BACKGROUND

[0001] Nucleic acid amplification and detection Is a technique utilized in research, medical diagnostics, and forensic testing. The ability to amplify a small quantity of a sample of a nucleic add to generate copies of the nucleic acid in the sample can permit research, medical diagnostic, and forensic tests that would not otherwise be permissible from the small quantity of the sample, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

[0001] FIG. 1 graphically illustrates an example microfluidic nucleic acid amplification system in accordance with the present disclosure;

[0002] FIG. 2 graphically illustrates an example microfluidic nucleic acid amplification system in accordance with the present disclosure;

[0003] FIG. 3 graphically illustrates an example microfluidic nucleic acid amplification system in accordance with the present disclosure;

[0004] FIG. 4 graphically illustrates an example microfluidic nucleic acid amplification system in accordance with the present disclosure; and

[0005] FIG. 5 is a flow diagram illustrating an example method of detecting nucleic acid amplification in accordance with the present disclosure.

DETAILED DESCRIPTION

[0006] Nucleic acid amplification can include denaturing, annealing, and extending nucleic acid chains. During denaturing, an increased temperature can cause hydrogen bonds between bases in a double-stranded nucleic acid sample to break apart resulting in two single strands realized from a formerly double-stranded nucleic acid. During annealing, the heated sample can then be cooled, enabling single stranded nucleic acid oligomers, such as primers, to attach to the complimentary nitrogen bases on the single strands of the nucleic acid. During extending of the nucleic acid chain, the temperature may be increased, for example, to enable a polymerase enzyme to extend the nucleic add strand by adding nucleic acid bases.

[0007] The present disclosure is drawn to a microfiuidic nucleic acid amplification. A microfiuidic nucleic acid amplification system (“system’’), for example, can include a microfiuidic device and a chamber particle. The microfiuidic device can include a microfiuidic amplification region and can have a heating element positioned to heat and directly interface with a fluid when present in the microfiuidic amplification region. The chamber particle can have an external diameter of from about 10 pm to about 200 pm. The chamber particle can include chamber particle walls with dried reagent associated therewith. The chamber particle walls can define a chamber therein that can be sized to allow a plurality of nucleic acid molecules to enter the chamber, in an example, the microfiuidic amplification region can have a volume for containing the fluid that can range from about ten times to about one-thousand times the size of the chamber particle by volume. In another example, the dried reagent can be covalently conjugated or lyophiiized on the chamber particle walls. The dried reagent can include primer, polymerase enzyme, dNTP, cofactor, intercalating dye, TaqMan probe, or a combination thereof. In yet another example, the chamber particle can be magnetic. In a further example, the system can further include a water-immiscible fluid to load into the microfiuidic amplification region as a carrier fluid for the chamber particle. The water-immiscible fluid can include a C5 to a C18 hydrocarbon, a f!uorinated hydrocarbon, a hydrocarbon acid, fatty acid, fatty acid ester, mineral oil, silicone oil, or an admixture thereof. In one example, the heating element can include a thin film foil, a metal film, wire array, mesh, or a perforated metal film. In another example, the system can further include an illumination source positioned or positionable to emit light into the microfluidic amplification region, and an optical detector positioned or positionable to receive and detect light emitted from the illumination source through the microfluidic amplification region. In yet another example, the system can further include an optical filter to block light emitted from the illumination source having a first wavelength outside of a detection range of the optical detector and to pass light from the illumination source having a second wavelength within the detection range of the optical detector, wherein the light emitted from the illumination source and the light received by the optical detector are wavelength-shifted, in one example, the microfluidic amplification region can be partially defined by an optically clear wail relative to a second wavelength within the detection range and can also be partially defined by a diffusing wall including light diffusing material. In another example, the illumination source and the optical detector can both be arranged to face into the microfluidic amplification region and can also face at a right angle with respect to one another, in the same direction as one another, or towards one another. In yet another example, the system can further include a dichroic mirror to bend light toward the optical detector.

[0008] Also presented herein is a method of detecting nucleic acid amplification. The method can include loading water or an aqueous media and a water-immiscible fluid into a microfluidic amplification region of a microfluidic device to create a waveguide interface. The microfluidic amplification region can include a heating element positioned to heat and directly interface with the water-immiscible fluid when present in the microfluidic amplification region. The method can further include loading a sample fluid including nucleic acid molecules into the microfluidic amplification region to form an amplification fluid that can include the sample fluid and the water-immiscible fluid, and loading a chamber particle into the microfluidic amplification region, where the chamber particle has a particle size from about 10 pm to about 300 pm. The chamber particle can include chamber particle walls with dried reagent associated therewith, where the chamber particle walls can define a chamber therein that can be sized to allow a plurality of nucleic acid molecules to enter the chamber. The method can further include thermally cycling the heating element to a heating temperature ranging from about 75 °C to about 100 °C and cooling to a cooling temperature ranging from about 40 °C to about 70 °C, where a temperature differential when cycling between the heating temperature and the cooling temperature can be from about 20 °C to about 60 °C; illuminating the amplification fluid which can have the chamber particle dispersed therein with light at a location within the microfluidic amplification region; and optically detecting luminescence upon interaction with nucleic acid molecules after the light passes through the microfluidic amplification region, in another example, the method can include loading a plurality of chamber particles that can independently have an affinity to interact with different nucleic acid molecules. The individual chamber particles can be independently barcoded with a marker selected from a fluorescent marker, an absorbent marker, a Raman marker, an infrared marker, or a combination thereof, and optically detecting can include distinguishing between the independently barcoded marker.

[0009] Also presented herein is a material set. The material set can include a water-immiscible fluid and a chamber particle. The water-immiscible fluid can be selected from a C5 to a C18 hydrocarbon, a f!uorinated hydrocarbon, a hydrocarbon acid, fatty acid, fatty acid ester, mineral oil, silicone oil, or an admixture thereof. The chamber particle can have a particle size from about 10 pm to about 300 μm. The chamber particle can include chamber particle walis sized to allow a plurality of nucleic acid molecules to enter the chamber. The chamber particle walls can further include dried reagent covalently conjugated or lyophiilzed thereon. The dried reagent can include primer, polymerase enzyme, dNTP, cofactor, intercalating dye, TaqMan probe, or a combination thereof, in one example, the chamber particle can be magnetic.

[0010] It is also noted that when discussing the microfiuidic nucleic acid amplification system, the method of detecting nucleic acid amplification, or the material set, such discussions of one example are to be considered applicable to the other examples, whether or not they are explicitly discussed in the context of that example. Thus, in discussing a chamber particle in the context of the microfluidic nucleic acid amplification system, such disclosure is also relevant to and directly supported in the context of the method of detecting nucleic acid amplification, the material set, and vice versa,

[0011 ] T urning now to the FIGS, for further detail, as an initial matter, there are several components of the microfluidic nucleic acid amplification systems shown that are common to multiple examples, and thus, the common reference numerals are used to describe various features. Thus, a general description of a feature in the context of a specific FIG. can be relevant to the other example FIGS, shown, and as a result, individual components need not be described and then re-described in context of another figure, in the following example descriptions, FIGS. 1-4 can be considered simultaneously in the description of the FIGS, to the extent relevant by a common reference numeral, for example.

Microfluidic Nucleic Acid Amplification Systems

[0012] A microfluidic nucleic acid amplification system is illustrated in FIG. 1. The microfluidic nucleic acid amplification system can include a microfluidic device 110 that can include a microfluidic amplification region 120 and a heating element 130 positioned to heat and directly interface with a fluid when present in the microfluidic amplification region. The system can further include a chamber particle 150 that can have an external diameter of from about 10 pm to about 200 pm. The chamber particle can include chamber particle walls 152 with a dried reagent (not shown) associated therewith. The dried reagent may be associated with the interior of the chamber particle walls, the exterior of the chamber particle walls, or a combination thereof. The chamber particle walls can define a chamber 154 that can be sized to allow a plurality of nucleic acid molecules to enter the chamber.

[0013] The microfluidic device, in further detail, can include a microfluidic amplification region that can be shaped and/or configured to receive fluid and chamber particles. The microfluidic amplification region can be an area in a microfluidic channel, a conical chamber, a cylindrical chamber, a cubed chamber, a polygonal prism chamber, or the like. The microfluidic amplification region can be a U-shape or V-sbape cut-out in a substrate. An interior area of the microfluidic amplification region is not particularly limited; however, the interior area can hold a volume of fluid and the chamber particles. In some examples, the microfluidic amplification region can have a volume for containing a fluid that can range from about ten times to about one-thousand times the size of the chamber particle by volume. In yet other examples, the microfluidic amplification region can have a volume for containing a fluid that can range from about ten times to about five-hundred times the size of the chamber particle by volume, in another example, the microfluidic amplification region can have a volume for containing a fluid that can range from about fifty times to about two-hundred and fifty times the size of the chamber particle by volume, in an example, the interior area of the microfluidic amplification region can have a diameter at the widest cross-section that can range from about 100 pm to about 5 mm, from about 100 pm to about 500 pm, from about 500 pm to about 3 mm, from about 1 mm to about 2 mm, from about 2 mm to about 3 mm, or from about 3 mm to about 5 mm. A volume of the interior area of the microfluidic amplification region can range from about 5 pL to about 500 pL, from about 5 pL to about 10 pL, from about 5 pL to about 75 pL, from about 30 pL to about 80 pL, from about 50 pL to about 150 pL, from about 150 pL to about 500 pL, from about 200 pL to about 400 pL, or from about 300 pL to about 500 pL.

[0014] The microfiuidic amplification region can be formed in a substrate. A material of the substrate can include glass, silicon, polydimethylsiloxane (PDM8), polystyrene, polycarbonate, po!ymethyl methacrylate, poly-ethylene glycol diacryiate, perflouroaloxy, fluorinated ethyienepropylene, polyfiuoropolyether diol methacrylate, polyurethane, cyclic olefin polymer, teflon, copolymers, and combinations thereof. In one example, the material can include a hydrogel, ceramic, thermoset polyester, thermoplastic polymer, or a combination thereof, in another example, the material can include silicon. In yet another example, the material can include a low-temperature co-fired ceramic.

[0015] At least one wall that forms the microfiuidic amplification region can be opticaiiy dear. The microfiuidic amplification region can be partially defined by an optically clear wall relative to a wavelength within the detection range of an optical detector, in yet other examples, the microfluidic amplification region can be partially defined by a light diffusing wall that can include a light diffusing material, in some examples a material that forms a wall of the microfluidic amplification region can be a heat diffusing wall including a heat diffusing material, in some examples, a diffusing material can form the wail of the microfluidic amplification region or can be a coating on a wail of the microfluidic amplification region. In some examples, a combination of different materials can define or coat different walls of the microfluidic amplification region. For example, the microfluidic amplification region can include an optically clear wall, a light diffusing wall, a heat diffusing wail, or a combination thereof.

[0016] In some examples, the microfluidic device can be configured to include an inlet port and an outlet port that can be fluidly connected to the microfluidic amplification region. The Inlet port and the outlet port can be used to provide fluid to (via the inlet port) and pass fluid from (via the outlet port) the microfluidic amplification region. It is noted that the terms “inlet” and “outlet” do not infer that these ports interact with the microfiuidic amplification region in one direction, though that could be the case. In some Instances, there may be occasion for the fluid to flow “backwards” or “bi-directionally,” and thus the terms “inlet port” and “outlet port” can be used because at some point during operation, these two ports act as inflow of fluid and outflow of fluid, respectively, relative to the microfiuidic amplification region.

[0017] The microfiuidic device can further include a heating element positioned to heat and directly interface with a fluid when present in the microfiuidic amplification region. The heating element may be thermally isolated and may be positioned along and form a floor of the microfiuidic amplification region. The heating element can include a resistive heating element, a field-effect transistor, a p-n junction diode, a thin film heater, a thermal diode, an indium tin oxide film, a foil metal film, a foil film with perforations, wire array, mesh, or a combination thereof, in one example, the heating element can include a resistive heating element, in another example, the heating element can include an indium tin oxide film, in one example, the heating element can include silver. In another example the heating element can include a thin film foil, a metal film, wire array, mesh, or a perforated metal film. In some examples, the heating element can include a thin film foil, or a metal film. The thin film foil or the metal film can include platinum, aluminum, copper, gold, silver, tantalum, titanium, nickel, tin, zinc, chromium, oxides, alloys, and combinations thereof. In one example, the heating element can include silver.

[0018] In one example, the heating element can be operable to permit pulsified heating of a fluid and can create a temperature wave in a fluid. The heating element can be thermally coupled to the microfluidic amplification region to heat a fluid in the microfluidic amplification region at a rate of about 10 °C/s to about 100 °C/s or from about 30 °C/s to about 100 °C/s (e.g., pulses of heat ranging in duration from the order of hundreds of nanoseconds to milliseconds), in another example, the heating element can be positioned to elevate a temperature of a fluid loaded in the microfluidic amplification region by about 10 °C to about 40 °C when pulsed on for 0.1 ps to 1 second. The heating element may be dimensionally as large as or larger in surface area than the microfluidic amplification region. In some examples, the heating element may be dimensionally as large or larger in surface area than one of the surfaces defining the microfluidic amplification region, e.g. a floor surface, a side wall surface, a celling surface, etc.

[0019] In some examples, a thermal resistive layer can be thermally coupled to the microfluidic amplification region to diffuse heat from a water-immiscible fluid in the microfluidic amplification region at a rate of 10 °C/s to 100 °C/s. in one example, the thermal resistive layer can define a portion of a boundary of the microfluidic amplification region. The thermal resistive layer can include a heat diffusing material and can be located along a side wail of the microfluidic amplification region, under the heating element, or a combination thereof. The heat diffusing material of the thermal resistive layer can include silicon dioxide, silicon nitride, non-electrically conductive oxides, nitride, ceramic materials, plastic, diamond, copper, aluminum, silicon, beryllium oxide, barium nitride, or a combination thereof. The heat diffusing layer can have an average thickness from about 1 pm to about 1 ,000 pm, but more typically from about 1 pm to about 200 mhi, from about 5 pm to about 20 pm, from about 10 pm to about 50 pm, or from about 50 pm to about 150 pm. In some examples, the heat diffusing layer can include a reflective material, such as a mirror, that can allow light to reflect or scatter off the layer.

[0020] In some examples, the microfluidic device can include multiple microfluidic amplification regions and heating elements. The additional microfluidic amplification regions can be arranged in parallel, in series, or a combination thereof. In some examples, the microfluidic device can further include integrated electrical elements. The integrated electrical elements can include circuitry, resistors, transistors, capacitors, inductors, diodes, light emitting diodes, transistors, converters, conductive wires, conductive traces, photosensitive components, thermal sensitive components, semiconductors, and the like. The integrated electrical components can be in electrical communication with circuity or other components inside or outside of the microfluidic device via a wire, a trace, a network of wires, a network of traces, an electrode, a conductive pad, and/or any other electrical communication structure that may or may not be embedded in the microfluidic device. The microfluidic detection device may be an on-chip, internally controlled, !ab-on-a-chip device.

[0021] The system can further include a chamber particle. The chamber particle can be cylinder which may include a hollow space, e.g. a chamber. An internal diameter of the chamber can range from about 1 pm to about 190 pm, from about 50 pm to about 150 pm, from about 1 pm to about 100 pm, from about 25 pm to about 75 pm, from about 100 pm to about 180 pm, from about 50 pm to about 150 pm, or from about 125 pm to about 190 pm. An external diameter of the chamber particle can range from about 10 pm to about 200 pm, from about 100 pm to about 200 pm, from about 50 pm to about 150 pm, or from about 25 pm to about 150 pm. A height of the chamber particle can be from about 0.5 times to 5 times an external diameter of the chamber particle, in yet other examples the height of the chamber particle can be from about 0.5 times to about 3 times, from about 2 times to about 4 times, or from about 3 times to about 5 times an exterior diameter of the chamber particle.

[0022] The walls of the chamber particle, e.g. chamber particle walls, may be formed of any suitable material. For example, the chamber particle walls can be formed of glass, silicate glass, crystalline silicone, poiycrystal!ine silicone, polymer, epoxy, SU8, or a combination thereof, in some examples, the wails can be formed of 8U8, The chamber particle walls may be magnetic and may include a magnetic material. For example, the chamber particle wails can include iron, iron oxide, steel, nickel, cobalt, particles thereof, or combinations thereof.

[0023] In yet other examples, the chamber particle walls can be barcoded with a marker or set of markers. The marker can be used to identify a particle type indicating the dried reagents that are associated therewith. The markers can be collectively referred to herein as a barcode. The markers may be selected from a fluorescent marker, an absorbent marker, a Raman marker, an infrared marker, colored dies, color absorbent markers, or a combination thereof. The incorporation of markers can allow for multiple dried reagents and multiple targets to be analyzed, amplified, or the like simultaneously, within a single assay.

[0024] The chamber particles can be configured as hollow particles with an opening at opposing ends of the chamber particle walls, like a straw, in yet other examples, the chamber particles can be configured as a U-shaped or V-shaped well with a single opening at one end of the chamber and can include both chamber particle walls and a chamber particle floor. An Interior of the chamber particle wails may be substantially straight or may be tapered towards the chamber particle floor or particle opening. The chamber particle can include a chamber particle floor, a chamber particle ceiling, and chamber particle walls, however, the chamber particles can be configured to allow for diffusion of nucleic acid molecules into the chamber therein.

[0025] The chamber particles can have dried reagents associated with the chamber particle walls. The dried reagents may be covalently conjugated onto the chamber particle walls. In another example, the dried reagents can be lyophiiized onto the chamber particle walls. Lyophilizing can remove the water from a reagent mixture and immobilize the reagents on the chamber particie walls while preserving the integrity of the reagents. The dried reagents can Include reagents for performing nucleic acid amplification and analysis. The reagents can be selected from master mix, amplification enzymes such as DNA polymerase, deoxynudeoside triphosphates, buffer, cofactor, primer, probe, or a combination thereof. Deoxynudeoside triphosphates can serve as the building blocks of a nucleic acid. DNA polymerase is an amplification enzyme that can cause a target segment of DNA to be replicated and assembled. Buffers may provide a suitable environment for the activity and stability of the DNA polymerase. Cofactor can be a chemical such as magnesium chloride that can activate the enzymatic activity of the DNA polymerase. Primers can be short single stranded DNA fragments that can form a complementary sequence to a target region of the DNA sample. In some examples, the dried reagents can include amplification indicators, such as a fluorescent intercalating dye. A fluorescence can increase when the dye intercalates with a nucleic acid, in one example, the dried reagents can include primer, polymerase enzyme, deoxynudeoside triphosphates, cofactor, intercalating dye, TaqMan probe, or a combination thereof.

[0026] In further examples, the chamber particles can include a delayed delivery film disposed over the dried reagents. The delayed delivery film can include sucrose, dextrose, trehalose, or an admixture thereof. In yet another example, the delayed delivery film may be a polyactide. Delayed delivery films may be used to delay solvation of the dried reagents when the chamber particles are in contact with a fluid.

[0027] In yet other examples, the system can further include a fluid stack. The fluid stack can include water or an aqueous media and a water-immiscible fluid. The aqueous media can include from about 85 wt% to about 99 wt% wafer and any combination of reagents, enzymes, buffers, sample fluid, and the like. The solutes or dispersions in the aqueous media may vary based on the reaction and the dried reagents associated with the chamber particles. The water-immiscible fluid can be any fluid capable of forming an interface with the water or the aqueous media, in some examples, the water-immiscib!e fluid can be selected from a C5 to C18 hydrocarbon, a fluorinated hydrocarbon, a hydrocarbon acid, fatty add, fatty add ester, mineral oil, silicone oil, or an admixture thereof. The fluid stack or a portion thereof, may act as a heat sink to cool the chamber particles down. The water, aqueous media, and/or water-immiscible fluid can be loaded or loadable into the microfiuidic amplification region and can act as a carrier fluid for the chamber particle, a sample, a reagent, or a combination thereof. An interface between the water or the aqueous media and the water-immiscible fluid can act as a wave guide causing light to bounce within the water-immiscible fluid in the microfiuidic amplification region. Light may pass from the water or aqueous media into the water-immiscible fluid; however, light does not pass through from the water-immiscible fluid through the interface into the water or aqueous media.

[0028] The system may also include an illumination source that can be positioned or positionabie to emit light into the microfiuidic amplification region of the microfiuidic device. The illumination source can be any light source capable of emitting light. Example Illumination sources can include an infrared light source, a near infrared light source, laser, light emitting diode, xenon arc lamp, mercury arc lamp, focused sunlight, halogen lamp, or the like. In some examples, the illumination source can emit blue light, in other examples, the illumination source can emit green light.

[0029] The system can also include an optical detector positioned or positionabie to receive and detect light emitted from the microfiuidic amplification region. The optical detector can include a pin-photodiode, an avalanche photodiode, a phototransistor, a multi-junction photodiode, a charge coupling device, a complimentary metal-oxide semiconductor, a photo-sensor, a photo-resistor, a pyroelectric detector, a thermopile, or a combination thereof. In another example, the optical detector can include a pin-photodiode. In yet another example, the optical sensor can include a multi-junction photodiode.

[0030] The optical detector can detect light that can be wavelength-shifted from the light emitted by the illumination source. For example, the illumination source can emit blue light and the optical detector can detect green light. In yet another example, the illumination source can emit green light and the optical detector can detect red light.

[0031] In some examples, the illumination source and the optical detector can be arranged to face the microfluidic amplification region. The arrangement can vary; however, the optical detector can be arranged to face to water-immiscible fluid. Example arrangements are shown in FIGS. 2-4. For example, the illumination source 210 and the optical detector 220 can be arranged towards one another on opposite sides of the microfluidic amplification region such that they face one another, as shown in FIG. 2. Also illustrated in FIG. 2 are an optical filter 230, a microfluidic device 110 with a heating element 130, chamber particle 150, water or an aqueous media 320, and a water-immiscible fluid 310. The water or aqueous media can become encapsulated in the chamber particle by surface tension and can remain in the chamber particle as it passes into and resides in the water-immiscible fluid. The water or aqueous media is illustrated in the chamber particle by hatching. The dashed lines in the figure illustrate light emitted from the illumination source and light emitted from the chamber particle.

[0032] In yet another example, as illustrated in FIG. 3, the illumination source and the optical detector can be arranged in the same direction as one another, on the same side of the microfluidic amplification region. In this arrangement a dichroic reflector 240 can be used to direct light emitted from the chamber particle towards the optical detector or the optical filter and the optical detector. In yet another example, as shown in FIG. 4, the illumination source can be located over the microfluidic amplification region and the optica! defector can be located along a side of the microfluidic amplification region. In yet another arrangement, which is not illustrated, the illumination source and the optical detector can be arranged at a right angle from a central point in the microfluidic amplification region with respect to one another, thereby, minimizing scattered light, in this arrangement the optical detector or the optical filter and the optical detector can be positioned along a front or rear facing wall of the microfluidic amplification region; whereas, the illumination source can be positioned along a side wall of the microfiuidic amplification region. Accordingly, light emitted from the chamber particle can exit the microfiuidic amplification region along the z axis, in yet another arrangement, also not illustrated, If the heater is semi-transparent or transparent to wavelengths detected by the optical detector (such as ITO heater) then the optical detector or the optical filter and the optical detector may be positioned beneath the microfiuidic amplification region and the illumination source may be located along a side, front, or back wall of the microfiuidic amplification region, or above the microfiuidic amplification region.

[0033] In yet other examples, the system can further include an optical filter, as mentioned in the arrangements above. The optical filter can be arranged between the microfiuidic amplification region and the optica! detector. The optica! filter can be operable to block light emitted outside of a wavelength in the detection range for the optical detector and can allow light having a wavelength in the detection range of the optical detector to pass therethrough. For example, the optical filter can block light emitted from the illumination source having a first wavelength outside of the detection range of the optical detector and can allow light having a second wavelength within the detection range of the optica! detector to pass therethrough. In some examples, the optical filter can reflect or absorb and contain wavelengths ranging from about 350 nm to about 700 nm, from about 350 nm to about 510 nm, or from about 560 nm to about 700 nm and can transmit wavelengths from about 510 nm to about 560 nm. In another example, the optical filter can reflect or absorb ail wavelengths of light of less than about 510 nm.

[0034] The optica! filter can be selected from a dichroic filter, absorptive filter, monochromatic filter, bandpass filter, Fabry-Perot etaion, antirefiective coating, bandstop filter, or a combination thereof. In some examples, the optical filter can be selected from a dichroic filter, a bandpass filter, or a bandstop filter, in yet another example, the optica! filter can include a dichroic filter,

[0035] When the optical filter is a dichroic filter, the dichroic filter can include alternating material layers of optically transparent materials, in some examples, the dichroic filter can include from 4 to 250 material layers, from 6 to 200 material layers, from 10 to 100 material layers, from 10 to 50 material layers, from 10 to 20 material layers, from 4 to 40 materia! layers, or from 4 to 20 material layers. The alternating material layers can include different optically transparent materials. When there are more than two “alternating” material layers, what Is meant is that the same layer is not applied twice, but does not infer that the multiple layers be applied sequentially and in an alternating manner, though they may be applied sequentially and repetitively. The optically transparent materials can be chosen for their optical properties, structural properties, chemical properties, or a combination thereof, for example. In an example, the optically transparent materials can be selected from titanium dioxide, zirconium oxide, hafnium oxide, aluminum oxide, indium oxide, tin (IV) oxide, tantalum oxide, silicon carbide, silicon dioxide, silicon nitride, titanium nitride, or a combination thereof.

[0036] In a further example, the system can include a dichroic reflector such as a dichroic mirror to reflect and direct light. The dichroic reflector can be used to bend and direct light exiting the microfluidic amplification region towards an optical detector.

[0037] In yet another example, the system can further include a magnetic field generator that can generate a magnetic field for moving chamber particles that are magnetic. In some examples, the magnetic field generator can be a magnet, a ring magnet, or a current carrying wire. Applying the magnetic field, magnetic field motion, and/or differing magnetic field gradients can attract chamber particles that are magnetic. The magnetic field may be turned on and off by introducing electrical current/voltage to the magnetic field generator. The magnetic field generator can be permanently placed, can be movable along the microfluidic device or can be movable in position and/or out of position to effect movement of the chamber particles. The magnetic field generator may create a force capable of pulling the chamber particles downward in the microfiuidic amplification region toward a floor surface and the heating element of the microfiuidic device. [0038] The microfluidic systems presented herein can be utilized for fluorescing biological assays. Examples of fluorescing biological assays can include nucleic acid micro-assays, bio-sensing assays, cell assays, PCR, drug delivery research, energy transfer-based assays, fluorescence in situ hybridization (FISH), fluorescent reporter assays, fluorescent spectroscopy, quantum dot detection of cancer markers/cells, detection of reaction oxygen species, protein interactions, prion research, detection of viral antigens, detection of pathogens, detection of toxins, protein/immunological assays, chemi-fluorescent enzyme-linked immunosorbent assays (ELISA), antibody micro-assays, protein micro-assays, giycin e/lectin assays, and the like for example. In some examples, the microfiuidic system can be configured as a micro-reactor assembly. For example, the microfiuidic system can be configured as a PCR micro-reactor.

Material Sets

[0039] Also presented herein, are material sets that can be used with the microfiuidic device described above and/or as part of the method of detecting nucleic acid amplification herein. The material set can include a water-immiscible fluid and a chamber particle. The water-immiscible fluid can be selected from a C5 to a C18 hydrocarbon, a fluorinated hydrocarbon, a hydrocarbon acid, fatty acid, fatty acid ester, mineral oil, silicone oil, or an admixture thereof. The water-immiscibie fluid, in some examples, can be a C5 to a C18 hydrocarbon such as pentanes, hexane, octane, decane, dodecane, tetradecane, hexadecane, or a combination thereof. In another example, the water-immiscibie fluid can be a hydrocarbon acid such as oleaic add, silicone oil, immiscible engineered oils, or a combination thereof. Engineered oils can include methoxy-nonafluorobutane, segregated hydrofiuoroether, ethoxy dodecafluoro trifiuoromethyl-hexane, perfluorocarbon, fluorocarbon, or an admixture thereof. Examples of commercially available engineered oils can include FC-40, FC-75, Novec™ HF E7100, Novec™ HFE7300, Novec™ HFE7500, or a combination thereof (all available from 3M™, USA). The material set can also include a chamber particle. The chamber particle may be as described above.

Methods of Detecting Nucleic Acid Amplification

[0040] In accordance with yet other examples, as shown in FIG. 5, a method of detecting nucleic acid amplification is presented 500. The method can include loading 510 water or an aqueous media as well as a water-immiscible fluid to form a fluid stack having a waveguide interface into a microfiuidic amplification region of a microfiuidic device. The waveguide interface can occur where the water or aqueous media interfaces with the water-immiscible fluid, for example. The microfiuidic amplification region can include a heating element positioned to heat and directly interface with a fluid when present in the microfiuidic amplification region. The method can further include loading 520 a sample fluid including nucleic acid molecules info the microfiuidic amplification region and loading 530 a chamber particle into the microfiuidic amplification region. The chamber particle can have an external diameter of from about 10 pm to about 200 pm and can include chamber particle walls with dried reagent associated therewith. The chamber particle walls can define the chamber therein that can be sized to allow a plurality of nucleic acid molecules to enter the chamber. The nucleic acid molecules can enter the chamber particle as the chamber particle passes from the water or aqueous media into the water-immiscible fluid.

[0041] The method can further include thermally cycling 540 the heating element to a heating temperature ranging from about 75 °C to about 100 °C and cooling to a cooling temperature ranging from about 40 °C to about 70 °C, where a temperature differential when cycling between the heating temperature and the cooling temperature can be from about 20 °C to about 60 °C. The method can also include illuminating 550 the amplification fluid having the chamber particle dispersed therein with light at a location within the microfiuidic amplification region and optically detecting 560 luminescence upon interaction with nucleic acid molecules after the light passes through the microfiuidic amplification region.

[0042] The loading, in further detail, can include placing the water or aqueous media, the water-immiscible fluid, the sample fluid, and the chamber particles into the microfluidic amplification region. The loading, in an example, can include first loading the water-immiscible fluid, followed by the water or aqueous media, the sample fluid, and then the chamber particles into the microfluidic amplification region, such that the water-immiscible fluid resides adjacent to the heating element of the microfluidic device and the water or aqueous media resides above the water-immiscible fluid. In yet other examples, the loading can be simultaneous in that the water or aqueous media, the water-immiscible fluid, the sample fluid, the chamber particles, or the combination thereof may be combined in a secondary vessel before loading them in the microfluidic amplification region. However, if the loading occurs simultaneously, a density of the water-immiscible fluid and the water or aqueous media can cause segregation and arrangement such that the water-immiscible fluid resides ciosest to the heating element in the microfluidic amplification region.

[0043] Upon loading, the chamber particles can pass through water or an aqueous media. Nucleic acid moiecu!es can enter the chamber particles in the water or the aqueous media. The chamber particles can pass from the water or the aqueous media into the water-immiscible fluid. A portion of the water or aqueous media can become trapped in the chamber particles as they pass due to surface tensions. The passing of the chamber particles from the water or the aqueous media into the water-immiscible fluid can occur by gravity or some other force such as centrifugal force or magnetic force. Once the chamber particles are in the water-immiscible fluid, thermal cycling can occur.

[0044] Thermal cycling of the heating element, in further detail, can include alternating heating and cooling of the water-immiscible fluid. The thermal cycling may include turning the heating element on and off. In one example, the thermal cycling involves elevating a temperature of the water-immiscible fluid to a heating temperature ranging from about 75 °C to about 100 °C when pulsed on for 0.1 ps to 10 ps and diffusing heat from the water-immiscible fluid. The diffusing can occur due to the water-immiscible fluid and/or the water or aqueous media acting as a heat sink, a thermal resistive layer along a portion of the microfiuidic amplification region, the heating element being turned off and atmospheric conditions, ora combination thereof. The thermal cycling can include allowing the water-immiscible fluid to cool to a cooling temperature ranging from about 40 °C to about 70 °C. A temperature differential between heating and cooling when thermal cycling can be from about 20 °C to about 60 °C. For example, the heating can be to a heating temperature of about 90 °C to about 100 °C and the cooling can be to a temperature of about 50 °C to about 60 °C. A time period between pulses can be from about 1 ps to about 100 ms. Thermal cycling temperatures and duration may depend on the component of interest in the sample fluid.

[0045] The method can further include illuminating the amplification fluid (the sample fluid and the water-immiscible fluid) using an illumination source, as described above, and optically detecting luminescence upon interaction with nucleic acid molecules. Optically detecting can include positioning and reading an optical detector.

[0046] In some examples, the loading can include loading a plurality of chamber particles which can independently have an affinity to interact with different nucleic acid molecules into the microfluidic amplification region. The individual chamber particles can be independently barcoded with a marker selected from a fluorescent marker, an absorbent marker, a Raman marker, an infrared marker, or a combination thereof. The barcoded markers can correspond with the affinity of the chamber particles. The method can further include optically detecting the barcoded markers to distinguish between the independently barcoded markers of the chamber particles.

[0047] In yet other examples, the chamber particles can be magnetic and the method can further include pulling the chamber particles from the water or aqueous media into and through the water-immiscible fluid towards the heating element of the microfluidic device. The pulling of the chamber particles can occur by creating a magnetic field that can draw the chamber particles using a magnetic field generator. ί Definitions

[0048] It is noted that, as used in this specification and the appended claims, the singular forms ”a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0049] The term "about" as used herein, when referring to a numerical value or range, allows for a degree of variability in the value or range, for example, within 10%, or, in one aspect within 5%, of a stated value or of a stated limit of a range. The term “about” when modifying a numerical range is also understood to include as one numerical subrange a range defined by the exact numerical value indicated, e.g., the range of about 1 wt% to about 5 wt% includes 1 wt% to 5 wt% as an explicitly supported sub-range.

[0050] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though the individual member of the list is identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on presentation in a common group without indications to the contrary.

[0051] Concentrations, amounts, and other numericai data may be expressed or presented herein in a range format. A range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include individual numerical values or sub-ranges encompassed within that range as if numericai values and sub-ranges are explicitly recited. As an illustration, a numericai range of “1 wt% to 5 wt%” should be interpreted to Include not only the explicitly recited values of about 1 wt% to about 5 wt%, but also to include individual values and sub-ranges within the indicated range. Thus, included in this numericai range are individual values such as 2, 3.5, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc. This same principle applies to ranges reciting one numerical value. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described. EXAMPLE

[0052] The foliowing illustrates an example of the present disclosure. However, it is to be understood that the following is illustrative of the application of the principles of the present disclosure. Numerous modifications and alternative compositions, methods, and systems may be devised without departing from the present disclosure.

Nucleic Acid Amplification and Detection

[0053] A microfluidic nucleic acid amplification system as described herein is obtained. A nasopharyngeal swab is collected and admixed with an XpressAMP™ lysis buffer, available from Promega Corporation, USA, and incubated at room temperature for ten minutes to prepare the sample. The sample is admixed with XpressAMP™ Solution, also available from Promega Corporation, USA, to form a sample fluid. 100 pL of silicon oil, a water-immiscible fluid, is added to the microfluidic amplification region of a microfluidic device. The sample fluid is loaded over the water-immiscible fluid. Chamber particles with dried reagents including primer, polymerase enzyme, dNTP, cofactor, intercalating dye, and TaqMan probe are loaded into the sample fluid. The chamber particles are allowed to settle into the water-immiscible fluid. The heating element of the microfluidic device is used to heat the water-immiscible fluid to about 94 °C for 100 milliseconds cycled off and the water-immiscible fluid is allowed to cool to about 58 °C over a period of 400 milliseconds. The heating and cooling is repeated. An illumination source is used to illuminate the microfluidic amplification region and an optical detector is used to detect the amplification.