WO2017223026A1 - Detection of rna using ligation actuated loop mediated amplification methods and digital microfluidics - Google Patents

Abstract

Apparatuses and methods for detecting a target RNA sequence or sequences using a ligation assisted loop mediated isothermal amplification (LA LAMP) assay. In particular, described herein are methods for LA LAMP including digital micro fluidics (DMF) implementation of LA LAMP.

Description

DETECTION OF RNA USING LIGATION ACTUATED LOOP MEDIATED AMPLIFICATION

METHODS AND DIGITAL MICROFLUIDICS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application may be related to International Patent Application No.

PCT/US2014/072802, filed July 9, 2015, titled "SYSTEMS, COMPOSITIONS AND METHODS FOR DETECTING AND ANALYZING MICRO-RNA PROFILES FROM A BIOLOGICAL SAMPLE", Publication No. WO2015/103293, which claims priority to U.S. Provisional Patent Application No. 61/921 ,761 , filed Dec. 30, 2013 and U.S. Provisional Patent Application No. 62/068,589, filed Oct. 24, 2014, each of which is herein incorporated by reference in its entirety. This application may also be related to International Patent Application No. PCT/US2016/036015, filed Jun. 6, 2016, titled "AIR- MATRIX DIGITAL MICROFLIUIDICS APPARATUSES AND METHODS FOR LIMITING EVAPORATION AND SURFACE FOULING" and/or to International Patent Application No.

PCT/US2016/036022, filed Jun. 6, 2016, titled "EVAPORATION MANAGEMENT IN DIGITAL MICROFLUIDIC DEVICES", each of which is herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

FIELD

[0003] Described herein are methods and apparatuses for detecting RNA, and in particular, short sequences of RNA referred to as microRNAs. For example, described herein are methods and apparatuses (and in particular, digital microfliuidics methods and apparatuses) that use a modified version of loop mediated isothermal amplification (LAMP) adapted to detect RNA, and particularly microRNA.

BACKGROUND

[0004] Genetic materials are becoming increasingly more useful for medical diagnostics, be it genetic material from a gene, a part of a gene, or a nucleotide sequence found in a living organism, cellular extract from the living organism, or other biological sample. Genetic material is typically characterized by specific nucleotide sequence, and researchers continue to search for ways to identify and detect the presence of specific nucleotide sequences.

[0005] These detection methods may be applied to a wide variety of systems. For example, identifying specific polynucleotide sequences maybe used to detect pathogens, the presence of certain alleles, the presence of lesions in a host genome, modifications of native sequences in host cell, and so forth. Typically, once the specific nucleotide sequence is known, the nucleic acids may be extracted from a sample to determine whether the specific sequence is present.

[0006] In recent years, research has shown that certain small, non-coding microRNAs to be extremely adept at regulating cellular processes and expression patterns have shown the potential of microRNAs as diagnostic, prognostic, and treatment response biomarkers. MicroRNAs (miRNAs) are small (typically 18- 25 nucleotides) non-coding RNAs that are important in regulating gene expression by binding to mRNA transcripts and influencing their stability or translation efficiency. MiRNAs have been shown to circulate within blood and appear to be relatively stable in the plasma and serum.

[0007] Research in the past 10 years has provided evidence that microRNA expression profiling can distinguish subtypes of cancer, even their stages, in much higher accuracy than genomics, transcriptomics or proteomics analyses. MicroRNAs can be found deregulated in the bloodstream (cell free or in exosomes or other vesicles), in patterns that can accurately reflect a wide range of pathophysiology, including many types of cancer, metabolic, psychiatric and cardiovascular diseases. Interestingly, a significant fraction of the microRNAs and other small RNAs found in circulation with biological implications and capability to reflect disease are in fact derived from exogenous sources, namely species of animals and plants that are part of the human diet such as plants, fungi, bacteria or even viruses all considered "exogenous RNA molecules" yet able to be associated with disease.

[0008] For example, there may be significant overexpression of miR-141 in individuals with prostate cancer compared with normal individuals. At a miRNA- 141 level of above 2,500 copies per μΐ, of serum, individuals with prostate cancer have been identified with 100% clinical specificity and 60%> clinical sensitivity. miRNA levels in individuals diagnosed with cancer have been shown to be moderately correlated with their PSA levels with Pearson and Spearman (rank) correlation coefficients of +0.85 and 1 0.62. Numerous other correlations between microRNAs and disease states have been suggested.

[0009] To date, various nucleic acid assay technologies have been used to identify and characterize miRNAs, such as microarray- and polymerase chain reaction (PCR)-based assays. Particularly for miRNAs that are present in low amounts, amplification techniques such as quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) or isothermal NASBA, (nucleic acid sequence based amplification) has been used to amplify the targets of interest. One major hurdle to overcome in identifying and characterizing miRNAs is being able to obtain enough sample concentration for either detection or characterization. Unfortunately, immobilization of probes or target amplification decreases assay sensitivities and increases cost and time requirements. Currently proposed methods are less effective and may be difficult to reliably interpret due in part to excessive background.

[0010] Adaptations of pre-existing molecular profiling methods (such as microarray, qPCR and massive parallel sequencing) to detect small RNAs and in particular miRNAs in tissues, cells and biofluids have been proposed. However, the chemistries available to date are not affordable for routine check-up diagnostic purpose, nor are the instruments necessary to collect the data (with the most affordable costing around $20,000 USD). Thus, high investment costs hamper the growth of global miRNA market. In addition, lack of skilled professionals who can execute complex molecular biology protocols behind each of the aforementioned techniques, also obstructs the growth of global miRNA market.

[0011] One technique, loop-mediated isothermal amplification (LAMP) has been extremely promising in developing point of care diagnostic tests based on amplification of nucleic acid sequences that code for pathogens but still possess some drawbacks that prevent the technique from being a widely- used diagnostic tool. The LAMP method amplifies nucleic acids with high sensitivity by using an enzyme with strand displacement activity under isothermal conditions. LAMP uses four to six specially designed primers recognizing six to eight regions of the target DNA sequence; the four to six primers afford LAMP its specificity. Although LAMP is known for having a fairly robust mechanism for amplification, specificity may still be a factor especially in detecting samples that are natively low in concentration such as microRNAs. Thus, improvements in specificity would be extremely beneficial for using a LAMP-based amplification method on microRNAs.

[0012] Although the use of LAMP techniques to modify mircoRNA has been suggested, it has proven extremely difficult to apply LAMP, particularly to multiple types of microRNAs. In general, LAMP requires a DNA template that is many times larger than the microRNA to be amplified, and includes sites for the 4-6 primers used for LAMP. As a result, the microRNAs to be detected by LAMP must first be processed into template that can be amplified. The accuracy and sensitivity of this detection method therefore depends critically on the ability to form the proper template material from a sample including microRNAs to be detected. When multiple microRNAs are to be detected out of a single sample, it is also critical that the template formation and LAMP method amplify uniformly between different microRNAs.

[0013] We have previously proposed a technique for the formation of LAMP templates using an enzyme such as SplintR ligase, also known as PBCV-1 DNA Ligase or Chlorella virus DNA Ligase, which catalyzes the ligation of adjacent, single-stranded DNA splinted by a complementary RNA strand (such as the microRNA). SplintR has been shown to be superior to other single-stranded DNA

("splinting") ligases, including in particular T4 RNA Ligase 2. See, e.g., Jin et al., Nucleic Acids Research (May 6, 2016), "Sensitive and specific miRNA detection method using SplintR Ligase".

Unfortunately, the use of SplintR ligase entails challenges with regard to the compatibility of the optimal buffer used for the ligation with the LAMP procedure (incompatible with some LAMP reaction detection methods such as calcein), as well as challenges with non-specific activity when the reaction is saturated with ligatable ends or sub optimal performance when the concentration of SplintR in a reaction is below l OnM.

[0014] Described herein are methods and apparatuses that may address the issues raised above. SUMMARY OF THE DISCLOSURE

[0015] In general, described herein are methods for detecting RNAs within a sample. In particular, the methods described herein are adept at targeting a single or many microRNAs of interest for amplification and detection within a single sample. The methods described herein improve upon existing methods of detecting RNA, specifically microRNAs using a modified LAMP-based technique.

[0016] The overall process for obtaining an amplified target sequence that is able to be detected includes an annealing step, a ligation step, several rounds of nucleotide strand displacement and hybridization syntheses, and finally amplification steps that are similar to the amplification steps of LAMP.

[0017] In general, the sample may or may not contain the target RNA sequence of interest. If the sample does contain the predicted target sequence, the methods described below will be able to amplify the target sequence so that it may be detectable using standard detection methods. When the sample does not contain the target sequence, the annealing step will fail and no sequences will be subsequently produced. In the event that a sequence similar to the target sequence is produced in the earlier steps, subsequent interrogation steps are provided to add another level of specificity not available in previously described LAMP techniques to prevent these sequences from being amplified.

[0018] Samples being tested using the methods described herein may be any biological sample where it is desirable to know if a target RNA sequence is present or absent. The samples may be purified or partially purified prior to performing the assay or may be a crude sample obtained from a subject.

[0019] For example, described herein are methods for detecting target RNAs, specifically target microRNAs where the first steps may include designing the primers and the template sequences needed for ultimately detecting the target sequence or sequences. An algorithm may be used to provide primers and templates that lead to greater specificity in the subsequent assay for the target sequence or sequences. The target RNA of known nucleotide sequence may be used to create both primers and recognition regions of the template nucleotide sequence.

[0020] Once the primers are created, any sample (e.g., patient sample) may be tested to see if the target RNA (e.g., microRNA) sequence is present. Template sequences (e.g., "Trap A" and "TrapB") may be introduced into the sample where the template sequences each contain regions that are complimentary to adjacent portions of the target RNA sequence at either their 3' end or at their 5' end. If the target RNA (e.g. mRNA) sequence is present, it will anneal with the template sequence and with the aid of a ligating agent, bring together the two template sequences having the complete target RNA sequence where the ligated template now contains not only the binding regions but also the target sequence. Through a series of strand displacement nucleotide synthesis and hybridization steps, a structure having ability to self-interrogate is generated prior to exponential amplification. This interrogation steps increase specificity for amplifying the target sequence or sequences and is a tremendous improvement on prior LAMP-based assays. Finally, in some instances, a positive sample may be run prior to running the actual samples to ensure that the system is functioning properly.

[0021] Described herein are Ligation-actuated loop mediated isothermal amplification (LA-LAMP) methods and apparatuses that have been optimized for performance so that they may reliably be used for diagnostic testing. Diagnostic testing with isothermal reactions is particularly difficult because it is often difficult to avoid errors in the amplification process (e.g., miss-hybridization of primers, target, template, etc.) or ligation and annealing. It may has also proven difficult to operate in very small volume (e.g., less than 20 μΐ,, less than 15 μΐ, less than 10 μΐ, less than 9 μΐ, less than, 8 μΐ,, less than 7 μί, less than 6 μί, less than 5 μί,, less than 4 xL, less than 3 μί, less than 2 μί, less than 1 μΐ, less than, etc. Finally, multiple, parallel (multiplexed) reactions have also proven very difficult to reliably and reproducibly create using such systems.

[0022] For example, described herein are methods of detecting a target microRNA sequence from a sample using a modified loop mediated isothermal amplification (LAMP) technique, the method comprising: forming a mixture by combining the sample with a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B), wherein a 3' end of Trap A is a ribonucleotide, further wherein the 3' end of Trap A is complementary to a first portion of the target microRNA, and wherein the 5' end of Trap B is complementary to a second portion of the target microRNA; ligating the 3' end of Trap A to the 5' end of Trap B by annealing Trap A and Trap B to the target microRNA using T4 RNA Ligase 2 to form a target ligation product (Trap C), wherein Trap C comprises a plurality of primer recognition polynucleotide sequences including: a B3 site that is 5' to a B2 site , wherein the B2 site is 5' to a B l site, wherein the B l site is 5' to an Fl c site, wherein the Fl c site is 5' to an F2c site, wherein the F2c site is 5' to an F3c site; and amplifying Trap C by loop mediated isothermal amplification by combining the target ligation product with: an F3 primer that is complementary to the F3c site, a B3 primer comprising the B3 site, a FlPsel primer comprising an Fl c site and an F2 site, wherein the F2 site is complementary to the F2c site, further wherein the 5' end of the FlPsel primer corresponds to at least the last two

polynucleotides of the 3' end of Trap A adjacent to a region that is complementary to the second portion of the target microRNA, and a BIPsel primer comprising a B2 site and a B l c site, wherein the B l c site is complementary to the B l site, further wherein the 5' end of the BIPsel primer corresponds to a region that is complementary to at least the first two polynucleotides of the 5' end of Trap B adjacent to a region having the sequence of the first portion of the target microRNA.

[0023] In some variations, ligating comprises exposing the mixture to a heating cycle followed by a cooling cycle. For example, ligating may comprises exposing the mixture to a heating cycle followed by a cooling cycle, wherein the heating cycle comprises heating to between 30-90 degrees Celsius. Ligating may comprise exposing the mixture to a heating cycle followed by a cooling cycle, wherein the cooling cycle comprises cooling to between 20-37 degrees Celsius.

[0024] In some variations, the mixture comprises DMSO, Pluronic F-127 and NaCl.

[0025] The method may include diluting the mixture comprising Trap C prior to amplifying. Trap A may comprise a hydroxyl group on its 5' end and Trap B may comprise a phosphate group on its 3' end. Trap A may comprise the B3, B2, and B l sites arranged from its 5' end to its 3' end. Trap B may comprise the Fl c, F2c, and F3c sites arranged from its 5' end to its 3' end.

[0026] Amplifying Trap C may comprise amplifying at a temperature between 58-68°Celsius.

Amplifying Trap C may comprise diluting the mixture in an amplification buffer comprising 5-50 niM Tris-HCl, 0-50 mM of a salt, and Bst polymerase or other isothermal polymerase. Any of these methods may include detecting amplification of Trap C, including detecting amplification of Trap C by fluorescence or electrochemistry.

[0027] Also described herein are methods of detecting a target microR A sequence from a sample using a modified loop mediated isothermal amplification (LAMP) technique, the method comprising: forming a mixture by combining the sample with a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B), wherein a 3' end of Trap A is a ribonucleotide, further wherein the 3' end of Trap A is complementary to a first portion of the target microRNA, and wherein the 5' end of Trap B is complementary to a second portion of , the target microRNA, wherein the mixture further comprises dimethyl sulfoxide (DMSO), Pluronic F-127 and a salt; heating the mixture to a temperature between 30 and 80°Celsius; cooling the mixture to a temperature between 20 and

37°Celsius; ligating the 3' end of Trap A to the 5' end of Trap B between 20 and 37°C by annealing Trap A and Trap B to the target microRNA using T4 RNA Ligase 2 to form a target ligation product (Trap C), wherein Trap C comprises a plurality of primer recognition polynucleotide sequences including: a B3 site that is 5' to a B2 site , wherein the B2 site is 5' to a B l site, wherein the B l site is 5' to an Fl c site, wherein the F 1 c site is 5' to an F2c site, wherein the F2c site is 5' to an F3c site; and amplifying Trap C by loop mediated isothermal amplification by combining the target ligation product with: an F3 primer that is complementary to the F3c site, a B3 primer comprising the B3 site, a FlPsel primer comprising an Fl c site and an F2 site, wherein the F2 site is complementary to the F2c site, further wherein the 5' end of the FlPsel primer corresponds to at least the last two polynucleotides of the 3' end of Trap A adjacent to a region that is complementary to the second portion of the target microRNA, and a BIPsel primer comprising a B2 site and a B l c site, wherein the B l c site is complementary to the B l site, further wherein the 5' end of the BIPsel primer corresponds to a region that is complementary to at least the first two polynucleotides of the 5' end of Trap B adjacent to a region having the sequence of the first portion of the target microRNA.

[0028] A method of detecting a target microRNA sequence from a sample using a modified loop mediated isothermal amplification (LAMP) technique may include: forming a mixture by combining the sample with a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B), wherein a 3' end of Trap A is a ribonucleotide, further wherein the 3' end of Trap A is complementary to a first portion of the target microRNA, and wherein the 5' end of Trap B is complementary to a second portion of the target microRNA; ligating the 3' end of Trap A to the 5' end of Trap B by annealing Trap A and Trap B to the target microRNA using T4 RNA Ligase 2 to form a target ligation product (Trap C), wherein Trap C comprises a plurality of primer recognition polynucleotide sequences including: a B3 site that is 5' to a B2 site , wherein the B2 site is 5' to a B l site, wherein the B l site is 5' to an Fl c site, wherein the Fl c site is 5' to an F2c site, wherein the F2c site is 5' to an F3c site; wherein Trap A comprises the B3, B2, B 1 and Fl c sites arranged from its 5' end to its 3' end; and amplifying Trap C by loop mediated isothermal amplification by combining the target ligation product with: an F3 primer that is complementary to the F3c site, a B3 primer comprising the B3 site, a FlPsel primer comprising an F l c site and an F2 site, wherein the F2 site is complementary to the F2c site, and a BIPsel primer comprising a B2 site and a B 1 c site, wherein the B 1 c site is complementary to the B 1 site.

[0029] A method of detecting a plurality of microRNAs in parallel from a patient sample containing microRNA using a multiplexed ligation and detection technique may include: combining the patient sample with a first mixture to form a multiplexing mixture comprising a plurality of pairs of Trap A template and Trap B template, wherein each pair of Trap A and Trap B template is specific to a target microRNA of the plurality of microRNAs because a 5' end of Trap B and a 3' end of Trap A in each pair comprise regions that are reverse complimentary to adjacent portions of the target microRNA and further wherein one or both of the 3' end of Trap B and the 5' end of Trap A comprises one or more nucleotide sequence that is specific to the pair of Trap B and trap A template; heating the multiplexing mixture to denature the microRNA, and cooling the multiplexing mixture to anneal pairs of Trap B and Trap A template to specific target microRNA if the target microRNA is present in the multiplexing mixture; ligating the annealed pairs of Trap B template and Trap A template using a ligase to form Trap C template specific to target microRNA; placing a portion of the multiplexing mixture into each of a plurality of separate regions; performing, in parallel, loop-mediated isothermal amplification of Trap C template specific to a different target microRNA in each of the plurality of separate regions, wherein each separate region comprises a polymerase having strand displacement activity and primers for the loop mediated amplification, wherein one or more of the primers for the loop mediated amplification includes the nucleotide sequence that is specific to a reverse complement to the target microRNA sequence of the plurality of microRNAs.

[0030] A method of detecting a plurality of microRNAs in parallel from a patient sample containing microRNA using a multiplexed ligation and detection technique may include: combining the patient sample with a first mixture to form a multiplexing mixture comprising a plurality of pairs of Trap A template and Trap B template, wherein each pair of Trap A and Trap B template is specific to a target microRNA of the plurality of microRNAs because a 5' end of Trap B and a 3' end of Trap A in each pair comprise regions that are reverse complimentary to adjacent portions of the target microRNA and further wherein each Trap A template comprises a B3 region at a 5' end of the Trap A template, a B2 region 3' to the B3 region, and a B l region that includes the reverse complimentary region of the microRNA 3' to the B2 region, wherein each Trap B comprises an F3c region at the 3' end of the Trap B template, an F2c region 5' to the F3c region, and an Fl c region that includes the reverse complimentary region of the microRNA 5' to the F2cc region, and wherein each pair of Trap A and Trap B templates includes one of: at least a B l and Fl c unique sequences that are different for each Trap A and Trap B pair specific to each different target microRNA; B 1 , B2, Fl c and F2c unique sequences that are different for each Trap A and Trap B pair specific to each different target microRNA; a sequence for each pair of Trap A and Trap B specific to a particular target microRNA that is unique from other pairs of Trap As and TrapBs for at least one of: the B3 region, the B2 region, an LB region, the B l region, the F3c region, the F2c region, an LFc region, and the Fl c region; heating the multiplexing mixture to denature the microRNA, and cooling the multiplexing mixture to anneal pairs of Trap B and Trap A template to specific target microRNA if that specific target microRNA is present in the multiplexing mixture; ligating the annealed pairs of Trap B template and Trap A template using a ligase to form Trap C template specific to target microRNA;

placing a portion of the multiplexing mixture into each of a plurality of different regions; performing, loop-mediated isothermal amplification of each of the plurality of different regions in parallel, wherein each different regions is associated with one specific target microRNA from the plurality of microRNAs and comprises a combination of primers that complement or include the unique sequence or sequences that is different from the other pairs of the plurality of pairs of Trap A template and Trap B template for at least one of the B3 region, the B2 region, the B l region, the F3c region, the F2c region, and the Fl c region of the template specific to target microRNA.

[0031] In general, also described herein are methods of detecting a target microRNA from a patient sample using an air-matrix digital microfluidic (DMF) apparatus. In any of these method, detecting may include: forming a reaction droplet in an air gap region of the air-matrix DMF apparatus from a portion of the patient sample including patient microRNA and first mixture comprising a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B); evaporating more than 20% of the reaction droplet by heating the reaction droplet within a ligation zone of the air gap to ligate the Trap A and Trap B to a target microRNA; combining the reaction droplet with a ligase enzyme to form a combined droplet and incubating the combined droplet; mixing the combined droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes to ligate the Trap A to Trap B and form Trap C in the presence of the target microRNA; adding a polymerase and a plurality of primers configured to selectively amplify Trap C to the combined droplet or a portion of the combined droplet and coating the combined droplet or portion of the combined droplet with a protective material to prevent evaporation of the combined droplet within a LAMP zone of the air gap; incubating the combined droplet or portion of the combined droplet at between 55-70°C to allow amplification of Trap C by loop mediated isothermal amplification (LAMP); and detecting amplification of Trap C.

[0032] A method of detecting a target microRNA from a patient sample using an air-matrix digital microfluidic (DMF) apparatus may include: forming a reaction droplet in an air gap region of the air- matrix DMF apparatus from a portion of the patient sample including patient microRNA and first mixture comprising a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B); mixing the reaction droplet by applying energy to at least one actuation electrode of a plurality of actuation electrodes adjacent to the air matrix; evaporating between 20% and 90% of the reaction droplet by heating the reaction droplet to greater than 70°C (e.g., greater than 75 °C, greater than 80°C, greater than 85°C, greater than 89°C, 90°C, etc.) and cooling the droplet to less than 40°C within a ligation zone of the air gap to ligate the Trap A and Trap B to a target microRNA; combining the reaction droplet with a ligation droplet comprising a ligase enzyme to form a combined droplet and incubating the combined droplet; mixing the combined droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes to ligate the Trap A to Trap B and form Trap C in the presence of the target microRNA; adding a polymerase and primers configured to selectively amplify Trap C to the combined droplet or a portion of the combined droplet and coating the combined droplet or portion of the combined droplet with a wax to prevent evaporation of the combined droplet within a LAMP zone of the air gap; incubating the combined droplet or portion of the combined droplet at between 55-70°C to allow amplification of Trap C by loop mediated isothermal amplification (LAMP); and detecting amplification of Trap C.

[0033] A method of detecting a target microRNA from a patient sample using an air-matrix digital microfluidic (DMF) apparatus, the method comprising: forming a reaction droplet in an air gap region of the air-matrix DMF apparatus from a portion of the patient sample including patient microRNA and first mixture comprising a first single-stranded DNA probe (Trap A), a second single-stranded DNA probe (Trap B), dimethyl sulfoxide and poloxamer, wherein forming comprises dispensing a first droplet of the first mixture from a reservoir in fluid connection with the air gap region and applying energy to a plurality of actuation electrodes to combine the first droplet with a sample droplet containing the portion of the patient sample; mixing the reaction droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes adjacent to the air matrix; evaporating between 20% and 90% of the reaction droplet by heating the reaction droplet to greater than 80°C and cooling the droplet to less than 40°C within a ligation zone of the air gap to ligate the Trap A and Trap B to a target microRNA, wherein evaporating the reaction droplet comprises monitoring the reaction droplet to determine when the volume of the reaction droplet falls below a threshold; combining the reaction droplet with a ligation droplet comprising a ligase enzyme to form a combined droplet and incubating the combined droplet; mixing the combined droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes to ligate the Trap A to Trap B and form Trap C in the presence of the target microRNA; adding a polymerase and primers configured to selectively amplify Trap C to the combined droplet or a portion of the combined droplet and coating the combined droplet or portion of the combined droplet with a paraffin wax material to prevent evaporation of the combined droplet within a LAMP zone of the air gap; incubating the combined droplet or portion of the combined droplet at between 55-70°C to allow amplification of Trap C by loop mediated isothermal amplification (LAMP); and detecting amplification of Trap C.

[0034] In any of these methods the first mixture may further comprises chemical additives including dimethyl sulfoxide (DMSO) and poloxamer, and forming may comprise forming a droplet of less than 10 μL·. Forming may comprise dispensing a first droplet of the first mixture from a reservoir in fluid connection with the air gap region and applying energy to the plurality of actuation electrodes to combine the first droplet with a sample droplet containing the portion of the patient sample.

[0035] In general, any of the steps described herein may include mixing using the DMF apparatus by actuating electrodes under (e.g., adjacent to) the droplet within the air matrix. For example, mixing the reaction droplet may comprise continuously moving the droplet within the air matrix or splitting and recombining the reaction droplet within the air matrix. Mixing may comprise mixing for 10 or more seconds at room temperature.

[0036] In any of these methods, evaporating the reaction droplet may comprise incubating the reaction droplet for between 1 and 10 min at greater than 80 °C followed by cooling the reaction droplet to less than 35 °C at a rate of between 0.1 to 0.3 °C/s. Evaporating the reaction droplet may comprise mixing the reaction droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes to move the reaction droplet within the ligation zone or to split and recombine the reaction droplet within the ligation zone. Evaporating the reaction droplet may comprise monitoring the reaction droplet to determine when the volume of the reaction droplet falls below a threshold.

[0037] Any of these methods may also include diluting the combined droplet prior to moving all of a portion to the LAMP zone.

[0038] Combining may comprise applying energy to one or more of the actuation electrodes to move the ligation droplet from a reservoir in fluid contact with the air gap through the air gap to merge with the reaction droplet. Combining the reaction droplet may comprise incubating the reaction droplet for greater than 10 minute at between 20-37°C.

[0039] In general, the protective material may be any coating material (e.g., wax or oil), and particularly material such as waxes that are solids at room temperature but melt above room temp (e.g., above 37°C, above 40°C, above 45°C, above 50°C, etc.). For example, the protective material may be paraffin wax.

[0040] Adding the polymerase and primers may comprise first splitting the combined droplet into a plurality of combined droplets that are processed in parallel.

[0041] In general, amplification from the trap C may be detected in any appropriate manner, such as optical (e.g., colorimetric, etc.) and/or electrical (e.g., detection of a change in an electrical property of the droplet within which the amplification is occurring, including electrical properties of the bulk solution) and/or electrochemically detecting amplification (e.g., electrochemical detection of DNA binding, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0043] FIG. 1 A is a flowchart showing an exemplary workflow for a ligation actuated loop mediated isothermal amplification (LA-LAMP) method as described herein.

[0044] FIG. 1 B schematically illustrates the formation of template DNA ("TrapC") from a target RNA (microR A) for detection of the target microRNA by LAMP. In FIG. 1 A, the microRNA target "splints" a first and a second single-stranded DNA (top), Trap A and Trap B, so that they can be ligated into a single-stranded DNA, Trap C, for amplification by LAMP.

[0045] FIG. 2 shows an exemplary LAMP detection method, illustrating a series of strand displacement steps for amplification of a single-stranded DNA template (Trap C) having the microRNA target sequence using four primers. [0046] FIG. 3 shows another exemplary version of the LAMP detection method, illustrating a series of strand displacement steps using four primers for a single-stranded DNA having the microRNA in a second location.

[0047] FIG. 4 illustrates the use of a LAMP primer that includes an "overhang" region spanning multiple nucleotides spanning the Trap A and Trap B ligation site, which has been shown herein to increase specificity of the LAMP method described. If the overhang region fails to recognize the specific target microRNA sequence (e.g., in the absence of the specific ligation due to recognition of a particular microRNA) the overhang region does not hybridize, preventing operation of the polymerase, and amplification is terminated early, greatly decreasing the overall background of the assay. In a multiplexing mixture, the miss-primed/non-specific ligation leading to incorrect pairs of traps that cannot form a miRNA specific template will not be amplified, by preventing polymerase activity, terminating amplification early, preventing false positives.

[0048] FIG. 5 illustrates a schematic the use of one or more spacers in the primers and/or template components (TrapA, TrapB). In FIG. 5 the upper diagram shows a forward inner primer (e.g., primer FlPsel) without a spacer, and the lower diagram shows a FlPsel with a spacer between its two recognition regions (F22 and Fl c). This example also includes an overhang region 505 on the inner primer shown (similar to that described in FIG. 4), where the overhang spans the ligation site and can interrogate the correct target microRNA sequence, effectively adding an extra layer of specificity in assay performance.

[0049] FIG. 6A illustrates one example of a digital microfluidics (DMF) apparatus (e.g., a cartridge portion of a DMF apparatus) that can be used to perform LA-LAMP as described herein. In this example, the DMF apparatus includes reservoir regions that may hold the starting sample, mixtures (heat/anneal mix, ligation enzyme mix, dilution buffer, LAMP mix, etc.) or lyophilized versions of these mixtures, which may be resuspended, as well as thermally regulated (heating/cooling) regions (ligation zone, LAMP zone, etc.). The DMF apparatus may generally include a plurality of actuation electrodes.

[0050] FIG. 6B illustrates a section through a thermally-regulated region showing the upper plate, air gap, and lower plate. A thermal regulator (heater/cooler) may be included in the upper or lower plate as well as one or more sensors, such as thermal sensors.

[0051] FIG. 6C shows a time sequence illustrating the use of LA-LAMP in an air-gap DMF apparatus such as the one shown in FIG. 6A.

[0052] FIG. 6D schematically illustrates one example of an apparatus (e.g., system) for detection of a polynucleotide (e.g., microRNA) by LA-LAMP using air-matrix DMF as described herein. In this example, a separate (and in some variations, disposable or alternatively reusable) air-matrix DMF cartridge may be connected to a reader (size not to scale) to electrically control the DMF cartridge (e.g., temperature, fluid movement, etc.) and/or to detect an amplification product. Optionally the reader may be in wireless (or alternatively wired) communication with one or more remote processors.

[0053] FIG. 7 is a graph illustrating amplification of miRNA-451 by an LA-LAMP method as described herein using DMF. Duplicate pairs of both a hemolysis sample and a non-hemolysis sample were examined, showing good agreement. [0054] FIG. 8 is another example of a graph illustrating amplification of miRNA-451 by a conventional method. Duplicate pairs of both a hemolysis sample and a non-hemo lysis sample were examined.

[0055] FIG. 9 is an example of a portion of a DMF (e.g., air-matrix DMF) in which the DMF apparatus includes a plurality of differently-sized electrodes that may be particularly useful when evaporating a sample (e.g., during ligation). In FIG. 9, electrodes smaller than the ones holding the original droplet before the evaporation may be provided in the vicinity of the evaporation site. These smaller droplet regions may allow more precise determination of droplet size, detection of evaporation level and/or rate, and processing of reduced-volume droplets.

[0056] FIGS. 1 OA- IOC, 1 1A-1 1C, 12A-12C, and 13 A- 13C schematically illustrate a multiplexing method in which a single sample (or parallel aliquots of sample) may be combined with pooled templates (Trap A and Trap B) to detect multiple (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 1 1 or more, 12 or more, 15 or more, 20 or more, 15 or more, 30 or more, 35 or more, 50 or more, 100 or more, etc.) different microRNAs,

[0057] FIGS. I OA- I OC show a schematic outline of a first example of a method using a multiplexing technique in which the Trap A and Trap B samples have universal B2, B3, LB (optional), LFc (optional), F2c and F3c regions and the reverse compliment of each of the target microRNAs are split and incorporated within the B l and Fl c regions.

[0058] FIGS. 1 1 A- 1 1 C show a schematic outline of a second example of a method using a multiplexing technique in which the Trap A and Trap B samples have universal B3, LB (optional), LFc (optional) and F3c regions and the reverse compliment of each of the target microRNAs are split and incorporated within the B l and Fl c regions. The B2 and F2c region may also be specific for each target micro RN A.

[0059] FIGS. 12A-12C show a schematic outline of a third example of a method using a multiplexing technique in which the Trap A and Trap B samples have universal B 1 , B2, B3, LB

(optional), LFc (optional) and F3c regions, and the reverse compliment of each of the target microRNAs are split and a portion appended to the B l regions and the other portion is incorporated within the Fl c regions for each target micro RN A.

[0060] FIGS. 13A-13C show a schematic outline of a fourth example of a method using a multiplexing technique in which the Trap A and Trap B samples have universal B l , B2, B3, LB

(optional), LFc (optional), F2c and F3c regions, and the reverse compliment of each of the target microRNAs are split and a portion appended to the Bl regions and the other portion is incorporated within the Fl c regions for each target microRNA. DETAILED DESCRIPTION

[0061] Described herein are apparatuses and methods for detecting and analyzing microRNAs, including diagnostic detection of microRNAs. These methods and apparatuses may include apparatuses and methods for performing ligation actuated loop mediated isothermal amplification (LA-LAMP) detection of microRNAs, and in particular may descried digital microfluidics (DMF) apparatuses and methods for performing LA-LAMP to detect microRNAs. Such detection and analysis may be useful for diagnosing, prognosticating (e.g. predicting a risk for getting), or treating a disease or syndrome, or analyzing response to a disease treatment, or performing another analysis. MicroRNAs have been implicated in almost every aspect of biology including where the disruption of microRNA function contribute to many human diseases, particularly, but not limited to, cardiovascular disorders, cancer, and genetic diseases.

[0062] MicroRNAs (or miRNA) are produced from DNA found in animal and plant cells and in viruses. MiRNAs are small (e.g., about 22 nucleotides) non-coding RNA molecules. Hundreds of such microRNAs have been identified and sequenced thus far. They have been shown to be involved in various biological processes such as gene expression and post-transcriptional modification. The action of a single miRNA may have an effect on dozens of different genes, RNAs, or proteins.

[0063] As mentioned, microRNA expression may be helpful in diagnosing, prognosticating (e.g. predicting a risk for getting), or treating a disease or syndrome, or analyzing response to a disease treatment, or performing another analysis. In some cases, it is desirable that a health care profession or technician analyze an expression of microRNAs (e.g., from a blood or other patient sample) very quickly, such as at the time the patient is being treated, referred to as the point of care (POC). In such cases, health care decisions may be quickly made and implemented. Although a having an assay that gives results quickly in order to provide results useful at the time of care, including within 30 min to 4 hours from taking a sample, is helpful, such assays should also ideally be relatively easy to perform and not require extensive training of personnel or complicated machinery. An assay should be cost effective and minimize cost per assay. Finally, such an assay should be both specific and sensitive as a false positive or a missed diagnosis may have significant negative consequences and could result in delayed or unnecessary treatment that could be costly or dangerous. It is highly challenging to provide an assay that can meet some or even most of these goals. In particular, even a seemingly small improvement in any of these or other metrics for an assay for making health care or other decisions is highly desirable.

[0064] In the past, performing an RNA assay generally required expensive reagents, specialized and expensive equipment, extensive technician training, and a significant amount of time, making them less than desirable as quick and reliable assays. Improvements in one metric often meant comprising on another one. For example, while it is less expensive (at least initially) to perform an assay using a very small patient sample and relatively smaller amounts of expensive reagents, stochastic (random) events begin to become important and skew the results, making the assay less reliable. For example, the reverse transcription polymerase chain reaction (RT-PCR) which is commonly used to analyze RNA, is notorious for creating artifacts, and much care has gone into improving its reliability, often at the expense of comprising other desirable factors. One improvement that reduces random events with RT-PCR and its associated problems is referred to as digital PCR, which involves dividing a sample into multiple portions and performing a PCR reaction on each individual portion in parallel. Although the reliability of the overall RT-PCR was improved, the assay is considerably more complex as a result of these modifications. [0065] Described herein are methods for detecting and analyzing microRNAs. However, the methods and apparatuses described herein may be adapted for use with general polynucleotide detection and/or amplification. The LA-LAMP procedure decribed herein and methods of performing it include numerous surprising improvements that individually and collectively result in highly accurate and reliable detection and/or amplification. For example, the methods described below provide improved specificity for the detection of target RNA such as microRNA. The methods described below may be easily incorporated into kits or devices to provide low costs ways of assaying for target sequences that may be associated with a wide host of conditions, and conditions.

[0066] In some variations, the assays described herein may take advantage of desirable properties of enzymes that are able to recognize polynucleotides. In some variations, the method and apparatus may include the user of hybrid DNA/RNA templates (e.g., partial DNA templates that include one or more RNA polynucleotides at the end, which may improve recognition sensitivity by an RNA polymerase, effectively "fooling" it in to operating on a primarily DNA polynucleotide. As will be described herein, the target RNA (micro RNA) may be hybridized to a pair of partial DNA templates and ligated to form a single piece of DNA that can be readily be amplified. The assays also take advantage of an amplification procedure utilizing a simple isothermal procedure, loop-mediated isothermal amplification procedure to quickly amplify the DNA .

[0067] As used herein, "amplify" in reference to a nucleic acid sequence refers to increasing the number of copies of a nucleic acid sequence.

[0068] As used herein, "nucleic acid sequence" refers to a string of nucleotide bases attached by phosphodiester bonds, for example DNA has deoxynucleotides, i.e. combinations of adenine (A), guanine (G), cytosine (C), and thymine (T) molecules attached by covalent phosphodiester bonds, RNA, mRNA, and microRNA has ribonucleotide, i.e. combinations of adenine (A), guanine (G), cytosine (C), and uracil (U) nucleotide molecules attached by covalent phosphodiester bonds.

[0069] As used herein, the term "target" in reference to an amplified nucleic acid sequence (e.g., microRNA) may refer to the source or original nucleic acid in a sample, such that when an amplified nucleic acid sequence is detected by the devices and methods described herein the target is found in the sample. For example, a particular microorganism can have a target nucleic acid, which when detected by the devices and methods described herein signifies that the microorganism is present in the sample. As another example, the target can be a cancer marker, amplification of a nucleic acid encoding the cancer marker, identifies that the cancer marker is present in the sample. The term "target," when used in reference to microRNA may refer to the molecules (e.g., nucleic acid) to be detected. Thus, the "target" is sought to be sorted out from other molecules (e.g., nucleic acid sequences) or is to be identified as being present in a sample through its specific interaction.

[0070] As used herein, a "microRNA" or "miRNA" typically refers to a ribonucleic acid (RNA) molecule, for one example, approximately 22 nucleotides in length. In one embodiment, miRNA sequences bind to complementary sequences in the 3' UTR of target mRNAs, usually resulting in silencing of the target mRNA, so that the target mRNA is not translated. [0071] As used herein, the term "primer" may refer to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer may first be treated to separate its strands before being used to prepare extension products. A primer may be an oligodeoxyribonucleotide. The primer may be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method. A primer may also be referred to as a probe.

[0072] As used herein, the term "probe" may refer to a molecule (e.g., an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification), that is capable of hybridizing to another molecule of interest (e.g., another

oligonucleotide). When probes are oligonucleotides they may be single-stranded or double-stranded. Probes may be useful in the detection, identification and isolation of particular targets (e.g., gene sequences).

[0073] As used herein, "conventional QPCR" and "QPCR" refer to "quantitative PCR," that for the purposes of the present disclosure is a real-time PCR analysis, such as real-time PCR reactions that are performed by a Taqman® thermal cycling device and reaction assays by Applied Biosystems. As used herein, "conventional PCR" and "PCR" refer to a non-quantitative PCR reaction, such as those reactions that take place in a stand-alone PCR machine without a real-time fluorescent readout.

[0074] As used herein, "isothermal amplification" refers to an amplification step that proceeds at one temperature and does not require a thermocycling apparatus.

[0075] The terms "sample" and "specimen" are used herein in their broadest sense, and may include a biological sample and an environmental sample. Patient samples may include all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, as well as solid tissue. Biological samples may be animal, including human, fluid or tissue.

[0076] As used herein, the term "oligonucleotides" or "oligos" refers to short sequences of nucleotides.

[0077] As used herein, the terms "thermal cycler" or "thermal cycler" refer to a programmable thermal cycling machine, such as a device for performing PCR.

[0078] As used herein, the term "amplification reagents" may refer to those reagents (such as DNA polymerase, deoxyribonucleotide triphosphates, buffer, etc.), necessary for nucleic acid sequence amplification.

[0079] The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid or protein with which it is ordinarily associated in its natural state or source. An isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA and or protein found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell genome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or

polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0080] As used herein the term "coding region" when used in reference to a structural gene refers to the nucleotide sequences that encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" that encodes the initiator methionine and on the 3' side by one of the three triplets that specify stop codons (i.e., TAA, TAG, and TGA).

[0081] As used herein the term "portion" when in reference to a nucleotide sequence or nucleic acid (as in "a portion of a given nucleotide sequence" or a "portion of a nucleic acid") refers to fragments of that sequence or that nucleic acid. The fragments may range in size from four nucleotides to the entire nucleotide sequence or nucleic acid minus one nucleotide.

[0082] The term "gene" may refer to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor. It is intended that the term encompass polypeptides encoded by a full length coding sequence, as well as any portion of the coding sequence, so long as the desired activity and/or functional properties (e.g., enzymatic activity, ligand binding, etc.) of the full-length or fragmented polypeptide are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3 ' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences that are located 5' of the coding region and which are present on the mRNA are referred to as "5' untranslated sequences." The sequences that are located 3 ' (i.e.,

"downstream") of the coding region and that are present on the mRNA are referred to as "3 ' untranslated sequences." The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form of a genetic clone contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." A subset of gene is "virulence and marker" genes or VMGs that refers to genes associated with virulence or used as markers for any specific reason. Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0083] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5 Or 3' to the non- translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3 ' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

[0084] Nucleic acid (e.g., DNA) molecules are said to have "5' ends" and "3 ' ends" because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide or polynucleotide, referred to as the "5' end" if its 5' phosphate is not linked to the 3 ' oxygen of a mononucleotide pentose ring and as the "3 ' end" if its 3 ' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5 ' and 3 ' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements that direct transcription of a linked gene are generally located 5' or upstream of the coding region.

However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

[0085] As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements include splicing signals, polyadenylation signals, termination signals, etc.

[0086] As used herein, the terms "complementary" and "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification and hybridization reactions.

[0087] Equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).

[0088] When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

[0089] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G :C ratio within the nucleic acids.

[0090] As used herein, the term "Tm" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art.

[0091] As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that "stringency" conditions may be altered by varying the parameters just described either individually or in concert. With "high stringency" conditions, nucleic acid base pairing will occur restricted between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization under "high stringency" conditions may occur between homologs with about 85-100% identity, preferably about 70-100% identity). With medium stringency conditions, nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (e.g., hybridization under "medium stringency" conditions may occur between homologs with about 50-70% identity). Thus, conditions of "weak" or "low" stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.

[0092] As used herein, "amplification" in reference to a method or apparatus described herein typically refers to amplifying a template or target nucleic acid sequence comprising the steps of hybridizing an amplification nucleic acid, such as a primer, to its complementary target sequence or sample nucleic acid sequence, also termed template nucleic acid sequence, in the presence of amplification reagents, free nucleic acids, and a polymerase, for example a BST polymerase for loop- mediated isothermal amplification, which results in the duplication of said complementary nucleic acid sequence then repeating these steps until amplification is detected or stopped. Amplification may be detected by a device of the present disclosures as fluorescent molecules become incorporated into the amplifying sequence or amplified sequence, or by removal of an optical quenching element to allow optical detection. Amplification may have a start time or point and an end time or point. Amplification may be considered a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribonucleotide or

deoxyribonucleotide) specificity. Template specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.

[0093] As used herein, the term "template" may refer to a sequence (e.g., polynucleotide sequence) including the reverse compliment of a microRNA sequence originating from a sample that is analyzed for the presence of "target."

[0094] As used herein, "multiplexed" may refer to the simultaneous and grouped processing of multiple (e.g., more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 30, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90, etc.) targets, such as microRNAs within a single pool or collection. Multiplexed processing as described herein may be performed in parallel within the same container, typically without interference between the various targets, e.g., microRNAs.

[0095] As used herein a Trap A (also referred to herein as "TrapA") template may be an acceptor template DNA sequence that includes a portion (e.g., one half) of a template to be amplified by, e.g., LAMP. Trap A template may include, for example, three or more distinct regions of nucleotide sequences (e.g., B l , B2, B3 regions) used to form primers for LAMP amplification; a distinct 3 ^' region may include in reverse complement a portion (e.g., approximately half) of a target microRNA. In some variations the 5' end of TrapA can be modified with hydroxyl.

[0096] As used herein Trap B template (also referred to as "TrapB") may be a donor template pDNA sequence (phosphorylated at the 5 ^' end) that includes a portion (e.g., approximately one half) of the microRNA template to be amplified by LAMP. A donor template may include, for example, three or more distinct (non-overlapping) regions of nucleotide sequences (e.g., Fl c, F2c, F3c regions) that may be used to form primers for LAMP amplification; a distinct 5 ^' region may include in reverse complement a portion (e.g., approximately half) of a target microRNA. As will be described in more detail below, in some variations the Fl c region includes the Trap B reverse complement of a portion of the target miRNA; the FIP primer may contain an Fl c region which extends beyond the ligation site, e.g., hybridizing to Trap B Fl c region but also 1 -3 nucleotides of the Trap A 3' end. Similarly, the B l region may include the Trap A reverse complement of a portion of the target microRNA.

[0097] The other half (or remaining interrogated sequence) reverse complement of the target microRNA may be fused to the 3 ^' end of the acceptor template so that when the 3 ^' end of Trap A template is fused to the 5 ' end of Trap B template, the ligation of the two results in a full-length template (microRNA-specific template), called Trap C, for amplification by LAMP. This is illustrated in FIG. I B and the examples of FIGS. 2-3, described in greater detail below.

[0098] As used herein sets or "pairs" of Trap B template and Trap A template typically refer to an individual donor template sequence and acceptor template sequence between which a full-length sequence complementary to an individual or particular target microRNA may be formed by combining (ligating) the Trap B and Trap A templates of the pair as described herein in the presence of the particular target microRNA.

[0099] In general, the Trap A and Trap B templates include both a reverse compliment region at an end (e.g., at the 3' end of TrapA and at the 5' end of TrapB) and multiple sequence regions that may be incorporated as primer regions (e.g., in Trap A, B 1 , B2 and B3 and in Trap B, Fl c, F2c and F3c). These regions may be conserved across multiple templates (Traps), or they may be unique to a particular target microRNA or set of microRNAs (e.g., sharing similar properties such as melting temperature, etc.). In some variations a reverse compliment of the target (e.g., target microRNA) may be included as part of the primer region (e.g., Fl c, etc.). In some variations the primer regions (e.g., B l , Fl c, etc.) may span the junction between Trap A and Trap B. In some variations, a nucleotide region that is specific to a pair of Trap B and Trap A templates may include a sequence (and typically not a sequence that is identical to or complementary to the microRNA sequence that the pair is targeting) that is different and/or distinct from (e.g., having more than a few different nucleotide sequences) other similarly-located regions of other donor (Trap B), acceptor (Trap A) or full-length templates. For example, a 3 ' end of the donor template and the 5 ^' end of the acceptor template may comprise one or more nucleotide sequence that is specific to the pair of donor and acceptor template and distinct from other similarly-located (at nucleotide positions relative to other donor and/or acceptor templates. In some variations, all of the donor (Trap B) templates that are directed to different target microRNAs may include similar or identical polynucleotide sequences, with the exception of the 5 ' end including the region complementary to a portion (e.g., half) of the different target microRNAs, and, in some variations, one or more Fl c, F2c, and/or F3c regions at the 3 ' end of the donor template. In some variations, all of the acceptor templates that are directed to different target microRNAs may include similar or identical polynucleotide sequences, with the exception of the 3 ' end including the region complementary to a portion (e.g., half) of the different target microRNAs, and, in some variations, one or more B 1 , B2 and/or B3 regions at the 5 ' end of the acceptor template.

[0100] The methods and apparatuses (e.g., devices and systems) described herein may be adapted for multiplexing, so that multiple LA-LAMP detection reactions may be run off of the same sample mix. For example, in some variations (described in greater detail below with respect to FIGS. 1 OA-10C) all of the Trap A templates for different target microRNAs are identical with the exception of B l , which includes a reverse compliment of a portion of the target microRNA; similarly, all of the Trap B templates for different target microRNAs are identical with the exception of Fl c, which is a unique sequence that is specific to the remainder of the target microRNA, so that each Trap A/Trap B pair hybridizes to a specific microRNA. In the example shown in FIGS. 1 l A-1 1 C, at least B l , B2, Fl c and F2c regions are sequences unique to each different microRNA target. Alternatively, as described in reference to FIGS. 12A- 12C, the Trap A template regions B 1 , B2, B3 and LB are all shared by the Trap A's for different target microRNAs, and a reverse compliment of a portion of the target microRNA is added to the 3' end of the Trap A; the LFc and F3c regions may be shared between all of the Trap Bs for different target microRNAs while the sequence of the microRNA may be incorporated into specific F l c and F2c regions. In another example is shown in FIGS. 13A-13C, the B l , B2, B3 and LB regions are all common to the TrapAs for different target microRNAs and a reverse compliment portion of the target microRNA is appended to the B I region; while only the Fl c regions of the Trap Bs for different target microRNAs are specific (incorporating a reverse compliment of the target microRNA adjacent to the portion appended to the complimentary Trap A), and the LFc, F2c and F3c regions are all identical between different target pairs. In general, each Trap A and Trap B pair corresponding to a specific target microRNA may include a unique sequence that is different from any of the donor and target pair at one or more B l , B2, B3, F l c, F2c, F3d sites or regions.

[0101 ] In general, a ligase as described herein may ligate ssDNA oligonucleotides splinted by a ssRNA. The term "splint ligase" may refer to an enzyme that is capable of ligating at least two ssDNA polynucleotides splinted by a complementary ssRNA polynucleotide and is capable of achieving ligation in less than 6 hours at molar concentrations of enzyme that are not absolutely required to be in molar excess compared to substrate. For example, see U.S. patent application 2014/0179539. The RNA splint ligase, single stranded polynucleotide and/or splint RNA may be immobilized on a matrix such as a reaction surface, or a magnetic bead to facilitate automated protocols and multiplexing reactions. In particular, the enzyme described herein may be T4 RNA Ligase 2.

[0102] The term "polynucleotide" may include DNA, RNA or part DNA and part RNA. The polynucleotides when used in a ligation reaction with an RNA splint are preferably single stranded and may be partially or wholly complementary to at least a portion of the RNA splint. An example of a polynucleotide described herein is a ssDNA oligonucleotide comprising at least 8 nucleotides.

[0103] As used herein, a "fluorescent molecule" or "fluorophore" or "fluorophores molecule" or

"fluorescent dye" in general refers to a molecule capable of excitation, i.e. activation, under conditions for emitting an optical energy emission, i.e. signal, for example, synthetic dyes, orange fluorescent dyes (stain) having exemplary optimal excitation wavelengths (i.e. spectra) in the 530 nm to 570 nm range and exemplary emission wavelengths in the 545-583 nm range, such as orange SYTO® 81 , SYTO®-82, and cyanine dyes, asymmetrical cyanine dyes, green fluorescent dyes (stain), such as SYBR® dyes, i.e., SYBR Green I and II, and green SYTO® dyes, etc. For the purposes of the present disclosure, a fluorescent molecule is capable of binding to a nucleic acid sequence. In some embodiments, the biological sample comprises a fluorescent compound, wherein the fluorescent compound is selected from the group consisting of SYBR™ Brilliant Green, SYBR™ Green I, SYBR™ Green II, SYBR™ gold, SYBR™ safe, EvaGreen™, a green fluorescent protein (GFP), fluorescein, ethidium bromide (EtBr), thiazole orange (TO), oxazole yellow (YO), thiarole orange (TOTO), oxazole yellow homodimer (YOYO), oxazole yellow homodimer (YOYO-1 ), SYPRO® Ruby, SYPRO® Orange, Coomassie Fluor™ Orange stains, and derivatives thereof. These dyes are generally available commercially, and many of them can be made as described by Deligeorgiev et al., Recent Pat. Mat. Sci. 2: 1 -26 (2006). In any of the examples described herein, calcein (e.g., fluorexon, fluorescein complex, etc.) may be used for detection. In addition, pH dyes (e.g., 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, or BCECF;

Seminaphtharhodafluor, or SNARF-1 and/or SNARF-5, etc.) may be used, as the amplification of template (Trap C) may result in a drop in pH (leading to an exponential drop in fluorescent signal during the amplification) as more H+ is generated through polymerization of the Trap C.

[0104] As used herein, an "optically activated fluorescent molecule" "optically activated fluorescent molecule" refers to a fluorescent molecule illuminated (i.e. excitation) under conditions for releasing energy as emitted light (i.e. emission) measured spectrally as wavelengths i.e. spectral profiles. In other words, light comprising wavelengths capable of exciting a fluorescent molecule, i.e. excitation light, causing the molecule to release emission energy capable of detection, i.e. captured, using a device of the present disclosures.

Methods of detecting and analyzing microRNAs

[0105] Described herein are methods, apparatuses and compositions for detecting and analyzing small RNAs (e.g., microRNAs) that generally involve obtaining a sample containing RNA. FIG. 1 A shows a generic overview of the method for LA-LAMP detection of a target polynucleotide (e.g., microRNA). In FIG. 1 A, the first step is preparing the sample 101. As previous described, these methods generally involve annealing the RNA of interest in the sample to template DNA oligonucleotides complementary to the RNA of interest if the target RNA(s) are present; joining (ligating) the complementary DNA oligonucleotides to each other; and amplifying any joined (ligated) product to create multiple copies indicating the presence of the target RNA, and assaying an aspect of the amplification as an indication that the RNA of interest was present in the sample. Additional steps may be performed in addition to these steps and in some variations only a subset of these steps may be performed. Additionally, a sample may not contain an RNA of interest and an assay as described herein may be performed on such a sample to show that the RNA is not present in the sample. Such samples are generally assayed similarly as those containing an RNA of interest (although with different results) and are considered as samples or samples containing an RNA of interest as described herein.

[0106] The step of obtaining a sample 101 containing an RNA of interest may include obtaining a synthetic RNA or a naturally occurring RNA. A synthetic RNA may be, for example, synthesized in vitro using an enzyme, a nucleic acid synthesizer, etc. A naturally occurring RNA may come from any biological sample such as a biopsy, blood, cerebrospinal fluid, fecal, pericardial fluid, plasma, pleural fluid, saliva, sputum, urine, etc. but in some particular examples is a blood sample for performing a point- of-care assay (and/or an in-clinic, in the hospital or in the field assay; alternatively the sample may be taken and sent to a clinic or central location for processing). A sample may be handled or treated such as to purify or partially purify the sample or to preserve the sample and prevent sample degradation before a subsequent step is performed (e.g., before its RNA is annealed to the oligonucleotides). For example, a blood sample may be centrifuged to enrich (separate) a sample into a blood plasma (liquid) portion and a blood cell portion, and either sample may be used for analysis as described herein.

[0107] The ligation actuated loop mediated isothermal amplification (LA LAMP) methods described herein represent an improvement over other loop mediated isothermal amplification (LAMP) techniques, particularly in specificity, and even more specifically with respect to use with microRNA.

[0108] After obtaining a purified or partially purified sample thought to contain the target RNA (e.g. microRNA target), the sample may be processed by annealing 103 and ligation 107. For example, single- stranded DNA template (through RNA regions may be included, as described herein), denoted as Trap A and Trap B may be introduced to the sample. In some variations, multiple target microRNAs may be examined form a single sample pool by including Trap A and Trap B pairs that target different microRNAs. Trap A and Trap B both have unique features necessary to carry out the series of steps that lead to detection of the target RNA sequence. Trap A is a single stranded DNA sequence that includes a portion of the target RNA sequence at its 3 ^' end, while Trap B is a single stranded DNA sequence that includes a portion of the remaining RNA target sequence at its 5 ^' end. Trap A and Trap B may also include regions (e.g. B3, B2, B l from 5 ^' to 3 ' end for Trap A and Fl c, F2c, and F3c from 5 ^' to 3 ^' end for Trap B) from within their sequences that will be recognized by a series of pre-selected primers.

[0109] In general, the detection methods described herein include the formation of template (Trap C) that can then be amplified by LAMP. FIG. I B generically illustrates one variation of this method of forming the template by using the target microRNA (or multiple microRNAs when performed in parallel) as a splint, to ligate two portion (Trap A and Trap B) specific to each type of microRNA in the sample. The top portion of FIG. I B shows a schematic of what may occur in the sample when these two single- stranded DNA sequence are added. As is expected, even with a purified or partially purified sample, the sample will likely contain a mixture of sequences having varying sequence lengths. As FIG. I B shows, within the mixture to which the sample has been added, when the target RNA sequence binds properly to both Trap A and the Trap B sequences, the ligase (and in particular T4 RNA ligase 2) may then form Trap C, as show. Properly formed template (Trap C) may then be amplified by LAMP downstream. While there may be a possibility that Trap A and Trap B bind improperly to the target RNA sequences, mechanisms have been incoiporated into these methods that will eventually lead to blockage of amplification of any non-target RNA sequences even if the sequences are nearly identical. These additional fail-safes allow the methods described herein to provide even greater specificity than previously-described amplification methods.

[0110] The annealing of Trap A with Trap B further includes a buffer or buffers and enhancing additives (e.g., DMSO, Pluronics compounds, and salts) for that maintaining proper pH and ionic strength. In one example, the mixture of Trap A, Trap B, input RNA, buffer and additives, in a volume of approximately 3-8 μί, is heated to a temperature between 30-90 degrees Celsius, followed by cooling to a temperature between 20-30 degrees Celsius. In some variations, typical concentrations of Trap A and Trap B do not exceed Ι Ο^ηΜ. The final temperature may be important for proper binding of the probes to the target RNA. Any of these conditions, including temperature, may be optimized for different RNA targets, or may be optimized generally for multiple microRNA targets. It should also be noted that Trap A and Trap B concentrations may be optimized for different RNA target sequences. In some instances, e.g., where the sample is rare or of small quantity such that optimization on the actual sample may not be feasible, substitute standard samples may be used to optimize concentrations prior to using the actual sample.

[0111] In any of the variations described herein, and particularly when ligating DNA strands with T4 RNA Ligase 2, one or both of the ends of DNA being ligated (e.g., the 3' end of Trap A) may include one or more ribonucleotides.

[0112] As mentioned, following the annealing process, the 3 ^' end of Trap A and the 5 ^' end of Trap B may be ligated together using an appropriate ligase. Described herein are optimized methods of forming template in which T4 RNA Ligase 2 has been optimize for robust operation using microRNA to form Trap C template. The Applicants have surprisingly found that the use of T4 RNA Ligase 2 is superior to other types of ligases, despite strong evidence that it would not work, or not work satisfactorily in these situations and conditions. See, e.g., J Jin et al., Nucleic Acids Research (May 6, 2016), "Sensitive and specific miRNA detection method using SplintR Ligase", in which T4 RNA Ligase 2 was reported to have virtually no activity for RNA splintered DNA ligation. Surprisingly, the Applicants have identified conditions for the use of T4 RNA Ligase 2 (despite being used to ligate DNA rather than RNA) that provide both robust and sensitive ligation of Trap A and Trap B constructs using microRNAs. These conditions are described and illustrated herein.

[0113] The bottom portion of FIG. 1 B shows a circle D drawn around Trap A and Trap B where the 3 ^' end of Trap A and the 5 ^' end correspond to the target RNA site. As mentioned, a dsRNA ligase 2 (e.g. T4 RNA ligase 2) or SplintR ligase may be used. The 5 ' end of Trap A may be modified with a hydroxyl and the 3' end of Trap B may be modified with a phosphate to prevent non-specific reactions (e.g., circularization of Trap A and Trap B). When dsRNA ligase 2 is used, it has been found to be particularly helpful to replace the nucleotide at the 3 ^' end of Trap A with a ribonucleotide (enhancing the RNA ligase recognition and/or binding to the target sequence). In FIG. 1 , the black arrow at the connection point of Trap A and Trap B shows were Trap A is linked to Trap B. The resulting single-stranded DNA is denoted Trap C.

[0114] As mentioned, the 5 ^' end of Trap A may be modified with a hydroxyl group and the 3 ' end of Trap B may be modified with a phosphate group. The presence of an additional hydroxyl group on the 5 ^' end of Trap C and a phosphate group at its 3 ' end does not appear to hinder subsequent steps of LA LAMP.

[0115] In some examples, the preferred ligation enzyme is T4 RNA ligase 2. This ligase may bind to the ligation site independent of target or probe sequence and the Applicants have found that the ligation buffer is compatible with other components of the assay. Surprisingly, this enzyme has reduced nonspecific activity compared to SplintR enzyme. [0116] In some examples, the ligation step is performed at a temperature that has been optimized for the particular RNA target. The temperature of ligation may range approximately between 20 and 37 degrees Celsius, and the time of ligation may range approximately between 1 and 120 minutes.

[0117] Following the ligation step, a series of strand displacement steps and sequence interrogation occur prior to exponential amplification. Two examples of the strand displacement steps are shown in FIGS. 2 and 3. As can be seen in FIG. 2, showing one example, the Trap C sequence includes former Trap A (white segment) and former Trap B (black segment). As mentioned earlier, the Trap A portion of Trap C includes three recognition regions B3, B2, and B 1 (from the 5 ' to the 3 ' direction) while the Trap B portion of Trap C includes Fl c, F2c, and F3c (also from the 5 ' to the 3 ' direction). In most instances, there will be at least one nucleotide that separates the recognition (primer sites or regions), e.g. a spacer. There may be several nucleotides that separate the recognition regions. This is so that further down the line, loops on the previously linear DNA strands may form properly. The number of spacer nucleotides may range between a few nucleotides to tens of nucleotides (e.g., 10, 12, 14, 16, 18, 20, 25, 30, etc.). Also, the number of nucleotides that separate the recognition regions of a single sequence may differ from each other.

[0118] In the first strand replacement step, primer FlPsel, which includes a F2 region linked to a F1 C region is able to binds to Trap C through coupling of the F2 region to Trap C's F2C region. With the addition of enzyme such as Bst DNA polymerase or other isothermal polymerase, FlPsel will initiate complementary strand synthesis starting at the F2 region, reading from the 3 ' to 5 ' end of Trap C. Outer primer F3, which is shorter than FlPsel and in some instances, lower in concentration than FlPsel, will also slowly hybridize to F3c of Trap C and initiate strand displacement DNA synthesis, and release a FIP- linked complementary strand. Next, the B2 region of primer BIPsel will bind to the B2c region on the complementary strand obtained after the first step and initiate another complementary strand synthesis starting at the B2c region and moving from the 5 ' end to the 3 ' end. Along with this, a B3 primer, like that of primer F3, will slowing hybridize to B3c region of the resulting complementary strand formed from the first complementary strand synthesis and proceed with another strand separation synthesis. Free strand E formed after B3 synthesis may be further hybridized with FlPsel to form another complementary strand F (as shown in FIG. 2). Strands E and F contain recognition sites Fl and Bl , respectively that are able to interrogate the ligation site for the presence of the correct target sequence. Recall that the ligation site contains the reverse complement of the target RNA sequence.

[0119] The interrogation of the ligation site for non-specific nucleic acid sequences is crucial for the increased specificity of this modified LAMP technique and not provided for in previously described LAMP type techniques. Because the Fl c and the B l c regions of the FlPsel and the BIPsel primers are designed to include sequences that overlap with the ligation site, only sequences that are complementary to these regions will go on to be amplified. In other words, if Trap A or Trap B are ligated to non-specific nucleic acid sequences, or if un-ligated Trap A and Trap B are spuriously amplified during the initial phase of LAMP, the 3 ' ends of Fl and B l will not anneal and therefore will not be extended by the polymerase. This prevents the non-specific Trap A and Trap B amplicons from entering the exponential amplification phase of LAMP.

[0120] In some variations, including Trap A or Trap B templates that including only one or two primer sites (e.g., including only B3 and B2 sites in Trap A, with Trap B including B l , Fl c, F2c, and F3c; or including B3, B2, B l and Fl c in Trap A and F2c and F3c in Trap B) may enhance specificity as well. In these variations, the target microRNA may be split between Trap A and Trap B between either the Bl and B2 sites or between the Fl c and F2c sites, for example (or spanning B2 or F2c). The relative sizes of the Trap A and Trap B are very different. In addition, the smaller Trap A or Trap B may be prevented from nonspecifically amplifying. For example, FIG. 3 shows one example of such a variation where the target site (complementary to the target microRNA) is not in the middle region of Trap C, and the lengths of the original Trap A and Trap B are not the same. Similar to what is shown in FIG. 2, the first series of steps prior to exponential amplification involves strand displacement nucleotide synthesis from the 3 ^' direction toward the 5 ' end using one of the four primers in solution with the annealed Trap C sequence; in this case, primer FIP. This will result in a FIP linked complementary strand (that is able for a loop out structure at one end). Primer F3 will also begin to initiate a slower hybridization of Trap C starting also from the 3 ' end and moving in the 5' direction. Next, the complementary strand previously formed now serves as a template for the BIP primer and subsequent B3 primed strand displacement nucleotide synthesis that leads to the production of dumb bell form structure. The strand formed from BIP primer further serves as a template for complementary sequence synthesis by FIP. As in the previous scenario, sequences E and F are then able to self-interrogate to ensure that only the sequences containing the target sequence are amplified.

[0121] In any of the variations described herein, additional specificity may be achieved by including a portion of one or both inner primers (FIP, BIP) that overlaps with either side of the ligation site (e.g., spanning the two halves of the microRNA in Trap A and Trap B). In this example, if the BIPsel (for selective BIP) and/or FlPsel (for selective FIP) cannot fully anneal at the ends (e.g., 5' end), it cannot initiate amplification. This is illustrated in FIG, 4. FIG. 4 shows three scenarios where the FlPsel sequences (corresponding to the Fl c portion of FlPsel) is taken from the sequence region across the ligation position of the Trap C (the joining of Trap A and Trap B). In the scenario on the far left, the primer interrogates (binds to) the nucleotides spanning the ligation region between Trap A and Trap C, to find that they are complementary and the polymerase allows the synthesis to continue. In the middle and in the scenario on the left, extension of the sequences cannot occur. In the middle scenario, there are no sequences beyond the arrow and thus, no further extension can occur. In the case shown on the left, extension of the sequence also fails because the 5' end does not specifically bind, as the sequence beyond the arrow does not match with the primer sequence. Thus, in order for the amplification process to continue, the target sequence has to be present in the sequence being interrogated. In practice two or more nucleotides spanning the joining site (the site where the microRNA was divided) on either side is sufficient to provide greatly enhanced specificity. This offers a very elegant way to ensure that only sequences having the target sequence continues through to the amplification process. In general, the problem of non-specific binding amplification has plagued previous attempt to use LAMP to detect microRNAs particularly in biological samples. The use of overhangs (in which two or more nucleotide spanning the joining site are incorporated into primers such as the BIPsel and FlPsel primers) dramatically reduce the non-specific binding. Although in some variations having only a single nucleotide of overlap may work, in the context of the LAMP techniques described herein, there may be insufficient reduction of the noise.

[0122] The steps described have been optimized to obtain the most robust signal from a target RNA sequence. The primer sequence is optimized to specifically amplify Trap C in amplification conditions defined by having the following conditions: a temperature of 58-68 degrees Celsius, 5-50 mM Tris-HCl pH 8.8 buffer, 0-50mM potassium chloride (or other salt), 2-12mM MgS04, 0.4-2M Betaine, 0.4-5mM dNTPs different concentrations of LAMP buffer additives which is dependent on the target sequence of interest, and 0.5-12 units of Bst or other isothermal polymerases.

[0123] In any of the variations described herein, the primers may include sequence spacers of specific length and sequence between primer components leading to increase in amplification efficiency, specificity, and proper structure formation prior to amplification (FIG. 5). Further, Trap A and Trap B may include sequence spacers of specific length and sequence leading to increase in amplification efficiency and specificity.

[0124] Returning now to FIG. 4, as mentioned above, when the FlPsel, BIPsel, F3 and B3 primers (and in some variations, additional primers) are combined with the template (Trap C) and enzyme (e.g., Bst or other isothermal polymerase) and reaction mix (e.g., nucleotides, buffers, etc.) complimentary strands having looped ends ("barbell ends") that may self-prime are formed when a recognized target microRNA is present. Once the dumbbell structures have been formed, they will self-replicate using the enzyme (e.g., isothermal polymerase) and amplify through normal LAMP amplification.

[0125] It should be noted that even though the steps described above have been directed to detecting a single target RNA sequence, it is also possible for this process to be used in detecting multiple target sequences. In the case with multiple target sequences, different Trap A and Trap B sequences may be included in the reaction mixture. Further, the reaction may also include different primers that are created to recognize and interrogate the different target sequences.

[0126] For example, FIGS. l OA-l OC, 1 1 A-1 1 C, 12A-12C and 1 3A-13C all illustrate multiplexing methods for pooling primer sets that recognize different target microRNAs, and for amplification (by LAMP) to determine (e.g., in parallel or sequentially, in separate reaction locations, droplets, chambers, etc.) the presence of a particular microRNA.

[0127] For example, in FIG. 1 OA, the top region illustrates the multiplexing mixture, including Trap A and Trap B templates that may be paired to recognized a plurality of different target microRNAs. In this example the B l and Fl c regions of each set of Trap A and Trap B, respectively, are different and incorporate reverse complimentary adjacent portions of different target microRNAs. The B3 and B2 regions of the Trap A (as well as the optional LB region) are conserved between Trap As targeting different microRNAs; similarly the F2c and F3c regions are also conserved between different TrapBs. This means that a "universal" or generic primers for the LBc, LFc, B3 and F3 may be used, while target microRNA specific BIPsel and FlPsel primers may be used, as indicated in FIG. 10B. Prior to the addition of the primers (and particularly the target-specific primers), the sample and template mixture may be divided into multiple aliquots and the LAMP reaction mixture (including primers) may be added for LAMP to proceed using this multiplexed mixture.

[0128] Another multiplexing example is shown in FIGS. 1 1 A-l 1 C. In this example, in addition to the target microRNA-specific B l and Fl c regions (which each incorporate a reverse compliment portion of the target microRNA), the B2 and F2c regions may also be unique for each target microRNA. In this example, the same universal primers used in the example of FIGS. 1 OA- I OC may be used, but the additional unique B2 and F2c regions may provide an additional level of specificity.

[0129] FIGS. 12A-12C also illustrates a multiplexing method that may be used, similar to the example shown in FIGS. l OA-l OC, except that the B l region does not include the reverse compliment of the target microRNA sequence, and instead the portion of the reverse compliment of the target microRNA is appended to the Trap A template adjacent to the B l region. In addition, a unique sequence for each target microRNA is included in the F2c region of Trap B. Thus, in this example, an additional universal primer is shared between all of the templates for LAMP, the "BIP" primer, in addition to the LBc, LFc, B3 and F3 primers, but a target microRNA-specific FlPsel for each target microRNA is used, and added with the universal primers to detect the target microRNA in a aliquot taken from the pooled, multiplexed mixture. The target microRNA-specific FlPsel includes the unique Fl c and F2c regions.

[0130] FIGS. 13A- 13C show another example of a multiplexing LA-LAMP mixture in which, as in FIGS. 12A-12C, the B l and Fl c regions are generic, and the unique reverse compliment of a particular target microRNA is appended to the B l region of all of the Trap As, and the adjacent reverse compliment sequence of the target microRNAs is incorporated into the Fl c region of the Trap Bs. In this example, the F2c region is also generic. Thus, the same universal primers as in FIGS. 12A-12C may be used, but the FlPsel may include both the target microRNA-specific (partial reverse compliment of the target microRNA) and the generic F2c region.

[0131] In any of the LA-LAMP variations described herein, including both multiplexing and non- multiplexing variations, the amplification is isothermal and may be allowed to occur for a sufficiently long time to permit reliable detection. For example, the isothermal amplification may run for between 30 minutes and 5 hours (e.g., between 1 hour and four hours, between 2 hours and 3.5 hours, between 2.5 hours and 3.5 hours, etc.). Finally, the accumulation of DNA copies having the target sequence after amplification may be detected by either fluorescence or electrochemical methods. The time of threshold amplification is employed to determine the presence of target in the sample.

[0132] All sequence elements (e.g., Trap A, Trap B, FlPsel, BIPsel, F3, and B3, etc.) may be generated de novo and may be specifically designed for detection of each target microRNA. This is important for two reasons, one, to eliminate any background signal from genomic DNA which may be present in our assay and two, to avoid unspecific binding of the target nucleic acid to any position other than the ligation site. Furthermore, in order to increase reaction specificity two steps of selectivity are applied; the first during the ligation step and the second, during the LAMP phase, where FlPsel and BIPsel primers are overlapping and are specifically designed for each microRNA target, (FIGS. 2 and 3).

[0133] As just described above, in general the LA-LAMP methods described herein may include two separate enzymatic reactions. The first enzymatic reaction comprises target recognition by two DNA probes (Trap A and Trap B) that, upon binding the target microRNA (see, e.g., FIG. 1 A, 103) become covalently linked (during ligation 105). The resulting linked DNA probes (Trap Cs) are amplified to detectable levels during the second enzymatic reaction, e.g., the LAMP procedure 107, so that the presence of a target microRNA can be detected.

[0134] As described above, the initial input of a typical assay is RNA (or any nucleic acid), purified from any biological source, and consisting of a mixture of sequences and strand lengths. The target RNA may be specifically recognized by two single-stranded DNA probes, Trap A and Trap B. Each probe overlaps a section of the target and discriminates the target from other strands through base pair recognition. Both probes must be bound in correct orientation for the assay to proceed. To achieve this specificity, the region of the probe that overlaps target RNA may be optimized using bioinformatic analysis and experimental validation, and the conditions for binding the probes to the target may be optimized.

[0135] The binding of the probes may be performed during a heating and cooling process. In one example, the mixture of Trap A, Trap B, input RNA, buffer and additives (e.g., annealing mixture), in a volume of 3-8 μΐ, is heated to a temperature between 30-90 °C, followed by cooling to a temperature between 20-30 °C. The final temperature may be important for proper binding of the probes to the target RNA and is optimized for each target. The annealing mixture may comprise buffer and enhancing additives, including DMSO, Pluronics, and NaCl.

[0136] The inventors have improved the specific recognition of target RNA in the LA-LAMP methods and apparatuses described herein by including one or more of the following: optimized base pairing of probes to the target in correct orientation, with the 3' end of Trap A immediately adjacent to the 5' end of Trap B; optimized concentration of TrapA and TrapB; optimized heating temperature to separate nucleic acid strands, followed by optimized cooling temperature to bind the probes to the complementary sequences of the target; incorporation of buffer and additives, including dimethyl sulfoxide, Pluronic F- 127, and sodium chloride; and optimized volume of the reaction.

[0137] In general, after the annealing step, correctly bound Trap A and Trap B may be linked together through the enzymatic process of ligation, forming Trap C. As described above, the position of this linkage can be between B l and Fl c regions (e.g., see FIG. 2, join site 205), or upstream of F2c region (e.g., FIG. 3, join site 305).

[0138] As mentioned above, any appropriate ligate may be used, such as SpIintR

(https://www.neb.com/products/m0375-splintr-ligase). Surprisingly, the use of T4 RNA ligase 2 (dsRNA ligase) (https://www.neb.com/pi ducts/m0239-t4-rna-ligase-2-dsrna-ligase) has been found by the inventors herein to be both unexpectedly and surprisingly better, particularly where one or both template portions (Trap A and/or Trap B) include one or more ribonucleic acid. The enzyme 1 19 (shown as D in FIG. I B) is used to ligate the 3' end of Trap A to 5' end of Trap B. The 5' end of Trap B may be modified with a phosphate to allow its ligation to the 3' end of Trap A. This process generates new single stranded DNA (Trap C).

[0139] In some cases, the preferred enzyme is T4 RNA ligase 2 because this enzyme binds to the ligation site independent of target or probe sequence, and the ligation buffer is compatible with other components of the assay. Finally, the T4 RNA ligase 2 has surprisingly reduced non-specific activity compared to SplintR enzyme.

[0140] In some variations, improvements to the LA-LAMP ligation may include blocking nonspecific reaction leading to circularization of Trap A and Trap B by modifications of the sequence ends. For example, the 5' end of Trap A may be modified with hydroxyl and the 3' end of Trap B is modified with phosphate. As mentioned, the last nucleotide (or last few nucleotides) of Trap A DNA may be substituted with a ribonucleotide to enhance the ligation efficiency by RNA ligase 2 enzyme. In addition, the ligation may be performed at a temperature that has been optimized for the target. In general, the temperature of ligation ranges between 20 and 37 °C. The time of ligation may range between 1 and 120 minutes.

[0141] After ligation, the reaction may be diluted with water. A fraction of this mixture may be used in each LAMP/detection step of the assay. As will be described in greater detail below, any of the methods (e.g., the LA-LAMP methods described herein) may be performed and optimized for use with digital microfluidics (DMF) apparatus or method. Dilution may be helpful to dilute down former buffer components that may interfere with the amplification by LAMP.

[0142] As an alternative, in some variations used with DMF as described herein, one of the partial templates (e.g., Trap A or Trap B) may be attached to a matrix, such as a hydrogel, magnetic beads, or discs, etc. In this case, the bound template (including ligated template Trap C to be amplified) may be washed (by DMF manipulation) to remove the ligation buffer together and unligated partial template. Following washing, the bound template may be resuspended (e.g., in water), which may significantly boost sensitivity; these steps are possible using DMF, but might otherwise result in the loss of too much template to reliably detect a signal when performed using traditional means.

[0143] Once Trap C is formed (in the presence of the right target microRNA), it may be detected by amplification, e.g., through a process of isothermal amplification (such as LAMP) and conversion to a detectable signal.

[0144] During LAMP, extensive copying of Trap C may lead to the production of a sufficient amount of DNA to be detected (FIGS. 2 and 3). Several modifications of LAMP have been implemented to enhance the efficiency and specificity of the reaction. For example, any of the primers may be designed to include an element that suppresses amplification of non-specific products. In LAMP, the reverse-complement sequences of FIP and BIP (forward inner primer and backward inner primer, respectively) form intramolecular loops that are extended by the polymerase to generate DNA strands of increasing length. This step is the initiation of exponential amplification phase of the assay, shown in FIGS. 2 and 3. Here, the F l c and B l c regions of the FlPsel and BIPsel primers, respectively, include sequences overlapping the ligation site, as shown in FIGS. 4 and 5. As discussed above, upon intramolecular loop formation by Fl and Bl (sequences complementary to Flc and Bl c), the ligation site may be interrogated by nucleobase complementarity for linkage between Trap A and Trap B, as shown. If Trap A or Trap B are ligated to non-specific nucleic acid sequences, or if un-ligated Trap A and Trap B are spuriously amplified during the initial phase of LAMP, the 3' ends of Fl and Bl will not anneal and therefore will not be extended by the polymerase. This prevents the non-specific Trap A and Trap B amplicons from entering the exponential amplification phase of LAMP. As shown in FIG. 3, if a ligation between Fl c and F2c 305 is used for annealing to target, then the F2 region of FIP primer is designed to overlap the ligation site and discriminate against non-specific ligation during the initial step of LAMP.

[0145] The primer sequence is may be optimized to specifically amplify TrapC in amplification conditions, such as: temperature between 58-68 °C, 5-50 mM Tris-HCl buffer, 0-50mM potassium chloride (or other salt), different concentrations of LAMP buffer additives, Bst or other isothermal polymerases. The primers may be designed to include sequence spacers (see, e.g., FIG. 5, as discussed above) of specific length and sequence between primer components leading to increase in amplification efficiency and specificity. Trap A and Trap B may be designed to include sequence spacers to increase in amplification efficiency and specificity.

[0146] As mentioned, in some variations the amplification reaction employs Bst polymerase (or other isothermal polymerase, including https://www.neb.com/products/m0275-bst-dna-polymerase-large- fragment, https://www.neb.com/products/m0538-bst-20-warmstart-dna-polymerase,

https://www.neb.com/products/m0537-bst-20-dna-polymerase, https://www.neb.com/products/m0374- bst-3-0-dna-polymerase), optimized buffer conditions and amplification temperature to enhance signal from target detection and suppress signal from non-specific sequence detection.

[0147] Detection of the amplification may be performed optically (e.g., color change, light absorption change, etc.), chemically (reaction product, solubility change, etc.), electrically (change in an electrical property, etc.) or any combination thereof. The detection of amplification may be based on detection of the actual amplification product, and/or it may be based on consumption of a component that is used up by successful amplification, and may be direct or indirect (e.g., change in pH, etc.).

[0148] In some variations, the accumulation of DNA copies during amplification may be detected by fluorescence or electrochemistry. The time course of detection may be used, such as the time to reach a preset threshold of amplification may be employed to determine the presence of target in the sample.

Integration of LA-LAMP in a Digital Microfluidics

[0149] Digital microfluidics (DMF) has emerged as a powerful preparative technique for a broad range of biological and chemical applications. DMF enables real-time, precise, and highly flexible control over multiple samples and reagents, including solids, liquids, and harsh chemicals, without need for pumps, valves, or complex arrays of tubing. In DMF, discrete droplets of nanoliter to microliter volumes are dispensed from reservoirs onto a planar surface coated with a hydrophobic insulator, where they are manipulated (transported, split, merged, mixed) by applying a series of electrical potentials to an embedded array of electrodes2-4. For many applications it is most convenient to carry out DMF on an open surface, such that the matrix surrounding the droplets is ambient air. A DMF apparatus in which the droplet(s) are moved in air (instead of in oil) may be referred to as air-matrix DMF. Air-matrix DMF permits evaporation, which has limited it's use with small volumes and at high temperatures (e.g., greater than 30°C, greater than 40°C, greater than 50 °C, etc.).

[0150] A typical air-matrix DMF apparatus may include two parallel plates separated by an air gap; one of the plates (typically the bottom plate) may contain a patterned array of individually controllable electrodes, and the opposite plate (e.g., the top plate) may include a continuous grounding electrode. Alternatively, grounding electrode(s) can be provided on the same plate as the actuating/high-voltage electrodes. The surfaces of the plates in the air gap may include a dielectric insulator with a hydrophobic material to decrease the wettability of the surface and to add capacitance between the droplet and the control electrode. The droplets may be manipulated in the air gap space between the plates, and may include or have access to a starting material or materials and any reaction reagents. The air gap may be divided up into regions, as some regions of the plates may include heating/cooling (e.g., by Peltier device, resistive heating, convective heating/cooling, etc. in thermal contact with the region) localized to that region. Detection (including imaging or other sensor-based detection) may also be provided over one or more localized regions; in some variations imaging may be provided over all or the majority of the reaction region (air gap space).

[0151] In general, any of the methods described above for LA-LAMP (including sample preparation) may be performed on a DMF apparatus (and in particular, but not limited to, an air-gap DMF apparatus) that is configured as described herein.

[0152] A DMF apparatus as described herein may include one or a series of thermal zones or regions that are in thermal communication that region, including in contact with the plates and/or with the actuation electrodes and therefore the plates.

[0153] The actuation electrodes are able to move droplets within the air gap. The actuation electrodes may divide the working region within the air-gap into discrete regions, such that each electrode corresponds to a unit region. In the examples provided herein, these unit regions are shown as relatively uniform in size and shape (e.g., square) corresponding to the electrode shapes and sizes; it should be understood that they may be any appropriate shape and/or size (e.g., including non-square shapes, such as round, oval, rectangular, triangular, hexagonal, etc., including irregular shapes, and also including any combination of shapes and/or sizes), as illustrated in FIG. 9, described below. The unit regions, each corresponding to a single electrode, may be grouped together functionally (thermally, electrically, etc.) and/or structurally to form regions including cooling/heating regions (thermal zones), imaging regions, etc. Thermal zones may be heated or cooled to temperatures necessary for performing a desired reaction. Thermoelectric components (e.g., Peltier devices, resistive heaters, convective heaters, etc.) and/or temperature detectors (e.g., resistive temperature detectors, RTDs, etc.) may be used to provide heating or cooling and detection of the temperature on the DMF device. The apparatus may also include insulated (thermally insulated) separation regions between different regions, including thermal voids that insulate one thermal zone from another.

[0154] Although the majority of the devices described herein are air-matrix DMF apparatuses that include two parallel pates forming the air gap, any of the techniques (methods and apparatuses) may be adapted for operation as part of a one-plate air-matrix DMF apparatus. In this case, the apparatus includes a single plate and may be open to the air above the single (e.g., first) plate; the "air gap" may correspond to the region above the plate in which one or more droplet may travel while on the single plate. The ground electrode(s) may be positioned adjacent to (e.g., next to) each actuation electrode, e.g., below the single plate. The plate may be coated with the hydrophobic layer (and an additional dielectric layer maybe positioned between the hydrophobic layer and the electrode).

[0155] FIG. 6A illustrates one example of an air-matrix DMF apparatus configured to automates the LA-LAMP workflow described above. This apparatus may allow a single, hands-free protocol for LA- LAMP to be carried out in ambient air. The air-matrix DMF apparatus described may be reconfigured and reprogrammed to accommodate different temperatures and other experimental conditions for a wide range of complex protocols and may be scalable with respect to the volume and number of samples, allowing efficient processing of precious samples.

[0156] In FIG. 6A, the apparatus may be configured as a cartridge (e.g., cassette, plate, etc.). When configured as a cartridge, the cartridge may be pre-loaded with the reaction components (e.g., heat/anneal mix 635, ligase enzyme 637, dilution buffer 639, LAMP mix 641 , etc.), and may allow a user to load (e.g., through a port) the sample 633 to be analyzed. In any of the apparatuses and methods described herein, the reagents or reaction components (e.g., in the reservoirs) may be lyophilized and reconstituted, e.g., buffer, prior to performing the methods described herein. Alternatively, the apparatus may be configured to operate on a 'raw' (e.g., blood, or any other bodily fluid) sample and process it to extract, expose, and/or isolate the sample (e.g., micro RNA). The cartridge may include the processor (e.g., controller) including software and/or hardware for controlling the operation of the device, including heating, cooling, detecting and moving droplets (including mixing) by applying power to the actuation electrodes 609. Alternatively or additionally, the processor may be separate from the cartridge and the cartridge may interface (e.g., connect, couple, etc.) with a processor. For example, the cartridge may be configured to connect or couple to a controller (e.g., reader, etc.). In some variations the cartridge makes an electrical connection with a processor via one or more electrical contacts.

[0157] Thus, any of the air-matrix DMF apparatuses described herein (e.g., the cartridge device portion) may be disposable and/or single-use. Alternatively, multi-use air-matrix DMF cartridges may be used; the air gap region may either be washed, refurbished, or different regions may be used (sharing some components or region, or alternatively in isolation from other regions.

[0158] As mentioned, herein any of the component reagents, e.g., enzyme, buffer, salt,

polynucleotides (e.g., trap A, trap B, primers, etc.), etc., may be lyophilized or dried and reconstituted during operation of the apparatus. The lyophilized reagents may be stored in any portion of the air-gap region, including within a reservoir area (region) or outside of a reservoir area, such as on an actuation electrode. In some variations the reaction droplet(s) may be used to reconstitute the lyophilized reagent and/or droplets of buffer may be used. In some variations, the lyophilized reagent is present in a zone (such as the ligation zone, the LAMP zone, etc.) where the reagent will be mixed or combined with the reaction droplet and/or a droplet derived from the reaction droplet (e.g., a "combined" droplet).

[0159] In FIG. 6A, the air-matrix DMF apparatus includes a plurality of fluid reservoirs for holding the sample 633, and different mixtures (e.g., heat/anneal mix 635, ligase enzyme 637, dilution buffer 639, LAMP mix 641 , etc.). The air-matrix DMF apparatus may also include a plurality of adjacent sub- regions (units) that are each adjacent to an actuation electrode 609. As shown in FIG. 6B, a section through the air-matrix DMF apparatus illustrates one arrangement of an apparatus including a first (e.g., upper or top) plate 601 that includes an inner surface with a hydrophobic and/or dielectric coating 603 (separate hydrophobic and dielectric coatings may be used). In some variations a ground or return electrode may be included as well. Either or both the first plate and the second plate may be transparent (e.g., clear) or translucent. In some variations the plate (top and/or bottom) is glass or plastic. A second plate 615 is opposite from the first plate 601 , and may also include a hydrophobic and/or dielectric coating 607 (the hydrophobic coating may also be a dielectric coating and/or a separate outer hydrophobic coating may overlay a dielectric coating). An air gap region 617 may be positioned between the first and second plates; the droplets being manipulated as described herein are held within this air gap region. The size of the air-gap region may be fixed or it may be varied (e.g., by increasing or decreasing the separation between the upper and lower plates) which may allow control of droplet volume. In some variation the spacing between the upper and lower plates may be adjustable.

[0160] The second (e.g., bottom or lower) plate 615 may include a substrate, such as a printed circuit board (PCB) substrate, onto which the actuation electrodes are supported. In some variations, and in some regions, the lower plate also supports or thermally contacts a thermal controller such as a thermoelectric module 61 1 (e.g., Peltier device), resistive heater, or the like. One or more sensors (e.g., temperature sensors, e.g., RTD) 613 may be secured to (e.g., the underside of) the substrate 610. The substrate may also include one or more electrical traces connecting to the actuation electrodes. In some variations additional circuitry, including control circuitry, may be included and/or electrical connections to connect to a separate controller/processor may be included.

[0161] The DMF apparatus 600 may include regions such as a LAMP zone 623 or ligation zone 621 made up of a plurality of different unit cells (each corresponding to a separate actuation electrodes). Each region or zone may be temperature controlled so that the air gap region is separately controlled. One or more sensors (e.g., optical, electrical, etc.) sensors may be included in an air-matrix DMF cartridge, or the sensor(s) may be present on a separate reader portion (see, e.g., FIG. 6D).

[0162] For example, in FIG. 6D, a schematic illustration of an apparatus including a separate cartridge 600' that may interface with a controller (reader 690) is illustrates. The cartridge may be a plate, cassette, etc. and may be secured in the reader by engaging with a dock, cradle, holder, etc. The reader in this example, includes an electrical connector 689 that may allow the reader to electrically control and/or sense from the electrodes (e.g., actuation electrodes 609). The cartridge may be secured to the reader and allow electrical interconnection between the two. In some variations the reader includes a controller 695 that controls functions such as heating, moving (including mixing and combining droplets, etc. by regulating the actuation electrodes in appropriate sequence), and in some cases sensing. Alternatively or additionally, one or more sensors 697 (e.g., optical sensors such as cameras, CCDs, etc.) may be optionally included in the reader (in some variations they are also or alternatively in the cartridge). In any of these variation the cartridge and/or a reader may include a communication module or sub-system that may include wireless circuitry for wirelessly communication 688 (e.g., by Bluetooth, Zigby, WiFi, etc.) with a remote server 693. A remote server may control/regulate the operation and/or may receive data from the apparatus. In some variations the remote server may interface with a hospital and/or other patient health software for storing (e.g., in a patient medical record) data from the apparatus. The reader may also include a user interface (e.g., controls and/or displays, including touchscreens, buttons, etc.) for controlling operation of the apparatus, entering patient information, security protocols (passwords, etc.), and/or displaying output (e.g., results, error messages, etc.).

[0163] A prototype apparatus such as the one shown in FIG. 6 A was constructed and configured to perform an LA-LAMP protocol described herein. In these tests, human RNA/miRNA were prepared and analyzed using the LA-LAMP protocol summarized schematically in FIG. 1. Key steps included:

annealing, ligation, LAMP and detection. The apparatus (similar to that shown in FIG. 6A) was an air- matrix DMF apparatus, including an array of 1 12 actuation electrodes interfaced 609 with five fluid reservoirs (one for starting material 633, four 635, 637, 639, 641 for reaction reagents), a ligation zone 621 , a LAMP zone 623 (housing a "Paraffin wall") and waste zone. Precise temperature control of the ligation and LAMP zones is obtained by using thermoelectric 61 1 and resistive temperature detectors 613 (RTD). The operation of the apparatus was configured to support execution of an LA-LAMP protocol to detect several high-value miRNAs. FIG. 7 illustrates the results of an exemplary assay for detection of miR-451 (FIG. 8 shows a detection using LA-LAMP without using DMF).

[0164] FIG. 6C shows a time course of images illustrating the operation of air-matrix DMF apparatus performing the LA-LAMP as described herein using the apparatus of FIG. 6A. In this example, each of images 1 -9 is taken through the top plate of the apparatus, and show a plurality of unit cells and different zones comprising subsets of these unit cells. In the upper left of FIG. 6C, the image shows a ligation zone 621 that is temperature controlled. A 2 μί, droplet of sample 633 (RNA sample) and a 4 droplet of heat/anneal mix 635' (including a Trap A and Trap B and chemical additives such as DMSO and Pluronics) are shown; these droplets were dispensed from their respective reservoirs 633, 635, and moved by DMF actuation by sequentially applying energy to the actuation electrodes to move them into the ligation zone 621 , where there are combined (shown in FIG. 6C, middle top, image 2) and mixed. Active mixing (e.g., 15 seconds at room temperature) was achieved by continuously moving the droplet in a circular fashion and/or splitting droplets in equal volumes and merging them to the opposite-ends of RNA droplet; this may be performed by the apparatus controller by applying energy to the appropriate actuation electrodes underlying the unit cell(s) holding or adjacent to the droplet(s). The mixture was then incubated for 2 min at 93 °C, followed by cooling to 25 °C at a rate of 0.1 up to 0.3 °C/s. The DMF apparatus controller may regulate the temperature as described above. During this incubation step, the DMF apparatus may continuously or periodically mix of the heat/anneal droplet 641 to maximize molecular interactions between the target miRNA and its respective Trap A and Trap B templates within the droplet. After completion of the annealing step, the final volume of the annealed mix 643 is decreased by -50% (from the original volume) because it was allowed to evaporate. Surprisingly, inventors have found that (when using an air-matrix DMF), evaporation is desirable and may be used to controllably lower volume, which may enhance ligation reaction efficiency. Data supports that this controlled evaporation using air-matrix DMF boosts the sensitivity and specificity of LA-LAMP dramatically. The size of the droplet can be monitored as it is evaporating, either by comparing it with a smaller electrode size (could serve as a threshold size to stop the evaporation), or monitoring the size by other means, such as capacitance measurements using the actuation electrodes under/adjacent the droplet (or a separate electrode). Size monitoring can be used to guide the reaction timing.

[0165] The annealed mix may then be merged with a droplet (e.g., 2 μί) of ligase enzyme 637' (as shown in FIG 6C, upper right, image 3) and incubated, e.g., for 20 min at 20-37 °C, shown in FIG. 6C, middle left, image 4. During this incubation step, the DMF apparatus may continuously or periodically mix the ligation droplet to maximize ligation efficiency. This contactless mixing of small volumes that is possible using the air-matrix DMF apparatus described herein is a unique feature of DMF and provides a substantial boost in sensitivity of LA-LAMP.

[0166] After ligation, a droplet (e.g., 2 μί) was split 647, 647' (shown in FIG. 6C, middle, image 5) from the ligated mix (the remaining ~4 μL may be used to feed more reactions, thanks to the multiplexing feature of LA-LAMP chemistry-distinct ligation products from different target miRNAs), diluted by half after merging with a. 2 μΐ_^ droplet of dilution buffer 649 (shown in FIG. 6C in the middle right, image 6), and then split again into two equal volume droplets (2 μΐ. ea.); each droplet destined to a distinct pre- LAMP zone.

[0167] In the next step, LAMP is performed using the DMF apparatus. As shown in FIG. 6C, bottom left, image 7, a 2-μί droplet 649 from the ligation zone (or multiple droplets running in parallel) is merged with a 6 μΐ_^ LAMP mix 641 '. The LAMP mix may contain unique primers capable of amplifying one of the multiple ligation products generated in the ligation step for each distinct target miRNA for which Trap A and Trap B pairs were present to capture. The LAMP reaction droplet may then be driven to the LAMP zone 623 and merged with a wax (e.g., paraffin) wall 625. The zone may then conditionally be heated to 60-65 °C, thus melting the wax around the reaction droplet and preventing evaporation for the remainder of the 2 hr reaction.

[0168] The use of DMF to perform the LA-LAMP protocol may provide a number of unexpected advantages compared to traditional techniques for performing standard LAMP. For example, the use of evaporation in the Heat/Anneal step to decrease the total volume of the ligation reaction volume was surprisingly shown to enhance ligation reaction efficiency. This step also boosts the sensitivity and specificity of LA-LAMP. In addition, active mixing during annealing, ligation and LAMP steps may ensure maximal exposure of reagents to successfully interact with one another. This feature of the DMF apparatus and method of use has been found to boost sensitivity of LA-LAMP.

[0169] During the ligation step of the LA-LAMP procedure, the method and apparatus may readily allow molecular multiplexing; for example, traps for more than one miRNA target may be combined in the same droplet during the annealing step, yielding a ligation product of heterogeneous trap C content. The ability to split multiple droplets from the same ligation product mix introduces a new layer of physical multiplexing. All these droplets are destined for multiple parallel LAMP reactions in distinct LAMP zones.

[0170] In addition, the final LAMP steps of the reaction may be configured to prevent evaporation, including ensuring zero evaporation during 120 mm (or longer) incubation at 65°C for Loop Mediated Isothermal Amplification by protecting the droplet, e.g., combing the droplet with an oil or wax, e.g., paraffin wax, that surrounds the reaction droplet. Optimal performance of LAMP when reaction volume is so tightly controlled.

[0171] Finally, because the procedure may be easily automated and controlled by the controller, there minimal handling errors may be introduced and can be easily limited. The inventors have found that this DMF-mediated implementation of the LA-LAMP procedures described herein may generate results matching and exceeding in performance when compared to conventional execution of event the optimized protocol in tubes using PCR cyclers for temperature controlled steps and RT-PCR for product detection (see, e.g., FIG. 8). The integrated LA-LAMP DMF method has the potential to enable hands free (fully automated) execution of a complex protocol that would otherwise require great expertise and multiple highly accurate pipetting steps, as well as greatly improving the LA-LAMP sensitivity and specificity for detection of miRNAs and other RNA species.

[0172] When a feature or element is herein referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

[0173] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/".

[0174] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0175] Although the terms "first" and "second" may be used herein to describe various

features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[0176] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising" means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term "comprising" will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0177] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or

"approximately," even if the term does not expressly appear. The phrase "about" or "approximately" may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1 % of the stated value (or range of values), +/- 1 % of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[0178] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0179] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term "invention" merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMS What is claimed is:

1. A method of detecting a target microRNA sequence from a sample using a modified loop

mediated isothermal amplification (LAMP) technique, the method comprising:

forming a mixture by combining the sample with a first single-stranded DNA probe (Trap

A) and a second single-stranded DNA probe (Trap B), wherein a 3' end of Trap A is a ribonucleotide, further wherein the 3' end of Trap A is complementary to a first portion of the target microRNA, and wherein the 5' end of Trap B is complementary to a second portion of the target microRNA;

ligating the 3' end of Trap A to the 5' end of Trap B by annealing Trap A and Trap B to the target microRNA using T4 RNA Ligase 2 to form a target ligation product (Trap C), wherein Trap C comprises a plurality of primer recognition polynucleotide sequences including: a B3 site that is 5' to a B2 site , wherein the B2 site is 5' to a B l site, wherein the B l site is 5' to an Fl c site, wherein the F l c site is 5' to an F2c site, wherein the F2c site is 5' to an F3c site; and

amplifying Trap C by loop mediated isothermal amplification by combining the target ligation product with:

an F3 primer that is complementary to the F3c site, a B3 primer comprising the B3 site,

a FlPsel primer comprising an Fl c site and an F2 site, wherein the F2 site is complementary to the F2c site, further wherein the 5' end of the FlPsel primer corresponds to at least the last two polynucleotides of the 3' end of Trap A adjacent to a region that is complementary to the second portion of the target microRNA, and

a BIPsel primer comprising a B2 site and a B 1 c site, wherein the B 1 c site is complementary to the B l site, further wherein the 5' end of the BIPsel primer corresponds to a region that is complementary to at least the first two polynucleotides of the 5' end of Trap B adjacent to a region having the sequence of the first portion of the target microRNA.

2. The method of claim 1 , wherein ligating comprises exposing the mixture to a heating cycle

followed by a cooling cycle.

3. The method of claim 1 , wherein ligating comprises exposing the mixture to a heating cycle

followed by a cooling cycle, wherein the heating cycle comprises heating to between 30-90 degrees Celsius.

4. The method of claim 1 , wherein ligating comprises exposing the mixture to a heating cycle followed by a cooling cycle, wherein the cooling cycle comprises cooling to between 20-37 degrees Celsius.

5. The method of claim 1 , wherein the mixture comprises DMSO, Pluronic F-127 and NaCl.

6. The method of claim 1 , further comprising diluting the mixture comprising Trap C prior to

amplifying.

7. The method of claim 1 , wherein Trap A comprises a hydroxyl group on its 5' end and wherein

Trap B comprises a phosphate group on its 3' end.

8. The method of claim 1 , wherein Trap A comprises the B3, B2, and B l sites arranged from its 5' end to its 3' end.

9. The method of claim 1 , wherein Trap B comprises the Fl c, F2c, and F3c sites arranged from its 5' end to its 3' end.

10. The method of claim 1 , wherein amplifying Trap C comprises amplifying at a temperature

between 58-68°Celsius.

1 1. The method of claim 1 , wherein amplifying Trap C comprises diluting the mixture in an

amplification buffer comprising 5-50 mM Tris-HCl, 0-50 mM of a salt, and Bst polymerase or other isothermal polymerase.

12. The method of claim 1 , further comprising detecting amplification of Trap C.

13. The method of claim 1 , further comprising detecting amplification of Trap C by fluorescence or electrochemistry.

14. A method of detecting a target microRNA sequence from a sample using a modified loop

mediated isothermal amplification (LAMP) technique, the method comprising:

forming a mixture by combining the sample with a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B), wherein a 3' end of Trap A is a ribonucleotide, further wherein the 3' end of Trap A is complementary to a first portion of the target microRNA, and wherein the 5' end of Trap B is complementary to a second portion of the target microRNA, wherein the mixture further comprises dimethyl sulfoxide (DMSO), Pluronic F-127 and a salt;

heating the mixture to a temperature between 30 and 80°Celsius;

cooling the mixture to a temperature between 20 and 37°Celsius;

ligating the 3' end of Trap A to the 5' end of Trap B between 20 and 37°C by annealing Trap A and Trap B to the target microRNA using T4 RNA Ligase 2 to form a target ligation product (Trap C), wherein Trap C comprises a plurality of primer recognition polynucleotide sequences including: a B3 site that is 5' to a B2 site , wherein the B2 site is 5' to a B l site, wherein the B l site is 5' to an Fl c site, wherein the Fl c site is 5' to an F2c site, wherein the F2c site is 5' to an F3c site; and

amplifying Trap C by loop mediated isothermal amplification by combining the target ligation product with:

an F3 primer that is complementary to the F3c site, a B3 primer comprising the B3 site,

a FlPsel primer comprising an Fl c site and an F2 site, wherein the F2 site is complementary to the F2c site, further wherein the 5' end of the FlPsel primer corresponds to at least the last two polynucleotides of the 3' end of Trap A adjacent to a region that is complementary to the second portion of the target microRNA, and

a BIPsel primer comprising a B2 site and a B l c site, wherein the B l c site is complementary to the B l site, further wherein the 5' end of the BIPsel primer corresponds to a region that is complementary to at least the first two polynucleotides of the 5' end of Trap B adjacent to a region having the sequence of the first portion of the target microRNA.

15. A method of detecting a target microRNA sequence from a sample using a modified loop

mediated isothermal amplification (LAMP) technique, the method comprising:

forming a mixture by combining the sample with a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B), wherein a 3' end of Trap A is a ribonucleotide, further wherein the 3' end of Trap A is complementary to a first portion of the target microRNA, and wherein the 5' end of Trap B is complementary to a second portion of the target microRNA;

ligating the 3' end of Trap A to the 5' end of Trap B by annealing Trap A and Trap B to the target microRNA using T4 RNA Ligase 2 to form a target ligation product (Trap C), wherein Trap C comprises a plurality of primer recognition polynucleotide sequences including: a B3 site that is 5' to a B2 site , wherein the B2 site is 5' to a B l site, wherein the B l site is 5' to an Fl c site, wherein the Fl c site is 5' to an F2c site, wherein the F2c site is 5' to an F3c site;

wherein Trap A comprises the B3, B2, B l and Fl c sites arranged from its 5' end to its 3' end; and

amplifying Trap C by loop mediated isothermal amplification by combining the target ligation product with:

an F3 primer that is complementary to the F3c site, a B3 primer comprising the B3 site, a FlPsel primer comprising an Fl c site and an F2 site, wherein the F2 site is complementary to the F2c site, and

a BIPsel primer comprising a B2 site and a Bl c site, wherein the B l c site is complementary to the B 1 site.

16. A method of detecting a plurality of microRNAs in parallel from a patient sample containing microRNA using a multiplexed ligation and detection technique, the method comprising: combining the patient sample with a first mixture to form a multiplexing mixture comprising a plurality of pairs of Trap A template and Trap B template, wherein each pair of Trap A and Trap B template is specific to a target microRNA of the plurality of microRNAs because a 5' end of Trap B and a 3' end of Trap A in each pair comprise regions that are reverse complimentary to adjacent portions of the target microRNA and further wherein one or both of the 3' end of Trap B and the 5' end of Trap A comprises one or more nucleotide sequence that is specific to the pair of Trap B and trap A template;

heating the multiplexing mixture to denature the microRNA, and cooling the

multiplexing mixture to anneal pairs of Trap B and Trap A template to specific target microRNA if the target microRNA is present in the multiplexing mixture; ligating the annealed pairs of Trap B template and Trap A template using a ligase to form Trap C template specific to target microRNA;

placing a portion of the multiplexing mixture into each of a plurality of separate regions;

performing, in parallel, loop-mediated isothermal amplification of Trap C template specific to a different target microRNA in each of the plurality of separate regions, wherein each separate region comprises a polymerase having strand displacement activity and primers for the loop mediated amplification, wherein one or more of the primers for the loop mediated amplification includes the nucleotide sequence that is specific to a reverse complement to the target microRNA sequence of the plurality of microRNAs.

17. A method of detecting a plurality of microRNAs in parallel from a patient sample containing microRNA using a multiplexed ligation and detection technique, the method comprising: combining the patient sample with a first mixture to form a multiplexing mixture comprising a plurality of pairs of Trap A template and Trap B template, wherein each pair of Trap A and Trap B template is specific to a target microRNA of the plurality of microRNAs because a 5' end of Trap B and a 3' end of Trap A in each pair comprise regions that are reverse complimentary to adjacent portions of the target microRNA and further wherein each Trap A template comprises a B3 region at a 5' end of the Trap A template, a B2 region 3' to the B3 region, and a

B l region that includes the reverse complimentary region of the microRNA 3' to the B2 region, wherein each Trap B comprises an F3c region at the 3' end of the Trap B template, an F2c region 5' to the F3c region, and an F l c region that includes the reverse complimentary region of the microRNA 5' to the F2cc region, and wherein each pair of Trap A and Trap B templates includes one of:

(a) at least a B l and Fl c unique sequences that are different for each Trap A and Trap B pair specific to each different target microRNA;

(b) B l , B2, Fl c and F2c unique sequences that are different for each Trap A and Trap B pair specific to each different target microRNA;

(c) a sequence for each pair of Trap A and Trap B specific to a particular target microRNA that is unique from other pairs of Trap As and TrapBs for at least one of: the B3 region, the B2 region, an LB region, the B l region, the F3c region, the F2c region, an LFc region, and the Fl c region;

heating the multiplexing mixture to denature the microRNA, and cooling the

multiplexing mixture to anneal pairs of Trap B and Trap A template to specific target microRNA if that specific target microRNA is present in the multiplexing mixture;

ligating the annealed pairs of Trap B template and Trap A template using a ligase to form Trap C template specific to target microRNA;

placing a portion of the multiplexing mixture into each of a plurality of different regions;

performing, loop-mediated isothermal amplification of each of the plurality of

different regions in parallel, wherein each different regions is associated with one specific target microRNA from the plurality of microRNAs and comprises a combination of primers that complement or include the unique sequence or sequences that is different from the other pairs of the plurality of pairs of Trap A template and Trap B template for at least one of the B3 region, the B2 region, the B l region, the F3c region, the F2c region, and the Fl c region of the template specific to target microRNA. 8. A method of detecting a target microRNA from a patient sample using an air-matrix digital micro fluidic (DMF) apparatus, the method comprising:

forming a reaction droplet in an air gap region of the air-matrix DMF apparatus from a portion of the patient sample including patient microRNA and first mixture comprising a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B);

evaporating more than 20% of the reaction droplet by heating the reaction droplet within a ligation zone of the air gap to ligate the Trap A and Trap B to a target microRNA; combining the reaction droplet with a ligase enzyme to form a combined droplet and incubating the combined droplet;

mixing the combined droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes to ligate the Trap A to Trap B and form Trap C in the presence of the target microRNA;

adding a polymerase and a plurality of primers configured to selectively amplify Trap C to the combined droplet or a portion of the combined droplet and coating the combined droplet or portion of the combined droplet with a protective material to prevent evaporation of the combined droplet within a LAMP zone of the air gap; incubating the combined droplet or portion of the combined droplet at between 55-70°C to allow amplification of Trap C by loop mediated isothermal amplification (LAMP); and

detecting amplification of Trap C.

19. A method of detecting a target microRNA from a patient sample using an air-matrix digital microfluidic (DMF) apparatus, the method comprising:

forming a reaction droplet in an air gap region of the air-matrix DMF apparatus from a portion of the patient sample including patient microRNA and first mixture comprising a first single-stranded DNA probe (Trap A) and a second single-stranded DNA probe (Trap B);

mixing the reaction droplet by applying energy to at least one actuation electrode of a plurality of actuation electrodes adjacent to the air matrix;

evaporating between 20% and 90% of the reaction droplet by heating the reaction droplet to greater than 80°C and cooling the droplet to less than 40°C within a ligation zone of the air gap to ligate the Trap A and Trap B to a target microRNA;

combining the reaction droplet with a ligation droplet comprising a ligase enzyme to form a combined droplet and incubating the combined droplet;

mixing the combined droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes to ligate the Trap A to Trap B and form Trap C in the presence of the target microRNA;

adding a polymerase and primers configured to selectively amplify Trap C to the combined droplet or a portion of the combined droplet and coating the combined droplet or portion of the combined droplet with a wax to prevent evaporation of the combined droplet within a LAMP zone of the air gap;

incubating the combined droplet or portion of the combined droplet at between 55-70°C to allow amplification of Trap C by loop mediated isothermal amplification (LAMP); and

detecting amplification of Trap C.

20. A method of detecting a target microRNA from a patient sample using an air-matrix digital microfluidic (DMF) apparatus, the method comprising:

forming a reaction droplet in an air gap region of the air-matrix DMF apparatus from a portion of the patient sample including patient microRNA and first mixture comprising a first single-stranded DNA probe (Trap A), a second single-stranded DNA probe (Trap

B), dimethyl sulfoxide and poloxamer, wherein forming comprises dispensing a first droplet of the first mixture from a reservoir in fluid connection with the air gap region and applying energy to a plurality of actuation electrodes to combine the first droplet with a sample droplet containing the portion of the patient sample;

mixing the reaction droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes adjacent to the air matrix;

evaporating between 20% and 90% of the reaction droplet by heating the reaction droplet to greater than 80°C and cooling the droplet to less than 40°C within a ligation zone of the air gap to ligate the Trap A and Trap B to a target microRNA, wherein evaporating the reaction droplet comprises monitoring the reaction droplet to determine when the volume of the reaction droplet falls below a threshold;

combining the reaction droplet with a ligation droplet comprising a ligase enzyme to form a combined droplet and incubating the combined droplet;

mixing the combined droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes to ligate the Trap A to Trap B and form Trap C in the presence of the target microRNA;

adding a polymerase and primers configured to selectively amplify Trap C to the combined droplet or a portion of the combined droplet and coating the combined droplet or portion of the combined droplet with a paraffin wax material to prevent evaporation of the combined droplet within a LAMP zone of the air gap; incubating the combined droplet or portion of the combined droplet at between 55-70°C to allow amplification of Trap C by loop mediated isothermal amplification (LAMP); and

detecting amplification of Trap C.

21 . The method of claim 18 or 19, wherein the first mixture further comprises chemical additives including dimethyl sulfoxide (DMSO) and poloxamer.

22. The method of claim 18, 19 or 20, wherein forming comprises forming a droplet of less than 10 uL.

23. The method of claim 1 8 or 19, wherein forming comprises dispensing a first droplet of the first mixture from a reservoir in fluid connection with the air gap region and applying energy to the plurality of actuation electrodes to combine the first droplet with a sample droplet containing the portion of the patient sample.

24. The method of claim 18, 19 or 20, wherein mixing the reaction droplet comprises continuously moving the droplet within the air matrix or splitting and recombining the reaction droplet within the air matrix.

25. The method of claim 18, 19 or 20, wherein mixing comprises mixing for 10 or more seconds at room temperature.

26. The method of claim 18, 19 or 20, wherein evaporating the reaction droplet comprises incubating the reaction droplet for between 1 and 10 min at greater than 80°C followed by cooling the reaction droplet to less than 35 °C at a rate of between 0.1 to 0.3 °C/s.

27. The method of claim 18, 19 or 20, wherein evaporating the reaction droplet comprises mixing the reaction droplet by applying energy to at least one actuation electrode of the plurality of actuation electrodes to move the reaction droplet within the ligation zone or to split and recombine the reaction droplet within the ligation zone.

28. The method of claim 18 or 19, wherein evaporating the reaction droplet comprises monitoring the reaction droplet to determine when the volume of the reaction droplet falls below a threshold.

29. The method of claim 18, 19 or 20, further comprising diluting the combined droplet prior to moving all of a portion to the LAMP zone.

30. The method of claim 18, 19 or 20, wherein combining comprises applying energy to one or more of the actuation electrodes to move the ligation droplet from a reservoir in fluid contact with the air gap through the air gap to merge with the reaction droplet.

31. The method of claim 18, 19 or 20, wherein combining the reaction droplet comprises incubating the reaction droplet for greater than 10 minute at between 20-37°C.

32. The method of claim 18, wherein the protective material is a wax.

33. The method of claim 18 or 19, wherein the protective material is a paraffin wax.

34. The method of claim 18, 19 or 20, wherein adding the polymerase and primers comprises first splitting the combined droplet into a plurality of combined droplets that are processed in parallel.

35. The method of claim 18, 19 or 20, wherein detecting comprises optically detecting.

36. The method of claim 1 8, 19 or 20, wherein detecting comprises electrically detecting.

37. The method of claim 18, 19, or 20, wherein detecting comprises electrochemically detecting.

38. The method of claim 18, 19, or 20, further comprising reconstituting a lyophilized reagent within one or more regions of the air-gap matrix.