WO2022186818A1

WO2022186818A1 - Chamber particles for nucleic acid processing

Info

Publication number: WO2022186818A1
Application number: PCT/US2021/020379
Authority: WO
Inventors: Viktor Shkolnikov; Alexander Govyadinov
Original assignee: Hewlett-Packard Development Company, L.P.
Priority date: 2021-03-02
Filing date: 2021-03-02
Publication date: 2022-09-09

Abstract

The present disclosure is drawn to a chamber particle for nucleic acid processing. The chamber particle can include a chamber defined by chamber particle walls and sized to allow a plurality of nucleic acid molecules to enter the chamber and dried reagents immobilized on the chamber particle walls. The dried reagents can include a nuclease enzyme.

Description

CHAMBER PARTICLES FOR NUCLEIC ACID PROCESSING BACKGROUND [0001] Nucleic acid processing, including but not limited to, amplification and detection are techniques utilized in research, medical diagnostics, forensic testing, and food chemistry. The ability to detect, target, process, and/or amplify a small quantity of a nucleic acid from a sample can permit research, medical diagnostic, forensic, and food product reliability testing that would not otherwise be permissible. BRIEF DESCRIPTION OF THE DRAWINGS [0002] FIG.1 graphically illustrates a perspective view of an example chamber particle in accordance with the present disclosure; [0003] FIG.2 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0004] FIG.3 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0005] FIG.4 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0006] FIG.5 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0007] FIG.6 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0008] FIG.7 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0009] FIG.8 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0010] FIG.9 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0011] FIG.10 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0012] FIG.11 graphically illustrates a cross-section of an example chamber particle in accordance with the present disclosure; [0013] FIG.12 graphically illustrates an example microfluidic nucleic acid processing system in accordance with the present disclosure; [0014] FIG.13 graphically illustrates an example microfluidic nucleic acid processing system in accordance with the present disclosure; [0015] FIG.14 graphically illustrates an example microfluidic nucleic acid processing system in accordance with the present disclosure; and [0016] FIG.15 is a flow diagram illustrating an example method of detecting a nucleic acid in accordance with the present disclosure. DETAILED DESCRIPTION [0017] Nucleic acid processing and testing allows for the detection and identification of a particular nucleic acid and/or the amplification of the nucleic acid. Nucleic acid detection can allow for early diagnosis, evaluation of food quality, identity testing, and the like. Amplification can include denaturing, annealing, and extending nucleic acid chains. During denaturing, hydrogen bonds between bases in a double-stranded nucleic acid sample can break apart resulting in two single strands realized from a formerly double-stranded nucleic acid. During annealing, single stranded nucleic acid oligomers, such as primers, can attach to the complimentary nitrogen bases on the single strands of the nucleic acid. During extending of the nucleic acid chain, a polymerase enzyme to extend the nucleic acid strand can be by adding nucleic acid bases. Amplification can result in multiple strands of a sample which can allow for additional testing and can increase detection limits. [0018] The present disclosure is drawn to a chamber particle for nucleic acid processing. The chamber particle can include a chamber that can be defined by chamber particle walls and sized to allow a plurality of nucleic acid molecules to enter the chamber. The chamber particle can further include dried reagents immobilized on the chamber particle walls, where the dried reagents can include a nuclease enzyme. In one example, the dried reagents can be covalently conjugated or lyophilized on the chamber particle walls. In another example, the nuclease enzyme can include a cas-enzyme that can be selected from families of Cas9 enzymes, Cas12 enzymes, Cas13 enzymes, Cas14 enzymes, or a combination thereof. In yet another example, the dried reagents can further include a reporter nucleic acid, guide RNA, an indicator nucleic acid, a nuclease enzyme, a master mix, a buffer salt, or a combination thereof. In a further example, the reporter nucleic acid can be present and can include a fluorophore and a quencher at opposing ends. In an example, the chamber particle can further include a delayed delivery film disposed over the dried reagents. In yet another example, the chamber particle can be in the shape of a hollow cylinder and an internal diameter of the chamber can range from about 1 μm to about 190 μm. In a further example, the chamber particle walls can include a label including a set of markers selected from fluorescent markers, absorbent markers, Raman markers, infrared markers, colored dyes, color absorbent markers, or a combination thereof. [0019] Also presented herein, is a microfluidic nucleic acid processing system. The system can include microfluidics, chamber particles, and an optical detection device. The microfluidics can include a microfluidic processing region and a microfluidic detection region fluidly coupled to the microfluidic processing region. The chamber particles can be loaded or loadable in the microfluidic processing region. The chamber particles can include chamber particle walls that can define a chamber that can be sized to allow a plurality of nucleic acid molecules to enter the chamber. The chamber particle walls can include dried reagents including a nuclease enzyme immobilized thereon. The optical detection device can be optically coupled to the microfluidic detection region to detect the nucleic acid molecules. In an example, a fluid to be introduced into the microfluidics can include a water immiscible fluid. In another example, the system can include a second microfluidic processing region with a second plurality of chamber particles loaded or loadable therein. The second plurality of chamber particles can have a different loading of dried reagents thereon. In yet another example, the microfluidic processing region can be a microfluidic amplification region. The microfluidic amplification region can include a heating element positioned to heat cycle a fluid containing the chamber particles loaded with nucleic acid molecules. [0020] Further presented herein, is a method of detecting a nucleic acid. The method can include loading a microfluidic processing region of a microfluidic nucleic acid processing system with chamber particles. The chamber particles can include a chamber sized to allow nucleic acid molecules to enter the chamber and chamber particle walls with dried reagents including a nuclease enzyme associated therewith. The method can further include introducing a sample fluid to the microfluidic processing region, where the sample fluid provides nucleic acid molecules that can enter the chamber with the chamber particles, processing the nucleic acid molecules carried by the chamber particles, flowing the chamber particles carrying processed nucleic acid molecules to a microfluidic detection region, and optically detecting the processed nucleic acid molecules. In one example, the sample fluid and the chamber particles carrying the nucleic acid molecules can be present in a water-immiscible carrier fluid, and the processing of the nucleic acid molecules can include amplifying the nucleic acid molecules with isothermal amplification at a temperature ranging from about 30 °C to about 45 °C. In yet another example, the dried reagents can further include reporter nucleic acid, and the method can include detecting cleavage of the reporter nucleic acid by a fluorescence from labeled markers. [0021] It is also noted that when discussing the chamber particle for nucleic acid processing, the microfluidic nucleic acid processing system, and the method of detecting a nucleic acid, 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 dried reagents in the context of the chamber particle for nucleic acid processing, such disclosure is also relevant to and directly supported in the context of the microfluidic nucleic acid processing system, the method of detecting a nucleic acid, and vice versa. [0022] Turning now to the FIGS. for further detail, as an initial matter, there are several components of the chamber particles for nucleic acid processing 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.1-14 can be considered simultaneously in the description of the FIGS. to the extent relevant by a common reference numeral, for example. Chamber Particles for Nucleic Acid Processing [0023] A chamber particle for nucleic acid processing is illustrated in FIG. 1. The chamber particle 100 can be defined by chamber particle walls 102 and sized to allow a plurality of nucleic acid molecules to enter the chamber 108. Dried reagents, not illustrated, can be immobilized on a surface of the chamber particle walls. The dried reagents may include a nuclease enzyme. [0024] The chamber particle, in further detail, can be a cylinder, cube, cone, sphere, or polygonal prism which may include a hollow space, e.g. a chamber. In an example, the chamber particle can include a cylinder. In another example, the chamber particle can include a sphere. In yet another example, the chamber particle can include a polygonal prism. The chamber particle may be configured as a hollow particle 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 well or a 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 walls may be substantially straight or may be tapered towards the chamber particle floor or particle opening. In some examples, 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. Example chamber particle configurations are illustrated in cross-sectional views in FIGS.2-12. As shown in FIGS.2-12, a chamber particle 100 can have a configuration that may include chamber particle walls 102 with dried reagent(s) therein. In some examples, there may also be a chamber particle floor 104, chamber particle ceiling 106, a delayed delivery film 130, or a combination thereof. In some examples, the delayed delivery film can form an enclosure preventing fluid from entering the chamber of the chamber particles until dissolution thereof, as illustrated in FIG.12. [0025] A structural surface of the chamber particle, e.g. chamber particle walls, chamber particle floor, and/or chamber particle ceiling, may be formed of any suitable material. For example, a structural surface of the chamber particles can be formed of glass, silicate glass, crystalline silicone, polycrystalline silicone, polymer, epoxy, SU8, or a combination thereof. In some examples, the structural surface can be formed of SU8. The chamber particle floor, the chamber particle ceiling, or a combination thereof may be formed of the same material or a different material than the chamber particle walls. In other examples a structural surface of the chamber particles may be magnetic and may include a magnetic material. For example, the chamber particles can include iron, iron oxide, steel, nickel, cobalt, particles thereof, or combinations thereof. [0026] An internal diameter of the chamber can range from about 1 μm to about 190 μm, from about 50 μm to about 150 μm, from about 1 μm to about 100 μm, from about 25 μm to about 75 μm, from about 100 μm to about 180 μm, from about 50 μm to about 150 μm, or from about 125 μm to about 190 μm. An external diameter of the chamber particle can range from about 10 μm to about 200 μm, from about 100 μm to about 200 μm, from about 50 μm to about 150 μm, or from about 25 μm to about 150 μm. 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. [0027] In yet other examples, the chamber particle walls can be labeled with a marker or set of markers. The markers 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 label. 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. [0028] The chamber particles can have dried reagents associated with the chamber particle walls, the chamber particle floor, the chamber particle ceiling, or a combination thereof. 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 dried reagents may be covalently conjugated onto the chamber particle walls. In another example, the dried reagents can be lyophilized onto the chamber particle walls. Lyophilizing can remove the water from a reagent mixture and immobilize the reagents on the chamber particle walls while preserving the integrity of the reagents. [0029] The dried reagents can include nuclease enzymes. Nuclease enzymes, such as cas-enzymes are proteins which cut and cleave nucleic acids at a specific location. Cas-enzymes can induce target specific, site-directed double strand breaks in nucleic acids. The cleavage can allow for the integration of a spacer crRNA to a nucleic acid target sequence’s protospacer. A double stranded nucleic acid can unwind in the area of the integration. The cas-enzymes of the dried reagents, in an example, can be selected from families of Cas9 enzymes, Cas12 enzymes, Cas13 enzymes, Cas14 enzymes, or a combination thereof. In an example, the cas-enzymes can include Cas12-detector, Cas14-detector, Cas9-flash, Cas13-sherlock, or a combination thereof. [0030] The chamber particles can include further dried reagents. The additional dried reagents, in an example, can include guide RNA, guide crRNA, transactivating crRNA, tracrRNA, enzymes, and the like. In some examples, the dried reagents can include labeling indicators, such as a fluorescent intercalating dye. Fluorescence can increase when the dye intercalates with a nucleic acid. In yet other examples, the dried reagents can include a fluorophore and a quencher at opposing ends of a reporter nucleic acid. When intact, the quencher can quench fluorescence of the fluorophore and when cleaved, the fluorophore and the quencher can separate therefore allowing the fluorophore to fluorescence. [0031] In yet other examples, 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, deoxynucleoside triphosphates, buffer, cofactor, primer, probe, or a combination thereof. Deoxynucleoside 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. Cofactors 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 one example, the dried reagents can include primer, polymerase enzyme, deoxynucleoside triphosphates, cofactor, intercalating dye, TaqMan probe, or a combination thereof. In another example, the dried reagents can further include a reporter nucleic acid, guide RNA, an indicator nucleic acid, enzyme, a master mix, a buffer salt, or a combination thereof. [0032] The chamber particles can include a delayed delivery film disposed over the dried reagents to block an opening of hollow chamber particles. Delayed delivery films may be used to delay solvation of the dried reagents when the chamber particles are in contact with a fluid. A thickness of the delayed delivery film can vary a release time of the dried reagents into a fluid. In some examples, the delayed delivery film can be composed of several layers which can alternate with specific dried reagents. For example, the delayed delivery film can include a first layer that can cover a nuclease enzyme with guide RNA and a second layer that can cover reagents needed for nucleic acid amplification. This can allow for separate delivery of the nuclease enzymes from reagents for amplification. 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. Microfluidic Nucleic Acid Processing System [0033] In some examples, the chamber particles can be part of a microfluidic nucleic acid processing system. A microfluidic nucleic acid processing system 200, as illustrated in FIG.13, can include microfluidics including a microfluidic processing region 210, and a microfluidic detection region 220 that can be fluidly coupled to the microfluidic processing region. The chamber particles 100 may be loaded or loadable into the microfluidic processing region and can be as described above. The system can further include an optical detection device 300 that can be optically coupled to the microfluidic detection region to detect the nucleic acid molecules. [0034] The microfluidics can be formed in a substrate. A material of the substrate can include glass, silicon, polydimethylsiloxane (PDMS), polystyrene, polycarbonate, polymethyl methacrylate, poly-ethylene glycol diacrylate, perflouroaloxy, fluorinated ethylenepropylene, polyfluoropolyether 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. [0035] The microfluidic processing region, the microfluidic detection region, or the combination thereof can be shaped and/or configured to receive fluid and chamber particles. The microfluidic processing region, the microfluidic detection region, or the combination thereof 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 processing region, the microfluidic detection region, or the combination thereof can include an opening in a form of a U-shape or V-shape cut-out in a substrate. An opening of the microfluidic processing region, the microfluidic detection region, or the combination thereof is not particularly limited, however, the opening can hold a volume of fluid and the chamber particles. In some examples, the microfluidic processing region, the microfluidic detection region, or the combination thereof can have an opening for containing a volume of fluid that can range from about 100 times to about 10¹¹ times the size of the chamber particle by volume. [0036] In an example, the opening of the microfluidic processing region, the microfluidic detection region, or the combination thereof can have a diameter at the widest cross-section that can range from about 100 μm to about 5 mm, from about 100 μm to about 500 μm, from about 500 μm 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 fluid that can be contained in the opening can range from about 1 nL to about 500 μL, from about 1 nL to about 100 nL, from about 100 nL to about 5 μL, from about 5 μL to about 500 μL, from about 5 μL to about 10 μL, from about 5 μL to about 75 μL, from about 30 μL to about 60 μL, from about 50 μL to about 150 μL, from about 150 μL to about 500 μL, from about 200 μL to about 400 μL, or from about 300 μL to about 500 μL. [0037] The microfluidic processing region, in further detail, can be an area where nucleic acids can be allowed to enter the chamber particles and processing of a sample can occur therein. The processing may vary depending on the dried reagents and the purpose of the nucleic acid detection and/or amplification. The microfluidic processing region can, in some examples, be configured as a series of microfluidic processing regions for subsequent processing of a sample. For example, there can be several microfluidic processing regions such as separate chambers or designated sections in a microfluidic flow channel. The microfluidic processing regions can be arranged in parallel, in series, or a combination thereof. In some examples, the microfluidic processing regions can be loaded or loadable with different types of chamber particles in individual microfluidic processing regions. The chamber particles can be different in that they can include different concentrations of dried reagents, different dried reagents associated therewith, or a combination thereof. In one example, the system can further include a second microfluidic processing region with a second plurality of chamber particles loaded or loadable therein, wherein the second plurality of chamber particles can have a different loading of dried reagents thereon. In one example, the different chamber particles in different microfluidic processing regions can have different dried reagents thereon, and individual areas may be designed to interact with a sample fluid in a different manner. For example, the chamber particles may be arranged in separate microfluidic processing regions to permit multi-step nucleic acid detection and/or analysis. In yet other examples, the chamber particles can be arranged in separate microfluidic processing regions to permit a different set of assays to occur in the areas. [0038] For example, as shown in FIG.14, the microfluidic nucleic acid processing system 200 can include a microchannel 202 connecting areas 212 and 214 of the microfluidic processing region 210. The individual areas can include chamber particles 100A and 100B having different dried reagents immobilized on the chamber particle walls. A sample fluid can pass through areas in the microfluidic processing region before passing into a fluidically coupled microfluidic detection region 220. In some examples, a heating element 230 can be used to permit nucleic acid amplification. The system can further include a water-immiscible fluid inlet 250 fluidically connected to the microchannel feeding the microfluidic detection region that can permit a water-immiscible fluid to be loaded therein downstream of the microfluidic processing region. [0039] The microfluidic detection region, in further detail, can include a wall or multiple walls that can be optically clear to a wavelength within the detection range of the optical detection device. The microfluidic detection region may also be partially defined by a light diffusing wall that can include a light diffusing material. In some examples, a combination of different materials can define or coat different walls of the microfluidic detection region. For example, the microfluidic detection region can include an optically clear wall, a light diffusing wall, a heat diffusing wall, or a combination thereof. [0040] In some examples, the microfluidics can be part of a microfluidic device and the microfluidic device can be configured to include an inlet port, an outlet port, or a combination thereof. The inlet port can be fluidly connected to the microfluidic processing region. The outlet port can be fluidically connected to the microfluidic detection region. In some examples, the microfluidic device can include multiple inlet ports, such as a secondary inlet port, that can permit loading of additional fluids, such as a secondary fluid, into the microfluidic device. [0041] The microfluidic device may further include a heating element positioned to heat and directly interface with the fluid containing chamber particles loaded with nucleic acids. In some examples, the heating element may be associated with the microfluidic processing region, a microfluidic channel, the microfluidic detection region, or a combination thereof. The heating element can be operable to allow for microfluidic amplification and can form a microfluidic amplification region. The heating element may be thermally isolated and 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. [0042] In some examples, the microfluidics that are part of a 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 device may also include fluid pumps operable to move a fluid and chamber particles through the device. In yet other examples, the microfluidic device may include membranes and/or gates to maintain separation between fluids in the device. The microfluidic device may be an on-chip, internally controlled, lab-on-a-chip device. In an example, the microfluidic device may be a point of care detection device. [0043] The system can also include an optical detector positioned or positionable to receive and detect light emitted in the microfluidic detection 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. [0044] In yet other examples, the system can further include an optical filter. The optical filter can be arranged to intercept light emitted from the microfluidic detection region. The optical filter can be operable to block light emitted outside of a wavelength in the detection range for the optical detection device and can allow light having a wavelength in the detection range of the optical detection device to pass therethrough. For example, the optical filter can block light having a first wavelength outside of the detection range of the optical detection device and can allow light having a second wavelength within the detection range of the optical detection device 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 all wavelengths of light of less than about 510 nm. [0045] The optical filter can be selected from a dichroic filter, absorptive filter, monochromatic filter, bandpass filter, Fabry-Perot etalon, antireflective 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 optical filter can include a dichroic filter. [0046] 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 about 4 to about 250 material layers, from about 6 to about 200 material layers, from about 10 to about 100 material layers, from about 10 to about 50 material layers, from about 10 to about 20 material layers, from about 4 to about 40 material layers, or from about 4 to about 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. [0047] In yet other examples, the system can further include a fluid. The fluid can include water, an aqueous media, a water-immiscible fluid, or a combination thereof. The aqueous media can include from about 85 wt% to about 99 wt% water 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-immiscible fluid can be selected from a C5 to C18 hydrocarbon, a fluorinated hydrocarbon, a hydrocarbon acid, fatty acid, fatty acid ester, mineral oil, silicone oil, or an admixture thereof. The water-immiscible 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-immiscible fluid can be a hydrocarbon acid such as oleaic acid, silicone oil, immiscible engineered oils, or a combination thereof. Engineered oils can include methoxy-nonafluorobutane, segregated hydrofluoroether, ethoxy dodecafluoro trifluoromethyl-hexane, perfluorocarbon, fluorocarbon, or an admixture thereof. Examples of commercially available engineered oils can include FC-40, FC-75, Novec^TM HF E7100, Novec^TM HFE7300, Novec^TM HFE7500, or a combination thereof (all available from 3M^TM, USA). The fluid may act as a heat sink to cool the chamber particles down in a microfluidic amplification region. The water, aqueous media, and/or water-immiscible fluid can be loaded or loadable into the microfluidics and can act as a carrier fluid for the chamber particle, a sample, a reagent, or a combination thereof. [0048] 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 through and from the microfluidic processing region, to and through the microfluidic detection region, or a combination thereof. [0049] 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, low temperature amplification assays, 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, glycine/lectin assays, and the like for example. In one example, the microfluidic system can be used for low temperature amplification assays which are compatible with the operating temperatures of nuclease enzymes, such as loop-mediated isothermal amplification, nucleic acid sequence-based amplification, helicase-dependent amplification, strand-displacement amplification, recombinase polymerase amplification, rolling circle amplification, exonuclease III-assisted signal amplification, hybridization chain reaction, or the like. Methods of Detecting Nucleic Acid Amplification [0050] Further presented herein, as illustrated in FIG.15, is a method of detecting a nucleic acid. The method 500 can include loading 510 a microfluidic processing region of a microfluidic nucleic acid processing system with chamber particles including a chamber sized to allow nucleic acid molecules to enter the chamber and chamber particle walls having dried reagents including a nuclease enzyme associated therewith; introducing 520 a sample fluid to the microfluidic processing region, where the sample fluid provides nucleic acid molecules that enter the chamber of the chamber particles; processing 530 the nucleic acid molecules carried by the chamber particles; flowing 540 the chamber particles carrying processed nucleic acid molecules to a microfluidic detection region; and optically detecting 550 the processed nucleic acid molecules. [0051] In some examples, the chamber particles can be pre-loaded into the microfluidic processing region or may be loaded into the microfluidic processing region by the end user. Loading of the chamber particles can include positioning the particles in the microfluidic processing region by dropping the chamber particles therein or by flowing the chamber particles in a fluid to the microfluidic processing region. Following the loading of the chamber particles, or simultaneously therewith, the sample fluid can be loaded into the microfluidic processing region. The sample fluid may be loaded directly into a fluid present in the microfluidic processing region, or may be loaded with a fluid. The fluid can include water, an aqueous medium, a water-immiscible fluid, or a combination thereof, as described above. [0052] Nucleic acid molecules from the sample can enter the chamber particles in a fluid. If the fluid includes the water or the aqueous media, a portion of the water or aqueous media can become trapped in the chamber particles due to surface tension. The nucleic acid molecules and the water or the aqueous media can remain trapped in the chamber particles as they enter a water-immiscible fluid, if present. [0053] The processing can vary depending on the reaction. In some examples, the processing can include initiating a nuclease reaction. The nuclease reaction can include detecting a presence of a target nucleic acid amplifying nucleic acids, inactivating interfering species, absorbing interfering species, or a combination thereof. Detecting a presence of a target nuclease can include cleaving a portion of the nucleic acid by a nuclease enzyme and detecting fluorescence. The fluorescence may occur as a reporter nucleic acid which includes a fluorophore and a quencher at opposing ends becomes cleaved such that the quencher no longer quenches fluorescence from the fluorophore. The amplification can be a low temperature amplification that works within a heating temperature of operability for the nuclease enzyme or can include higher temperature amplification if the nuclease enzyme has completed its purpose in the processing before the higher temperature is applied. Amplification can be by any of the low temperature amplification methods described above, or once the nuclease enzyme has completed its purpose, by polymerase chain reaction. In one example, the processing of the nucleic acid molecules can include amplifying the nucleic acid molecules using isothermal amplification at a temperature ranging from about 30 °C to about 45 °C. [0054] The chamber particles and nucleic acid molecules may flow to a microfluidic detection region where a luminescence may occur which can be optically detected. Optically detecting the luminescence can include positioning and reading an optical detector. In some examples, the individual chamber particles can be independently labeled with a marker selected from a fluorescent marker, an absorbent marker, a Raman marker, an infrared marker, colored dyes, color absorbent markers, or a combination thereof. The labeled markers can correspond with the affinity of the chamber particles. The optically detecting can include detecting a fluorescence of reporter nucleic acids due to a cleavage and correlating the detected fluorescence from labeled markers. [0055] In yet other examples, the chamber particles can be magnetic and the method can further include pulling the chamber particles through the microfluidics from the microfluidic processing region to the microfluidic detection region. In some examples, the pulling can include moving the chamber particles from the water or aqueous media into and through the water-immiscible fluid towards a heating element of a 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. Definitions [0056] 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. [0057] 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. [0058] 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. [0059] Concentrations, amounts, and other numerical 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 numerical values and sub-ranges are explicitly recited. As an illustration, a numerical 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 numerical 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.

Claims

CLAIMS What Is Claimed Is: 1. A chamber particle for nucleic acid processing, comprising: a chamber defined by chamber particle walls and sized to allow a plurality of nucleic acid molecules to enter the chamber; and dried reagents immobilized on the chamber particle walls, wherein the dried reagents include a nuclease enzyme.

2. The chamber particle of claim 1, wherein the dried reagents are covalently conjugated or lyophilized on the chamber particle walls.

3. The chamber particle of claim 1, wherein the nuclease enzyme includes a cas-enzyme that can be selected from families of Cas9 enzymes, Cas12 enzymes, Cas13 enzymes, Cas14 enzymes, or a combination thereof.

4. The camber particle of claim 1, wherein the dried reagents further include a reporter nucleic acid, guide RNA, an indicator nucleic acid, enzyme, a master mix, a buffer salt, or a combination thereof.

5. The chamber particle of claim 4, wherein the reporter nucleic acid is present and includes a fluorophore and a quencher at opposing ends.

6. The chamber particle of claim 1, further including a delayed delivery film disposed over the dried reagents.

7. The chamber particle of claim 1, wherein the chamber particle is in the shape of a hollow cylinder and wherein an internal diameter of the chamber ranges from about 1 μm to about 190 μm.

8. The chamber particle of claim 1, wherein the chamber particle walls further include a label including a set of markers selected from fluorescent markers, absorbent markers, Raman markers, infrared markers, colored dyes, color absorbent markers, or a combination thereof.

9. A microfluidic nucleic acid processing system, comprising: microfluidics including: a microfluidic processing region, and a microfluidic detection region fluidly coupled to the microfluidic processing region; chamber particles loaded or loadable in the microfluidic processing region, wherein the chamber particles include chamber particle walls that define a chamber that is sized to allow a plurality of nucleic acid molecules to enter the chamber, the chamber particle walls including dried reagents including a nuclease enzyme immobilized thereon; and an optical detection device optically coupled to the microfluidic detection region to detect the nucleic acid molecules.

10. The microfluidic nucleic acid processing system of claim 9, wherein fluid to be introduced into the microfluidics includes a water immiscible fluid.

11. The microfluidic nucleic acid processing system of claim 9, further comprising a second microfluidic processing region with a second plurality of chamber particles loaded or loadable therein, wherein second plurality of chamber particles have a different loading of dried reagents thereon.

12. The microfluidic nucleic acid processing system of claim 10, wherein the microfluidic processing region is a microfluidic amplification region, wherein the microfluidic amplification region includes a heating element positioned to heat cycle a fluid containing the chamber particles loaded with nucleic acid molecules.

13. A method of detecting a nucleic acid, comprising: loading a microfluidic processing region of a microfluidic nucleic acid processing system with chamber particles including a chamber sized to allow nucleic acid molecules to enter the chamber and chamber particle walls with dried reagents including a nuclease enzyme associated therewith; introducing a sample fluid to the microfluidic processing region, wherein the sample fluid provides nucleic acid molecules that enter the chamber of the chamber particles; processing the nucleic acid molecules carried by the chamber particles; flowing the chamber particles carrying processed nucleic acid molecules to a microfluidic detection region; and optically detecting the processed nucleic acid molecules.

14. The method of claim 13, wherein the sample fluid and the chamber particles carrying the nucleic acid molecules are present in a water-immiscible fluid, and processing the nucleic acid molecules includes amplifying the nucleic acid molecules with isothermal amplification at temperature ranging from about 30 °C to about 45 °C.

15. The method of claim 13, wherein the dried reagents further include reporter nucleic acid, and the method includes detecting cleavage of the reporter nucleic acid by a fluorescence from labeled markers.