Nucleic acid hybridization cascade enhanced by droplet evaporation

Abstract

The present disclosure relates to novel methods for the detection of nucleic acid sequences.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 253,722, filed October 8, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Government Interests This invention was made with Government support under Primary No. U54 EB027049 from the National Institutes of Health Grant No. 60058448. The Government has certain rights in this invention.

[0003] The present disclosure relates to the fields of molecular biology and diagnostics, including nucleic acid-based detection. [Background technology]

[0004] Nucleic acid-based detection methods are used to detect specific nucleic acid sequences and thereby detect and identify specific species or subspecies of organisms (e.g., pathogens) or physiological / pathological states of cells (e.g., cancerous cells). From cancer screening to the diagnosis of acute viral infections, sensitive and accurate detection of nucleic acid sequences plays a key role. The most widely used assays for nucleic acid detection, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), rely on environmentally sensitive and expensive enzymes that are difficult to maintain in point-of-care (POC) applications. Enzyme-free nucleic acid amplification strategies, such as those based on DNA hairpin assembly, have become increasingly attractive in biological sensors due to their ability to significantly amplify signals from single nucleic acid targets. However, enzyme-based nucleic amplification assays are still much more sensitive than DNA hairpin assembly. Attempts to improve the sensitivity of DNA hairpin copolymer assembly include DNAzymes. 1 , nuclease 2 , Enzyme-Mediated Nucleic Acid Enhancement 3 , and gold nanoparticles 4、5These include the incorporation of ELISA kits. However, each of these approaches requires the addition of additional components to the reaction, increasing the overall complexity of the assay workflow, assay costs, and longer reaction times. Thus, there is a need for rapid, simple, and more cost-effective diagnostics. Summary of the Invention

[0005] The present disclosure addresses the above needs in several aspects.

[0006] In one aspect, the disclosure provides a method for detecting a target nucleic acid in a test sample, the method comprising: (i) providing a substrate having a reaction area where a test sample and a detection solution are deposited on the substrate to form a reaction droplet, the detection solution including a detection agent having a sequence that is complementary to a strand of the target nucleic acid, (ii) incubating the reaction droplet under conditions that allow evaporation thereof, and (iii) detecting a hybridization complex of the target nucleic acid and the sequence in the reaction droplet. The presence of the hybridization complex indicates the presence of the target nucleic acid in the test sample.

[0007] In the above method, the providing step may include (a) disposing a sample droplet containing the test sample on the reaction area, and (b) contacting the reaction area on the substrate or the test sample with a detection solution. The method may further include drying the sample droplet on the reaction area (e.g., in a drying chamber) prior to the contacting step.

[0008] In the above method, the reaction area can be hydrophilic or hydrophobic. It can be surrounded by a hydrophobic area. In an embodiment, the reaction area or substrate can be surrounded by an impermeable boundary. The reaction area or substrate can include a material selected from the group consisting of glass, plastic, metal, silicon, and paper. In some embodiments, the substrate includes a columnar or pedestal-like structure with a circular top that serves as the reaction area. The circular top can have a diameter of about 0.5 μm to about 4 mm (e.g., about 1 μm to about 3 mm, about 100 μm to about 2 mm, or about 500 μm to about 1 mm).

[0009] In the above method, the reaction droplets or sample droplets can be about 1 to 25 μl (eg, about 2 to 20 μl, about 3 to 15 μl, or about 4 to 10 μl).

[0010] In some embodiments, the detection solution may include a hybridization chain reaction (HCR) mixture, which is described in detail below.

[0011] The detection solution contains about 15 mM to about 800 mM (e.g., about 20 mM to 500 mM or about 50 mM to 200 mM) Na + and K + The detection solution may contain any combination of ions. The detection solution may contain about 0.1x to about 5.0x SSC (e.g., about 0.1x to about 2x, about 0.2x to 1.0x, or about 0.25x to 0.5x), about 1mM to about 80mM, for example, 1mM to about 15mM (e.g., about 2mM to 15mM, about 5mM to 10mM), Mg 2+ or Zn 2+ , PBS, TBS, a detergent, glycerol, and sucrose.

[0012] The above conditions may include one or more of a humidity of about 5% to about 95% (e.g., about 10% to about 80%, about 20% to about 50%, or about 30% to about 40%), a temperature of about 10°C to about 50°C (e.g., about 15°C to about 45°C, about 20°C to about 40°C, or about 30°C to about 40°C), and a duration of about 10 to about 300 minutes (e.g., about 30 to about 240 minutes, about 60 to about 180 minutes, or about 90 to about 120 minutes).

[0013] The target nucleic acid can be DNA or RNA. In one embodiment, the target nucleic acid is the nucleic acid of a pathogen, such as a virus, e.g., HIV, dengue, SARS-CoV-2, or Ebola. Non-limiting examples of viruses include reoviruses that have a significant impact on human health, including Norwalk, Rotavirus, Poliovirus, Ebola virus, Marburg virus, Lassa virus, Hantavirus, Rabies, Influenza, Yellow Fever virus, Coronavirus, SARS, SARS-CoV-2, West Nile virus, Hepatitis A virus, Hepatitis C virus (HCV) and Hepatitis E virus, Dengue virus, Toga (e.g. rubella), Rhabdo (e.g. rabies and VSV), Picorna (polio and rhinovirus), Myxo (e.g. influenza), Retro (e.g. HIV, HTLV), Bunya, Corona, and Hepatitis D viruses and plant RNA viruses, and viroid-like viruses such as Tobus virus, Luteovirus, Tobamovirus, Potexvirus, Tobravirus, Comovirus, Nepovirus, Almovirus, Cucumovirus, Bromovirus, and Iraluvirus. The test sample may include a blood sample, a sputum sample, a urine sample, a urine swab sample, or a saliva sample.

[0014] In another aspect, the present disclosure further provides a kit comprising one or more of the substrates and detection solutions described above. The kit may further comprise one or more probes having a sequence complementary to the target sequence. In one embodiment, the kit comprises the HCR reaction mixture described herein.

[0015] The details of one or more embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will become apparent from the description and the claims. [Brief description of the drawings]

[0016] [Figure 1]Figure 1 shows a schematic of a hybridization chain reaction (HCR) using Forster resonance energy transfer (FRET) for fluorescent detection of target trigger or initiator sequences. H1 has a region complementary to the initiator sequence, causing it to unfold from its secondary structure and hybridize to the initiator, exposing the complementary sequence to H2. H2 continues the cascade by hybridizing to H1 and exposing the complementary sequence to H1. The intensity of the H2 acceptor fluorophore depends on the length of the H1 / H2 HCR product. Reactions can be incubated in sessile droplets (bottom). [Figure 2A-2B] Photographs of agarose gels showing HCR products at various trigger concentrations incubated in cuvettes at room temperature for 2.5 hours (FIG. 2A) and in 5 μL sessile droplets until dry (approximately 1.5 hours at ambient conditions) (FIG. 2B). [Figure 3A-3C] Photograph showing custom 3D printed sticky droplet pedestals containing 3 μL HCR mixture droplets (FIG. 3A), and fluorescence microscopy images of dried HCR-FRET sticky droplets at 0 nM (FIG. 3B) and 1 nM (FIG. 3C) trigger concentrations. [Figure 4] Photograph showing a direct comparison of sessile droplet-enhanced HCR (S) using 0.25x SSC buffer containing 10 mM MgCl2 with HCR (L) incubated in a cuvette. Trigger concentrations decrease from left to right from 100 nM trigger to 0 nM trigger. [Diagram 5] FIG. 13 shows the relative FRET intensities of HCR and anchorage-enhanced HCR incubated in a liquid cuvette at 0 nM and 100 nM trigger concentrations using 0.25×SSC buffer containing 10 mM MgCl 2 . [Figure 6] Photographs showing sessile droplet-enhanced hybridization chain reaction incubated in buffers containing various concentrations of sodium and magnesium. Each box shows a comparison of HCR products between 0 pM and 500 pM trigger concentrations and their corresponding buffers. [Figure 7A-7B]Photographs showing examples of sessile droplet experimental devices and setups. Figure 7A shows three PDMS rings containing 1.5 mm diameter pedestals surrounded by desiccant. A windowed lid is placed over the pedestals to control evaporation. Figure 7B shows a magnified image of one ring containing six HCR sessile droplets suspended on the pedestals. [Figure 8A] 1 is a series of photographs of an agarose gel showing enhanced hybridization of HCR via sessile droplet incubation compared to traditional cuvette incubation. [Figure 8B-8C] Spectrofluorometer data showing normalized FRET intensity of HCR products from synthetic proviral DNA targets (FIG. 8B) and synthetic viral RNA targets (FIG. 8C) when incubated in both sessile droplets and cuvettes are shown. [Figure 9] Agarose gel showing HCR incubated against a synthetic DNA trigger in two different conditions: wet (W) and dry (D). [Figure 10A] HCR sticky droplet incubations with 0, 1, and 2 mismatches in the synthetic DNA trigger sequence are shown. [Figure 10B] Fluorescence FRET results of HCR incubated in sessile droplets with synthetic trigger DNA and a 5:1 mass ratio of salmon DNA to synthetic trigger DNA are shown, demonstrating high resistance to off-target hybridization. [Figure 11A] FRET intensity of sessile droplet HCR products when incubated in buffers with different ionic concentrations is shown. [Figure 11B] 1 shows the experimental melting temperatures of the H1 hairpin at different sodium concentrations. [Figure 12A] A 1.5 mm diameter pad was cut and bonded to a PDMS pedestal to allow for the formation of a sessile droplet on the hydrophilic material. The target nucleic acid was allowed to dry on the paper disc. [Figure 12B]5 μL of HCR solution (H1 and H2) was applied as a sessile drop to each pedestal and allowed to dry completely. [Figure 12C] The dried paper was then imaged using a fluorescence microscope equipped with a FRET cube. Image intensities were analyzed using ImageJ. p values ​​were calculated using a two-tailed Student's t-test in JMP Pro 16. [Fig. 12D-12E] Sessile droplet HCR FRET intensity detecting synthetic HIV RNA (FIG. 12D) and proviral DNA (FIG. 12E) directly on filter paper (Fusion5, Cytiva) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure relates to molecular biology and diagnostics, including nucleic acid-based detection methods, devices, and systems. The diagnostic methods described herein are simple to perform, do not require instrumentation or expensive enzymes, are inexpensive to manufacture, are stable in a variety of environmental conditions, and can be easily interpreted by unskilled end users. Because most conventional nucleic acid detection tests suffer from extreme sensitivity to thermal storage conditions and reduced performance at low target concentrations, the methods, devices, and systems described herein have considerable advantages over conventional approaches and are suitable for point-of-care (POC) applications, especially as rapid diagnostics in non-industrialized areas around the world.

[0018] Certain aspects of the present disclosure are based, at least in part, on the unexpected discovery that the efficiency of hybridization of nucleic acid structures can be enhanced by performing the reaction in low volume droplets (e.g., picoliters to microliters) of solution, referred to as sessile droplets, that are exposed to atmospheric conditions and / or allowed to evaporate. The sessile droplets described herein offer the advantage of reduced internal currents and volume upon evaporation that allow for enhanced molecular interactions. It is believed that the Marangoni effect (i.e., evaporation-induced currents generated by thermodynamic dispersion across the droplet) creates a self-mixing environment in the sessile droplet, increasing molecular interactions in otherwise diffusion-limited reactions. 7、8

[0019] Sessile droplets have been used to enhance bimolecular interactions but not for complex assembly processes such as HCR. For example, the use of sessile droplets has been shown to increase reaction rates and decrease overall reaction times in the context of enzyme-driven colorimetric assays. 6 However, so far this has been limited to simple interactions between two molecules. The methods, devices, and systems disclosed in this disclosure take advantage of sessile droplet evaporation to increase copolymer hybridization of nucleic acid nanostructures, thereby improving the capabilities of enzyme-free nucleic acid detection.

[0020] Substrates and Devices The detection methods described herein involve carrying out a nucleic acid hybridization reaction in sessile droplets present on a substrate, which are exposed to atmospheric conditions and allowed to evaporate.

[0021] The use of evaporation allows for the concentration of detection targets, significantly shortening reaction times and enhancing detection sensitivity. In addition, natural convection currents resulting from Marangoni currents during evaporation mix the solutions in the droplets, which speeds up assay reactions, shortens assay times and increases detection limits. Differences in evaporation rates result in movement in sessile droplets. Depending on the contents of the droplet (e.g., salts) and various environmental factors that affect evaporation (e.g., temperature differences), evaporation can generate primary radial flow or Marangoni flow. Varying these control factors to generate one or the other is a major design consideration in low-source assays that rely on droplet deposition. Thus, design parameters including substrate material and solution components can be optimized to promote Marangoni flow. Depending on design considerations, this flow pattern can be axisymmetric, radially outward along the air-liquid interface, radially inward along the substrate, or directed in the opposite direction.

[0022] The surface tension gradient that creates the Marangoni flow field is believed to be caused by a temperature gradient resulting from the cooling effect caused by the non-uniform evaporation flux along the drop surface. The evaporation rate of the drop is greatest at the contact line because the close location of the surrounding unsaturated gas results in non-uniform evaporation along the air-liquid interface (Deegan et al., Physical Review E, 62, (1), 756-765, 2000.). The extent to which the non-uniform evaporative cooling effect results in a temperature gradient along the drop surface is determined in part by the heat transfer rate from the isothermal substrate to the air-liquid interface (Ristenpart et al., Physical Review Letters, 99, (23), 2007). These heat transfer rates are, in part, a function of both the drop height and the thermal conductivity of the substrate and the liquid.

[0023] If the thermal conductivity of the substrate is low enough, evaporative cooling will dominate, generating the lowest temperature at the contact line. Conversely, a highly thermally conductive substrate will facilitate sufficient heat transfer at the contact line to overcome the evaporative cooling effect, resulting in the highest temperature at the edge of the drop and the lowest temperature at the center. These temperature gradients will cause surface tension gradients that drive Marangoni flow. If the substrate has, for example, a thermal conductivity less than 1.45 times that of the liquid, which results in fluid flowing radially outward along the air-liquid interface and radially inward along the substrate interface to the center of the drop, the drop will be cooled most at the contact line (Ristenpart et al., Physical Review Letters, 99, (23), 2007 and US Patent No. 10101323). That is, the fluid flows along the substrate toward the center of the drop, then turns toward the air-liquid interface and flows along the drop surface in the direction of the contact line. If the substrate has, for example, a thermal conductivity more than twice that of the liquid, the flow direction will be reversed. Furthermore, these Marangoni flow fields are axisymmetric about the drop centre, resulting in a toroidal configuration when viewed from above the drop.

[0024] Thus, substrates that can be used herein can include those with suitable thermal conductivity such that a temperature gradient can induce a sufficient surface tension gradient, which drives Marangoni flow.

[0025] The substrate can be permeable or impermeable to water or aqueous solutions. It has a reaction area adapted to receive nucleic acids, or a sample composition containing nucleic acids, or a solution for a hybridization reaction. In some embodiments, the reaction area is flat and impermeable to water or aqueous solutions. In some other embodiments, a substrate that is permeable to water or aqueous solutions can be used, as long as enough liquid can saturate the permeable substrate and generate a sessile droplet on the surface of the substrate. In that case, initial capillary forces can draw some nucleic acid (such as target and DNA probe) material into the permeable substrate, but shortly after, evaporation forces in the droplet (not necessarily Marangoni forces) can theoretically pull material out of the substrate. A thin permeable layer, such as a section of Fusion5 filter paper, can support a sessile droplet and can be imaged using a fluorescent microscope in a similar manner to a non-porous, hydrophobic surface. Thicker permeable substrates such as TFN (blood spot transfer paper) may also be used under the conditions described above, and reactants may then be eluted from the permeable dots and analyzed using any suitable method (e.g., fluorescence microscopy, gel electrophoresis, etc.).

[0026] The substrate can be made of or coated with virtually any type of material that provides the necessary fluid dynamics and adhesion of the sample composition or nucleic acid. Examples of such materials include glass, plastic (e.g., polystyrene), latex, metal, polymer, silica, metal oxide, ceramics, or any other substance suitable for binding with nucleic acid or other materials with suitable thermal conductivity and contact angle (hydrophobicity). Regions can be derivatized to facilitate the use of different solvents and detection methods. Substrates with high thermal conductivity and / or high contact angle (hydrophobicity) are desirable. In some embodiments, the droplet can form a contact angle of 70° or more.

[0027] For example, the nucleic acid reaction area may be formed from glass, plastic, metal, silicon, paper, cloth, textile, or non-woven cellulose substrates. In some embodiments, the reaction area may be formed from paper, such as commercially available filter paper (e.g., Whatman paper, nitrocellulose, or fiberglass). In some embodiments, the reaction area may be functionalized to facilitate attachment of nucleic acid. For example, the nucleic acid receiving area may be functionalized to be positively charged, for example, with chitosan or polyethyleneimine (PEI). As used herein, the term "functionalized" or "chemically functionalized" refers to the addition of functional groups to the surface of a material by chemical reaction. As will be readily understood by those skilled in the art, functionalization may be employed for surface modification of a material to achieve desired surface properties, such as biocompatibility and wettability.

[0028] The substrate or reaction area on the substrate can be of various geometric shapes and sizes. For example, the substrate or reaction area can have a circular, semicircular, triangular, square, rectangular, pentagonal, or hexagonal shape. In some embodiments, the substrate or reaction area can have a circular shape and a diameter of, for example, about 0.5 μm to about 4 mm (e.g., about 1 μm to about 2 mm, about 2 μm to about 1 mm, and about 5 μm to about 500 μm). In some embodiments, the substrate or reaction area is provided in the form of a microarray suitable for high-throughput assays.

[0029] The amount of fluid sample applied to the substrate or reaction area may vary as long as it is sufficient to provide the desired volume and maneuverability of the assay. In some embodiments, the substrate or reaction area is adapted for small volumes of sample, for example, from about 1 μL to about 25 μL (e.g., from about 2 μL to about 20 μL, from about 3 μL to about 15 μL, and about 10 μL).

[0030] The substrates described above can be included in a biosensor device for concentrating and detecting biomarker molecules such as nucleic acids. The device can include the substrate and optionally other components such as a support for holding the substrate, a hydrophobic surface surrounding the substrate, or a chamber for containing the substrate. For example, such a device can include a support, one or more substrates disposed on the support, one or more reaction regions disposed on the substrate, and a hydrophobic surface surrounding the substrate.

[0031] The device may also include a chamber housing all other components for controlling the reaction between the target biomarker molecules and the detection agent. Shown in Figure 7A is an exemplary device. The device includes a housing for three PDMS ring-shaped supports. Each support has nine pedestals (1.5 mm diameter) surrounded by desiccant. A windowed lid is placed over the pedestals to control evaporation. Figure 7B shows a magnified image of one ring containing six HCR sessile droplets suspended on the pedestals.

[0032] The reaction area can be configured to receive a test sample and / or a detection solution. The reaction area can also be structured to immobilize one or more detection agents (e.g., probes or other agents) for detecting molecules of a target biomarker. The reaction area can also be configured to attract an array of droplets of a biomarker solution containing the target biomarker molecules. The hydrophobic surface surrounding the reaction area can include nanostructures with rough surfaces to concentrate the target biomarker molecules in the reaction area by enhancing evaporation of the array of droplets resulting in an array of concentrated droplets having an increased concentration of the target biomarker molecules compared to before evaporation.

[0033] In some embodiments, the substrate may include a reaction region surrounded by an impermeable boundary. In some embodiments, the reaction region may be hydrophilic and surrounded by a hydrophobic region. For example, the substrate may include a hydrophilic (e.g., SiO2, metal, dielectric, or other hydrophilic material) pattern surrounded by a micro- or nano-patterned, hydrophobic or superhydrophobic, black silicon surface.

[0034] A superhydrophobic surface can generate large contact angles for a droplet of aqueous solution, much more than 90 degrees or 150 degrees. For black silicon and nanostructured polydimethylsiloxane (PDMS) surfaces, the contact angle of a droplet of water can exceed 150 degrees. In one embodiment for achieving a superhydrophobic surface, the device can include an array of hydrophilic micro islands or a microarray of hydrophilic islands surrounded by superhydrophobic black silicon fabricated on a Si wafer. To form the black silicon, an exemplary Bosch etching process can be employed in a reactive ion etcher, for example, in the manner described in US 2016 / 0258020, the disclosure of which is incorporated by reference. The etching process includes alternating cycles of etching (SF6) and passivation (C4F8), forming nanopillar structures. In the areas protected by the lithographically defined SiO2 pattern, no etching or passivation occurs, so the hydrophilic properties are retained after the black silicon process.

[0035] In another embodiment for achieving a superhydrophobic surface, the device can include an array of hydrophilic regions, islands, pedestals, or pillars surrounded by superhydrophobic PDMS regions formed using 3D printing or nanoimprinting or hot embossing, for example in a manner as described in US 2016 / 0258020, the disclosures of which are incorporated by reference.

[0036] Samples containing nucleic acids can be concentrated and hybridized on such devices via rapid evaporation followed by the formation of self-assembled droplets (e.g., microliter, nanoliter, or smaller). In the droplets, the hybridization reaction proceeds rapidly to completion due to the reduction in volume, resulting in accelerated hybridization times. Furthermore, the precise volume control provided by the self-assembled droplet (e.g., microliter, nanoliter, or smaller) arrays allows for broad control over the concentration rate, resulting in higher sensitivity and extended dynamic range.

[0037] Nucleic Acid Detection Various methods for detecting nucleic acids are known in the art and can be used herein. In some embodiments, nucleic acid copolymer assemblies such as those made by hybridization chain reaction (HCR) can be used to significantly amplify the signal from the target nucleic acid without the use of enzymes or thermal cycling, making them ideal for point-of-care diagnostics. Carrying out the reaction in sessile droplets that are exposed to atmospheric conditions and evaporate further enhances the efficiency of copolymer hybridization.

[0038] HCR Described herein is a novel and rapid method for enhancing hybridization kinetics to increase nucleic acid detection sensitivity by forming hybridization within a sessile droplet without adding extra assay reagents or reaction time. In some embodiments, detection of one or more nucleic acids in a biological sample is performed based on a non-enzymatic method of nucleic acid amplification called hybridization chain reaction, shown in FIG.

[0039] HCR, a nucleic acid amplification technique, utilizes naturally occurring DNA self-assembly to form extended notch dsDNA assemblies. HCR does not require enzymes and can operate isothermally. Various forms of HCR are known in the art and can be used in the methods described herein. See, for example, Dirks, R. and Pierce, N. Proc. Natl. Acad. Sci. USA 101(43):15275-15278 (2004), U.S. Patent Nos. 8,105,778 and 8,507,204, U.S. Patent Publication Nos. 2019 / 02186080 and 2018 / 0010166, each of which is incorporated herein by reference in its entirety.

[0040] HCR may include an initiator sequence and two or more strategically designed metastable hairpin monomers (e.g., H1 and H2 shown in FIG. 1), whose secondary structure forms a metastable hairpin. The hairpin monomers each have at least one single-stranded toehold, a single-stranded loop, and a double-stranded stem. The energy to drive the self-assembly cascade is stored in the single-stranded loop and toehold segments of the hairpin. Each monomer is caught in a kinetic trap, preventing the system from rapidly equilibrating. That is, pairs of monomers cannot hybridize to each other in the absence of the initiator. Upon introduction of the initiator strand, the monomers undergo a chain reaction of hybridization events to form a nicked double-stranded polymer. HCR can be used to detect the presence of an analyte of interest in a sample, for example, by detecting the analyte with a probe carrying an HCR initiator, which in turn triggers HCR signal amplification. HCR signal amplification makes it possible to increase the signal-to-background ratio for molecular detection and imaging applications by boosting the signal above the background arising from the sample.

[0041] As used herein, a "monomer" is an individual nucleic acid oligomer. Typically, at least two monomers are used in a Hybridization Chain Reaction, although three, four, five, six, or more monomers may be used. Typically, each monomer contains at least one region that is complementary to at least one other monomer used in the HCR reaction.

[0042] An "initiator" or "trigger" is a molecule that can initiate polymerization of a monomer. An exemplary initiator can include a nucleic acid region that is complementary to the initiator complementary region of an HCR monomer. The initiator can be, for example, an analyte of interest, or a nucleic acid that can contact the first monomer (e.g., HI in FIG. 1) only in the presence of an analyte of interest, as discussed in more detail below. For example, the initiator can be a nucleic acid (e.g., viral RNA) that is itself to be detected.

[0043] As shown in Figure 1, H1 has a region complementary to the initiator (trigger) sequence, causing it to unfold from its secondary structure and hybridize with the initiator, exposing the complementary sequence to H2. H2 continues the cascade by hybridizing with H1 and exposing the sequence complementary to H1 (Figure 1). For the FRET experiments described herein, H1 can be conjugated with a donor fluorophore and H2 can be conjugated with an acceptor fluorophore. The intensity of the H2 acceptor fluorophore depends on the length of the resulting H1-H2 HCR product.

[0044] In one example, a single copy of a target nucleic acid acts as an initiator sequence and is contacted with a reaction solution or reaction mixture that includes an HCR probe set that includes two DNA hairpin probes, H1 and H2. Upon contact, the target nucleic acid / initiator sequence triggers the opening of the first H1 DNA hairpin through duplex formation, which then triggers the opening of the first H2 DNA hairpin. This released H2 then binds to a region of the second H1, which opens this second H1, and the cascade continues. Each hairpin can be labeled at its terminus or end with a donor or acceptor fluorophore, as described above, and thus as the cascade proceeds, the binding and opening of each hairpin triggers FRET, increasing the signal for quantification by an imaging device, e.g., a smartphone camera, whereby resonance energy is transferred from the electron of the donor fluorophore to the acceptor fluorophore, which is emitted as a photon having a longer wavelength than the photon emitted by donor fluorophore A.

[0045] In some embodiments, the reaction mixture comprises an HCR mixture. In some embodiments, the reaction solution / mixture comprises one or more probes (e.g., an HCR probe set) having a sequence that is complementary to a target sequence of a nucleic acid.

[0046] An "HCR probe set" or "HCR initiator / hairpin set" may include one or more initiator strands of nucleic acid capable of forming a hybridization chain reaction polymer with one or more metastable HCR monomers, such as nucleic acid hairpins. HCR probes may be synthesized using standard methods, such as chemical nucleic acid synthesis, including commercial sources, such as Integrated DNA Technologies (IDT, Coralville, Iowa), WMKeck Foundation Oligo Synthesis Resource (New Haven, Connecticut), or Molecular Instruments (Pasadena, California). Alternatively, HCR probes can be synthesized and / or amplified using standard enzymatic methods, such as PCR followed by lambda exonuclease digestion of one strand to obtain ssDNA (see, e.g., Current Protocols in Molecular Biology (2014):14-23, incorporated herein by reference in its entirety), or in vitro transcription followed by reverse transcription to obtain ssDNA (see, e.g., Chen et al., Science 348:6233 (2015):aaa6090, incorporated herein by reference in its entirety).

[0047] In one embodiment, to enhance enzyme-free nucleic acid detection for rapid diagnostic applications, a target nucleic acid sequence ("target"), such as a sequence of mRNA or genomic DNA or RNA, is applied and / or captured in a reaction zone consisting of either a hydrophilic circular pad or a circular post surrounded by a hydrophobic material. The target is concentrated after application to the reaction zone because evaporation reduces the droplet volume. The target is dried or mostly dried on the reaction zone before the sessile droplet of DNA hairpin reaction solution / mixture is applied. Evaporation of the reaction solution / mixture simultaneously causes internal mixing of the reaction solution / mixture, increasing the reaction concentration due to the reduction in the droplet volume. More H1 and H2 hairpins can hybridize during the evaporation process, increasing the HCR product and subsequently increasing the signal output of the assay. In the case of the example described herein, the signal output is the intensity of the acceptor fluorophore of the H1 / H2 FRET pair.

[0048] In certain embodiments, the evaporation process of the droplets may be facilitated by a hydrophobic or superhydrophobic surface, and the resulting droplets (e.g., nanoliter volume or smaller) may be encapsulated by droplets of water-immiscible liquid (e.g., oil) to form stable reaction chambers (e.g., nanoliter or microliter volumes) in the manner described in U.S. Patent Publication No. 2016 / 0258020, which is incorporated by reference.

[0049] The test sample, the reaction solution / mixture, or a mixture of both, may be applied to the reaction area or site using any convenient protocol, for example, via a dropper, pipette, syringe, or the like.

[0050] In one example, the test sample is mixed with the reaction solution / mixture before being applied to the reaction area, hi another example, the test sample may be applied to the reaction area or site simultaneously with, before, or after the reaction solution / mixture is applied to the reaction area.

[0051] In addition, the test sample of the reaction solution / mixture may be applied to the region with any suitable liquid, such as, for example, a buffer, to provide suitable reaction or hybridization conditions. Examples of suitable liquids include, but are not limited to, buffers. Examples of buffers include Tris, Tricine, SSC, MOPS, HEPES, PIPES, MES, PBS, TBS, and the like.

[0052] In one example, a suitable liquid may be mixed with the test sample or reaction solution / mixture prior to application to the reaction area, hi another example, a suitable liquid may be applied to the reaction area or site simultaneously with, prior to, or after application of the sample.

[0053] Given the distinct advantages of HCR in this droplet format, it has broad utility in molecular diagnostics as an alternative to PCR. In one embodiment, this format of HCR can be used for POC or home viral load monitoring for pathogens such as HIV. Of global importance, the sessile droplet assay format may have particular utility in resource-limited environments such as sub-Saharan Africa, where costs, environmental conditions, and assay complexity make standard enzyme-based nucleic acid amplification strategies difficult.

[0054] Thus, further provided in the present disclosure is a method for identifying a patient infected with or suspected of being infected with a virus, which may include (i) obtaining a patient sample (e.g., a blood sample), (ii) determining the level of viral load (e.g., the level of HIV RNA) in the blood sample by the methods described above, (iii) comparing the determined level of viral load to a control level and determining whether the determined level is elevated compared to the control level, and (iv) identifying the patient as having a viral infection if the determined level of viral load is elevated compared to the control level.

[0055] The terms "patient," "individual," and "subject" are used interchangeably and generally refer to any living organism in which the disclosed methodology is utilized to obtain a bodily fluid sample to perform the diagnostic or monitoring methods described herein. A patient may be an animal, such as a human. A patient may also be a domestic or livestock animal. A "patient" or "individual" may also be referred to as a subject.

[0056] As used herein, a "control" level of a pathogen, e.g., a viral load, in some embodiments, refers to a level of the pathogen obtained from a sample obtained from one or more individuals not suffering from the disease or disorder of interest in the study, e.g., a viral infection such as HIV infection. The level may be measured on an individual basis or may be measured on a population basis, such as an average. A "control" level may also be determined by analysis of a population of individuals who have the disease or disorder but have not experienced an acute phase of the disease or disorder. To obtain such a "control" level, a "control" sample may be used. A control sample may be obtained from one or more individuals not suffering from the disease or disorder of interest in the study. A control sample may also be obtained from a population of individuals who have the disease or disorder but have not experienced an acute phase of the disease or disorder. In some embodiments, the control level is from the same individual as the individual whose diagnosis is sought or whose condition is being monitored, but obtained at a different time. In certain embodiments, a control level or sample may refer to a level or sample obtained from the same patient at an earlier time, e.g., weeks, months, or years ago.

[0057] As used herein, "the determined level is elevated compared to a control level" refers to a positive change in value from the control level.

[0058] detection One of the key advantages of the assays disclosed herein is the use of simple and inexpensive detection methods that do not require sophisticated equipment or trained personnel, however, the assays are also applicable to such more sophisticated procedures that are within the scope of the present disclosure.

[0059] The product of HCR can be easily detected by methods known to those skilled in the art for detecting nucleic acids, including, for example, agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, and gel-filled capillary electrophoresis. Because the polymer contains nucleic acids, it can be visualized by standard techniques, such as staining with ethidium bromide. Other methods may also be suitable, including light scattering spectroscopy, such as dynamic light scattering (DLS), viscosity measurement, colorimetric systems, mass spectrometry, and fluorescence and fluorescence polarization spectroscopy. As described in more detail, in some methods for in situ imaging and detection, the HCR product is fluorescently labeled.

[0060] Visual detection is one of the simplest detection approaches. Visual detection can rely on color (presence or absence), color change, luminescence, or fluorescence. The present disclosure contemplates that the appearance, color, or light of the product generated from the droplet evaporation provides an easily recognizable feature that allows for home and field use of the assay with minimal instructions (i.e., package insert with instructions and figures).

[0061] In some embodiments, an optical microscope or light microscope can be used. An optical microscope or light microscope involves passing visible light that is transmitted or reflected off a sample through a single or multiple lenses to allow for an expanded view of the sample. The resulting image can be detected directly by eye, imaged on a photographic plate, or captured digitally. A single lens and its accessories, or a system of lenses and imaging equipment, along with appropriate lighting equipment, sample stage, and support, constitute a basic optical microscope. One example is a digital microscope that uses a CCD camera to focus on the object of interest. The camera can be attached to it via a USB port so that the image can be shown on a computer screen. Another example is a smartphone equipped with a digital camera. Any suitable imaging microscope can also be used, including bright field, dark field, and fluorescent microscopes.

[0062] In some embodiments, HCR is monitored by FRET. Certain monomers are labeled with fluorescent dyes, so that the structural changes caused by HCR can be monitored by detecting changes in fluorescence. In one embodiment, one hairpin molecule (e.g., H1 in FIG. 1) is labeled with a donor fluorophore and another hairpin molecule (e.g., H2 in FIG. 1) is labeled with an acceptor fluorophore. Upon polymerization, the donor and acceptor fluorophores align in close proximity within the condensed nucleic acid structure, thereby providing FRET. In this case, the presence of a single initiator is amplified by a chain of fluorescent events triggered by HCR. In the context of in situ imaging, the presence of a single target molecule can be amplified by a chain of fluorescent events.

[0063] Samples and Diagnostic Targets The methods, devices, and systems disclosed herein can be used for the amplification and detection of biomarker molecules such as DNA and RNA without the need for any amplification step to increase the copy number of those molecular markers. Such methods, devices, and systems are particularly attractive for a variety of applications, including, but not limited to, gene expression analysis by estimating the copy number of DNA or RNA transcripts present in a sample, molecular detection applications, nucleic acid biomarker quantification for clinical, laboratory, and point-of-care diagnostic purposes, development for detecting pathogens and infectious diseases by direct identification of endogenous sequences, including fragmented microbial genomes (DNA) and RNA sequences, the ability to quantify miRNA and other forms of circulating epigenetic markers for early diagnosis of disease and injury, and the use thereof for mutation analysis of cancer and genetic diseases.

[0064] A variety of samples can be used in the methods disclosed herein. As used herein, the term "sample" refers to anything that can be analyzed by the methods provided herein. In some embodiments, a sample contains or is suspected to contain one or more nucleic acids that can be analyzed by the methods. Preferably, the sample contains nucleic acids (e.g., DNA, RNA, cDNA, microRNA, mitochondrial DNA, etc.). A sample can be a complex or mixed sample (e.g., host and pathogen nucleic acids, mutant and wild-type species, heterogeneous tumors) that contain nucleic acids that include multiple different nucleic acid sequences. A sample can contain nucleic acids from more than one source (e.g., different species, different subspecies, etc.), subject, and / or individual. In some embodiments, the methods provided herein optionally include purifying the sample or purifying nucleic acids from the sample. In some embodiments, the sample can contain purified nucleic acids. In some embodiments, the sample is derived from biological, clinical, environmental, research, forensic, or other sources.

[0065] In some embodiments, the methods, compositions, systems, and devices disclosed herein utilize a sample that includes a nucleic acid template. The sample may be obtained from any suitable source and may be obtained for purposes related to any field, including but not limited to diagnostics, research, forensics, epidemiology, pathology, archaeology, and the like. The sample may be biological, environmental, forensic, veterinary, clinical, and the like. The sample may include nucleic acid from any suitable source, including eukaryotes, prokaryotes (e.g., infectious bacteria), plants, mammals, humans, non-human primates, dogs, cats, cattle, horses, pigs, mice, viruses, and the like. The sample may include, for example, whole organisms, organs, tissues, cells, organelles (e.g., chloroplasts, mitochondria), synthetic nucleic acids, cell lysates, and the like. The nucleic acids present in a sample (e.g., target nucleic acids, template nucleic acids, non-target nucleic acids, contaminating nucleic acids) can be of any type, e.g., genomic DNA, RNA, plasmids, bacteriophage, synthetic origin, natural origin, and / or artificial sequences (not occurring in nature), synthetically produced but naturally occurring sequences, etc.

[0066] Biological specimens may involve, for example, solid matter such as feces or tissue (including biopsies), tissue extracts, or liquids including whole blood, lymph, serum, plasma, buccal, sweat, tears, saliva, sputum, cerebrospinal (CSF) fluid, amniotic fluid, semen, vaginal discharge, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudate, exudate, cyst fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, swabs, aspirates (bone marrow, fine needle aspirate), or lavage fluids (e.g., oral, nasopharyngeal, bronchial, bronchoalveolar, ocular, rectal, intestinal, vaginal, epidermal, etc.), and / or other fresh, frozen, cultured, preserved (PAXgene, RNAlater, RNasin, etc.) or archived (formalin fixed paraffin embedded (FFPE), fixed cell / lymphocyte pellet, etc.) biological specimens. Such samples may be solubilized or diluted as necessary to perform the assays of the present disclosure. Solvents used to solubilize or dilute the sample include water, acetone, methanol, toluene, ethanol, and the like.

[0067] Other samples include manufactured, industrial, or environmental samples that may or may not contain living cells or organisms. Such samples may include soil, water, food, alcoholic beverages, building products, bulk chemicals, or reagents including drugs. Again, such samples may be solubilized or diluted as necessary to perform the assays of the present disclosure.

[0068] In a preferred embodiment, the assay of the present disclosure can be used to detect various infectious agents. Infection refers to any condition in which an abnormal collection or population of viable intracellular or extracellular microorganisms is present in a subject. Various types of microorganisms can cause infections, including bacteria, viruses, fungi, and parasites. Detection of nucleic acids associated with these microorganisms is within the scope of the present disclosure.

[0069] Examples of viruses include, but are not limited to, Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 (also referred to as HTLV-III, LAV, or HTLV-III / LAV, or HIV-III; and other isolates, e.g., HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus, Bunyaviridae (e.g., Hantavirus, Bungavirus, Phlebovirus, Nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, rotavirus); Bornaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex viruses) pesviruses (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses; Poxviridae (variola virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); unclassified viruses (e.g., the causative agent of hepatitis delta (thought to be a defective satellite of hepatitis B virus), hepatitis C; Norwalk and related viruses, astrovirus); and respiratory syncytial virus (RSV).

[0070] Examples of bacteria include pneumococci, streptococci, e.g., S. pyogenes, S. agalactiae, S. equi, S. canis, S. bovis, S. equinus, S. anginosus, S. sanguis, S. salivarius, S. mitis, S. mutans, other viridan streptococci, pepto streptococci, other related species of streptococci, enterococci, e.g., Enterococcus faecalis, Enterococcus faecium, staphylococci, e.g., Staphylococcus epidermidis, Staphylococcus aureus, Hemophilus influenzae, Pseudomonas species, e.g., Pseudomonas aeruginosa, Pseudomonas pseudomallei, Pseudomonas mallei, Brucella species, e.g., Brucella melitensis, Brucella suis, Brucella abortus, Bordetella pertussis, Borellia species, e.g., Borellia burgedorferi, Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, Corynebacterium pseudodiphtheriticum, Corynebacterium urealyticum, Corynebacterium hemolyticum, Corynebacterium equi, etc., Listeria monocytogenes, Nocordia asteroides, Bacteroides spp., Actinomycetes spp., Treponema pallidum, Leptospirosa spp., Haemophilus spp., Helicobacter spp. including Helicobacter pylori, Treponema spp., and related organisms.The present invention may also be useful against gram-negative bacteria, such as Klebsiella pneumoniae, Escherichia coli, Proteus, Serratia species, Acinetobacter, Yersinia pestis, Francisella tularensis, Enterobacter species, Bacteroides and Legionella species, Shigella species, Mycobacterium species (e.g., Mycobacterium tuberculosis, Mycobacterium bovis or other Mycobacteria infections), Mycobacterium avium complex (MAC), Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium kansaii, Yersinia infections (e.g., Yersinia pestis, Yersinia enterocolitica, or Yersinia pseudotuberculosis), and the like.

[0071] Examples of parasites include Cryptosporidium, Entamoeba, Plasmodium spp., e.g., Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax, as well as Toxoplasma gondii, Giardia, Leishmania, Trypanasoma, Trichomonas, Naegleria, Isospora belli, Trichomonas vaginalis, Wunchereria, Ascaris, Schistosoma species, Cyclospora species, e.g., Chlamydia trachomatis, and other Chlamydia infectives, e.g., Chlamydia psittaci, or Chlamydia pneumoniae. Of course, it should be understood that the present invention can be used for any pathogen for which a specific probe can be made, e.g., the H1 and H2 hairpin probes described herein.

[0072] Fungal and other bacterial pathogens, some of which have been described in Human Mycoses (1979); Opportunistic Mycoses of Man and Other Animals (1989); and Scrip's Antifungal Report (1992), are also contemplated as targets for diagnosis. Fungal diseases contemplated in the context of the present invention include, but are not limited to, aspergillosis, nematode mycosis, candidiasis, nematode mycosis, cryptococcosis, onychomycosis, or otitis externa (otomycosis), brown fungus, phycomycosis, tinea versicolor, tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea verruca, tinea pedis, tinea unguium, torulopsis, axillary trichophyton, leiomycosis, and their synonyms for severe systemic or opportunistic infections, such as, but not limited to, tinea versicolor ... Known fungal and bacterial pathogens include, but are not limited to, actinomycosis, aspergillosis, candidiasis, chromomycosis, coccidioidomycosis, cryptococcosis, entomophthorosis, geotrichum, histoplasmosis, mucormycosis, mycetoma, nocardiosis, North American blastomycosis, paracoccidioidomycosis, pheochromocytoses, phycomycosis, pneumocystis pneumonia, pythium infection, sporotrichosis, and torulopsis, and their synonyms, some of which can be fatal. Known fungal and bacterial pathogens include, but are not limited to, the genera Absidia, Actinomadura madurae, Actinomyces sp., Allescheria boydii, Alternaria sp., Anthopsis deltoidea, Apophysomyces elegans, Arnium leoporinum, Aspergillus sp., Aureobasidium pullulans, Basidiobolus ranarum, Bipolaris sp., Blastomyces dermatitidis, Candida sp., Cephalosporium sp., Chaetoconidium sp., Chaetomium sp., Cladosporium sp., Coccidioides immitis, Conidiobolus sp., Corynebacterium tenuis, Cryptococcus sp., Cunninghamellabertholletiae, Curvularia, Dactylaria, Epidermophyton, Epidermophyton floccosum, Exserophilum, Exophiala, Fonsecaea, Fusarium, Geotrichum, Helminthosporium, Histoplasma, Lecythophora, Madurella, Malassezia furfur, Microsporum spp., Mucor spp., Mycocentrospora acerina, Nocardia spp., Paracoccidioides brasiliensis, Penicillium spp., Phaeosclera Dematioides, Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp., Phoma spp., Piedraia hortai, Pneumocystis carinii, Pythium insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizopus spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella boppii, Torulopsosis spp., Trichophyton spp., Trichosporon spp., Ulocladium chartarum, Wangiella dermatitidis, Xylohypha spp., Zygomyetes spp., and synonyms thereof. Other fungi with pathogenic potential include, but are not limited to, Thermomucor indicae-seudaticae, Radiomyces spp., and other species of known pathogenic genera.

[0073] Other medically relevant microorganisms have been described extensively in the literature, see, for example, Medical Microbiology (1983), the entire contents of which are incorporated herein by reference.

[0074] In other embodiments, the methods and devices described herein can be used for early cancer / precancerous detection from circulating extracellular vesicles (EVs) encapsulated miRNA in blood, biological fluids, or cerebrospinal fluid (CSF), allowing for rapid and accurate diagnosis at a fraction of the current cost. The disclosed detection technology is highly sensitive, reliable, and does not require amplification, thus eliminating any bias associated with the amplification process.

[0075] The disclosed devices are compact, easy to use, low cost, and rapidly generate test results from small sample volumes. Thus, the disclosed detection techniques and devices provide a platform technology applicable to miRNA and DNA biomarkers for a variety of pathogens and cancer types / subtypes.

[0076] Reaction solution Solutions and compositions for probe-target in hybridization applications are known in the art. Such compositions may include, for example, buffers, enhancers, chelators, salts, surfactants, and blocking agents. Marangoni flow can be achieved relatively easily in droplets of organic solvents, but may be more difficult in aqueous solutions, such as the types of liquid solutions used in bioassays involving nucleic acid hybridization. To facilitate Marangoni flow in aqueous droplets, various enhancing additives such as surface active agents and detergents can be used. Other additives can also be added to enhance visualization of the product (e.g., with FRET pairs under a fluorescent microscope) or to stabilize the droplets. Examples include glycerol, salts (e.g., NaCl and LiCl), and polymers (e.g., FICOLL®, a copolymer of sucrose and epichlorohydrin).

[0077] Examples of buffers may include SSC, HEPES, SSPE, PIPES, TMAC, TRIS, SET, citrate, phosphate buffers such as potassium phosphate or sodium pyrophosphate. The buffers may be present at a concentration of 0.01× to 50×, e.g., 0.01×, 0.1×, 0.5×, 1×, 2×, 5×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, or 50×. Typically, the buffers are present at a concentration of 0.1× to 10×.

[0078] Examples of enhancers may include polymers such as FICOLL, PVP, heparin, dextran sulfate, proteins such as BSA, glycols such as ethylene glycol, glycerin, 1,3 propanediol, propylene glycol, or diethylene glycol, combinations thereof such as Denhardt's solution and BLOTTO, and organic solvents such as formamide, dimethylformamide, DMSO, and the like. The enhancer may be present in a concentration of 1% to 80% or 0.1x to 10x, for example, 0.1% (or 0.1x), 0.2% (or 0.2x), 0.5% (or 0.5x), 1% (or 1x), 2% (or 2x), 5% (or 5x), 10% (or 10x), 15% (or 15x), 20% (or 20x), 25% (or 25x), 30% (or 30x), 40% (or 40x), 50% (or 50x), 60% (or 60x), 70% (or 70x), or 80% (or 80x). Typically, formamide is present in a concentration of 25% to 75%, e.g., 25%, 30%, 40%, 50%, 60%, 70%, or 75%, while DMSO, dextran sulfate, and glycol are present in a concentration of 5% to 10%, e.g., 5%, 6%, 7%, 8%, 9%, or 10%.

[0079] Examples of chelating agents may include EDTA, EGTA, etc. The chelating agent may be present at 0.1 mM to 10 mM, e.g., 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. Typically, the chelating agent is present at a concentration of 0.5 mM to 5 mM, e.g., 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM.

[0080] Examples of salts may include sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, magnesium chloride, magnesium phosphate, etc. The salts may be present at a concentration of 1 mM to 800 mM, for example, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, or 750 mM. Typically, the salts are present at a concentration of 10 mM to 500 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500 mM.

[0081] Examples of detergents may include TWEEN, SDS, TRITON, CHAPS, deoxycholic acid, etc. The detergent may be present at a concentration of 0.001% to 10%, such as 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Typically, the detergent is present at a concentration of 0.01% to 1%, such as 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

[0082] Examples of nucleic acid blocking agents may include, for example, yeast tRNA, homopolymeric DNA, denatured salmon sperm DNA, herring sperm DNA, total human DNA, COT1 DNA, etc. Nucleic acid blocking agents may be present at concentrations from 0.05 mg / mL to 100 mg / mL. However, the compositions and methods of the present invention surprisingly exhibit significantly reduced background levels without the need for blocking agents.

[0083] The above compositions may include any of the above components in combination with at least one polar aprotic solvent. The components may be present in the same concentration as used in conventional denaturing solutions, or may be present in higher or lower concentrations, or may be omitted entirely. In some embodiments, one or more components may be applied to a substrate or reaction area at a much lower concentration, since sessile droplets offer the advantage of reduced internal current and volume upon evaporation, which allows for enhanced molecular interactions.

[0084] The solution may have some salt / sugar present to prevent non-specific interactions, but the concentration may be low enough that complete evaporation of the droplet will not cause enough crystallization to impair the assay. For example, a concentration of ≦50 mM Na+ may be sufficient to screen for non-specific interactions and will not cause deleterious crystallization upon complete evaporation of the droplet. Also, a NaCl concentration of 50 mM would be sufficient to create flow patterns in the droplet of evaporated liquid carrier on the PDMS.

[0085] kit In another aspect, the present disclosure provides kits containing various reagents useful for carrying out the methods described herein. The kits may include suitable substrates in derivatized or underivatized form, reagents for sample isolation, purification or preparation, probes, hybridization solutions, liquid carriers, hygroscopic materials, salts, wash solutions, blocking agents, HCT reaction mixtures, probes, reporter molecules, and means for detecting the probes or reporter molecules.

[0086] The components of the kit may be packaged either in aqueous media or in lyophilized form. When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that a solvent may also be provided.

[0087] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which a component may be placed, and preferably appropriately aliquoted. If there is more than one component in the kit, the kit will also generally include a second, third, or other additional container into which the additional component may be separately placed. However, various combinations of components may be included in the vial. The kits of the present invention will also typically include a means for sealingly containing the reagent vials and other kit components for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0088] Regardless of the number or type of containers, the kits of the present invention may also include or be packaged with instructions for use of the various reagents. In some embodiments, the kits also include containers containing the compositions and, optionally, informational materials. The informational materials may be descriptive, instructional, marketing, or other materials related to the methods and / or uses of the agents described herein. The informational materials of the kits are not limited in form. In some embodiments, the informational materials may include information regarding the manufacture of the compositions, concentration, expiration date, batch or manufacturing site information, and the like. The information may be provided in a variety of formats, including printed text, computer readable materials, video recordings, or audio recordings, or information that includes links or addresses to the substantive materials. The kits optionally include a device suitable for carrying out the methods disclosed herein.

[0089] definition As used herein, the term "liquid" or "droplet" refers to a small volume of liquid that is immiscible with its surroundings (e.g., gas, liquid, surface, etc.). A droplet may be present on a surface or encapsulated by an immiscible fluid (e.g., an emulsion, a continuous phase of a gas (e.g., air, nitrogen)), or a combination thereof. Droplets are typically spherical or substantially spherical in shape, but may be non-spherical. The shape of an otherwise spherical or substantially spherical droplet may be altered by deposition on a surface or shrinkage within a small diameter capillary channel. A droplet may be a "simple droplet" or a "compound droplet," where one droplet encapsulates one or more additional smaller droplets. The volume of a droplet and / or the average volume of a set of droplets provided herein is typically less than about 100 microliters (e.g., 100 μL, 10 μL, 1 μL, 100 nL, 10 nL, 1 nL, 100 pL, 10 pL, 16 L, 100 fL, 10 fL, or 1 fL). The diameter of a droplet and / or the average diameter of a set of droplets provided herein is typically less than about 1 millimeter (e.g., 1 mm, 100 μm, 10 μm, or 1 μm). The droplets may be formed by any suitable technique (e.g., emulsification, microfluidics, injection, etc.) and may be monodisperse (e.g., substantially monodisperse) or polydisperse.

[0090] A sessile droplet refers to a droplet that resides on the surface of a solid or semi-solid substrate.

[0091] An "aqueous solution" should be understood as a solution that contains water, even a small amount of water. For example, a solution that contains 1% water should be understood as an aqueous solution.

[0092] "Hybridization reaction," "hybridization assay," "hybridization experiment," "hybridization procedure," "hybridization technique," "hybridization method," and the like, should be understood to refer to any process involving hybridization of nucleic acids. Unless otherwise specified, the terms "hybridization" and "hybridization step" should be understood to refer to the reannealing step, as well as the denaturation step (if present), of the hybridization procedure.

[0093] "Reaction solution" refers to a solution or composition for carrying out a reaction. If the reaction involves detection or hybridization, the reaction solution is also referred to as a "detection solution" (i.e., a solution or composition for carrying out a reaction such as a detection assay) or a hybridization solution (i.e., a solution or composition for carrying out a reaction such as a hybridization assay).

[0094] "Hybridization solution" refers to an aqueous solution for use in a hybridization composition. Hybridization solutions are discussed herein and can include, for example, buffers, facilitating agents, chelating agents, salts, surfactants, and blocking agents.

[0095] "Hybridization composition" refers to an aqueous solution for carrying out a hybridization procedure, for example, to bind a probe to a nucleic acid sequence. The hybridization composition may, for example, include at least one polar aprotic solvent, at least one nucleic acid sequence, and a hybridization solution. The hybridization composition does not include other components, such as enzymes or deoxynucleoside triphosphates (dNTPs), for amplifying nucleic acids in a biological sample.

[0096] The term "contacting" as used herein, when used with respect to any set of components, includes any process in which the components to be contacted are mixed in the same mixture (e.g., added to the same compartment or solution) and does not necessarily require actual physical contact between the mentioned components. The mentioned components may be contacted in any order or in any combination (or subcombination), and may also include situations in which one or some of the mentioned components are subsequently removed from the mixture, optionally prior to the addition of other mentioned components. For example, "contacting A with B and C" includes any and all of the following situations: (i) A is mixed with C, and then B is added to the mixture; (ii) A and B are mixed into the mixture, B is removed from the mixture, and then C is added to the mixture; and (iii) A is added to a mixture of B and C.

[0097] Nucleic acid or polynucleotide refers to a DNA molecule (such as, but not limited to, cDNA or genomic DNA) or an RNA molecule (such as, but not limited to, mRNA, rRNA, tRNA, miRNA, or siRNA), including DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. DNA or RNA molecules can contain non-naturally occurring moieties, such as modified bases, modified backbones, deoxyribonucleotides in RNA, etc. Nucleic acid molecules can be single-stranded or double-stranded.

[0098] The term "disease" as used herein is intended to be generally synonymous with, and is used interchangeably with, the terms "disorder" and "condition" (in medical conditions), in that both reflect an abnormal condition of the human or animal body or parts thereof that impairs normal functioning, is typically manifested by clear signs and symptoms, and reduces the duration or quality of human or animal life.

[0099] As used herein, the term "approximately" or "about" when applied to one or more subject values, refers to a value similar to the reference value mentioned. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value mentioned in either direction (greater or smaller), unless otherwise stated or unless otherwise clear from the context (except when such number exceeds 100% of the possible values). Unless otherwise indicated in the specification, the term "about" is intended to include values, such as weight percentages, close to the reference range that are equivalent in terms of the functionality of the individual components, compositions, or embodiments.

[0100] When values ​​and ranges are provided herein, it should be understood that all values ​​and ranges subsumed within those values ​​and ranges are intended to be encompassed within the scope of the disclosure. Moreover, all values ​​that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the application. EXAMPLES

[0101] Example 1 This example describes the materials and methods used in Examples 2-7.

[0102] material All DNA oligos were purchased from IDT Integrated DNA Technology (Coralville, IA, USA). All 3D printed materials were designed in SOLIDWORKS 2018-2019 Student Edition and printed using polylactic acid (PLA) and a Prusa i3 MK3S purchased from Prusa (Prague, Czech Republic). Droplets were evaporated using a Drierite from WA Hammond Drierite Co. (Xenia, OH, USA). Gel electrophoresis was performed using a HE33 Mini Horizontal Agarose Electrophoresis Unit and a MIGHTY SLIM SX250 power supply from Hoefer (Holliston, MA, USA). UltraPure™ agarose, Sybr Safe DNA gel stain, sodium chloride, magnesium chloride, sodium citrate, and Amplitron® II thermocycler were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Water was purified using a Barnstead™ NANOpure II from Thermo Fisher Scientific. 10× Tris-borate EDTA (TBE) was purchased from Bio-Rad Laboratories (Hercules, CA, USA), as was the CHEMIDOC MP used for imaging the agarose gel. Sylgard™ 184 Silicone Elastomer Kit was purchased from Dow (Midland, MI, USA). A FLUOROMAX-4 spectrofluorometer from Horiba Scientific (Piscataway, NJ, USA) was used for acquiring spectral data. A UV-1800 spectrophotometer from Shimadzu (Kyoto, Japan) was used for DNA hairpin melting analysis. Data were analyzed using JMP Pro 16.

[0103] DNA Hairpin Annealing The H1 and H2 DNA hairpins were incubated at 95° C. for 5 min in a thermocycler and then cooled to room temperature for 30 min.

[0104] Fabrication of the pedestal A 1.5 mm pedestal ring mold and drying chamber were designed using computer software and 3D printing. The silicone elastomer kit was mixed in a 7:1 ratio (part A to part B) and poured into the pedestal mold to create a polydimethylsiloxane (PDMS) pedestal. The PDMS in the mold was degassed and cured overnight. The cured PDMS pedestal was removed from the mold and placed in a drying chamber.

[0105] HCR preparation Annealed H1 and H2 DNA hairpins were diluted to 1500 nM concentration and initiators were diluted to 3x their final concentration in the specified assay buffer. For the fluorescent assay, H1 and H2 have 3' ATTO 550N and 5' ATTO 647N fluorophore conjugations, respectively. All buffers were made by diluting 5x saline sodium citrate (SSC) (750 mM sodium chloride and 75 mM sodium citrate) with water and adding magnesium chloride as indicated. The diluted hairpins and initiators were then mixed in a 1:1:1 ratio in a 0.5 μL cuvette. The final concentrations of H1 and H2 were 500 nM and the initiator concentrations are as indicated in the results figures. Two 5 μL droplets for each reaction were dispensed onto the pedestal and 10 μL of each reaction was left in the cuvette as a liquid incubation control. Desiccant was added to the drying chamber, the experiment was covered with the chamber lid, and allowed to evaporate completely for 1 hour. 5 μL of water was dispensed onto each dried sessile droplet pedestal and left covered to resuspend the dried material. After 5 minutes, the droplets were drawn into a pipette and two droplets (two separate pedestals) per reaction were added to a cuvette for a total final volume of 10 μL per reaction.

[0106] Agarose gel electrophoresis A 3% agarose gel was prepared by boiling 1.2 grams of agarose in 40 mL of 1×TBE via microwave. 4 μL of SYBR SAFE was added to the melted solution and mixed thoroughly before pouring into a gel mold and setting. 10 μL of sample was prepared for gel electrophoresis by adding 3.5 μL of water and 1.5 μL of 40% sucrose in water, then run in an agarose gel in 0.5×TBE running buffer at 150 volts for 40 minutes.

[0107] Forster Resonance Energy Transfer (FRET) For the fluorescent HCR assay, 10 μL of sample was diluted with 690 μL of water. 700 μL of the diluted solution was added to a quartz cuvette, and the spectral emission intensity was collected by a spectrofluorometer by exciting the sample at a wavelength of 560 nm and scanning the emission intensity from 567 nm to 750 nm. The spectral intensity was normalized to the peak emission intensity and graphed using MATLAB R2020b.

[0108] Example 2 In this example, HCR was used to demonstrate significant enhancement of extended DNA hairpin hybridization when incubated in sessile droplets compared to conventional liquid cuvette incubation. In summary, HCR assays were performed in both sessile droplets (S) and cuvettes in the manner described above. The resulting hybridization products were visualized and examined in the manner described above. The results are shown in Figures 2, 3, and 4.

[0109] As shown in Figures 2A and 2B, significantly more HCR products were formed by the hairpin probe when incubated in sessile droplets compared to conventional incubation conditions in a cuvette, especially at low trigger concentrations (50 nM). Both experiments in Figures 2A and B were prepared in an identical manner using 0.5x SSC buffer containing 10 mM MgCl2.

[0110] Figure 3A shows a custom designed, 3D printed array of 2 mm diameter pedestals used to dry the HCR sessile droplets, and FRET fluorescence images obtained from the HCR product are shown in Figures 3B and C.

[0111] Figure 4 shows a direct comparison between HCR experiments incubated in sticky droplets (S) and cuvettes (L) with various concentrations of trigger sequences. Each sticky droplet lane had distinct higher molecular weight banding at higher intensity than its liquid cuvette complement, indicating increased hybridization activity with sticky droplet incubation.

[0112] Additional results are shown in Figures 8A, 8B, and 8C. The agarose gel results shown in Figure 8A empirically demonstrated enhanced hybridization of HCR via sticky droplet incubation when compared to conventional cuvette incubation. The addition of Tween 20 detergent to the hybridization buffer (+) also found enhanced HCR product in both sticky and cuvette incubation via hybridization buffer without Tween 20 (-). Enhanced hybridization of HCR via sticky droplet incubation is shown by spectrofluorometer data shown in Figures 8B and 8C, where the FRET intensities of HCR products from synthetic proviral DNA targets (Figure 8B) and synthetic viral RNA targets (Figure 8C) were normalized using the FRET ratio.

[0113] Example 3 In this example, we further demonstrated the effect of sticky droplet-induced hybridization via FRET-HCR. The increase in hybridization leads to more H1 and H2 FRET pairs within a communicable distance from each other. More specifically, when excited at the donor fluorophore excitation wavelength (560 nm), the hybridized H1 fluorophore transfers resonance energy to the hybridized H2 fluorophore and emits fluorescence at a much longer wavelength (about 664 nm) than that emitted by the unhybridized H1 fluorophore. As shown in Figure 5, the 100 nM trigger sticky droplet experiment had significantly higher H2 intensity than the 100 nM trigger liquid cuvette experiment, indicating a proportional increase in HCR hybridization with sticky incubation.

[0114] Example 4 In this example, an assay was performed to examine the effect of buffer ion concentration on hybridization efficiency. Buffers containing high sodium and magnesium concentrations are known to enhance DNA hybridization. However, as the volume of the evaporating sessile droplets decreases, the ion concentration of the buffer increases, affecting the hybridization of DNA oligos. We used various concentrations of SSC buffers with or without magnesium chloride to demonstrate the effect of ions on sessile droplet-enhanced hybridization. 5x concentration of SSC is commonly used for hybridization experiments. However, as shown in Figure 6, the buffer with the highest ion concentration (0.5x SSC with 10 mM MgCl2) had the largest HCR hybridization product, but it also had significantly more uninitiated hybridization in its 0 nM control lane than the lower ion buffer. 0.25XSSC with 10 mM MgCl2 appeared to have higher molecular weight band formation at the 500 pM trigger concentration and significantly less band formation in the 0 nM control lane, suggesting that a lower salt buffer may have a beneficial effect on the sticky droplet hybridization assay.

[0115] Similar assays were performed in the presence of different concentrations of NaCl or MgCl2, or both, to examine the effect of ionic concentration on hybridization efficiency. The results are shown in Figure 11A. As shown, higher ionic concentrations reduced overall hybridization.

[0116] Additional assays were performed to examine the experimental melting temperature of the H1 hairpin at different sodium concentrations. The results are shown in Figure 11B. As the salt concentration increased, the melting temperature of the hairpin increased, indicating that here the volume of the sessile droplets was decreasing due to evaporation. The volume concentration relative to the salt was calculated using 75 mM NaCl as the starting concentration. The increase in hairpin stability with increasing salt concentration supports the results in Figure 11A, indicating that higher salt concentrations may decrease hybridization. The salt concentration in the buffer increased as the droplets evaporated, highlighting the importance of the buffer composition during sessile droplet incubation. All buffers in Figure 11A contained 0.1% Tween 20. The buffers in Figure 11B do not contain Tween 20 because excessive micelle formation would corrupt the UV / vis data. Data was analyzed using JMP Pro 16.

[0117] Example 5 HCR assays were performed on synthetic DNA triggers under two different conditions. In the first, "wet" condition (W), the sessile droplets were rehydrated every 20 min to maintain their original volume of 5 μL. In the second, "dry" condition (D), the sessile droplets were allowed to evaporate completely. Both droplets were incubated for 70 min and then analyzed by agarose gel. The results shown in Figure 9 show a significant increase in HCR product in the dry condition (D), demonstrating the importance of reducing the volume of the sessile droplets.

[0118] Example 6 The HCR sticky droplet assay described herein was carried out in the presence of three synthetic DNA triggers with 0, 1, and 2 mismatches: AGGTTTGGGGAAGAGACA (SEQ ID NO: 1), AGGTTTGGGGAGGAGACA (SEQ ID NO: 2), and AGGTCTGGGGTAGAGACA (SEQ ID NO: 3). The results are shown in FIG. 10A. It was found that there was no significant increase in HCR products with 1 and 2 mismatches compared to the 0 nM trigger control. This result demonstrates the high specificity of the HCR sticky droplet assay.

[0119] The HCR sticky droplet assay described herein was carried out in the presence of non-specific DNA such as salmon DNA with a mass ratio of salmon DNA to synthetic trigger DNA of 5:1. All data was analyzed using JMP Pro 16, and p-values ​​were calculated using a two-tailed Student's t-test. The results are shown in Figure 10B. As shown in Figure 10B, the HCR sticky droplet assay shows high resistance to off-target hybridization.

[0120] Example 7 The HCR sticky droplet assay described herein was performed to detect synthetic HIV RNA and proviral DNA directly on filter paper (Fusion5, Cytiva). The assay setup is shown in Figures 12A, 12B, and 12C. Briefly, 1.5 mm diameter pads were cut and attached to PDMS pedestals to allow sticky droplets to form on the hydrophilic material (Figure 12A). The target nucleic acid was allowed to dry on the paper disk. 5 μL of HCR solution (H1 and H2) was then applied as sticky droplets to each pedestal and allowed to dry completely (Figure 12B). The dried paper was then imaged using a fluorescent microscope equipped with a FRET cube (Figure 12C). Image intensities were analyzed using ImageJ. p-values ​​were calculated using a two-tailed Student's t-test. The results are shown in Figures 12D and 12E. As shown in the figure, the assay was successful in detecting as little as 2.5 fmole of synthetic HIV RNA and 0.75 fmole of proviral DNA.

[0121] References 1. Luo J, Xu Y, Huang J, Zhang S, Xu Q, He J. Enzyme-free amplified detection of circulating microRNA by making use of DNA circuits, a DNAzyme, and a catalytic hairpin assembly. Microchim Acta 2017 1851. 2017;185(1):1 - 6. doi:10.1007 / S00604-017-2565-9 2. Guixiu Dong, Jianyuan Dai, Limin Jin, et al. A rapid room-temperature DNA amplification and detection strategy based on nicking endonuclease and catalyzed hairpin assembly. Anal Methods. 2019;11(19):2537 - 2541. doi:10.1039 / C9AY00507B 3. Dong Q, Liu Q, Guo L, et al. A signal-flexible gene diagnostic strategy coupling loop-mediated isothermal amplification with hybridization chain reaction. Anal Chim Acta. 2019;1079:171 - 179. doi:10.1016 / j.aca.2019.06.048 4. Wang W, Nie A, Lu Z, Li J, Shu M, Han H. Catalytic hairpin assembly-assisted lateral flow assay for visual determination of microRNA-21 using gold nanoparticles. Microchim Acta. 2019;186(9):1 - 9. doi:10.1007 / s00604-019-3743-8 5.Wang W-J,Li J-J,Rui K,Gai P-P,Zhang J-R,Zhu J-J.Sensitive Electrochemical Detection of Telomerase Activity Using Spherical Nucleic Acids Gold Nanoparticles Triggered Mimic-Hybridization Chain Reaction Enzyme-Free Dual Signal Amplification.AnalChem.2015;87(5):3019-3026.doi:10.1021 / AC504652E 6.Hernandez-Perez R,Fan ZH,Garcia-Cordero JL.Evaporation-Driven Bioassays in Suspended Droplets.AnalChem.2016;88(14):7312-7317. doi:10.1021 / acs.analchem.6b01657 7.Hu H,Larson RG.Marangoni effect reverses coffee-ring depositions.J Phys Chem B.2006;110(14):7090-7094.doi:10.1021 / jp0609232 8.Mitre E,Schulze M,Cumme GA,Roβler F,Rausch T,Rhode H.Turbo-Mixing in Microplates.J Biomol Screen.2007;12(3):361-369.doi:10.1177 / 1087057106297565.

[0122] The foregoing examples and descriptions of preferred embodiments should be understood as illustrative rather than limiting the present disclosure as defined by the claims. As will be readily understood, numerous variations and combinations of the above features may be utilized without departing from the present disclosure as set forth in the claims. Such variations are not to be considered as departing from the scope of the present disclosure, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. 1. A method for detecting a target nucleic acid in a test sample, comprising: providing a substrate having a reaction area on which the test sample and a detection solution are deposited to form a reaction droplet, the detection solution comprising a detection agent having a sequence complementary to a strand of the target nucleic acid, the detection solution further comprising a hybridization chain reaction (HCR) mixture; incubating the reaction droplet under conditions that allow evaporation thereof, The condition is Humidity of about 5% to about 95%; a temperature of about 10 to about 50°C, and / or incubating for a duration of about 10 to about 300 minutes; detecting a hybridization complex of the target nucleic acid and the sequence in the reaction droplet; whereby the presence of the hybridization complex indicates the presence of the target nucleic acid in the test sample.

2. The providing step: placing a sample droplet containing the test sample on the reaction area; and contacting the reaction area or the test sample on the reaction area with the detection solution.

3. The method of claim 2 , further comprising drying the sample droplet on the reaction area prior to the contacting step.

4. The method of any one of claims 1 to 3, wherein the reaction region is hydrophilic and surrounded by hydrophobic regions.

5. The method according to any one of claims 1 to 3, wherein the reaction area or the substrate is surrounded by an impermeable border.

6. The method of any one of claims 1 to 3, wherein the reaction area or the substrate comprises a material selected from the group consisting of glass, plastic, metal, silicon, and paper.

7. The method of any one of claims 1 to 3, wherein the substrate comprises a pillar-like or pedestal-like structure with a circular top that serves as the reaction area.

8. The method of claim 7, wherein the circular top has a diameter of about 0.5 μm to 4 mm.

9. The method of any one of claims 1 to 3, wherein the reaction droplets or sample droplets are about 1 to 25 μl.

10. 3. The method of claim 1, wherein the detection solution comprises any combination of Na ions and K ions at a concentration of about 15 to about 800 mM.

11. 11. The method of claim 10, wherein the detection solution comprises one or more of about 0.1 to about 5.0×SSC, about 1 to about 15 mM Mg2+, PBS, TBS, a surfactant, glycerol, and sucrose.

12. The method of claim 1 , wherein the target nucleic acid is DNA or RNA.

13. The method of claim 1 , wherein the target nucleic acid is a pathogen nucleic acid.

14. 14. The method of claim 13, wherein the pathogen is a virus, optionally wherein the virus is HIV, dengue, SARS-CoV-2, or Ebola.

15. 3. The method of claim 1 or 2, wherein the test sample comprises a blood sample, a sputum sample, a urine sample, a urine swab sample, or a saliva sample.

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