. This second step takes place at a temperature called the annealing temperature.

[0015] The reaction volume / mixture further contains DNA polymerase. In the third step, the DNA polymerase synthesizes a copy of the complement starting from the forward primer and a copy of the sense strand starting from the 5' end of the reverse primer. Through synthesis, a copy of the antisense strand also hybridizes with the sense strand, and a copy of the sense strand hybridizes with the antisense strand. This third step is called extension and is performed at a temperature called the extension temperature. After the extension step, the first, second, and third steps are repeated until the desired degree of amplification is achieved, creating multiple copies of the sense and antisense strands. As PCR proceeds, the generated nucleic acids themselves are used as templates for replication, initiating a chain reaction in which the original nucleic acid template is amplified exponentially.

[0016] PCR is typically performed in small reaction tubes (e.g., 0.2–0.5 ml (mL) in a thermal cycler) with volumes of 10–200 microliters (μL). A thermal cycler (also called a thermocycler, PCR machine, or DNA amplifier) ​​is a device used to amplify DNA segments via the PCR process. A thermal cycler typically has a thermal block with holes into which tubes holding the PCR reaction volume / mixture can be inserted. Heat is provided by a solid heater or infrared lamp. The thermal cycler raises and lowers the temperature of the thermal block in pre-programmed individual steps. The thermal cycler heats and cools the reaction tubes to achieve the required temperature at each step of the reaction (e.g., a cycle). Many modern thermal cyclers utilize the Peltier effect, which allows for both heating and cooling of the block holding the PCR tubes by reversing the current. Thin-walled reaction tubes allow for high thermal conductivity, enabling rapid thermal equilibrium. Since individual steps of PCR may be performed at different temperatures, it may be necessary to perform one or more heating steps and, where applicable, cooling steps during or between PCR steps in which the reaction volume or a portion thereof is cooled. According to various embodiments of the present disclosure, the reaction volume / mixture further comprises a heating medium containing photothermal nanoparticles that can locally and rapidly heat the reaction volume / mixture to a target temperature to accelerate the PCR cycling time.

[0017] In typical PCR, the denaturation temperature is chosen so that the single strands of nucleic acid are denatured without damaging, for example, the polymerase. A typical denaturation temperature is approximately 95°C. The optimal annealing temperature usually depends on the sequence and length of the primers. Typically, primers are designed for annealing temperatures of approximately 50°C to 65°C. The optimal extension temperature typically depends on the DNA polymerase used. For example, when using Taq DNA polymerase, an extension temperature of approximately 72°C is commonly used. After extension, the temperature is returned to 94°C to denaturate the double-stranded DNA into single-stranded DNA. This cycle of denaturation-annealing-extension is repeated several times, typically 20 to 40 times.

[0018] As mentioned above, several components and reagents are commonly used in PCR. These components include a nucleic acid template, such as a DNA template (e.g., double-stranded DNA) containing the target sequence to be amplified; an enzyme that polymerizes the new nucleic acid strand (e.g., a polymerase enzyme such as DNA polymerase, such as Taq DNA polymerase); two nucleic acid primers (oligonucleotides, e.g., single-stranded) complementary to the 3' (3-prime) ends of the sense and antisense strands of the nucleic acid target, namely nucleoside triphosphates (NTPs) such as deoxyribonucleotide triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs); and a buffer solution that provides a chemical environment suitable for amplification and optimal polymerase activity and stability. Certain buffer solutions often contain divalent cations such as magnesium (Mg) or manganese (Mn) ions and monovalent cations such as potassium (K) ions. Consistent with various examples of this disclosure, the reaction mixture may further include a heating medium containing photothermal nanoparticles having a specific geometric structure that allows for rapid heating of the reaction mixture. Adding a heating medium significantly reduces the time required for PCR cycling.

[0019] In various examples of this disclosure, the nucleic acid amplification method generally includes the steps of bringing a reaction volume / mixture containing a nucleic acid template into contact with photothermal nanoparticles and irradiating the nanoparticles using an activating light source so that the reaction volume / mixture reaches a desired temperature. Thus, instead of using a Peltier heater or infrared lamp to transfer heat to a container containing the reaction mixture, the reaction volume / mixture is heated using photothermal nanoparticles to heat the reaction of this disclosure to a desired temperature for PCR. In various examples, the entire reaction mixture, half of the reaction mixture, less than half of the reaction mixture, or a small amount of the reaction mixture is irradiated around the nanoparticles. In some examples of this disclosure, the method involves forming a reaction mixture comprising a nucleic acid template, a polymerase enzyme, and photothermal nanoparticles suspended in solution or immobilized on the surface of wells on a substrate such as a microchip or a multiwell plate. As described herein, the photothermal nanoparticles may be nanoparticles having a specific geometric shape and comprising one or multiple components. Multiple plasmon photothermal nanoparticles may have a specific range of resonance wavelengths for converting energy absorbed from a light source to sufficiently heat the volume of the composition for the transfer of new nucleic acid chains. For example, the specific geometric shape may be selected from the group consisting of polyhedra, spheroids, and annular shapes, among other shapes.

[0020] As will be more fully described herein, certain geometric shapes may include polyhedral nanoparticles. Polyhedral nanoparticles may be asymmetric. For example, a nanoparticle may have multiple faces, at least two of which have a different shape or size compared to the rest of the multiple faces. As a further example, a certain geometric shape may be a polyhedral nanoparticle having multiple faces, where at least three of which have a different shape or size compared to the rest of the multiple faces. Furthermore and / or, a mixture comprising multiple plasmon photothermal nanoparticles may include different types of polyhedral shapes. For example, the mixture may include a relative ratio of a first type of plasmon photothermal nanoparticle to a second type of plasmon photothermal nanoparticle for targeting a particular peak resonance wavelength.

[0021] The excitation of photothermal nanoparticles is carried out by alternating magnetic fields, such as alternating electromagnetic and / or optical fields. Excitation of photothermal nanoparticles occurs in the ultraviolet range of about 300 nm, the visible light range (e.g., 400 nm to 700 nm), the infrared range of 710 nm to 1000 nm, and radio waves above 1000 nm. Preferably, the excitation of photothermal nanoparticles occurs in the range of about 300 nm to 1500 nm. The wavelength of light at which peak excitation is achieved depends on the shape of the photothermal nanoparticles used in the reaction mixture / volume. For example, photothermal nanoparticles can be tuned / derived to have various peak wavelengths in the range of about 300 nm to 1500 nm. In various embodiments, photothermal nanoparticles can also be tuned / derived to have various peak wavelengths by adjusting the geometric structure of the particle shape. For example, the nanoparticles may be non-spherical and / or non-rod-shaped. Such non-spherical and / or non-rod-shaped nanoparticles have the following geometric structures, described in non-limiting examples: nanostars, nanotriangular / prisms, nanocubes, hexagons, triangular and spherical nanorings, nanocages, nanoquadrilateral plates, trioctahedrons, rhombic dodecahedrons, truncated tetrahedrons, tetrahexahedrons, octahedrons, decahedrons, icosahedrons, nanobars, and grain-like shapes.

[0022] In various embodiments, the reaction mixture / volume may include plasmon photothermal nanoparticles having a single geometric shape. Furthermore and / or, the reaction mixture / volume may include combinations of the geometric structures of nanoparticles. As an example, the reaction mixture / volume may include nanoparticles having a nanostar geometric structure, and / or the reaction mixture may include nanoparticles having a nanostar geometric structure and nanoparticles having an octahedral geometric structure (as an unrestricted example). Specific geometric structures of nanoparticles, and combinations of nanoparticles, may be selected to achieve specific peak absorption wavelengths. For example, specific combinations of the geometric structures of nanoparticles may be selected to achieve specific peak wavelengths such as in the range of approximately 300 nm to 1500 nm (e.g., used in PCR).

[0023] In various exemplary embodiments, nanoparticles are excited by a laser. More preferably, the laser light has a frequency that excites surface plasmon resonances in the nanoparticles. The laser can supply light continuously or as pulsed light. In non-limiting examples, the laser may be a gas laser, a diode laser, or a diode-pumped solid-state laser, among other types of lasers. The nanoparticles and / or PCR mixture may be irradiated for about 10 seconds, or for a time sufficient to otherwise raise the temperature from 45°C to 95°C. In various embodiments, the energy of the radiation source (e.g., laser) is transferred to the photothermal nanoparticles by absorption of electromagnetic waves by the photothermal nanoparticles. The radiation source used to excite the nanoparticles may also originate from sources such as LEDs, thermal radiators, flashlights, and / or ultrasound.

[0024] Through the excitation of nanoparticles, thermal energy is transferred from the excited nanoparticles to the surrounding reaction mixture / volume such that the temperature of the reaction mixture / volume is sufficient to denature the double-stranded nucleic acids in the reaction mixture / volume. In such embodiments, rapid PCR cycling times can be rapidly achieved (e.g., up to 75°C / sec) through the excitation of nanoparticles to perform denaturation. Furthermore, annealing and extension temperatures can also be achieved more rapidly by nanoparticle excitation, as only a small amount of energy needs to be transferred to excite the nanoparticles. Subsequently, the reaction volume is heated by the excitation of nanoparticles (e.g., the entire reaction volume, half of the reaction volume, less than half of the reaction volume, a small amount of the reaction volume, etc.). For example, by exciting nanoparticles to heat the reaction mixture / volume, and by using a specific geometric shape of the nanoparticles, the PCR reaction mixture / volume can be heated to up to 75°C / sec to complete more than 30 cycles in less than 15 minutes.

[0025] Various geometric shapes of photothermal nanoparticles can be used to heat reaction mixtures / volumes. Depending on the specific geometric shape of the nanoparticles and their chemical composition, photothermal nanoparticles that are stable under light irradiation and have a high efficiency of light-to-heat conversion can be provided. The geometric structure of photothermal nanoparticles is non-spherical and / or non-rod-shaped. For example, the geometric structure of photothermal nanoparticles can be selected from the following shapes: nanostar, nanotriangular / prism, nanocube, nanoring, nanocage, nanoquadrilateral plate, trioctahedron, rhombododecahedron, truncated tetrahedron, hexahedron, octahedron, decahedron, icosahedron, nanobar, and rice-like shapes.

[0026] The shape of nanoparticles can be defined by groups and / or categories. One group includes or relates to shapes having single-component polyhedral nanoparticles, and the other group includes or relates to shapes having multi-component (hybrid) polyhedral nanoparticles. The single-component polyhedral nanoparticle group includes subgroups or subcategories such as polyhedral shapes / nanoparticles (e.g., cubic, prism, octahedron, and / or regular polyhedron shapes) and polyhedral frames / hollow polyhedra (e.g., rings, cubic cages, and hollow structures). The multi-component group includes subgroups or subcategories such as alloys, core-shell shapes, and multi-shell shapes.

[0027] In various non-limiting exemplary embodiments, the nanoparticles described are composed of noble metals. Nanoparticles may also be composed of metals having an inorganic surface coating. Furthermore and / or, nanoparticles may be composed of alloys, core-shells, and multi-shells. Examples of such metals and / or alloys include, but are not limited to, gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), copper (Cu), aluminum (Al), zinc (Zn), and / or combinations thereof. Alloys can be coated with organic or inorganic surface coatings. For example, nanoparticles can be coated with organic and / or inorganic surface coatings to prevent interference with PCR-based reactions caused by bare particle surfaces and / or to enhance the stability of nanoparticles under irradiation and during storage. Non-limiting examples of coating materials include silica, silicon, synthetic polymers (e.g., PVP, thiol-terminated polystyrene, polyacrylamide, etc.), oligo or polyethylene glycol, peptides, polysaccharides, etc. Examples are not limited thereto, and in various embodiments, the coatings may include non-polymer materials.

[0028] Accordingly, the following description provides various specific details to illustrate the particular examples presented herein. However, it should be obvious to those skilled in the art that one or more other examples and / or variations of these examples may be carried out without all the specific details given below. In other examples, well-known features are not described in detail so as not to obscure the description of the examples herein. For the sake of convenience, the same reference numerals may be used in different figures to refer to the same element or further examples of the same element. Also, while embodiments and features may be described in individual figures in some cases, it will be understood that combinations may be made by combining features from one figure or embodiment with features from another figure or embodiment, even if they are not explicitly shown or described as combinations. In the invention disclosed herein, plasmon photothermal nanoparticles of various different shapes and / or combinations of different shapes are designed and prepared for use as a heating medium for PCR-based reaction mixtures. These nanoparticles absorb energy from a light source such as a laser or LED having a wavelength response of about 300 nm to about 1500 nm, converting this into heat to rapidly heat the PCR mixture at up to 75°C / second. These nanoparticles are stable under light irradiation and have high light-to-heat conversion efficiency. The photothermal nanoparticles described herein may consist of metals or alloys with or without inorganic or organic surface coatings. The shape and / or geometric structure of the photothermal nanoparticles affect the light-to-heat conversion efficiency and stability under laser irradiation or during storage, and determine the peak wavelength. In the invention herein, photothermal nanoparticles may consist of metals including, but not limited to, gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), copper (Cu), aluminum (Al), zinc (Zn), and combinations thereof. In certain examples, the nanoparticles are essentially made from gold or a gold-silver alloy. The geometric structures of the photothermal nanoparticles described herein are non-spherical and / or non-rod-shaped, including but not limited to nanostars, nanotriangular / prisms, nanocubes, nanorings, nanocages, nanoquadrilateral plates, trioctahedrons, rhombododecahedrons, truncated tetrahedrons, tetrahexahedrons, octahedrons, decahedrons, icosahedrons, nanobars, rice-like shapes, and combinations thereof. These geometric structures allow the absorption peak wavelength of the nanoparticles to be tuned from about 300 nm to about 1500 nm. The photothermal nanoparticles described herein may also be coated with organic or inorganic materials to prevent interference by PCR-based reaction components due to the bare particle surface and / or to enhance the stability of the nanoparticles under irradiation and during storage. These coating materials include, but are not limited to, silica, silicon, synthetic polymers (e.g., PVP, thiol-terminated polystyrene, polyacrylamide, etc.), oligo or polyethylene glycol, peptides, polysaccharides, and / or nonpolymer coatings.To ensure the photothermal properties of the nanoparticles, the light source may be a laser or LED, and the input power should be sufficient for a rapid heating gradient of up to 75°C / second. According to various exemplary embodiments, the photothermal nanoparticles have very different geometric structures, including shapes that allow for application to quantitative PCR (qPCR) by changing the peak wavelength of the nanoparticles from about 300 nm to 1500 nm. The described geometric structures, shapes, and / or surface coatings of the photothermal nanoparticles provide higher light-to-heat conversion efficiency and higher morphological stability during irradiation, thereby preventing melting of the nanoparticles during irradiation by the light source.

[0029] Figures 1A and 1B illustrate a photothermal test system for evaluating the light-to-heat conversion of nanoparticles for rapid nucleic acid amplification, according to the present disclosure. As shown in Figures 1A and 1B, the system comprises a light source 101, a PCR tube 103, a thermocouple 105, and a fan 109. The system includes a PCR tube 103 for containing a reaction mixture containing photothermal nanoparticles. The PCR tube 103 may include a container, chamber, assembly, or other structure adapted to contain the reaction mixture and nanoparticles and to allow light to reach them. As shown in Figure 1A, the thermocouple 105 can be used to measure the temperature change inside the PCR tube 103 containing the photothermal nanoparticles.

[0030] The system further includes a light source 101 for irradiating photothermal nanoparticles with an activation ray so that the photothermal nanoparticles release enough heat to heat the surrounding reaction mixture. In various embodiments, the light source 101 can be embodied by a laser or LED (light-emitting diode) that generates light at a wavelength tuned to match the plasmonic properties of the nanoparticles. As an example, the light source 101 may be a laser having the following specifications: output wavelength 808 nanometers (nm); output power 2 watts (W); adjustable divergence angle from 0.1 milliradians (mrad) to about 10 mrad; rectangular laser shape; exit pupil diameter 5 millimeters (mm); infrared optical aspherical glass lens optical system; adjustable spot specification of a > φ2 mm; DC operating voltage 3 volts (V); operating current 1.1 amperes (A); operating temperature -10℃ to about 50℃; storage temperature -40℃ to about 80℃; laser module dimensions φ25 mm × 40 mm, and circuit board dimensions φ60 mm × 40 mm × 24 mm. The light source 101 may be part of a photogenerating assembly that allows control of the optical parameters of the activation ray, such as wavelength, optical intensity, and, in embodiments where the ray is pulsed, duty cycle, spot size.

[0031] In various examples, photothermal nanoparticles are characterized by photothermal properties sufficient to raise the temperature of a mixture in contact with them. For example, photothermal nanoparticles may be characterized by photothermal properties sufficient to raise the temperature of a mixture from 55°C to 95°C in less than one second. Furthermore, photothermal nanoparticles may be characterized by photothermal properties sufficient to raise the temperature of a mixture from 55°C to 95°C in less than two seconds. Furthermore, photothermal nanoparticles may be characterized by photothermal properties sufficient to raise the temperature of a mixture from 55°C to 95°C in less than four seconds.

[0032] As a further example, photothermal nanoparticles may feature photothermal properties sufficient to raise the temperature of a mixture in contact with the photothermal nanoparticles at rates of up to 75°C per second, up to 40°C per second, up to 20°C per second, and / or up to 10°C per second. As used herein, photothermal properties relate to, or include, the shape, size, composition, and / or shell structure of the photothermal nanoparticles. For example, as discussed with respect to Figures 2A-2U, various shapes, compositions, and / or shell structures of photothermal nanoparticles can be used to select a specific heating rate for nucleic acid amplification. For example, in the illustrated embodiment, the light source 101 generates an activation ray having a wavelength of 808 nm that resonates with the photothermal nanoparticles in the PCR tube 103. As shown in Figure 1B, the system may include a fan 109 positioned near the PCR tube 103 (for example, as shown in Figure 1A, the fan 109 may be positioned 1 cm away from the PCR tube 103, and the PCR tube 103 may be positioned 5 cm away from the light source 101) which can be operated to accelerate the cooling of the reaction mixture during the cooling phase of thermocycling. A fan controller preferably allows control of the operation of the fan 109. A timing device may be used to measure the time between cycles of PCR reactions.

[0033] Figures 2A-2U show exemplary nanoparticles according to this disclosure. As mentioned above, nanoparticles can have various geometric shapes. Depending on the specific geometric shape of the nanoparticles and their chemical composition, photothermal nanoparticles are provided that are stable under light irradiation and have a high efficiency in converting light to heat. The shape of photothermal nanoparticles is non-spherical and / or non-rod-shaped. For example, nanoparticles may include shapes having four or more faces and spherical shapes. In certain embodiments, nanoparticles may have four or more faces and be polyhedral in shape. These faces may be planar, convex, and / or concave (e.g., diagonal) polygonal faces. Spherical shapes may include spheres, rings or annuluses, and curved shapes with fewer than four faces, such as ellipsoids. As will be further described below, nanoparticles may be hollow, solid, or core-shell in shape. The geometric structures of photothermal nanoparticles can generally be classified into groups. For example, nanoparticles can be single-component polyhedral nanoparticles. Single-component polyhedral nanoparticles relate to, or include, nanoparticles composed of a single material, such as platinum. Subgroups or subcategories of single-component polyhedral nanoparticles include, or relate to, the shape of the nanoparticles. For example, single-component polyhedral nanoparticles may include polyhedral shapes such as cubes, prisms, octahedra, and / or regular polyhedra. Furthermore and / or, single-component polyhedral nanoparticles may include hollow polyhedral shapes, such as when the nanoparticles are frame- or cage-like. Examples of such polyhedral frames or hollow polyhedra include cubic cages, regular rings, hexagonal rings, and triangular rings, among other hollow structures.

[0034] In various embodiments, polyhedral nanoparticles may have multiple components (e.g., multi-component nanoparticles). For example, nanoparticles may be composed of a combination of metallic elements (e.g., an alloy) and / or have several layers / shells. Nanoparticles may have a single shell surrounding a core metal (e.g., core-shell nanoparticles) and / or have multiple shell layers surrounding a core metal (e.g., multi-shell nanoparticles). Thus, photothermal nanoparticles can be multi-component polyhedral nanoparticles containing a plasmon core and one or more shell layers.

[0035] Each of the photothermal nanoparticles shown in Figures 2A-2U features photothermal properties sufficient to raise the temperature of the mixture in contact with the photothermal nanoparticle. For example, a photothermal nanoparticle may feature photothermal properties sufficient to raise the temperature of the mixture from 55°C to 95°C in less than 1 second, from 55°C to 95°C in less than 2 seconds, and / or from 55°C to 95°C in less than 4 seconds. Furthermore, a photothermal nanoparticle may feature photothermal properties sufficient to raise the temperature of the mixture in contact with the photothermal nanoparticle at a rate of up to 75°C per second, up to 40°C per second, up to 20°C per second, and / or up to 10°C per second.

[0036] More specifically, Figure 2A shows an exemplary nanostar-shaped nanoparticle. A nanostar shape includes, or is related to, a three-dimensional stellated polygon, such as a regular stellated polyhedron, having self-intersecting faces and / or self-intersecting edges. As shown, a nanostar shape can be a regular stellated polyhedron, such as a Kepler-Poinsot polyhedron, that includes faces that are pentagrams, triangles, or pentagons. For example, the nanostar-shaped nanoparticle shown in Figure 2A includes a self-intersecting triangular face 201. Examples are not limited thereto, and exemplary nanostar shapes include the small stellated dodecahedron, the great stellated dodecahedron, the great icosahedron, and the great dodecahedron. Other exemplary nanostar shapes include polyhedra such as the pentagonal prism, the pentagonal bipyramid, the great icosidodecahedron, and the great dodecicosacron dual. A homogeneous stellated polyhedron has regular faces or regular stellated polygonal faces. A doubly homogeneous stellated polyhedron has a vertex shape of regular faces or regular stellated polygons. Other exemplary stellated shapes include stellated polyhedra of convex polyhedra and their dual polyhedra, facets of dual polyhedra, stellated polytopes, and stellated polyhedra of stellated regions. Furthermore and / or, nanoparticles may have a nanohexapod shape. As used herein, a nanohexapod nanoparticle relates to or includes a nanoparticle having an octahedral core and six arms growing from its vertices. Nanoparticles having a nanohexapod shape are also referred herein to as nanoparticles having a nanohexapod geometric structure.

[0037] Figure 2B shows exemplary triangular prism or triangular plate-shaped nanoparticles. The triangular plate or triangular prism shape includes, or relates to, a polyhedron having two triangular faces 203-1, 203-2 or a base and three faces 205-1, 205-2, 205-3 (e.g., rectangular faces) joining the corresponding sides of the triangular faces. Other embodiments may include a triangular pyramid or tetrahedron, which includes, or relates to, a polyhedron having four triangular faces, six linear edges, and four vertex corners.

[0038] Figure 2C shows an exemplary nanocubic nanoparticle. A nanocubic shape includes, or is related to, a polyhedron having six square faces 207, three of which intersect at each vertex. As shown, a nanocube may have six faces, twelve edges, and eight vertices. Although not shown in Figure 2C, a nanocubic nanoparticle may be a concave nanocube, where each of the six square faces 207 is a concave surface. Figure 2D shows an exemplary ring-shaped nanoparticle. The ring shape includes a plane of revolution generated by a three-dimensional shape of a circle or ellipse above an axis that is coplanar with the ring, for example, a circle or ellipse. Figure 2E shows an exemplary nanocage-shaped nanoparticle. Nanocage-shaped nanoparticles are also referred to herein as nanoparticles having a nanocage geometric structure. The nanocage shape is similar to a nanocube, where the cube is hollow (e.g., without faces). The nanocage shape includes, or is related to, a polyhedron having six square faces defined by edges. As shown, the nanocage may have 12 edges and 8 vertices, which in such embodiments define “faces” that are openings. For example, each “face” includes a square perimeter 209 that defines the boundary of a hollow square opening 211 on each face.

[0039] Figure 2F shows an exemplary triangular ring-shaped nanoparticle. The triangular ring shape is similar to that of a triangular prism or triangular plate-shaped nanoparticle, where the prism is hollow. For example, the triangular ring shape includes or is related to a polyhedron having three physical faces 213-1, 213-2, 213-3 (e.g., the rectangular faces shown) that connect the corresponding sides forming a triangular shape. The triangular shape formed by the intersecting faces 213-1, 213-2, and 213-3 defines the boundary of the hollow triangular opening 215. Figure 2G shows an exemplary nanobar-shaped nanoparticle. The nanobar shape includes or is related to a polyhedron having six faces, three of which intersect at each vertex. Embodiments are not limited in this way, but at least two of the rectangular faces may be square and the remaining faces may be rectangular. For example, as shown in Figure 2G, faces 217-1 and 217-2 are rectangular, while faces 219-1 and 219-2 are square.

[0040] Figure 2H shows an exemplary octahedral nanoparticle. An octahedron includes, or is related to, a polyhedron having 8 faces, 12 edges, and 6 vertices. In certain embodiments, the 8 faces 221 may be triangles formed by the intersection of 4 faces at each vertex. Figure 2H shows a nanoparticle having a bipyramidal geometric structure, but examples are not limited thereto. As illustrated, the nanoparticles described herein may have a non-bipyramidal geometric structure. Figure 2I shows an exemplary icosahedral nanoparticle. An icosahedral shape includes, or is related to, a polyhedron having 20 faces, 12 vertices, and 30 edges. As shown, each of the 20 faces 223 may be triangular. Figure 2J shows an exemplary hexahedral nanoparticle. A hexahedral shape is defined as a polyhedron having 24 triangular faces 225, or related thereto, where 4 of the 24 faces correspond to cubic faces. Figure 2K shows an exemplary trioctahedral nanoparticle. A trioctahedral shape includes or is associated with a polyhedron having 24 triangular faces 227, three of which correspond to octahedral faces.

[0041] Figure 2L shows an exemplary rhombicosidodecahedron nanoparticle. A rhombicosidodecahedron shape includes, or is related to, a polyhedron having 20 triangular faces, 30 square faces, 12 regular pentagonal faces, 60 vertices, and 120 edges. Figure 2M shows an exemplary decahedral nanoparticle. A decahedron includes, or is related to, a polyhedron having 10 faces. In various embodiments, the decahedron may be, among others, an octagonal prism, a square antiprism, a square pylon, a pentagonal bipyramid, a lateral pyramidal pentagonal prism, a twisted bipentagonal pyramid, or a nonagonal pyramid. In various examples, the faces of the decahedron may include various geometric shapes. For example, faces 231-1 and 231-2 (e.g., the top and bottom faces) may each include a plurality of triangular faces 233, while the sides may each include a square or rectangular face 235 and a rectangular face 237 opposite the rectangular face 235. The various nanoparticles of the shapes described above can be solid, hollow, core-shell, or multi-shell particles. Core-shell particles include, or are related to, particles that have a composite structure with one or more concentric layers. Multi-core-shell particles include, or are related to, core-shell particles with two or more concentric layers. Such particles can be formed by coating nanoparticles with another type of nanomaterial.

[0042] Figure 2N shows an exemplary core-shell nanoprismatic nanoparticle. The core-shell nanoprismatic consists of two nanoprismatics, as described above, with one of the nanoprismatics 241 being internal and the other 243 formed around the internal nanoprismatics 241.

[0043] Figure 2O shows an exemplary hollow nanospherical nanoparticle. Nanospherical nanoparticles are also referred to herein as nanoparticles having a nanospherical geometric structure. The hollow nanospherical shape includes, or is related to, a sphere 245 having a hollow interior (e.g., a hollow core). Figure 2P shows an exemplary nanoparticle consisting of a single nanosphere core having multiple nanosphere shells. A nanosphere having a single core and multiple shells is related to three or more nanospheres having a solid interior. The innermost nanosphere 247 is the core, a second nanosphere 249 is formed around the first nanosphere 247, and a third (or more) nanosphere 251 is formed as an outer layer around the second nanosphere 249. Figure 2Q shows an exemplary grain-like nanoparticle. The grain-like shape includes or is related to a spheroid or a flattened sphere. As shown, the grain-like shape can be an elongated spheroid or an oblate spheroid. Figure 2R shows an exemplary hexagonal plate-shaped nanoparticle. A hexagonal plate shape includes, or is related to, a polyhedron having two hexagonal faces or bases (e.g., face 253 and the opposite hexagonal face not explicitly shown or numbered in Figure 2R) and six faces joining the corresponding sides of the two hexagonal faces. As shown in the figure, the six faces joining the corresponding hexagonal faces could be rectangular faces 255.

[0044] Figures 2S-2U show exemplary hexagonal ring-shaped nanoparticles. Hexagonal ring-shaped nanoparticles include, or are related to, hexagonal plate-shaped nanoparticles having a hollow center or cavity. For example, the hexagonal nanoparticle shown in Figure 2S includes a hollow center 257, the hexagonal nanoparticle shown in Figure 2T includes a hollow center 259, and the hexagonal nanoparticle shown in Figure 2S includes a hollow center 261. The hollow center or cavity may have a hexagonal or other shape. For example, the hollow center 257 in Figure 2S is hexagonal, while the hollow center 261 in Figure 2U is circular. Furthermore, the hollow centers of each hexagonal nanoparticle may have different diameters. For example, the hollow center 257 in Figure 2S has a diameter 258, which is larger than the diameter 260 of the hollow center 259 shown in Figure 2T. The portion of the hexagonal ring-shaped nanoparticle between the outer hexagonal face and the hollow center or cavity may be hollow or solid in various examples. As a non-limiting example, referring to Figure 2T, the hexagonal ring-shaped particle portion between the outer surface 262 and the inner surface 263 may be hollow or solid.

[0045] Some specific embodiments relate to systems and / or kits comprising one or more of the above-described photothermal nanoparticles, DNA polymerases, and reaction reagents. The DNA polymerase is thermally stable. The reaction reagent comprises dNTPs, buffer, Mg+ (e.g., magnesium chloride (MgCl2)), and optionally a stabilizer. The components of the kit can each be provided in separate volumes (e.g., containers) and / or combined into one or more volumes / containers / mixtures. For example, the nanoparticles, DNA polymerase, and reaction reagent may all be in a single container. In other embodiments, the DNA polymerase and reaction reagent are combined in a master mix and contained in a first container, while the nanoparticles are in a separate container. In various embodiments, plasmon photothermal nanoparticles may be suspended in solution or immobilized on a microfluidic chip (microchip) or plate surface for containing the reaction mixture. Thus, a PCR tube or PCR plate well may have a PCR reaction mixture comprising the nanoparticles as a heating medium as described herein, a nucleic acid template for carrying out PCR as described herein, and a polymerase enzyme. As an example, a PCR reaction mixture can be formed using a heating medium containing photothermal nanoparticles fabricated on a multiwell plate, the photothermal nanoparticles having a nanorod-like geometric structure and being essentially made from gold.

[0046] Nucleic acids that can be amplified using the present invention include, but are not limited to, DNA (in single-stranded, double-stranded, linear, covalently closed, supercoiled, and relaxed circular forms) or RNA (single-stranded, double-stranded, linear, or covalently closed) or combinations of DNA and RNA.

[0047] As used herein, the term “mixture” encompasses naturally occurring or chemically designed biological materials that include at least one nucleic acid in addition to other non-nucleic acid materials such as biomolecules (e.g., proteins, polysaccharides, lipids, low molecular weight enzyme inhibitors, oligonucleotides, primers, templates), polyacrylamide, trace metals, organic solvents, etc. Examples of naturally occurring mixtures include, but are not limited to, whole blood, plasma, and other bodily fluids, as well as tissue cell cultures obtained from humans, plants, or animals. Examples of chemically designed mixtures include, but are not limited to, nucleic acid samples eluted from lysates, agarose, and / or polyacrylamide gels, solutions containing multiple nucleic acid molecules obtained from any nucleic acid amplification method such as PCR amplification, reverse transcription polymerase chain reaction (RT-PCR) amplification, or RNA or DNA size selection procedures, as well as solutions obtained from post-sequencing reactions.

[0048] Terms such as upper / lower, left / right, top / bottom, and upward / downward may be used herein to describe the relative position of elements as shown in the figures. This phrasing is used solely for notational purposes, and in actual use, the disclosed structures may be oriented in directions other than those shown in the figures. Therefore, these terms should not be interpreted restrictively. Those skilled in the art will recognize that the various expressions used herein (including in the claims) imply the ordinary meaning in the art unless otherwise indicated. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” and “contain(s),” and their variations, when used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include multiple referents unless it is clearly indicated otherwise in the context. This disclosure also contemplates other embodiments that “include,” “consist of,” and “essentially consist of,” the embodiments or elements presented herein, whether expressly described or not.

[0049] As used herein, the term “sample” generally refers to any material containing nucleic acids, including, for example, food and similar products, clinical samples, and environmental samples. However, a sample is generally a biological sample, which may contain any viral or cellular material, including any prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts, and organelles. Thus, such biological material may include animal cells of all types of mammals and non-mammals, plant cells, algae including cyanobacteria, fungi, bacteria, protozoa, etc. Thus, a typical sample includes whole blood, as well as blood-derived products such as plasma, serum, and buffy coat, urine, feces, cerebrospinal fluid, or other body fluids, tissues, cell cultures, cell suspensions, etc. This sample may include lysates. A sample may also include relatively pure starting materials, such as PCR products or semi-pure preparations obtained by other nucleic acid recovery processes.

[0050] Based on the above discussion and examples, those skilled in the art will readily recognize that various modifications and changes can be made to various embodiments without strictly adhering to the exemplary embodiments and uses described herein. For example, methods illustrated in the figures may include steps performed in various orders, or fewer or more steps, while retaining one or more aspects of the embodiments herein. Such modifications will not depart from the true spirit and scope of the various aspects of this disclosure, including the aspects described in the claims.

[0051] More detailed embodiments of the experiment In relation to the embodiments of the experiment, as further shown below, photothermal cycling for rapid PCR was carried out using photothermal nanoparticles of different shapes and similar absorbances, as described herein. Specific examples include photothermal cycling using various photothermal nanoparticles: gold nanostars (e.g., gold nanostars coated with polyethylene glycol (PEG)) and silver nanoprismatics (e.g., silver triangular nanoplates).

[0052] As a specific example, Figure 3A shows the photothermal cycle of polyvinylpyrrolidone-coated silver nanoprisms according to this disclosure. Figure 3C shows the photothermal cycle of polyethylene glycol-coated gold nanostars according to this disclosure. In each graph shown, photothermal nanoparticles in GoTaq® G2 Hot Start PCR master mix solution (2 optical density (OD), 25 uL) were irradiated with a 2 W / cm2 laser, the photothermal cycle was recorded for 15 minutes, and then the degradation of the nanoparticles at 95°C was tested by continuously irradiating each solution. As shown in Figure 3A, the silver nanoprisms showed an average heating rate of 60.6 seconds (45°C to 95°C) and an average cooling rate of 39.1 seconds (95°C to 45°C). Different concentrations of photothermal nanoparticles (0–50 OD) can be used with different laser powers or different amounts of PCR master mix solution (5–50 uL) to achieve different heating rates. Figure 3B shows an exemplary transmission electron microscope (TEM) image of silver nanoprismatics according to this disclosure.

[0053] Similarly, Figure 3C shows an exemplary photothermal cycle of a polyethylene glycol-coated gold nanostar according to the present disclosure. As shown, the PEG-coated gold nanostar exhibited an average heating rate of 31.7 seconds (45°C to 95°C) and an average cooling rate of 45.3 seconds (95°C to 45°C). Figure 3D shows an exemplary TEM image of the gold nanostar according to the present disclosure. Similarly, Figure 3E shows an exemplary photothermal cycle of a polyethylene glycol-coated gold nanocage according to this disclosure. As shown, the PEG-coated gold nanocage exhibited an average heating rate of 2.5 seconds (45°C to 95°C) and an average cooling rate of 17 seconds (95°C to 45°C). Different concentrations of photothermal nanoparticles (0–50 0.D), laser power, or varying amounts of PCR master mix solution (5–50 uL) can be used to achieve different heating rates. Figure 3F shows an exemplary TEM image of a gold nanocage according to this disclosure. Figure 3G shows exemplary ultraviolet-visible spectra illustrating the maximum light absorption peaks of gold nanostars and silver nanoprisms according to this disclosure. As shown in Figure 3G, the silver nanoprisms exhibited an absorbance of 1.897 arbitrary units at a peak wavelength of 726 nm. The gold nanostars exhibited an absorbance of 1.975 arbitrary units at a peak wavelength of 798 nm.

[0054] Figures 4A and 4B show the results of gel electrophoresis from rapid nucleic acid amplification inhibition using photothermal nanocages according to this disclosure. In the examples shown in Figures 4A and 4B, PCR was performed using cDNA templates prepared with dT primers and RTX Exo+GoTaq® G2 Hot Start master mix. Two different 200 nM primers were used: beta-1 microglobulin (B2M) primer (left side of Figure 4B) and guanine nucleotide-binding protein subunit beta-2-like 1 (GNB2L1) primer (right side of Figure 4B). PEG-coated gold nanocages were added to the reaction mixture at concentrations from 0 to 16 O.D. The total volume of the reaction mixture was 25 μL. The solution was heated at 95°C for 2 minutes and then cycled 35 times according to the following pattern: 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C. As shown in Figure 4B, consistent results were achieved for each of the nanoparticle concentrations used during PCR, indicating that no significant PCR inhibition was observed as a result of adding gold nanocages to the PCR reaction mixture.

[0055] Figure 5 shows the results of gel electrophoresis from rapid nucleic acid amplification using photothermal nanocages according to this disclosure. In the example shown in Figure 5, a reaction mixture consisting of non-hot-start recombinant Taq DNA polymerase, PCR nucleotide mixture (deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP)), forward and reverse primers, PCR buffer, 50 ng of human genomic DNA, and 20.D PEGylated gold nanocages was prepared on ice. The PCR primers were designed to amplify the 337 base pair portion of the human betaglobin gene. 20 μl of the reaction mixture was added to a 0.2 ml PCR tube, topped with 15 μl of mineral oil, and placed on ice. Two-step photonic PCR cycling was performed in a prototype instrument. 35 cycles were performed as follows: Temperature 1 (T1) was set to approximately 94°C, and Temperature 2 (T2) was set to approximately 64°C. Both temperatures were pre-verified using thermocouple probes placed in the reaction mixture and cycling with a 70% laser. To achieve T1, the laser was activated / turned on to excite the nanoparticles and heat them, thereby heating the reaction mixture. An infrared (IR) sensor was used to measure the temperature, and after the reaction mixture reached the appropriate temperature, the laser was switched off and a fan was activated to cool the reaction mixture to the appropriate temperature, and then the laser was activated and another cycle was started. Three different T1 reaction temperatures were tested in three separate reaction tubes corresponding to 94°C (well 1), 90°C (well 2), and 86°C (well 3). Amplification was performed in less than 15 minutes. A control reaction was carried out by placing the reaction tubes in a conventional Peltier thermocycler and cycling over 35 cycles, each consisting of a denaturation step of 2 seconds at 95°C and an annealing step of 6 seconds at 65°C. This reaction cycle was used to positively confirm the ability of the reaction mixture to produce amplicons of appropriate size in the presence of nanoparticles. To confirm amplification, all four reactions were performed on a 2% agarose gel containing ethidium bromide and imaged using a UV camera system.

[0056] As shown in Figure 5, the amplified products were clearly visible in wells 2 and 3, demonstrating the effectiveness of the photothermal nanoparticle-mediated PCR reaction of the present invention. No product was observed in well 1, which may be due to a discrepancy between the temperature measured by the IR sensor and the actual temperature in the solution.

Claims

1. A method for forming a reaction mixture comprising a nucleic acid template, a polymerase enzyme, and a heating medium containing a plurality of photothermal nanoparticles suspended in solution, fabricated on a microchip, or fabricated on the surface of a well of a multiwell plate, wherein the photothermal nanoparticles are nanoparticles having a specific geometric shape and comprising single or multi-component components, and the plurality of photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonance wavelengths for converting energy absorbed from a light source to sufficiently heat the volume of the composition for the transfer of a new nucleic acid chain, and the specific geometric shape is selected from the group consisting of polyhedra, spheroids, rings, and hollow shapes.

2. A method comprising forming a suspension in which photothermal nanoparticles are contained in a solution, or immobilizing photothermal nanoparticles on the surface of a substrate.

3. A method for rapidly amplifying nucleic acid molecules by polymerase chain reaction, Forming a reaction mixture comprising a nucleic acid template, a polymerase enzyme, and a heating medium containing photothermal nanoparticles suspended in solution, fabricated on a microchip, or fabricated on the surface of a well of a multiwell plate, wherein the photothermal nanoparticles have a specific geometric shape and consist of one or multiple components, and the photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonance wavelengths for converting energy absorbed from a light source to sufficiently heat the volume of the composition for the transfer of a new nucleic acid chain, and the specific geometric shape is selected from the group consisting of polyhedra, ellipsoids, rings, and hollow shapes. By adjusting the concentration of photothermal nanoparticles, the reaction mixture is irradiated with wavelengths ranging from 300 nm to 1500 nm, depending on the specific geometric shape of the photothermal nanoparticles, which is sufficient to heat the reaction mixture at a rate of up to 75°C per second, and the amplified thermal cycle is completed in a time of 15 minutes or less. Methods that include...

4. The method according to claim 3, comprising forming a reaction mixture comprising a nucleic acid template, a polymerase enzyme, and a heating medium containing photothermal nanoparticles fabricated on a microchip, on a plate surface, or on the wells of a multiwell plate, wherein the photothermal nanoparticles have a specific geometric structure.

5. The method according to claim 3, comprising irradiating the photothermal nanoparticles to heat the reaction mixture for rapid amplification of nucleic acid molecules.

6. The method according to claim 5, comprising irradiating the entirety of the reaction mixture, half of the reaction mixture, less than half of the reaction mixture, or a small amount of the reaction mixture around the photothermal nanoparticles.

7. The method according to claim 3, wherein the reaction mixture is irradiated with a light source of a wavelength selected based on the geometric shape and chemical composition type of the photothermal nanoparticles.

8. The method according to claim 3, further comprising irradiating the reaction mixture with a light-emitting diode (LED) or laser having a wavelength range of 300 nm to 1500 nm.

9. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing photothermal nanoparticles having a specific geometric structure and a specific chemical coating sufficient to maintain stability under irradiation and to maintain a high efficiency of light-to-heat conversion.

10. The method according to claim 3, comprising forming the reaction mixture having a heating medium that includes the photothermal nanoparticles, which include a metal having a core shell of an organic and / or inorganic material.

11. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles, which include a metal having an organic surface coating.

12. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles, which include an alloy having an inorganic surface coating.

13. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles, which include an alloy having an organic surface coating.

14. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing photothermal nanoparticles comprising a metal selected from the group consisting of core shells, multi-shells, alloys, and combinations thereof of gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), copper (Cu), aluminum (Al), and zinc (Zn).

15. The method according to claim 14, comprising forming the reaction mixture having a heating medium containing photothermal nanoparticles comprising a metal selected from the group consisting of Au, Ag, Pt, Pd, Fe, Cu, Al, Zn, and combinations thereof.

16. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles having a non-spherical geometric structure.

17. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles having a non-rod-shaped geometric structure.

18. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles having a non-biconical geometric structure.

19. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing photothermal nanoparticles having a geometric structure selected from the group consisting of shapes such as nanostars, nanotriangles or prisms, nanocubes, nanorings, nanocages, hollow nanoparticles, nanoplates, polyhedra, concave nanocubes, nanobars, hexagonal plates, triangular rings, hexagonal rings, and grain-like shapes.

20. The method according to claim 3, comprising coating the photothermal nanoparticles with an organic material sufficient to prevent interference with the reaction.

21. The method according to claim 20, wherein the organic material is selected from the group consisting of synthetic polymers, oligos or polyethylene glycols, peptides, polystyrenes, and polysaccharides.

22. The method according to claim 3, comprising coating the photothermal nanoparticles with an inorganic material sufficient to prevent interference with the reaction.

23. The method according to claim 22, wherein the inorganic material is selected from silica and silicon.

24. The method according to claim 3, comprising coating the photothermal nanoparticles with an organic material sufficient to enhance the stability of the photothermal nanoparticles under irradiation and during storage.

25. The method according to claim 3, comprising coating the photothermal nanoparticles with an inorganic material sufficient to enhance the stability of the photothermal nanoparticles under irradiation and during storage.

26. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles having a nanostar geometric structure and being essentially made of gold.

27. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles having a nanocage or hollow nanoparticle geometric structure and being essentially made of gold or a gold-silver alloy.

28. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles having a nanoprismatic / nanotriangular geometric structure and being made essentially from silver, gold, gold-coated silver, or a gold-silver alloy.

29. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles having a nanoring geometric structure and being essentially made of gold or a gold-silver alloy.

30. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing photothermal nanoparticles having a hexagonal ring geometric structure with cavities of different shapes and diameters, and being made essentially of gold or a gold-silver alloy.

31. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles having a triangular ring geometric structure and being essentially made of gold or a gold-silver alloy.

32. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles having a hexagonal plate-shaped geometric structure and being essentially made of silver, gold, gold-coated silver, or a gold-silver alloy.

33. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing the photothermal nanoparticles having a nanohexapod geometric structure and being made essentially from silver, gold, gold-coated silver, or a gold-silver alloy.

34. The method according to claim 3, comprising forming the reaction mixture having a heating medium containing photothermal nanoparticles having a disk-like geometric structure and being essentially made of silver, gold, gold-coated silver, or a gold-silver alloy.

35. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a nanoring geometric structure and are essentially made of gold or a gold-silver alloy.

36. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a nanocage geometric structure and are essentially made of gold or a gold-silver alloy.

37. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a nanostar geometric structure and are essentially made of gold.

38. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a hexagonal ring geometric structure with cavities of different shapes and diameters, and are essentially made of gold or a gold-silver alloy.

39. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a triangular ring geometric structure and are essentially made of gold or a gold-silver alloy.

40. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a hexagonal plate-shaped geometric structure and are essentially made of silver, gold, gold-coated silver, or a gold-silver alloy.

41. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a nanohexapod geometric structure and are essentially made of silver, gold, gold-coated silver, or a gold-silver alloy.

42. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a nanorod geometric structure and are essentially made of gold.

43. The method according to claim 3, comprising forming the reaction mixture having the heating medium containing the photothermal nanoparticles fabricated on the multiwell plate, wherein the photothermal nanoparticles have a nanospherical geometric structure and are essentially made of gold.

44. A method for rapidly amplifying nucleic acid molecules by polymerase chain reaction, Forming a reaction mixture comprising a nucleic acid template, a polymerase enzyme, and a heating medium containing photothermal nanoparticles on a microchip or the surface of a plate, wherein the photothermal nanoparticles have a specific geometric shape and consist of one or multiple components, and the photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonance wavelengths for converting energy absorbed from a light source to sufficiently heat the volume of the composition for the transfer of a new nucleic acid chain, and the specific geometric shape is selected from the group consisting of polyhedra, ellipsoids, and rings and hollow shapes. By adjusting the concentration of photothermal nanoparticles, the reaction mixture is irradiated with wavelengths ranging from 300 nm to 1500 nm, depending on the specific geometric shape of the photothermal nanoparticles, which is sufficient to heat the reaction mixture at a rate of up to 75°C per second, and the amplified thermal cycle is completed in a time of 15 minutes or less. A method by which.

45. Photothermal nanoparticles having a specific geometric shape and comprising single-component or multi-component nanoparticles, wherein the photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonant wavelengths for converting energy absorbed from a light source to heat, and the specific geometric shape is selected from the group consisting of polyhedra, spheroids, rings, and hollow shapes.

46. The photothermal nanoparticle according to claim 45, wherein the specific geometric shape includes a polyhedron having at least one curved surface.

47. The photothermal nanoparticle according to claim 45, wherein the resonance wavelength is in the range of 300 nm to 1500 nm.

48. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a single-component polyhedral nanoparticle.

49. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a multi-component polyhedral nanoparticle.

50. The photothermal nanoparticles according to claim 45, wherein the photothermal nanoparticles are multi-component nanoparticles containing an alloy.

51. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a multi-component polyhedral nanoparticle comprising a plasmon core and one or more shell layers.

52. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a multi-component polyhedral nanoparticle comprising a plasmon core and at least one shell layer.

53. The photothermal nanoparticles according to claim 45, wherein the photothermal nanoparticles are spheroidal nanoparticles characterized by a grain-like shape.

54. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a polyhedral nanoparticle characterized by a nanostar shape.

55. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a polyhedral nanoparticle characterized by a nanocube shape.

56. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a polyhedral nanoparticle characterized by a nanoprismatic shape.

57. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a polyhedral nanoparticle characterized by a polyhedral frame having a hollow core.

58. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a polyhedral nanoparticle having a plurality of faces, and at least two of the plurality of faces have a different shape or size compared to the rest of the plurality of faces.

59. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a polyhedral nanoparticle having a plurality of faces, and at least three of the plurality of faces have a different shape or size compared to the rest of the plurality of faces.

60. The photothermal nanoparticle according to claim 45, wherein the photothermal nanoparticle is a polyhedral nanoparticle having at least two asymmetrical portions.

61. The photothermal nanoparticle according to claim 45, further characterized by photothermal properties sufficient to raise the temperature of a mixture in contact with the photothermal nanoparticle from 55°C to 95°C in less than one second.

62. The photothermal nanoparticle according to claim 45, further characterized by photothermal properties sufficient to raise the temperature of a mixture in contact with the photothermal nanoparticle from 55°C to 95°C in 2 seconds or less.

63. The photothermal nanoparticle according to claim 45, further characterized by photothermal properties sufficient to raise the temperature of a mixture in contact with the photothermal nanoparticle from 55°C to 95°C in 4 seconds or less.

64. The photothermal nanoparticle according to claim 45, further having photothermal properties sufficient to raise the temperature of a mixture in contact with the photothermal nanoparticle at a rate of up to 75°C per second.

65. The photothermal nanoparticle according to claim 45, further having photothermal properties sufficient to raise the temperature of a mixture in contact with the photothermal nanoparticle at a rate of up to 40°C per second.

66. The photothermal nanoparticle according to claim 45, further having photothermal properties sufficient to raise the temperature of a mixture in contact with the photothermal nanoparticle at a rate of up to 20°C per second.

67. The photothermal nanoparticle according to claim 45, further having photothermal properties sufficient to raise the temperature of a mixture in contact with the photothermal nanoparticle at a rate of up to 10°C per second.

68. A first type of plasmon photothermal nanoparticle having a specific geometric shape and comprising one or multiple components, wherein the first type of plasmon photothermal nanoparticle is further classified by surface plasmon resonance having a specific range of resonant wavelengths for converting energy absorbed from a light source for heating, and the specific geometric shape is selected from the group consisting of polyhedra, spheroids, rings, and hollow shapes, At least one second type of plasmon photothermal nanoparticle having a specific geometric shape different from that of the first type of plasmon photothermal nanoparticle, A composition containing the following:

69. The composition according to claim 68, wherein the second type of plasmon photothermal nanoparticles consists of single-component or multi-component nanoparticles, and the second type of plasmon photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonant wavelengths for converting energy absorbed from a light source for heating, and the specific geometric shape is selected from the group consisting of polyhedra, spheroids, rings, and hollow shapes.

70. The composition according to claim 68, further comprising a relative ratio of the first type of plasmon photothermal nanoparticles to the second type of plasmon photothermal nanoparticles for targeting a specific peak resonance wavelength.

71. The composition according to claim 68, further comprising a relative ratio of the first type of plasmon photothermal nanoparticles to the second type of plasmon photothermal nanoparticles for targeting a peak resonance wavelength sufficient for at least one polymerase chain reaction process.

72. A reaction reagent comprising deoxyribonucleotide triphosphate (dNTP) and a buffer solution, A polymerase enzyme configured to transcribe a new nucleic acid chain from dNTPs, A plurality of plasmon photothermal nanoparticles having a specific geometric shape and comprising a single or multiple component, wherein the plurality of plasmon photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonance wavelengths for converting energy absorbed from a light source to sufficiently heat the volume of the composition for the transfer of new nucleic acid chains, and the specific geometric shape is selected from the group consisting of polyhedra, spheroids, rings, and hollow shapes. A composition containing the following:

73. The composition according to claim 72, wherein the reaction reagent further comprises magnesium.

74. The composition according to claim 72, wherein the reaction reagent further comprises magnesium chloride.

75. The composition according to claim 72, wherein the reaction reagent further comprises a stabilizer.

76. A kit for transcribing nucleic acid strands, A first volume containing a reaction reagent comprising nucleoside triphosphate (NTP) and a buffer solution, A second volume containing a polymerase enzyme configured to transcribe a new nucleic acid chain from an NTP, A third volume comprising a plurality of plasmon photothermal nanoparticles having a specific geometric shape and comprising single or multi-component elements, wherein the plurality of plasmon photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonance wavelengths for converting energy absorbed from a light source to sufficiently heat the volumes of the first, second, and third volumes for the transfer of new nucleic acid chains, and the specific geometric shape is selected from the group consisting of polyhedra, ellipsoids, rings, and hollow shapes. A kit that includes this.

77. A kit for transcribing nucleic acid strands, A first volume containing a reaction reagent comprising nucleoside triphosphate (NTP) and a buffer solution, A polymerase enzyme configured to transcribe a new nucleic acid chain from NTPs, and A plurality of plasmon photothermal nanoparticles having a specific geometric shape and comprising one or multiple components, wherein the plurality of plasmon photothermal nanoparticles are further classified by surface plasmon resonance having a specific range of resonance wavelengths for converting energy absorbed from a light source to sufficiently heat the volumes of the first and second volumes for the transfer of new nucleic acid chains, wherein the specific geometric shape is selected from the group consisting of polyhedra, spheroids, rings, and hollow shapes, and the plurality of plasmon photothermal nanoparticles comprises a second volume containing the plurality of plasmon photothermal nanoparticles, A kit that includes this.

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