MICROFLUIDIC MICRO ARRAY DEVICES AND METHODS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of United States Provisional Application No. 62/621,014 filed on January 23, 2018, the content of which is incorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 768592000240SEQLIST.txt, date recorded: January 23, 2019, size: 2 KB).

FIELD OF THE INVENTION

[0003] The present invention relates to microfluidic microarray devices, methods of microarray synthesis, and methods of use thereof.

BACKGROUND OF THE INVENTION

[0004] DNA microarrays play a central role in nucleic acid detection, including clinical diagnostics and agricultural applications, by allowing screening of thousands of genetic loci simultaneously. Light-directed synthesis is a powerful tool for flexible fabrication of high- density microarrays. While various methods are known for the synthesis of microarrays on glass substrates, these methods suffer from limitations with respect to scalability, automation, speed, and cost. Historically, light-directed microarrays are fabricated on glass slides or wafers compressed against another material, then later processed and fitted with a hybridization chamber. There is a need for simple, reproducible, scalable, and cost-effective methods for synthesis of microarray devices, which have high success rates and are amenable to automation.

[0005] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides microfluidic devices, methods of preparation and methods of using the microfluidic devices.

[0007] One aspect of the present application provides a microfluidic device comprising: (a) a first plate comprising a first organic polymer substrate; (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface comprises a plurality of oligonucleotide probes; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the first plate is bonded to the second plate covalently or via entangled polymers.

[0008] In some embodiments according to any one of the microfluidic devices described above, the 5’ end of each oligonucleotide probe is attached to the first surface or the second surface of the microfluidic channel. In some embodiments, the 3’ end of each oligonucleotide probe is attached to the first surface or the second surface of the microfluidic channel. In some embodiments, each oligonucleotide probe is attached to the first surface or the second surface at a pre-determined position. In some embodiments, the microfluidic channel comprises about 2 to about 10 oligonucleotide probes. In some embodiments, the plurality of oligonucleotide probes are DNA probes. In some embodiments, the plurality of oligonucleotide probes comprises an oligonucleotide probe pair capable of detecting single nucleotide polymorphism (SNP) alleles in a gene of interest. In some embodiments, each oligonucleotide probe is about 10 to about 75 nucleotides long.

[0009] In some embodiments according to any one of the microfluidic devices described above, the microfluidic device further comprises a target nucleic acid hybridized to one or more oligonucleotide probes. In some embodiments, one or more oligonucleotide probes are extended by one or more nucleotides that are complementary to the target nucleic acid. In some embodiments, one or more oligonucleotide probes are ligated to an oligonucleotide

complementary to the target nucleic acid.

[0010] Another aspect of the present application provides a microfluidic device comprising:

(a) a first plate comprising a first organic polymer substrate; (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface of the microfluidic channel is capable of binding a nucleoside phosphoramidite, and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the first surface and/or the second surface of the microfluidic channel is bound to a nucleoside phosphoramidite. In some embodiments, the first plate is bonded to the second plate covalently or via entangled polymers.

[0011] In some embodiments according to any one of the microfluidic devices described above, the first organic polymer substrate is selected from the group consisting of

polydimethylsiloxane (PDMS), cyclic olefin copolymer (COC) and cyclo-olefin polymer (COP). In some embodiments, the second plate comprises a glass substrate. In some embodiments, the second plate comprises a second organic polymer substrate. In some embodiments, the second organic polymer substrate is selected from the group consisting of PDMS, COC COP. In some embodiments, the first organic polymer substrate is the same as the second organic polymer substrate. In some embodiments, the first organic polymer substrate is different from the second organic polymer substrate.

[0012] In some embodiments according to any one of the microfluidic devices described above, the first surface and/or the second surface of the microfluidic channel is silanized. In some embodiments, the first surface and/or the second surface of the microfluidic channel comprises a silane having a terminal hydroxyl group or a terminal amine group. In some embodiments, the first surface and/or the second surface of the microfluidic channel comprises one or more silanes selected from the group consisting of 3-[bis(2-hydroxyethyl)amino]propyl- triethoxysilane, N-(hydroxyethyl)-N,N-bis(trimethoxysilylpropyl)amine , N,N'-bis(2- hydroxyethyl)-N,N'-bis(trimethoxysilylpropyl)ethylenediamine , (3- aminopropyl)treiethoxysilane, 3-aminopropyidimethylethoxysilane, N,N-bis(2-hydroxyethyl)-3- aminopropyltriethoxysilane, 3-(4-semicarbazidyl)propyltriethoxysilane, triethoxysilylundecanal,

3-aminopropyldimethylethoxysilane, N-(6-aminohexyl)aminopropyltrimethoxysilane, N-(3- triethoxysilylpropyl)-4-hydroxybutyramide, hexadecafluorododec- 11 -en- 1 -yltrimethoxysilane,

4-amino-3,3-dimethylbutylmethyldimethoxysilane, 3-aminopropylmethyldiethoxysilane, 1- amino-2-(dimethylethoxysilyl)propane, 3-aminopropyldiisopropylethoxysilane, N-(6- aminohexyl)aminomethyltriethoxysilane, N-(2-aminoethyl)- 1 l-aminoundecyltrimethoxysilane, N-3-[(amino(polypropylenoxy)]aminopropyltrimethoxysilane, N-(2-aminoethyl)-3- aminopropyltrimethoxysilane, l,2-bis(trimethoxysilyl)decane, and l,8-bis(triethoxysilyl)octane.

[0013] In some embodiments according to any one of the microfluidic devices described above, the microfluidic channel is about 0.1 mm to about 300 mm (such as about 0.5 mm to about 100 mm) long. In some embodiments, the microfluidic channel is about O.lmm to about 300 mm (such as about 0.1 mm to about 10 mm) wide. In some embodiments, the microfluidic channel is about 1 mih to about 800 mih (such as about 5 mih to about 800 mih) deep. In some embodiments, the microfluidic channel has a volume of less than about 100 pL, such as less than about 50 pL, 20 pL, 10 pL or less.

[0014] In some embodiments according to any one of the microfluidic devices described above, the microfluidic channel is molded into the first organic polymer substrate and/or the second organic polymer substrate. In some embodiments, the microfluidic channel is embossed, etched, or 3D-printed into the first organic polymer substrate and/or the second organic polymer substrate.

[0015] In some embodiments according to any one of the microfluidic devices described above, the microfluidic channel is a rectangular prism.

[0016] In some embodiments according to any one of the microfluidic devices described above, the microfluidic device further comprises ports and fluidic connections for filling and emptying the microfluidic channel. In some embodiments, the ports and/or fluidic connections are pre-formed in the first and/or second organic polymer substrate.

[0017] In some embodiments according to any one of the microfluidic devices described above, the microfluidic device comprises a plurality of microfluidic channels, such as about 2 to about 100 microfluidic channels. In some embodiments, the plurality of microfluidic channels are parallel to each other, and wherein the distance between adjacent microfluidic channels is about 100 mih to about 1 mm.

[0018] Another aspect of the present application provides a method of preparing a microfluidic microarray device, comprising synthesizing a plurality of oligonucleotide probes at pre determined positions on the first surface and/or the second surface of the microfluidic channel of any one of the microfluidic devices described above.

[0019] In some embodiments, there is provided a method of preparing a microfluidic microarray device, comprising synthesizing a plurality of oligonucleotide probes at pre determined positions on a first surface and/or a second surface of a microfluidic channel in a microfluidic device, wherein the microfluidic device comprises: (a) a first plate comprising a first organic polymer substrate (e.g., PDMS); (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface of the microfluidic channel is capable of binding a nucleoside phosphoramidite, and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the plurality of oligonucleotide probes are synthesized at pre-determined positions in reaction volumes of less than about 100 mΐ _^ , such as less than about 50 mΐ _^ , 20 mΐ _^ ,

10 mί or less.

[0020] In some embodiments according to any one of the methods described above, the method comprises bonding a first plate comprising a first organic polymer substrate comprising a microfluidic channel to a second plate comprising a second organic polymer substrate or a glass substrate. In some embodiments, the method comprises molding, embossing or etching the microfluidic channel into the first organic polymer substrate and/or the second organic polymer substrate.

[0021] In some embodiments according to any one of the methods described above, the method further comprises passing a silane through the microfluidic channel prior to the synthesizing.

[0022] In some embodiments, there is provided a method of preparing a microfluidic microarray device, comprising synthesizing a plurality of oligonucleotide probes in a reaction volume of no more than about 100 pL (e.g., no more than about 50, 20, 10 or less pL) at pre determined positions on an interior surface of a microfluidic channel in a microfluidic device, wherein the microfluidic device comprises a glass substrate and/or an organic polymer substrate (e.g., PDMS), wherein the interior surface of the microfluidic channel is formed by the glass substrate or the organic polymer substrate, and wherein the microfluidic channel has a volume of no more than about 100 pL (e.g., no more than about 50, 20, 10 or less pL). In some

embodiments, the reaction volume is no more than about 20 pL. In some embodiments, the reaction volume is no more than about 10 pL.

[0023] In some embodiments according to any one of the methods described above, the plurality of oligonucleotide probes is synthesized in the 5’ to 3’ direction. In some embodiments, the plurality of oligonucleotide probes is synthesized in the 3’ to 5’ direction. In some embodiments, a portion of the plurality of oligonucleotide probes is synthesized in the 5’ to 3’ direction, and the remaining portion of the plurality of oligonucleotide probes is synthesized in the 3’ to 5’ direction.

[0024] In some embodiments according to any one of the methods described above, the plurality of oligonucleotide probes is synthesized by a light-directed method. In some

embodiments, the light-directed method uses UV light. In some embodiments, the light-directed method uses blue light and a triplet sensitizer. In some embodiments, the light-directed method uses two-photo infrared (IR) light. In some embodiments, the nucleoside phosphoramidite has a broad-wavelength deprotecting group. In some embodiments, the plurality of oligonucleotide probes is synthesized by maskless photolithography. In some embodiments, the plurality of oligonucleotide probes is synthesized using a series of photomasks.

[0025] In some embodiments according to any one of the methods described above, the synthesizing comprises: (i) providing the microfluidic device having a first nucleoside phosphoramidite or a plurality of oligonucleotides comprising a first nucleoside

phosphoramidite at the 5’ (or 3’) terminus, wherein the first nucleoside phosphoramidite or the plurality of oligonucleotides are attached to the first surface and/or the second surface of the microfluidic channel via the 3’ (or 5’) terminus, and wherein the first nucleoside

phosphoramidite comprises a photo-labile protective group at the 5’ (or 3’) position; (ii) deprotecting the first nucleoside phosphoramidite using a patterned light beam to provide deprotected first nucleoside phosphoramidite at pre-determined positions on the first surface and/or the second surface of the microfluidic channel; (iii) passing a second nucleoside phosphoramidite through the microfluidic channel to couple the second nucleoside

phosphoramidite to the deprotected first nucleoside phosphoramidite, wherein the second nucleoside phosphoramidite comprises a photo-labile protective group at the 5’ (or 3)’ position; optionally (iv) passing a capping composition through the microfluidic channel; optionally (v) passing an oxidizing solution through the microfluidic channel; and (vi) repeating steps (ii)-(v) for a pre-determined number of times, wherein the patterned light beam is programmed at each step according to the sequences of the plurality of oligonucleotide probes; thereby providing the plurality of oligonucleotide probes at the pre-determined positions on the microfluidic device. In some embodiments, the oxidizing composition and the capping composition comprise a solvent that does not swell the first organic polymer substrate. In some embodiments, the oxidizing composition and the capping composition do not comprise dichloromethane. In some embodiments, the oxidizing composition and the capping composition comprise acetonitrile. In some embodiments, the synthesizing is carried out in an automated oligonucleotide synthesis system comprising a digital micromirror device capable of producing the patterned light beam.

[0026] Further provided by the present application is a method of analyzing a sample comprising target nucleic acids, comprising: (a) contacting the sample with the plurality of oligonucleotide probes in any one of the microfluidic devices described above, wherein the microfluidic device comprises a plurality of oligonucleotide probes; (b) hybridizing the target nucleic acids to the plurality of oligonucleotide probes in the microfluidic channel to provide probe-target hybrids; and (c) detecting the probe-target hybrids. In some embodiments, step (a) comprises injecting the sample into the microfluidic channel of the microfluidic device. In some embodiments, probe-target hybrids on the first surface are detected. In some embodiments, the method further comprises removing the second plate prior to (c). In some embodiments, probe- target hybrids on the second surface are detected. In some embodiments, the method further comprises removing the first plate prior to (c). In some embodiments, the microfluidic channel is washed (such as soaked in water or a buffer overnight) prior to the detecting.

[0027] In some embodiments according to any one of the methods of analyzing described above, the microfluidic device is used for a biochemical assay. In some embodiments, the biochemical assay is selected from the group consisting of quantitative hybridization, quantitative annealing, hybridization-ligation, nuclease hybridization assay, allele specific primer extension, and short-read sequencing. In some embodiments, the method comprises sequencing of one or more nucleobases ( e.g ., no more than about any one of 50, 40, 30, 20, 10, 5 or fewer nucleobases) of a target nucleic acid hybridized to an oligonucleotide probe. In some embodiments, the short-read sequencing comprises sequencing by synthesis or sequencing by ligation.

[0028] In some embodiments according to any one of the methods of analyzing described above, the microfluidic device comprises a plurality of microfluidic channels each comprising a plurality of oligonucleotide probes, and wherein a different sample comprising target nucleic acids is injected into each microfluidic channel.

[0029] It is understood that aspects and embodiments of the invention described herein include “consisting” and/or“consisting essentially of’ aspects and embodiments.

[0030] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0031] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0032] The term“about X-Y” used herein has the same meaning as“about X to about Y.” [0033] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0034] These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention just as if each and every combination is individually and explicitly disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 illustrates an exemplary automated light-directed microarray synthesis method. In this example, PDMS microfluidic channels are formed using soft lithography techniques with an SET-8 master mold. The PDMS substrate is bonded to a glass substrate by activating both glass and PDMS using oxygen plasma or UV-ozone. Following bonding, the glass substrate and the PDMS substrate comprising the microfluidic channels are modified with a hydroxyl terminating silane, which can be coupled to oligonucleotides via phosphoramidite chemistry. This silanized microfluidic device is then loaded into a light-directed oligonucleotide synthesizer, which produces multiple microarrays per microfluidic device. After the in situ synthesis of the entire oligonucleotide probes, the exocyclic groups are removed using traditional deprotection chemistry. The microfluidic microarray device is then thoroughly washed and may be used for biochemical assays such as DNA synthesis or ligation.

[0036] FIG. 2 illustrates a schematic of light-directed synthesis of oligonucleotide probes on the microfluidic devices. This light-directed synthesis consists of a 4-step cyclical process. Each cycle includes light deprotection, oxidation, capping and coupling. Each cycle adds a single nucleotide to the growing oligonucleotide strand.

[0037] FIG. 3 shows an exemplary image projected by the Visitech FUXBEAM ^® lithography system to deprotect the photosensitive protection groups on the 5’ end of growing

oligonucleotides.

[0038] FIGS. 4A-4D show signals from template samples hybridized to three different oligonucleotide probes (HIV, HCV1 and HCV2) on a microfluidic microarray device. FIG. 4A demonstrates successful synthesis of all three oligonucleotide probes. FIGS. 4B-4D demonstrate specific detection of template DNA by each of the three oligonucleotide probes.

[0039] FIG. 5 shows phosphoramidites used for oligonucleotide probe synthesis in the 3’ ->5’ direction (left) or 5’ ->3’ direction (right).

[0040] FIGS. 6A-6B show detection of hybridization of template samples to four

oligonucleotide probes (TTT, AAA, CCC, and GGG probes) from glass surface only (FIG. 6A) or from both the glass surface and the PDMS surface (FIG. 6B).

[0041] FIG. 7 shows specific hybridization of TRB-91 to its complementary probe CCC. This image was taken from the PDMS surface only.

[0042] FIG. 8 shows specific hybridization (right) of template DNA to SNP oligonucleotide probes (A and B), and allele- specific extension of the hybridization product between the template DNA and the A probe (left).

[0043] FIG. 9 shows quantitative analysis of the images in FIG.8.

[0044] FIG. 10 shows extension of control probes and strand- specific probes hybridized to amplicons from Gyrase A of Neisseria gonorrhoeae.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present application provides microfluidic devices and methods for inexpensive, in situ ( e.g ., light-directed) synthesis of microarrays. The microfluidic devices described herein comprise one or more microfluidic channels that allow fluid flow for microarray synthesis as well as for subsequent hybridization and biochemical assays on the microfluidic device. The microfluidic channels minimize the volumes of expensive chemicals used in microarray synthesis, as well as precious biological samples used in the biochemical assays. In some embodiments, the microfluidic device comprises a first plate comprising an organic polymer substrate (e.g., polydimethylsiloxane,“PDMS”) bonded to a second plate comprising a glass substrate, wherein the microfluidic channels have a first surface in the organic polymer substrate and a second surface formed by the glass substrate, and wherein both the first surface and the second surface may be attached to oligonucleotide probes. Methods of preparing a microfluidic microarray device are provided herein, in which the oligonucleotide probes may be synthesized at pre-determined positions in small reaction volumes (e.g., less than about 20 pL) on the surface of the microfluidic channels in the 5’ to 3’ direction and/or the 3’ to 5’ direction. Such microfluidic devices can be fabricated at a lower cost than traditional microarray flow-cells which are made by bonding etched glass to glass. Light-directed oligonucleotide synthesis can be carried out using an automated oligonucleotide synthesizer system to prepare microfluidic microarray devices useful for a variety of biochemical assays. Compared to traditional spotted microarrays, the microfluidic devices, methods and systems described herein allow faster fabrication of oligonucleotide microarrays with higher density, and at a lower cost and greater sequence flexibility.

[0046] Accordingly, one aspect of the present application provides a microfluidic device comprising: (a) a first plate comprising a first organic polymer substrate (e.g., PDMS); (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface comprises a plurality of oligonucleotide probes; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the microfluidic device comprises a plurality of microfluidic channels.

[0047] One aspect of the present application provides a microfluidic device comprising: (a) a first plate comprising a first organic polymer substrate (e.g., PDMS); (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface is capable of binding or is bound to a nucleoside

phosphoramidite; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the microfluidic device comprises a plurality of microfluidic channels.

[0048] Another aspect of the present application provides a method of preparing a microfluidic microarray device, comprising synthesizing a plurality of oligonucleotide probes in a

microfluidic device, wherein the microfluidic device comprises: (a) a first plate comprising a first organic polymer substrate (e.g., PDMS); (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the plurality of oligonucleotide probes are synthesized at pre-determined positions on the first surface and/or the second surface of the microfluidic channel, and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the first surface and/or the second surface are silanized. In some embodiments, the microfluidic device comprises a plurality of microfluidic channels. In some embodiments, the plurality of oligonucleotide probes are synthesized by a light-directed method.

[0049] Also provided are microfluidic devices for in situ synthesis of microarrays, methods of using the microfluidic devices, kits and articles of manufacture comprising the microfluidic devices described herein.

Microfluidic devices

[0050] The present application provide microfluidic devices comprising one or more microfluidic channels comprising a plurality of oligonucleotide probes. In some embodiments, the one or more microfluidic channels each comprise one or more surfaces ( i.e ., interior surfaces) comprising the plurality of oligonucleotide probes. The microfluidic devices comprising oligonucleotide probes are also referred herein as“microfluidic microarray devices.”

[0051] One aspect of the present application provides a microfluidic device comprising a microfluidic channel comprising a first surface formed by a first organic polymer substrate and a second surface formed by a second organic polymer substrate or a glass substrate, wherein the first surface and/or the second surface comprises a plurality of oligonucleotide probes. In some embodiments, each of the first surface and the second surface comprises a plurality of oligonucleotide probes. In some embodiments, the microfluidic device comprises a plurality of microfluidic channels.

[0052] In some embodiments, there is provided a microfluidic device comprising: (a) a first plate comprising a first organic polymer substrate; (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface comprises a plurality of oligonucleotide probes; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, each of the first surface and the second surface comprises a plurality of oligonucleotide probes.

[0053] In some embodiments, there is provided a microfluidic device comprising: (a) a first plate comprising a first organic polymer substrate; (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a plurality of microfluidic channels each comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface comprises a plurality of oligonucleotide probes; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel.

[0054] In some embodiments, there is provided a microfluidic device comprising: (a) a first plate comprising a PDMS substrate; (b) a second plate comprising a glass substrate; and (c) a microfluidic channel comprising a first surface in the PDMS substrate and a second surface formed by the glass substrate, wherein the first surface and the second surface comprises a plurality of oligonucleotide probes; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the microfluidic device comprises a plurality of microfluidic channels.

[0055] In some embodiments, there is provided a microfluidic device comprising a microfluidic channel having a volume of no more than about 100 pL (such as no more than about 50, 20, 10 or less pL), wherein the microfluidic device comprises a glass substrate, wherein the microfluidic channel comprises one or more interior surface(s) formed by the glass substrate, and wherein the one or more interior surface(s) comprising a plurality of

oligonucleotide probes. In some embodiments, the microfluidic device comprises a plurality of microfluidic channels. In some embodiments, the microfluidic channel is etched into the glass substrate.

[0056] In some embodiments, the microfluidic microarray device further comprises a target nucleic acid hybridized to one or more oligonucleotide probes. In some embodiments, the one or more oligonucleotide probe is extended by one or more nucleotides at the terminus not attached to the first surface or the second surface. In some embodiments, the one or more oligonucleotide probe is ligated to a nucleic acid. In some embodiments, the microfluidic microarray device further comprises an enzyme ( e.g ., ligase, polymerase, nuclease, etc.). In some embodiments, the microfluidic microarray device further comprises a sequence-specific protein bound to one or more oligonucleotide probes.

[0057] Further provided are microfluidic devices comprising one or more microfluidic channels for in situ synthesis of oligonucleotide probes. The interior surface(s) of the one or more microfluidic channels comprise functional groups that can be linked to growing oligonucleotide chains, including nucleoside phosphoramidites. In some embodiments, the interior surface(s) of the one or more microfluidic channels are bound to oligonucleotide chains, including nucleoside phosphoramidites. In some embodiments, one or more surfaces of each microfluidic channel are silanized. The microfluidic devices described herein for synthesis of oligonucleotide probes are also referred herein as“microfluidic synthesis devices.”

[0058] One aspect of the present application provides a microfluidic device comprising a microfluidic channel comprising a first surface formed by a first organic polymer substrate and a second surface formed by a second organic polymer substrate or a glass substrate, wherein the first surface and/or the second surface is capable of binding a nucleoside phosphoramidite. In some embodiments, the first surface and/or the second surface is bound to a nucleoside phosphoramidite. In some embodiments, the first surface and/or the second surface of the microfluidic channel is silanized, e.g., by a silane having a terminal hydroxyl group.

[0059] In some embodiments, there is provided a microfluidic device comprising: (a) a first plate comprising a first organic polymer substrate; (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface of the microfluidic channel is capable of binding a nucleoside phosphoramidite; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, each of the first surface and the second surface is capable of binding a nucleoside phosphoramidite. In some embodiments, the first surface and/or the second surface of the microfluidic channel is silanized, e.g., by a silane having a terminal hydroxyl group. In some embodiment, the microfluidic device comprises a plurality of microfluidic channels.

[0060] In some embodiments, there is provided a microfluidic device comprising: (a) a first plate comprising a first organic polymer substrate; (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface of the microfluidic channel is bound to a nucleoside phosphoramidite; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, each of the first surface and the second surface is bound to a nucleoside phosphoramidite. In some embodiments, the first surface and/or the second surface of the microfluidic channel is silanized, e.g., by a silane having a terminal hydroxyl group. In some embodiment, the microfluidic device comprises a plurality of microfluidic channels. [0061] In some embodiments, there is provided a microfluidic device comprising: (a) a first plate comprising a PDMS substrate; (b) a second plate comprising a glass substrate; and (c) a microfluidic channel comprising a first surface in the PDMS substrate and a second surface formed by the glass substrate, wherein the first surface and the second surface are capable of binding to a nucleoside phosphoramidite; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the first surface and the second surface are bound to a nucleoside phosphoramidite. In some embodiments, the first surface and/or the second surface of the microfluidic channel is silanized, e.g., by a silane having a terminal hydroxyl group. In some embodiment, the microfluidic device comprises a plurality of microfluidic channels.

[0062] In some embodiments, there is provided a microfluidic device comprising: (a) a first plate, the first plate comprising of an organic polymer surface molded to contain one or more microfluidic channels capable of binding a nucleoside phosphoramidite; (b) a second plate, the second plate comprising of an organic polymer surface or glass substrate with or without microfluidic channels; and wherein the first plate is bonded to the second plate with covalent bonds or entangled polymers.

[0063] In some embodiments, there is provided a microfluidic device comprising a

microfluidic channel having a volume of no more than about 100 pL (such as no more than about 50, 20, 10 or less pL), wherein the microfluidic device comprises a glass substrate, wherein the microfluidic channel comprises one or more interior surface(s) formed by the glass substrate, and wherein the one or more interior surface(s) are capable of binding a nucleoside phosphoramidite. In some embodiments, the microfluidic device comprises a plurality of microfluidic channels. In some embodiments, the microfluidic channel is etched into the glass substrate.

First and second plates

[0064] In some embodiments, the microfluidic device is a single plate comprising an organic polymer substrate comprising one or more microfluidic channels. The microfluidic channels can be made in the organic polymer substrate by molding or by 3D printing.

[0065] In some embodiments, the microfluidic device comprises two or more plates. In some embodiments, the microfluidic device comprises two plates bonded to each other. As used herein,“bond,”“bonded” or“bonding” refers to establishment of permanent linkage between two substrates by forming chemical linkages ( e.g ., covalent bonds), using an adhesive substance, or applying mechanical forces ( e.g ., pressure). The two plates can be made of the same substrate or different substrates. In some embodiments, the first plate comprises a first organic polymer substrate, and the second plate comprises a different, second organic polymer substrate. In some embodiments, the first plate and the second plate comprise the same organic polymer substrate. In some embodiments, the first plate comprises an organic polymer substrate, and the second plate comprises a glass substrate.

[0066] The organic polymer substrate can be any suitable material or group of materials having a rigid or semi-rigid surface or surfaces, and the substrate is substantially inert to the various reagents for synthesizing or using the microarray. In some embodiments, the organic polymer substrate is optically transparent to allow fluorescence imaging (such as scan or microscopy). Suitable organic polymers for the first and/or second organic polymer substrate include, but are not limited to, polydimethylsiloxane (PDMS), cyclic olefin copolymer (COC) and cyclo-olefin polymer (COP). In some embodiments, the first plate comprises PDMS, and the second plate comprises a glass substrate. In some embodiments, the first plate comprises PDMS and the second plate comprises PDMS. Organic polymer substrates, such as PDMS, allow manufacture of microfluidic devices with microfluidic channels using rapid and inexpensive prototyping methods. However, certain organic polymer substrates may be prone to swelling in contact with organic fluids, and may auto-fluoresce. The methods of preparation described herein provide suitable conditions to allow oligonucleotide synthesis on the microfluidic device and subsequent biochemical assays using the microfluidic device.

[0067] The first plate and the second plate may be bonded to each other via covalent bonds or via entangled polymers. The bonding between the first plate and the second plate may be reversible or irreversible. The first plate or the second plate may be removed from the microfluidic microarray device prior to signal detection.

[0068] The first and second plates may have any suitable shapes and dimensions. In some embodiments, the first plate has the same dimensions as the second plate. In some embodiments, the first plate has different dimensions from the second plate. For example, the length and width of the plates can be at least about any one of 1 mm, 5 mm, lcm, 5cm, lOcm, 20cm, 30cm, 40cm, or 50cm. The thickness of each plate can be no more than about any one of 10, 5, 4, 3, 2, 1 cm or less. Microfluidic channels

[0069] The microfluidic devices describe herein may comprise a single microfluidic channel or a plurality of microfluidic channels, such as at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more microfluidic channels. In some embodiments, the microfluidic device comprises any one of about 1 to about 100, about 1 to about 10, about 10 to about 20, about 5 to about 50, about 20 to about 50 or about 50 to about 100 microfluidic channels. In some embodiments, each microfluidic channel comprises a plurality of

oligonucleotide probes, is capable or binding to a nucleoside phosphoramidite, or is bound to a nucleoside phosphoramidite. In some embodiments, one or more microfluidic channels do not comprise any oligonucleotide probes.

[0070] In some embodiments, the microfluidic channel is molded, heat embossed, etched, or 3D printed in the first organic polymer substrate. In some embodiments, the microfluidic channel is molded, heat embossed, etched, or 3D printed in the second organic polymer substrate or the glass substrate. In some embodiments, a portion (e.g., half) of the microfluidic channel is molded, heat embossed, etched, or 3D printed in the first organic polymer substrate, and the remaining portion (e.g., half) of the microfluidic channel is molded, heat embossed, etched, or 3D printed in the second organic polymer substrate or the glass substrate, and the full microfluidic channel is formed when the first plate comprising the first organic polymer substrate and the second plate comprising the glass substrate or the second organic substrate are bonded to each other.

[0071] In some embodiments, the microfluidic channel comprises a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate. As used herein, the“first surface” and the“second surface” refer to interior surfaces of the microfluidic channel. In some embodiments, the first surface and/or the second surface is flat or leveled to make surfaces substantially flat. In some embodiments, the first surface and/or the second surface is uneven. In some embodiments, the first surface and/or the second surface is curved. In some embodiments, the first surface and the second surface are parallel to each other. In some embodiments, the first surface and the second surface are opposite to each other. In some embodiments, both the first surface and the second surface are attached to oligonucleotide probes. In some embodiments, only the first surface is attached to oligonucleotide probes, is capable of binding, or is bound to to a nucleoside phosphoramidite. In some embodiments, only the second surface is attached to oligonucleotide probes, is capable of binding, or is bound to a nucleoside phosphoramidite.

[0072] In some embodiments, the first surface and/or the second surface is functionalized for binding to a nucleoside phosphoramidite or oligonucleotide probes. In some embodiments, a nucleoside phosphoramidite or a plurality of oligonucleotide probes is covalently linked to the first surface and/or the second surface. Methods of covalently linking a nucleoside

phosphoramidite or an oligonucleotide probe to a functionalized solid substrate are known in the art. For example, an organic polymer substrate or a glass substrate may be first derivatized with a silane reagent containing a functional group, such as sulfhydryl, amine, hydroxyl, or carboxylic acid group, which can be crosslmked to the 5’ terminal nucleotide or the 3’ terminal nucleotide of the oligonucleotide probe via a corresponding reactive functional linker. For example, a sulfhydryl reactive linker may contain a maleimide group. An amine reactive linker may contain a succinimidyl ester (NHS) or isothiocyanate (ITC) group. In some embodiments, the first/second surface and the oligonucleotide probe or nucleoside phosphoramidite are covalently linked to each other via a bifunctional linker. See, for example, US5,412,087 and Sheng H. and Ye BC. Appl. Biochem. Biotech. 2009 152(1): 54-56. In some embodiments, the oligonucleotide probe is attached to the solid substrate via noncovalent interactions between two binding moieties.

[0073] In some embodiments, the first surface and/or the second surface comprises a silane. In some embodiments, the silane comprises a terminal group that can be covalently linked to a nucleoside phosphoramidite. Silanes having a terminal hydroxyl group or a terminal amine group may be used. In some embodiments, the first surface and/or the second surface comprises one or more silanes selected from the group consisting of 3-[bis(2-hydroxyethyl)amino]propyl- triethoxysilane (“HEPTES,” CAS No. 7538-44-5), N-(hydroxyethyl)-N,N- bis(trimethoxysilylpropyl)amine (“Silane B,” CAS No. 264128-94-1), N,N'-bis(2-hydroxyethyl)- N,N'-bis(trimethoxysilylpropyl)ethylenediamine (“Silane A,” CAS NO. 214362-07-9), (3- aminopropyl)treiethoxysilane (“APTES”), 3-aminopropyIdimethyiethoxysilane (“APDES”), N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-(4- semicarbazidyl)propyltriethoxy silane, triethoxy silylundecanal, 3- aminopropyldimethylethoxysilane, N-(6-aminohexyl)aminopropyltrimethoxysilane, N-(3- triethoxysilylpropyl)-4-hydroxybutyramide, hexadecafluorododec- 11 -en- 1 -yltrimethoxysilane, 4-amino-3,3-dimethylbutylmethyldimethoxysilane, 3-aminopropylmethyldiethoxysilane, 1- amino-2-(dimethylethoxysilyl)propane, 3-aminopropyldiisopropylethoxysilane, N-(6- aminohexyl)aminomethyltriethoxysilane, N-(2-aminoethyl)- 1 l-aminoundecyltrimethoxysilane, N-3-[(amino(polypropylenoxy)]aminopropyltrimethoxysilane, N-(2-aminoethyl)-3- aminopropyltrimethoxysilane, l,2-bis(trimethoxysilyl)decane, and l,8-bis(triethoxysilyl)octane. In some embodiments, the first surface and/or the second surface comprises HEPTES. In some embodiments, the first surface and/or the second surface comprises HEPTES and a second silane. In some embodiments, the molar ratio between the HEPTES and the second silane is at least about any one of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1 or higher. In some embodiments, the first surface and the second surface comprises a mixture of HEPTES and Silane A, e.g., at a ratio of about 5:1 or 10:1. In some embodiments, the first surface and the second surface comprises a mixture of HEPTES and Silane B, e.g., at a ratio of about 5:1 or 10: 1.

[0074] The microfluidic channel(s) may have any suitable shape and dimensions. In some embodiments, the microfluidic channel has a rectangular, square, elliptical, or circular cross- section. In some embodiments, the microfluidic channel is straight. In some embodiments, the microfluidic channel has one or more bents. In some embodiments, the microfluidic channel is zig-zagged. In some embodiments, the microfluidic channel is a rectangular prism or a cubic prism.

[0075] In some embodiments, the microfluidic channel is at least about any one of 0.1, 0.2,

0.5, 1, 2, 5, 10, 20, 50, 100, 200, 300 or more mm long. In some embodiments, the microfluidic channel is any one of about 0.5 mm to about 10 mm, about 1 mm to about 5 mm, about 5 mm to about 50 mm, about 50 mm to about 100 mm, about 100 mm to about 300 mm, or about 0.1 mm to about 300mm long. In some embodiments, the microfluidic channel is at least about any one of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 300 or more mm wide. In some embodiments, the microfluidic channel is any one of about 0.1 mm to about 5 mm, about 0.2 mm to about 2 mm, about 2 mm to about 5 mm, about 5 mm to about 50 mm, about 50 mm to about 100 mm, about 100 mm to about 300 mm, or about 0.1 mm to about 300 mm wide. In some embodiments, the microfluidic channel is at least about any one of 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 800 or more mih deep. In some embodiments, the microfluidic channel is any one of about 5 mih to about 100 mih, about 10 mih to about 100 mih, about 50 mih to about 200 mih, about 20 mih to about 500 mih, about 400 mih to about 800 mih, or about 1 mih to about 800 mih deep. As used herein, for a rectangular prism microfluidic channel in a microfluidic device having two plates,“length” of the microfluidic channel refers to the longest dimension along the long axis of the microfluidic channel parallel to the first plate and the second plate;“depth” refers to the dimension perpendicular to the first plate and the second plate; and“width” refers to the dimension perpendicular to the depth. In some embodiments, the volume of the microfluidic channel is no more than about any one of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 20, 15, 10, 7.5, 5, 1, 0.5, 0.2, 0.1 or less mΐ. In some embodiments, the volume of the

microfluidic channel is about any one of 100 nl to about 1 ml, about 100 nl to about 1 mΐ, about 1 mΐ to about 10 mΐ, about 1 mΐ to about 20 mΐ, about 5 mΐ to about 20 mΐ, about 10 mΐ to about 50 mΐ, about 50 mΐ to about 100 mΐ, about 10 mΐ to about 100 mΐ, about 100 mΐ to about 500 mΐ, or about 500 mΐ to about 1 ml. In some embodiments, the volume of the microfluidic channel is less than about 20 mΐ. In some embodiments, the volume of the microfluidic channel is less than about 10 mΐ·

[0076] In some embodiments, wherein the microfluidic device has a plurality of microfluidic channels, the plurality of microfluidic channels can be arranged in any suitable pattern with respect to each other. In some embodiments, the plurality of microfluidic channels are parallel to each other. In some embodiments, the distance between adjacent microfluidic channels is no more than 5x, 2x, lx, ½, 1/5, 1/10, 1/20, 1/100 or a smaller fraction of the width of the microfluidic channels. In some embodiments, the distance between adjacent microfluidic channels is no more than about any one of 10 mm, 5 mm, 1 mm, 100 mhi, 50 mhi, 20 mhi, 10 mhi or less. In some embodiments, the distance between adjacent microfluidic channels is any one of about 10 mhi to about 1 mm, about 10 mhi to about 100 mhi, about 100 mhi to about 500 mhi, or about 500 mhi to about 1 mm.

[0077] The microfluidic channels may or may not be connected to each other. In some embodiments, each microfluidic channel has an inlet and an outlet, which can be independently controlled. In some embodiments, fluid flow through the microfluidic channel can be controlled using a syringe, a pipette, or a syringe pump. In some embodiments, the microfluidic device further comprises ports and fluidic connections for filling and emptying the microfluidic channels. In some embodiments, the ports and/or fluidic connections are pre-formed in the first and/or second organic polymer substrate. Oligonucleotide probe

[0078] The microfluidic microarray devices of the present application comprise arrays or patterns of oligonucleotide probes on one or more surfaces of the microfluidic channel. In some embodiments, wherein first surface is in a PDMS substrate and the second surface is in a glass substrate, each of the first surface and the second surface comprises a plurality of

oligonucleotide probes.

[0079] As used herein, an oligonucleotide probe refers to a collection of oligonucleotide molecules having the same sequence attached to an isolated test area, such as a spot or another defined pattern, attached to a surface of the microfluidic channel. In some embodiments, the oligonucleotide molecules in an oligonucleotide probe are arranged in a pattern that provides a pictorial readout of the name of the oligonucleotide probe upon hybridization to a target nucleic acid. In some embodiments, each test area comprises at least about any of 1, 2, 5, 10, 20, 50,

100, 200, 500, 1000 or more molecules of the oligonucleotide probe.

[0080] The microfluidic microarray device may comprise any suitable number of

oligonucleotide probes. Oligonucleotide probes at different positions may have the same sequence or different sequences. In some embodiments, the microfluidic microarray device comprises more than one (such as at least about any of 2, 5, 10, 20, 50, 100 or more) distinct test areas that comprise the same oligonucleotide probe. Test areas with the same oligonucleotide probe sequence on the microfluidic microarray device provide experimental replicate data, which may be averaged or analyzed statistically to enhance signal to noise ratio and improve accuracy. In some embodiments, the microfluidic microarray device comprises a plurality of oligonucleotide probes having different sequences. In some embodiments, the microfluidic microarray device comprises at least about any of 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 ⁴ , 2xl0 ⁴ , 5xl0 ⁴ , 10 ⁵ , 2xl0 ⁵ , 5xl0 ⁵ , 10 ⁶ , 2xl0 ⁶ , 5xl0 ⁶ , or 10 ⁷ oligonucleotide probes. In some embodiments, the microfluidic microarray device comprises any one of about 2-10, 10- 100, 100-1000, 1000- 10 ⁴ , 10 ⁴ -10 ⁵ , 10 ⁵ -10 ⁶ , 10 ⁶ -10 ⁷ , 2-10 ³ , 10 ³ -10 ⁵ , or 10 ⁵ -10 ⁶ oligonucleotide probes.

[0081] In some embodiments, the microfluidic microarray device is a high-density array. In some embodiments, the density of the oligonucleotide probes on the first surface or the second surface is at least about any one of 10 , 10 , 10 , 10 , 10 , 10 features/cm or higher.“Features” as used herein refer to regions comprising oligonucleotide probes with each region having identical oligonucleotide probe sequences. In some embodiments, the density of the oligonucleotide probes on the first or the second surface is at least about any one of 10 ¹ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ molecules/mm ² or higher.

[0082] Different microfluidic channels may contain the same set of oligonucleotide probes or different sets of oligonucleotide probes. Different microfluidic channels may contain

oligonucleotide probes arranged in the same pattern or different patterns. In some embodiments, wherein the microfluidic device comprises a plurality of microfluidic channels, each

microfluidic channel comprises the same set of oligonucleotide probes attached to the same positions. In such case, the plurality of microfluidic channels can be used for measurements in replicates, and/or for simultaneous measurements using different biological samples.

[0083] Each oligonucleotide probe may be attached to the first surface or the second surface via its 5’ terminus or its 3’ terminus. In some embodiments, the oligonucleotide probes are synthesized in situ on the surface(s) of the microfluidic channel. In some embodiments, the oligonucleotide probes are chemically synthesized and then attached to the surface(s) of the microfluidic channel.

[0084] In some embodiments, each oligonucleotide probe is attached at a pre-determined position ( i.e ., pre-determined test area) on the first surface or the second surface. In some embodiments, the oligonucleotide probes are attached to the same pre-determined positions on the first surface and the second surface. The positional information of each oligonucleotide probe on the microarray allows the user to retrieve the sequence information of each

oligonucleotide probe. In some embodiments, the microfluidic channel comprises reference oligonucleotide probes in certain test areas, which can serve as experimental controls, and to allow calibration of the background signal.

[0085] The oligonucleotide probes are single-stranded nucleic acids having a 5’ terminus and a 3’ terminus, in which a first terminus is attached to the first surface or the second surface, and the second terminus is free. The second terminus can be modified or manipulated in a

biochemical assay subsequent to hybridization of a target nucleic acid to the oligonucleotide probe.

[0086] The oligonucleotide probe may have any suitable length based on factors, including, but not limited to desired binding specificity, melting temperature, secondary structures, and complexity of the target nucleic acid. For example, for a target nucleic acid with relatively high complexity, i.e., a relatively large total length of unique sequence, the oligonucleotide probe is designed to contain a relatively longer sequence to avoid nonspecific binding. The oligonucleotide probe is also designed to have a suitable length to allow hybridization to the target nucleic acid under suitable experimental conditions (i.e., in a suitable temperature range and at suitable ionic strength). Longer oligonucleotide probes may be chosen to enhance the specificity of hybridization, but too long of a sequence may lead to undesirable consequences, such as binding to partial complements, formation of internal secondary structures, or difficulty in dissociating the target nucleic acid from the oligonucleotide probe. In some embodiments, the oligonucleotide probe is at least about any one of 4, 5, 6, 7, 8, 9, 1(3, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides long. In some embodiments, the oligonucleotide probe is about any one of 5- 15, 10-30, 20-30, 10-20, 25-50, 50-75, 75-100, 20-50, 50-100, or 10-75 nucleotides long.

[0087] In some embodiments, the sequence of the oligonucleotide probe is designed to specifically hybridize to an allele of interest, or a diagnostic portion thereof. In some

embodiments, the oligonucleotide probe comprises a sequence that is identical to the coding strand sequence of the allele or diagnostic portion thereof, or a sequence that is complementary to the coding strand sequence (i.e., identical to the noncoding strand sequence) of the allele or diagnostic portion thereof. An oligonucleotide probe comprising the identical sequence as the allele hybridizes to the noncoding strand of the allele, and an oligonucleotide probe comprising the complementary sequence of the allele hybridizes to the coding strand of the allele.

[0088] Exemplary diagnostic portions include, for example, nucleic acid sequences adjacent to or near, including, for example, immediately upstream (i.e., 5’ to) or immediately downstream (i.e., 3’ to) of, a typable locus in the allele.“Typable locus” refers to a location of genetic variation in an allele of interest, including, for example, single nucleotide polymorphisms (SNPs), mutations, variable number of tandem repeats (VNTRs) and single tandem repeats (STRs), other polymorphisms, insertions, deletions, splice variants or any other known genetic markers. Design of the sequence of oligonucleotide probes is accomplished using standard methods in the art. For example, sequences that have self-complementarity, such that the resulting oligonucleotides would either fold upon themselves, or hybridize to each other at the expense of binding to the target nucleic acid, are generally avoided.

[0089] The sequence of the oligonucleotide probe can be designed based on the known sequence information of SNPs and other genetic variations in public databases. In some embodiments, the oligonucleotide probe comprises a typable locus at the free terminus. For example, in some embodiments, the terminal nucleotide at the free terminus of the oligonucleotide probe corresponds to a SNP. In some embodiments, the oligonucleotide probe comprises a perfectly matching sequence immediately upstream of a typable locus (such as SNP). In some embodiments, the oligonucleotide probe comprises a perfectly matching sequence immediately downstream of a typable locus (such as SNP). The immediately upstream sequence or the immediately downstream sequence is chosen over the other to provide an oligonucleotide probe to enhance its hybridization properties, such as desirable specificity, suitable G/C content, and/or to reduce internal secondary structure. The directionality of the oligonucleotide probe sequence (/.« _? ., whether to be the same or complementary sequence as the coding sequence of the allele) may depend on whether the upstream or downstream sequence is chosen, and on which terminus (S’ or 3’) the oligonucleotide probe is attached to the solid substrate.

[0090] The oligonucleotide probe may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), or modified nucleotides thereof (e.g., nucleic acids containing modified bases, modified phosphate linkage, modified sugar moieties, labels, binding moieties, spacers, linkers, etc.). The oligonucleotide probe may further comprise additional chemical moieties of non-nucleotide nature, including, for example, linkers, fluorophores, quenchers, and minor groove binders. In some preferred embodiments, the oligonucleotide probe is a DNA probe, such an

oligonucleotide probe comprising only DNA nucleotides, or an oligonucleotide probe

comprising substantially DNA nucleotides.

[0091] In some embodiments, the oligonucleotide probe comprises one or more nucleotides containing a non-natural sugar moiety in the backbone. Exemplary sugar modifications include but are not limited to 2' modifications such as addition of halogen, alkyl, substituted alkyl, allcaryi, aralkyl, Q-allcaryi or O-aralkyl, SH, SC¾, OCN, Cl, Br, CN, CF _3, OCF ₃ , SOCH ₃ , S0 ₂ , CH3, ONO2, NO2, N ₃ , NH ₂ , heterocycloallcyl, heterocycloallcaryl, aminoallcylamino, polyallcylamino, substituted silyl, and the like. Similar modifications can also be made at other positions on the sugar, such as the 3' position of the sugar moiety.

[0092] The oligonucleotide probe may comprise native or non-native bases. For example, a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. Exemplary non-native bases that can be included in the oligonucleotide probe, whether having a native backbone or analog structure, include, without limitation, inosine, xathanine, hypoxathanine, isocytosine, isoguanine, 5-meihylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6- methyl guanine, 2-propyl guanine 2-propyl adenine, 2-thioLiracil, 2-ihiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8- thiol adenine or guanine, 8-ihioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine or the like. A particular embodiment can utilize isocytosine and isoguanine in a nucleic acid in order to reduce non-specific hybridization, as generally described in U.S. Pat. No. 5,681,702.

100931 The oligonucleotide probe may further comprise non-nucleotide moieties. A“non nucleotide moiety” refers to any agent or molecule that can be linked to an oligonucleotide probe at any specific pre-selected iocation(s) therein. The non-nucleotide moiety may be linked to either the backbone or the base of any nucleotide(s) in the oligonucleotide probe, or to the 5’ or 3’ terminus of the oligonucleotide probe. Suitable non-nucleotide moieties include, but are not limited to, linkers, fluorescent moieties, fluorophores, quenchers, chelators (e.g., minor groove binders), labels, etc.

[§094] The oligonucleotide probe is attached to the first surface and/or the second surface via either its 5’ terminus or its 3’ terminus. In some embodiments, all oligonucleotide probes on a microarray are attached to the surface(s) via the same termini (i.e., all 5’ termini, or all 3’ termini). In some embodiments, oligonucleotide probes in one microfluidic channel are attached to the first surface and/or the second surface via the 5’ terminus, and oligonucleotide probes in a second microfluidic channel in the same microfluidic device are attached to the first surface and/or the second surface via the 3' terminus. In some embodiments, a portion (e.g., any one of at least 10%, 20%, 30%, 40% . 50%, 60%, 70%.=, 80%, 90%= or more) of the plurality of oligonucleotide probes in a microfluidic channel are attached to the first surface and/or the second surface via the 5’ terminus, and the remaining portion of the plurality of oligonucleotide probes in the microfluidic channel are attached to the first surface and/or the second surface via the 3’ terminus. In some embodiments, a portion (e.g., any one of at least 10%=, 20%, 30%=, 40%, 50%, 60%, 70%, 80%=, 90% or more) of the plurality of oligonucleotide probes in a microfluidic channel are attached to the first surface and/or the second surface via the 3’ terminus, and the remaining portion of the plurality of oligonucleotide probes in the microfluidic channel are attached to the first surface and/or the second surface via the 5’ terminus. The choice of attachment at the 5’ or the 3’ terminus may be determined based on the usage of the microfluidic microarray device, for example, the enzymatic assay downstream of hybridization which is required for analyzing the target nucleic acid. For example, an extension-based assay requires a free 3’ -end, and thus, a microfluidic microarray device useful for extension-based assays (e.g., extension-based SNP assay, or short-read sequencing assay by extension) comprises

oligonucleotide probes each attached to the first surface and/or the second surface via the 5’ end, leaving the 3’ end free for extension by a polymerase. However, a microfluidic microarray device for a ligation-based assay (e.g., ligation-based SNP assay, or short-read sequencing by ligation) may comprise oligonucleotide probes each attached to the first surface and/or the second surface either at the 5 end or at the 3’ end. In some embodiments, a first plurality of oligonucleotide probes attached to the first surface and/or the second surface via the 5’ ends and a second plurality of oligonucleotide probes (having the same sequences or different sequences as the first plurality) attached to the first surface and/or the second surface via the 3’ ends are required for an assay.

[0095] Some embodiments of the present application provides microfluidic SNP microarray devices, which comprise oligonucleotide probes specific for SNP alleles. A“single nucleotide polymorphism” or“SNP” is a locus present in a population which displays a variation in the identity of a single nucleotide between members of the population. A variety of assays have been developed to detect SNPs using a microarray, including hybridization-based assays, and enzyme-based assays, such as extension-based or ligation-based assays. See, for example,

Gunderson KL et al Nature Genetics , 2005, 37:S5. The microfluidic microarray devices described in the present application can be compatible with any of the SNP assay formats known in the art .

[0096] In some embodiments, the plurality of oligonucleotide probes comprises one or more oligonucleotide probe pairs, wherein each oligonucleotide probe pair comprises a first probe and a second probe each comprising a matching sequence immediately upstream or immediately downstream of a single-nucleotide polymorphism (SNP), and wherein the terminal nucleotide at the free terminus of the first probe matches a first allele of the SNP and the terminal nucleotide at the free terminus of the second probe matches a second allele of the SNP.

[0097] hi some embodiments, the plurality of oligonucleotide probes comprises one or more oligonucleotide probes comprising a matching sequence upstream or downstream of an SNP. Such SNP oligonucleotide probes can be used in extension assays using fluorescently labeled nucleotides. Methods of preparation

[0098] One aspect of the present application provides methods of preparing microfluidic microarray devices, comprising synthesizing a plurality of oligonucleotide probes at pre determined positions on the first surface and/or the second surface of the microfluidic channel(s) of any of the microfluidic synthesis devices described herein.

[0099] In some embodiments, there is provided a method of preparing a microfluidic microarray device, comprising synthesizing a plurality of oligonucleotide probes in a

microfluidic device, wherein the microfluidic device comprises: (a) a first plate comprising a first organic polymer substrate ( e.g ., PDMS); (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface of the microfluidic channel is capable of binding a nucleoside phosphoramidite, wherein the first plate and the second plate are bonded to each other to form the microfluidic channel, and wherein the plurality of oligonucleotide probes are synthesized at pre-determined positions on the first surface and/or the second surface of the microfluidic channel. In some embodiments, the method further comprises molding the microfluidic channel in the first organic polymer substrate and/or the second organic polymer substrate. In some embodiments, the method further comprises bonding the first plate to the second plate. In some embodiments, the method further comprises passing a silane through the microfluidic channel. In some embodiments, the method comprises light-directed synthesis of the plurality of oligonucleotide probes. An exemplary method of preparing a microfluidic microarray device is shown in FIG.l.

[0100] In some embodiments, there is provided a method of synthesizing oligonucleotide probes at pre-determined positions on one or more interior surface(s) in a microfluidic device at a reaction volume of no more than about 100 pL (e.g., no more than about 50, 20, 10 or less pL), comprising: (a) providing a microfluidic device comprising a microfluidic channel having the surface capable of binding a nucleoside phosphoramidite, wherein the microfluidic channel has a volume of no more than about 100 pL (e.g., no more than about 50, 20, 10 or less pL); and (b) synthesizing oligonucleotide probes at pre-determined positions in the microfluidic channel, e.g., using light-directed synthesis. In some embodiments, the microfluidic device comprises a glass substrate. In some embodiments, the microfluidic device comprises a first plate comprising a first substrate (e.g., a first organic polymer substrate such as PDMS, or a glass substrate) and a second plate ( e.g ., a second organic polymer substrate such as PDMS, or a glass substrate), and wherein the first plate and the second plate are bonded to each other to form the microchannel.

In some embodiments, the method further comprises molding the microfluidic channel in the first substrate and/or the second substrate. In some embodiments, the method further comprises bonding the first plate to the second plate. In some embodiments, the method further comprises etching the microfluidic channel into the glass substrate. In some embodiments, the method further comprises passing a silane through the microfluidic channel. In some embodiments, the microfluidic device comprises a plurality of microfluidic channels.

[0101] In some embodiments, there is provided a method of preparing a microfluidic microarray device, comprising: (a) providing a microfluidic device comprising a microfluidic channel having a volume of no more than about 100 pL (such as no more than about 50, 20, 10 or less pL), wherein the microfluidic device comprises a glass substrate and/or an organic polymer substrate (e.g., PDMS), wherein the microfluidic channel comprises one or more interior surface(s) formed by the glass substrate or the organic polymer substrate, and wherein the one or more interior surface(s) are capable of binding a nucleoside phosphoramidite; (b) synthesizing a plurality of oligonucleotide probes in a reaction volume of no more than about 100 pL (e.g., no more than about 50, 20, 10 or less pL) at pre-determined positions on the one or more interior surfaces of the microfluidic channel. In some embodiments, the microfluidic device comprises a glass substrate. In some embodiments, the microfluidic device does not comprise an organic polymer substrate. In some embodiments, the microfluidic device comprises a glass substrate and an organic polymer substrate. In some embodiments, the microfluidic device does not comprise an organic polymer substrate.

[0102] The oligonucleotide probes can be synthesized using any suitable oligonucleotide synthesis methods known in the art that are compatible with the organic polymer substrates and the glass substrate. In some embodiments, the oligonucleotide probes are synthesized in the 3’ to 5’ direction. In some embodiments, the oligonucleotide probes are synthesized in the 5 to 3’ direction. For example, phosphoramidite chemistry can be used for the 3’ to 5’ synthesis or the 5’ to 3’ synthesis. FIG. 5 shows exemplary nucleoside phosphoramidites that can be used for light-directed synthesis of the oligonucleotide probes.

[0103] In some embodiments, light-directed oligonucleotide synthesis is used to synthesize the plurality of oligonucleotide probes. Light-directed combinatorial synthesis of oligonucleotide arrays on a solid substrate (such as glass surface) are known in the art using either mask-guided or maskless methods. See, for example, Fodor SP et al. Science. 1991 Feb 15; 25l(4995):767-73; and Nuwaysir EF et al. Genome Res. 2002 Nov; 12(11): 1749-55. Briefly, the solid substrate is derivatized with functional groups blocked by a photo-labile protection group to allow

photolysis through a photolithographic mask or using micro-mirrors to selectively expose the functional groups which are then ready to react with incoming 5' or 3’ photo-protected

nucleoside phosphoramidites. The phosphoramidites react only with those sites which are illuminated (and thus exposed by removal of the photo-labile blocking group). Thus, the phosphoramidites only add to those areas selectively exposed from the preceding step. These steps are repeated until the desired array of sequences have been synthesized on the solid surface. Combinatorial synthesis of different oligonucleotide analogues at different locations on the array is determined by the pattern of illumination during synthesis and the order of addition of coupling reagents.

[0104] FIG. 2 shows an exemplary synthesis cycle for 3’ to 5’ light-directed synthesis of oligonucleotide probes using phosphor amidite chemistry. Each cycle of synthesis comprises the steps of: light deprotection, oxidizing, coupling, and capping. An exemplary photo-protecting group that may be used is nitrophenylpropyloxycarbonyl (“NPPOC”).

[0105] Automated oligonucleotide synthesizer systems can be used for the synthesis. For example, commercial oligonucleotide synthesizers, such as DNA synthesizer EXPEDITE ^® 8909 can be used. A digital micromirror device can be coupled to the automated oligonucleotide synthesizer to allow maskless light-directed synthesis of oligonucleotide probes on the microfluidic synthesis device. For example, a digital micromirror device is used to project a series of pattern light beams to allow deprotection of the photo-labile protecting group at the growing end of an oligonucleotide chain at pre-determined positions on the surface(s) of the microfluidic channels. Programmable digital micromirror projectors are commercially available, including, for example, the Visitech LUXBEAM ^® lithography system.

[0106] Depending on the nature of the protecting group and the organic polymer substrates used in the microfluidic synthesis device, light of a suitable wavelength can be used for the light- directed synthesis. For example, UV light can be used for synthesis on a microfluidic synthesis device comprising PDMS. For microfluidic synthesis devices comprising COC or COP, however, blue light can be used for light-directed oligonucleotide synthesis. In some

embodiments, a triplet sensitizer can be used to shift blue light that pass through COC or COP substrate to UV light to allow synthesis of oligonucleotide probes in a microfluidic synthesis device comprising COC or COP. In some embodiments, the method uses two-photons of IR light for light-directed synthesis. Two-photon IR methods have certain advantages, such as non linearity and low noise. In some embodiments, the nucleoside phosphor amidite has a broad- wavelength protecting group.

[0107] In some embodiments, the method comprises synthesizing the plurality of

oligonucleotide probes comprising: (i) providing a microfluidic device according to any one of the microfluidic devices described herein having a first nucleoside phosphoramidite or a plurality of oligonucleotides comprising a first nucleoside phosphoramidite at the 5’ (or 3’) terminus, wherein the first nucleoside phosphoramidite or the plurality of oligonucleotides are attached to the first surface and/or the second surface of the microfluidic channel via the 3’ (or 5’) terminus, and wherein the first nucleoside phosphoramidite comprises a photo-labile protective group at the 5’ (or 3’) position; (ii) deprotecting the first nucleoside phosphoramidite using a patterned light beam to provide deprotected first nucleoside phosphoramidite at pre determined positions on the first surface and/or the second surface of the microfluidic channel; (iii) passing a second nucleoside phosphoramidite through the microfluidic channel to couple the second nucleoside phosphoramidite to the deprotected first nucleoside phosphoramidite, wherein the second nucleoside phosphoramidite comprises a photo-labile protective group at the 5’ (or 3)’ position; optionally (iv) passing a capping composition through the microfluidic channel; optionally (v) passing an oxidizing solution through the microfluidic channel; and (vi) repeating steps (ii)-(v) for a pre-determined number of times, wherein the patterned light beam is programmed at each step according to the sequences of the plurality of oligonucleotide probes; thereby providing the plurality of oligonucleotide probes at the pre-determined positions on the microfluidic device. In some embodiments, the method comprises the capping step (iv) in each nucleotide addition cycle of synthesizing the oligonucleotide probes. In some embodiments, the method comprises the capping step (iv) in a portion ( e.g ., at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more; such as every other cycle, every three cycles, or once halfway through the synthesis of the oligonucleotide probes and once at the end of the synthesis of the oligonucleotide probes) of the nucleotide addition cycles of synthesizing the oligonucleotide probes. In some embodiments, the method comprises passing a capping solution through the microfluidic channel after incorporating all nucleoside phosphoramidites into the oligonucleotide probes. In some embodiments, the method comprises the oxidization step (v) in each nucleotide addition cycle of synthesizing the oligonucleotide probes. In some embodiments, the method comprises the oxidization step (v) in a portion ( e.g ., at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more; such as every other cycle, every three cycles, or once halfway through the synthesis of the oligonucleotide probes and once at the end of the synthesis of the oligonucleotide probes) of the nucleotide addition cycle of synthesizing the oligonucleotide probes. In some embodiments, the method comprises passing an oxidizing solution through the microfluidic channel after incorporating all nucleoside phosphoramidites into the oligonucleotide probes. In some embodiments, the capping step (iv) is carried out prior to the oxidization step (v). In some embodiments, the capping step (v) is carried out prior to the oxidization step (iv). In some embodiments, the method comprises a sulfurization step comprising passing a sulfurizing solution through the microfluidic channel instead of an oxidization step (v), wherein the plurality of oligonucleotide probes comprise one or more phosphorothioate linkages.

[0108] In some embodiments, the method comprises synthesizing a first plurality of oligonucleotide probes followed by synthesizing a second plurality of oligonucleotide probes, wherein the first plurality of oligonucleotide probes and the second plurality of oligonucleotide probes are attached to the same pre-determined positions on the first surface and/or the second surface of the microfluidic device. In some embodiments, the first plurality of oligonucleotide probes have the same sequences as the second plurality of oligonucleotide probes at the same pre-determined position(s). In some embodiments, the first plurality of oligonucleotide probes have different sequences as the second plurality of oligonucleotide probes at the same pre determined position(s). In some embodiments, the first plurality of oligonucleotide probes are synthesized from the 3’ to 5’ direction and the second plurality of oligonucleotide probes are synthesized from the 5’ to 3’ direction. In some embodiments, the first plurality of

oligonucleotide probes are synthesized from the 5’ to 3’ direction and the second plurality of oligonucleotide probes are synthesized from the 3’ to 5’ direction.

[0109] In some embodiments, the plurality of oligonucleotide probes are synthesized on the surfaces formed by both the first organic polymer substrate and the glass substrate in the microfluidic channel of the microfluidic synthesis device. Unlike in situ oligonucleotide synthesis methods used for traditional microarrays, the methods described herein use chemical reagents that are compatible with the organic polymer substrate. In some embodiments, the oxidizing composition and the capping composition comprise a solvent that does not swell the first organic polymer substrate. In some embodiments, the oxidizing composition and the capping composition do not comprise dichlorome thane. In some embodiments, the oxidizing composition and the capping composition comprise acetonitrile. In some embodiments, the oxidizing composition and the capping composition are each passed through the microfluidic channel for a short amount of time, such as no more than about any one of 10 minutes, 5 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 15 seconds or less.

[0110] The plurality of oligonucleotide probes are synthesized in the microfluidic channels that have small reaction volumes. In some embodiments, the oligonucleotide probes are synthesized in a reaction volume of no more than about any one of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 20, 15, 10, 7.5, 5, 1, 0.5, 0.2, 0.1 or less mΐ. In some embodiments, the oligonucleotide probes are synthesized in reaction volumes of about any one of 100 nl to about 1 ml, about 100 nl to about 1 mΐ, about 1 mΐ to about 10 mΐ, about 1 mΐ to about 20 mΐ, about 5 mΐ to about 20 mΐ, about 10 mΐ to about 50 mΐ, about 50 mΐ to about 100 mΐ, about 10 mΐ to about 100 mΐ, about 100 mΐ to about 500 mΐ, or about 500 mΐ to about 1 ml. In some embodiments, the oligonucleotide probes are synthesized in a reaction volume of less than about 20 mΐ. In some embodiments, the oligonucleotide probes are synthesized in a reaction volume of less than about 10 mΐ.

[0111] The methods described herein may further comprise one or more steps for preparing the microfluidic synthesis device, including, but not limited to any one or more steps of: (i) making the microfluidic channel in the first organic polymer substrate and/or the second organic polymer substrate and/or the glass substrate; (ii) bonding the first plate with the second plate;

(iii) functionalizing (e.g., silanizing) the first organic polymer substrate and/or the second organic polymer substrate or the glass substrate; and (iv) washing and drying the microfluidic channel.

[0112] The microfluidic channel can be made in the microfluidic synthesis device using any known methods in the art, including molding, heat embossing, etching, and 3D printing.

[0113] Depending on the substrate materials of the first plate and the second plate, the two plates may be bonded to each other using any known methods in the art, e.g., by covalent bonding, or via entangled polymers. Methods of bonding PDMS to glass are well known in the art, and are less expensive than bonding etched glass to another glass substrate as in traditional microarrays. For example, a PDMS plate and a glass plate can be bonded to each other by first activating the two plates using oxygen plasma or UV-ozone, followed by incubation at an elevated temperature, such as a temperature between about 80°C to about l20°C for a suitable period of time (such as about 1 hour).

[0114] The organic polymer surface(s) and/or the glass surface of the microfluidic channel may be functionalized before bonding the two plates or after bonding the two plates. In some embodiments, both the first surface and the second surface of the microfluidic channel are silanized, rending both surfaces capable of binding to a nucleoside phosphoramidite. In some embodiments, the method comprises passing one or more silanes ( e.g ., HEPTES, Silane A or Silane B) through the microfluidic channel to functionalize the first surface and the second surface.

[0115] The microfluidic channel can be washed by injecting water, alcohol (such as ethanol), or buffer, or soaking the microfluidic synthesis device in water, alcohol or buffer. The microfluidic channel can be dried by injecting air, heating, or placing in a vacuum.

[0116] Further provided are microfluidic microarray devices prepared by any one of the methods of preparation described herein.

Methods of use

[0117] The microfluidic synthesis devices described herein can be used for synthesizing microfluidic microarray devices. The microfluidic microarray devices described herein can be used for a variety of biochemical assays. In some embodiments, the microfluidic microarray device is used in a hybridization-based assay. In some embodiments, the microfluidic microarray device is used for SNP detection. Suitable biochemical assays include, but are not limited to, quantitative hybridization, quantitative annealing, hybridization-ligation, nuclease hybridization assay, allele specific primer extension, and short-read sequencing.

[0118] In some embodiments, there is provided a method of analyzing a sample comprising a target analyte, comprising: contacting the sample with the plurality of oligonucleotide probes in any one of the microfluidic microarray devices described herein; and detecting binding of the target analyte to one or more oligonucleotide probes. In some embodiments, the target analyte is a target nucleic acid. In some embodiments, the target analyte is a sequence- specific protein, such as a sequence-specific enzyme, e.g., Cas9.

[0119] In some embodiments, there is provided a method of analyzing a sample comprising target nucleic acids, comprising: (a) contacting the sample with a plurality of oligonucleotide probes in a microfluidic device, wherein the microfluidic device comprises: (i) a first plate comprising a first organic polymer substrate; (ii) a second plate comprising a second organic polymer substrate or a glass substrate; and (iii) one or more microfluidic channels comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface of the one or more microfluidic channels comprises the plurality of

oligonucleotide probes; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel; (b) hybridizing the target nucleic acids to the plurality of oligonucleotide probes in the microfluidic channel to provide probe-target hybrids; and (c) detecting the probe-target hybrids.

[0120] In some embodiments, the first plate (or the second plate) is removed prior to depositing a sample onto the second surface (or the first surface), allowing contact of the sample to oligonucleotide probes on the second surface (or the first surface) only. However, disassembly of the microfluidic microarray device is not required for contacting the sample with the oligonucleotide probes.

[0121] The sample can be brought into contact to the oligonucleotide probes by injecting the sample into the microfluidic channel. In some embodiments, different samples are injected into different microfluidic channels on a microfluidic microarray device. Simultaneous analysis of multiple replicates of the same sample or different biological samples can be analyzed on the same microfluidic microarray device at the same time.

[0122] Signal from binding of the target analyte, such as hybridization of the target nucleic acid to an oligonucleotide probe, can be detected from either the first surface or the second surface. In some embodiments, the first organic polymer substrate is removed prior to detecting signals from the second surface (e.g., the glass substrate) only. In some embodiments, the second organic polymer substrate or the glass substrate is removed prior to detecting signals from the first surface ( i.e ., the first organic polymer substrate) only. However, disassembly of the microfluidic microarray device is not necessary for detection of signals. In some embodiments, signals are detected from both the first surface and the second surface in an intact microfluidic microarray device.

[0123] Signals of binding or hybridization to the oligonucleotide probes can be detected using any known methods in the art. For example, target molecules having fluorescent labels that are bound to the oligonucleotide probes can be detected by scanning the microfluidic microarray device, the first plate, or the second plate in any suitable slide scanner, or by imaging using a suitable fluorescence microscope or imager. In some embodiments, wherein signals are detected from both the first surface and the second surface in an intact microfluidic microarray device, the focal plane of the fluorescence scanner or imager is set at the first surface, the second surface, or a plane between the first surface and the second surface ( e.g ., when the first surface and the second surface are opposite to each other). In some embodiments, fluorescently labeled nucleotides are used in a primer extension assay to detect sequence-specific binding of the target nucleic acid to the oligonucleotide probes. In some embodiments, biotin-labeled nucleotides in a primer extension assay to detect sequence-specific binding of the target nucleic acid to the oligonucleotide probes, and the primer extension product is detected using fluorescently labeled avidin, streptavidin, or EXTRA VID IN ^® .

[0124] In some embodiments, signal is enhanced by washing or soaking the microfluidic channel using water or a binding buffer. In some embodiments, the microfluidic channel is washed with at least about any one of lx, 2x, 5x, lOx, 20x, 50x or more water or buffer prior to signal detection. In some embodiments, the microfluidic microarray device is soaked or dialyzed in water or a binding buffer (such as 300 mM sodium chloride, 30 mM sodium citrate, pH 7.0) for at least about any one of 2, 4, 6, 10, 12 hour or longer with stirring prior to the signal detection (e.g. imaging).

[0125] In some embodiments, the microfluidic microarray device is used for a nuclease hybridization assay. In some embodiments, step (b) is followed by a step comprising injecting a nuclease into the microfluidic channel to degrade single-stranded nucleic acids.

[0126] In some embodiments, the microfluidic microarray device is used for a hybridization ligation assay. In some embodiments, step (b) is followed by a step comprising injecting a ligase and a plurality of free adapter oligonucleotides into the microfluidic channel to provide ligated probe-target hybrids, and detecting the ligated probe-target hybrids.

[0127] In some embodiments, the microfluidic microarray device is used for an allele- specific primer extension assay. In some embodiments, step (b) is followed by a step comprises injecting a polymerase and nucleotides into the microfluidic channel to provide extended probe-target hybrids, and detecting the extended probe-target hybrids.

[0128] In some embodiments, the microfluidic microarray device is used for a short-read sequencing assay. “Short-read sequencing” refers to sequencing a region of the target nucleic acid that has no more than about 50 nucleobases. In some embodiments, the method comprises sequencing one or more nucleobases of a target nucleic acid bound to an oligonucleotide probe. In some embodiments, the method comprises sequencing about any one of 2, 5, 10, 15, 20, 30, 40, or 50 nucleobases. In some embodiments, the method comprises: (a) injecting a target nucleic acid into the microfluidic channel; (b) hybridizing the target nucleic acid to an oligonucleotide probes; (c) injecting a polymerase, nucleotides having a fluorescent label, and a terminator into the microfluidic channel, wherein each type of nucleotide has a differently colored fluorescent label, and wherein the oligonucleotide probes hybridized to the target nucleic acid is extended by one nucleotide; (d) detecting the fluorescent label incorporated to the oligonucleotide probe, thereby determining the identity of the incorporated nucleotide; and (e) removing the terminator ( e.g ., by injecting a de -protecting agent) and repeating steps (c)-(e) for a number of times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or more times), such that only one base is added a time, thereby determining the sequence of a short region of the target nucleic acid downstream to the hybridization site of the oligonucleotide probe on the target nucleic acid.

[0129] In some embodiments, the microfluidic microarray device is used for detection of single-nucleotide polymorphisms (SNP) using enzyme-based assays, such as extension-based assays or ligation-based assays. The primer extension assays, such as Single-base extension (SBE) and allele- specific primer extension (ASPE), and ligation assays used to detect SNPs in the target nucleic acids have been described in the art. See, for example, Gunderson KL el al. Nature Genetics, 2005, 37:85; US20080131894A1; US20020177141A1; and

US 20030016897 Al, which are incorporated herein by reference. Other SNP detection methods may alternatively be used with the microfluidic microarray device described herein, including, but not limited to, rolling circle-based detection methods, allele- specific oligonucleotide (ASO) hybridization and others.

[0130] The microfluidic microarray devices described in the present application may be used In a variety of applications, including gene expression analysis and genotyping in clinical diagnosis, agricultural, environmental, and forensic settings. In some embodiments, the microfluidic microarray device is used in diagnosis of a sexually transmitted disease, such as HIV, HCV, or gonorrhea.

[0131] The methods described herein may be used to analyze any sample of target nucleic acids from any source. The term“target nucleic acid” refers to a nucleic acid molecule which contains a sequence which has at least partial complementarity with at least an oligonucleotide probe. The target nucleic acid may comprise single- or double-stranded DNA or RNA. The term “sample” is used in its broadest sense to include any specimen or culture (e.g., microbiological cultures), as well as biological and environmental samples. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may he obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as cows, horses, fish, rodents, etc. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.

[§132] In some embodiments, the sample of target nucleic acids comprise genomic DNA or genomic DNA fragments. Genomic DNA can be isolated from one or more cells, bodily fluids or tissues. Known methods can be used to obtain a bodily fluid such as blood, sweat, tears, lymph urine, saliva, semen, cerebrospinal fluid, feces or amniotic fluid. Similarly known biopsy methods can be used to obtain cells or tissues such as buccal swab, mouthwash, surgical removal, biopsy aspiration or the like. Genomic DNA can also be obtained from one or more cell or tissue in primary culture, in a propagated cell line, a fixed archival sample, forensic sample or archeological sample, A genome fragment can be DNA, RNA, or an analog thereof. In some embodiments, the sample of target nucleic acids comprise cDNA or cDNA fragments. cDNAs may be prepared using any known methods in the art, including, for example, reverse

transcription from total RNA.

[0133] In some embodiments, the sample of target nucleic acids are further amplified to provide nucleic acid fragments prior to hybridization to the microfluidic microarray device. In some embodiments, the amplification is whole genome amplification. In some embodiments, the amplification is targeted amplification that enhances the presentation of certain alleles and loci of interest in the sample to he hybridized to the oligonucleotide probes. In some embodiments, the target nucleic acids are at least about any of 25, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more nucleotides long.

[§134] The sample of the target nucleic acids is hybridized to the oligonucleotide probes in the microfluidic microarray device. Depending on the application, complexity of the sample, and the multiplexity ( i.e ., number of different oligonucleotide probes) on the microarray, an appropriate stringency condition may be chosen for the hybridization step.“Stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. At“high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. At“weak” or“low” stringency, nucleic acids that are not completely complementary to one another will hybridize to one another. Because SNP alleles differ by only a single nucleotide, methods for detecting SNP alleles normally comprise hybridization of the sample to the microarray under high stringency conditions.“Hybridization” in vol ves the annealing of a complementary sequence to the target nucleic acid. The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon, and conditions for hybridization may be chosen and refined by a person skilled in the art.

[0135] The probe- target hybrids formed between the oligonucleotide probes and the target nucleic acids may be detected directly or indirectly. In some embodiments, the target nucleic acids are attached to a label that can be detected by any methods known in the art. In extension- based SNP detection methods, the oligonucleotide probe at the free terminus is extended by one or more nucleotides the terminal nucleotide in the oligonucleotide probe base pairs perfectly with the corresponding nucleotide in the target, indicating that a particular allele is present. Perfect complementarity between the rest of the oligonucleotide probe and the target nucleic acid enhances the rate of extension. Thus, labelled nucleotides, such as fluorescently labelled nucleotides or nucleotides attached to a hapten may be used to allow direct or indirect detection of an extended oligonucleotide probe, thereby allowing detection of the SNP allele in the target nucleic acid. In ligation-based SNP detection methods, the oligonucleotide probe on the microarray is ligated to a free adapter oligonucleotide when the target nucleic acid is perfectly complementary to the oligonucleotide probe and the free adapter oligonucleotide. Thus, the free adapter oligonucleotide may be labelled to allow detection of a ligated oligonucleotide probe on the microarray, thereby allowing detection of the SNP allele in the target nucleic acid.

[0136] In some embodiments, a fluorescent dye that specifically binds to double- stranded nucleic acids may be used to stain the modified probe-target hybrids. As extended probe-target hybrids have longer fragments of double-stranded nucleic acids than non-extended probe-target hybrids, signals from extended probe-target hybrids are stronger than unextended probe-target hybrids, thereby allowing detection of hybridized target nucleic acid or SNPs in the target nucleic acid. Dyes specific for single- stranded nucleic acids or double- stranded nucleic acids are known in the art, including, for example, SYBR ^® Gold, SYBR ^® Green, PICOGREEN ^® , OLIGREEN ^® and RIBOGREEN ^® . [0137] The term“label” as used herein refers to any atom or molecule which can be used to provide a detectable (and preferably quantifiable) signal, and which can be attached to a nucleic acid. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, oxidation potential, electrochemical properties and the like. In some embodiments, the label is a hapten.“Hapten” refers to a small molecule, such as drug, hormone, or synthetic compound. A hapten may be detected by staining with a labelled protein, such as an antibody, that specifically recognizes the hapten. Non-limiting examples of label moieties useful for detection in the present application include, without limitation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, b-galactosidase, or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin/biotin, avidin/biotin or an antigen/antibody complex including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue™, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthamide complexes such as those including Europium and

Terbium, Cy3, Cy5, molecular beacons and fluorescent derivatives thereof, as well as others known in the art as described, for example, in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the 6th Edition of the Molecular Probes Handbook by Richard P. Hoagland; a luminescent material such as luminol; light scattering or plasmon resonant materials such as gold or silver particles or quantum dots; or radioactive material include ¹⁴ C, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, Tc", ³⁵ S or ³ H. For high-density microarrays, fluorescent labels can be conveniently used for detection, as fluorescent signals from a large number of test areas on the microarray may be detected simultaneously using a fluorescence microscope or a fluorescence scanner.

Kits and articles of manufacture

[0138] The present application further provides kits and articles of manufacture comprising any one of the microfluidic microarray devices described herein, useful a variety of biochemical applications. Also provided are kits and articles of manufacture comprising any one of the microfluidic synthesis devices described herein, useful for in situ microarray synthesis.

[0139] In some embodiments, there is provided a kit comprising a microfluidic microarray device comprising: (a) a first plate comprising a first organic polymer substrate ( e.g ., PDMS); (b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface comprises a plurality of oligonucleotide probes; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the microfluidic microarray device comprises a plurality of microfluidic channels. In some embodiments, the kit comprises one or more parts or accessories that connect the microfluidic device to a fluidic system. In some embodiments, the kit is used for SNP detection using a primer extension assay or a ligation assay. In some embodiments, the kit is used for quantitative hybridization, quantitative annealing, hybridization-ligation, nuclease hybridization assay, allele specific primer extension, and short-read sequencing.

[0140] In some embodiments, there is provided a kit comprising a microfluidic synthesis device comprising: (a) a first plate comprising a first organic polymer substrate ( e.g ., PDMS);

(b) a second plate comprising a second organic polymer substrate or a glass substrate; and (c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface of the microfluidic channel is capable of binding a nucleoside phosphoramidite; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel. In some embodiments, the first surface and/or the second surface is bound to a nucleoside phosphoramidite. In some embodiment, the microfluidic synthesis device comprises a plurality of microfluidic channels. In some embodiments, the kit is used for synthesizing microarrays in the microfluidic channel. In some embodiments, the kit further comprises nucleoside phosphoramidites, and reagents for synthesizing oligonucleotides (e.g., capping agent, oxidizing agents, activators, etc.). In some embodiments, the kit further comprises one or more silanes, such as HEPTES, Silane A and/or Silane B.

[0141] The kits may contain one or more additional components, such as containers, buffers, enzymes (e.g., polymerase, ligase, etc.), reagents (e.g., nucleotides), cofactors, oligonucleotides, primers, or additional agents, such as agents for isolating nucleic acids from cells. The kits may also contain data analysis software or instructions for data analysis. The kit components may be packaged together and the package may contain or be accompanied by instructions for using the kit. [0142] It will be appreciated by persons skilled in the art the numerous variations, combinations and/or modifications may be made to the invention as shown without departing from the spirit of the inventions as broadly described.

Exemplary embodiments

[0143] Among the embodiments provided herein are:

1. A microfluidic device comprising:

(a) a first plate comprising a first organic polymer substrate;

(b) a second plate comprising a second organic polymer substrate or a glass substrate; and

(c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface comprises a plurality of oligonucleotide probes; and wherein the first plate and the second plate are bonded to each other to form the microfluidic channel.

2. The microfluidic device of embodiment 1, wherein the 5’ end of each oligonucleotide probe is attached to the first surface or the second surface of the microfluidic channel.

3. The microfluidic device of embodiment 1, wherein the 3’ end of each oligonucleotide probe is attached to the first surface or the second surface of the microfluidic channel.

4. The microfluidic device of embodiment 1, wherein a portion of the plurality of

oligonucleotide probes is attached to the first surface or the second surface of the microfluidic channel via the 5’ end, and the remaining portion of the plurality of oligonucleotide probes is attached to the first surface or the second surface of the microfluidic channel via the 3’ end.

5. The microfluidic device of any one of embodiments 1-4, each oligonucleotide probe is attached to the first surface or the second surface at a pre-determined position.

6. The microfluidic device of any one of embodiments 1-5, wherein the microfluidic channel comprises about 2 to about 10 oligonucleotide probes.

7. The microfluidic device of any one of embodiments 1-6, wherein the plurality of

oligonucleotide probes are DNA probes.

8. The microfluidic device of any one of embodiments 1-7, wherein the plurality of

oligonucleotide probes comprises oligonucleotide probes capable of detecting single nucleotide polymorphism (SNP) alleles. The microfluidic device of any one of embodiments 1-8, wherein each oligonucleotide probe is about 10 to about 75 nucleotides long.

The microfluidic device of any one of embodiments 1-9, further comprising a target nucleic acid hybridized to one or more oligonucleotide probes.

A microfluidic device comprising:

(a) a first plate comprising a first organic polymer substrate;

(b) a second plate comprising a second organic polymer substrate or a glass substrate; and

(c) a microfluidic channel comprising a first surface formed by the first organic polymer substrate and a second surface formed by the second organic polymer substrate or the glass substrate, wherein the first surface and/or the second surface of the microfluidic channel is capable of binding a nucleoside phosphoramidite, and

wherein the first plate and the second plate are bonded to each other to form the microfluidic channel.

The microfluidic device of embodiment 11, wherein the first surface and/or the second surface of the microfluidic channel is bound to a nucleoside phosphoramidite.

The microfluidic device of any one of embodiments 1-12, wherein the first plate is bonded to the second plate covalently or via entangled polymers.

The microfluidic device of any one of embodiments 1-13, wherein the first organic polymer substrate is selected from the group consisting of polydimethylsiloxane (PDMS), cyclic olefin copolymer (COC) and cyclo-olefin polymer (COP).

The microfluidic device of any one of embodiments 1-14, wherein the second plate comprises a glass substrate.

The microfluidic device of any one of embodiments 1-14, wherein the second plate comprises a second organic polymer substrate.

The microfluidic device of embodiment 16, wherein the second organic polymer substrate is selected from the group consisting of PDMS, COC COP.

The microfluidic device of embodiment 16 or 17, wherein the first organic polymer substrate is the same as the second organic polymer substrate.

The microfluidic device of embodiment 16 or 17, wherein the first organic polymer substrate is different from the second organic polymer substrate.

The microfluidic device of any one of embodiments 1-19, wherein the first surface and/or the second surface of the microfluidic channel is silanized. The microfluidic device of embodiment 20, wherein the first surface and/or the second surface of the microfluidic channel comprises one or more silanes selected from the group consisting of 3-[bis(2-hydroxyethyl)amino]propyl-triethoxysilane, N-(hydroxyethyl)-N,N- bis(trimethoxysilylpropyl)amine , N,N'-bis(2-hydroxyethyl)-N,N'- bis(trimethoxysilylpropyl)ethylenediamine, (3-aminopropyl)treiethoxysilane, 3- aminopropyldimethylethoxysilane, N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-(4-semicarbazidyl)propyltriethoxysilane, triethoxysilylundecanal, 3- aminopropyldimethylethoxysilane, N-(6-aminohexyl)aminopropyltrimethoxysilane, N-(3- triethoxysilylpropyl)-4-hydroxybutyramide, hexadecafluorododec- 11 -en- 1 - yltrimethoxysilane, 4-amino-3,3-dimethylbutylmethyldimethoxysilane, 3- aminopropylmethyldiethoxy silane, 1 -amino-2-(dimethylethoxysilyl)propane, 3- aminopropyldiisopropylethoxysilane, N-(6-aminohexyl)aminomethyltriethoxysilane, N-(2- aminoethyl)-l l-aminoundecyltrimethoxy silane, N-3-

[(amino(polypropylenoxy)]aminopropyltrimethoxysilane, N-(2-aminoethyl)-3- aminopropyltrimethoxysilane, l,2-bis(trimethoxysilyl)decane, and 1,8- bis(triethoxysilyl)octane.

The microfluidic device of any one of embodiments 1-21, wherein the microfluidic channel is about 0.1 mm to about 300 mm long.

The microfluidic device of any one of embodiments 1-22, wherein the microfluidic channel is about 0. lmm to about 300 mm wide.

The microfluidic device of any one of embodiments 1-23, wherein the microfluidic channel is about 1 mhi to about 800 mhi deep.

The microfluidic device of any one of embodiments 1-24, wherein the microfluidic channel is molded into the first organic polymer substrate and/or the second organic polymer substrate.

The microfluidic device of any one of embodiments 1-25, wherein the microfluidic channel is a rectangular prism.

The microfluidic device of any one of embodiments 1-26, further comprises ports and fluidic connections for filling and emptying the microfluidic channel.

The microfluidic device of any one of embodiments 1-27, wherein the microfluidic channel has a volume of less than about 20 mΐ _^ , such as less than about 10 mΐ _^ . The microfluidic device of any one of embodiments 1-28, comprising a plurality of microfluidic channels.

The microfluidic device of embodiment 29, comprising about 2 to about 100 microfluidic channels.

The microfluidic device of embodiment 29 or 30, wherein the microfluidic device comprises a plurality of microfluidic channels that are parallel to each other, and wherein the distance between adjacent microfluidic channels is about 100 mhi to about 1 mm.

A method of preparing a microfluidic microarray device, comprising synthesizing a plurality of oligonucleotide probes at pre-determined positions on the first surface and/or the second surface of the microfluidic channel of the microfluidic device of any one of embodiments 11-31.

The method of embodiment 32, comprising bonding a first plate comprising a first organic polymer substrate comprising a microfluidic channel to a second plate comprising a second organic polymer substrate or a glass substrate.

The method of embodiment 32 or 33, comprising molding the microfluidic channel into the first organic polymer substrate and/or the second organic polymer substrate.

A method of preparing a microfluidic microarray device, comprising synthesizing a plurality of oligonucleotide probes in a reaction volume of no more than about 20 pL (e.g., no more than about 10 pL) at pre-determined positions on an interior surface of a microfluidic channel in a microfluidic device, wherein the microfluidic device comprises a glass substrate and/or an organic polymer substrate (e.g., PDMS), wherein the interior surface of the microfluidic channel is formed by the glass substrate or the organic polymer substrate, and wherein the microfluidic channel has a volume of no more than about 20 pL (e.g., no more than about 10 pL).

The method of any one of embodiments 32-35, further comprising passing a silane through the microfluidic channel prior to the synthesizing.

The method of any one of embodiments 32-36, wherein the plurality of oligonucleotide probes are synthesized in the 5’ to 3’ direction.

The method of any one of embodiments 32-36, wherein the plurality of oligonucleotide probes are synthesized in the 3’ to 5’ direction.

The method of any one of embodiments 32-38, wherein the plurality of oligonucleotide probes are synthesized by a light-directed method. The method of embodiment 39, wherein the plurality of oligonucleotide probes is synthesized by maskless photolithography.

The method of embodiment 39, wherein the plurality of oligonucleotide probes is synthesized using a series of photomasks.

The method of any one of embodiments 39-41, wherein the synthesizing comprises:

(i) providing the microfluidic device comprising a first nucleoside phosphoramidite or a plurality of oligonucleotides comprising a first nucleoside phosphoramidite at the 5’ (or 3’) terminus, wherein the first nucleoside phosphoramidite or the plurality of oligonucleotides are attached to the first surface and/or the second surface of the microfluidic channel via the 3’ (or 5’) terminus, and wherein the first nucleoside phosphoramidite comprises a photo- labile protective group at the 5’ (or 3’) position;

(ii) deprotecting the first nucleoside phosphoramidite using a patterned light beam to provide deprotected first nucleoside phosphoramidite at pre-determined positions on the first surface and/or the second surface of the microfluidic channel;

(iii) passing a second nucleoside phosphoramidite through the microfluidic channel to couple the second nucleoside phosphoramidite to the deprotected first nucleoside phosphoramidite, wherein the second nucleoside phosphoramidite comprises a photo-labile protective group at the 5’ (or 3)’ position;

optionally (iv) passing a capping composition through the microfluidic channel;

optionally (v) passing an oxidizing solution through the microfluidic channel; and

(vi) repeating steps (ii)-(v) for a pre-determined number of times, wherein the patterned light beam is programmed at each step according to the sequences of the plurality of

oligonucleotide probes; thereby providing the plurality of oligonucleotide probes at the pre determined positions on the microfluidic device.

The method of embodiment 42, wherein the oxidizing composition and the capping composition comprise a solvent that does not swell the first organic polymer substrate.

The method of embodiment 43, wherein the oxidizing composition and the capping composition do not comprise dichloromethane.

The method of embodiment 43 or 44, wherein the oxidizing composition and the capping composition comprise acetonitrile. The method of any one of embodiments 42-45, wherein the synthesizing is carried out in an automated oligonucleotide synthesis system comprising a digital micromirror device capable of producing the patterned light beam.

The method of any one of embodiments 32-34 and 36-46, wherein the plurality of oligonucleotide probes are synthesized at pre-determined positions in reaction volumes of less than about 20 mΐ _^ , such as less than about 10 mΐ _^ .

A microfluidic microarray device prepared using the method of any one of embodiments 32- 47.

A method of analyzing a sample comprising target nucleic acids, comprising:

(a) contacting the sample with the plurality of oligonucleotide probes in the microfluidic device of any one of embodiments 1-10, 13-31 and 48;

(b) hybridizing the target nucleic acids to the plurality of oligonucleotide probes in the microfluidic channel to provide probe-target hybrids; and

(c) detecting the probe-target hybrids.

The method of embodiment 49, wherein step (a) comprises injecting the sample into the microfluidic channel of the microfluidic device.

The method of embodiment 49 or 50, wherein probe-target hybrids on the first surface are detected.

The method of embodiment 51, further comprising removing the second plate prior to (c). The method of embodiment 51 or 52, wherein probe-target hybrids on the second surface are detected.

The method of embodiment 53, further comprising removing the first plate prior to (c). The method of any one of embodiments 49-54, wherein the microfluidic channel is washed prior to the detecting.

The method of any one of embodiments 49-55, wherein the microfluidic device is used for a biochemical assay.

The method of embodiment 56, wherein the biochemical assay is selected from the group consisting of quantitative hybridization, quantitative annealing, hybridization-ligation, nuclease hybridization assay, allele specific primer extension, and short-read sequencing. The method of any one of embodiments 49-57, wherein the microfluidic device comprises a plurality of microfluidic channels each comprising a plurality of oligonucleotide probes, and wherein a different sample comprising target nucleic acids is injected into each microfluidic channel.

EXAMPLES

[0144] The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

Example 1: Synthesis of a single oligonucleotide probe in the 3’-^5’ direction

[0145] This example describes preparation of an exemplary microfluidic microarray device comprising 8 microfluidic channels each having a single type of oligonucleotide probe synthesized in the 3’ to 5’ direction. FIG. 1 provides an overview of the method.

1. Construction of the functionalized microfluidic chip

[0146] Slygard 184 PDMS was cast onto a master mold to form 8 microfluidic channels. The dimensions of the microfluidic channels were as follows: 2.2 mm in width, 50 mm in length, 500 micron spacing between adjacent channels, and a channel height of 40 microns. Each microfluidic channel has a flowcell volume of 6.7 microliters. Both the PDMS and glass substrates were then activated using oxygen plasma. The activated PDMS was then placed atop the glass substrate and the combined unit was then incubated at 80°C in an oven for 1 hour. The completed microfluidic device was then removed from the oven and cooled to room

temperature. This process yielded a microfluidic device with 8 channels, upon which

oligonucleotide probes were synthesized.

[0147] The microfluidic device was next cleaned by loading 10 flow-cell volumes of ethanol through the microfluidic channels using a micropipette. Subsequently, the ethanol was ejected by injecting air, then the microfluidic channels were filled with 6% 3-Bis(2- hydroxyethyl)amino]propyl-triethoxysilane (HEPTES) silane in 94.9% ethanol/5% water/0.1% acetic acid. After silanization, the microfluidic device was washed with ethanol as described above, and then placed into a vacuum oven at 110°C overnight. Finally, the microfluidic device was taken out of the oven, and used immediately for microarray synthesis.

2. Light-directed Oligonucleotide Synthesis System

[0148] A light-directed oligonucleotide synthesis system was constructed in house using a traditional DNA synthesizer EXPEDITE ^® 8909 and a Visitech LUXBEAM ^® lithography system that projected ultra-violet light at 365nm. As illustrated in FIG. 2, light-directed synthesis comprised the following steps: coupling, capping, oxidation and photo-deprotection. The EXPEDITE ^® 8909 is typically used to perform coupling, capping, oxidation and acid-labile deprotection on Luer columns containing modified controlled pore glass. Here, the EXPEDITE ^® 8909 was modified for use with the microfluidic device described above as well as the Visitech LUXBEAM ^® projector device for photo-deprotection.

3. On chip Synthesis of a Single Oligonucleotide probe 3’->5’

[0149] A single oligonucleotide was built on the glass and PDMS surfaces using

RAYDITES™ purchased from Sigma Aldrich. The synthesis of this probe comprised the following steps: photo-deprotection, oxidation, coupling and capping as shown in FIG. 2.

[0150] Traditional chemistry for the capping and oxidation steps in solid phase

oligonucleotide synthesis utilizes harsh chemicals, which swell PDMS, rendering the microfluidic device unusable. Here, we modified the oligonucleotide synthesis chemistry for compatibility with the PDMS-based microfluidic device. For example, typically,

dichloromethane (DCM) is the solvent used in the oxidizing solution and the capping solution. A microfluidic device comprising PDMS would not be able to withstand the swelling by DCM during typical oligonucleotide synthesis. Therefore, we used atypical capping and oxidizing solutions, which contained acetonitrile rather than DCM.

[0151] The solutions listed below in Table 1 were injected into the microfluidic channels during these steps. The oxidation and photo-deprotection steps required only one solution each, whereas, the coupling and capping steps each required a mixture of two solutions, i.e., activator and coupling solutions, or activator and capping solutions respectively, in equal parts.

Table 1. Solutions used in Oligonucleotide Synthesis.

[0152] An exemplary oligonucleotide probe TRB54-E was synthesized from the 3’ end to the 5’ end on the microfluidic device using the modified EXPEDITE ^® 8909 system according to a program known to those skilled in the art. The TRB54-E probe was 30 nucleotides long and complementary to a region encompassing an SNP in the TRB56 synthetic DNA template. The 5’ end of the TRB56 template was coupled to a phosphoramidite carrying a Cyanine-3 fluorescent dye.

[0153] During the photo-deprotection step of the cycle, an image was projected onto the microfluidic device via UV light from the Visitech LUXBEAM ^® system. At the positions exposed to the UV light on the surfaces of the PDMS and glass substrates, the 3’-protecting group from the 5’ end of the growing oligonucleotide was removed to expose a hydroxyl group on the 5’ end. A single nucleotide was added in the subsequent coupling step to the exposed 5’ end. A set of images were projected from the Visitech LUXBEAM ^® system based on the position and sequence of the oligonucleotide probe, with one image per cycle.

[0154] An exemplary image projected by the Visitech system is displayed in FIG. 3. In the white regions projected onto the microfluidic channels, the 3’-protecting group at the 5’ end of the growing oligonucleotide would be deprotected by a 365-nm UV light, while the black regions would have no UV light exposure and no nucleotide would be coupled to growing oligonucleotides in such regions in the subsequent coupling step. For the synthesis of a single oligonucleotide probe, the same image could be projected at every cycle of synthesis.

[0155] The oligonucleotide formed on the microfluidic device also contained typical exocyclic protecting groups on the adenosine, cytosine and guanosine residues. The exocyclic protecting groups were removed after synthesis of the entire oligonucleotide probe was completed. The deprotection was achieved by injecting a solution of 1:1 ethanol: ethylenediamine into the microfluidic channels, followed by incubation at room temperature for 2 hours. The microfluidic device was then washed with ethanol, then with deionized water, and then soaked in a beaker of deionized water maintained at 4 °C overnight with stirring. Immediately prior to use, the water was expunged by forcing air through the microfluidic channels.

Example 2: Synthesis of two different oligonucleotide probes in the 3’ -^5’ direction

[0156] In this example, two oligonucleotide probes having different sequences were synthesized in the microfluidic channels on the same microfluidic microarray device. The same synthesis method, chemical reagents, and program on the modified EXPEDITE ^® 8909 as described in Example 1 were used for the synthesis of two different oligonucleotide probes. However, the sequence input to the EXPEDITE ^® 8909 synthesizer took into account the sequences of both oligonucleotide probes. A growing oligonucleotide was photo-deprotected using the Visitech LUXBEAM ^® system only when based on the sequence of the oligonucleotide probe, the subsequent couple step was used to add a corresponding nucleotide to the growing oligonucleotide. Therefore, multiple images were used to synthesize two oligonucleotide probes, rather than projecting the same image for each cycle as in Example 1.

[0157] In this example, TRB54-E and TRB54-E(mod) probes were synthesized. Each probe was 30 nucleotides long. TRB54-E(mod) differed from TR54-E in 8 internal, non-consecutive nucleotide positions.

[0158] Each oligonucleotide probe was attached to the microfluidic channels at positions determined by the projected images provided by the Visitech LUXBEAM ^® system. The two oligonucleotide probes were synthesized simultaneously except for the nucleobases that differed, in which case those bases were synthesized sequentially. Using this technique, both probes were created at distinct positions within the same microfluidic channel.

Example 3: Detection of Human Immunodeficiency Virus, Hepatitis C Virus, and Multi- Drug Resistant Hepatitis C Virus Using a Microfluidic Microarray Device

[0159] A microfluidic microarray device having microfluidic channels each comprising three sets of three different oligonucleotide probes was prepared using the method as described in Example 2. A different set of images from those in Example 2 were projected by the Visitech LUXBEAM ^® system, and the sequence input to the EXPEDITE ^® 8909 synthesizer took into account the sequences of the three oligonucleotide probes. In this example, the three

oligonucleotide probes for: the Human Immunodeficiency Virus (HIV), the Hepatitis C Virus (HCV2) and a Multi-Drug Resistant Hepatitis C virus (HCV1). The oligonucleotide probes were 33 or 34 nucleotides long. After synthesis of the oligonucleotide probes, the exocyclic groups were removed from the oligonucleotide probes. The microfluidic microarray device was then washed in 4 °C deionized water overnight.

[0160] Template DNAs having a 3’ Cy3 dye were used in a hybridization experiment. Three template DNAs TRB97 (HCV1 template), TRB98 (HCV2 template) and TRB99 (HIV template) were used. Each template DNA was 60 nucleotides long, and had a Cy3 label at the 3’ end. Prior to hybridization, the microfluidic microarray device was washed with 50 flowcell volumes of 5X SSC buffer (IX SSC buffer is 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0). The SSC buffer was ejected by flowing l-flowcell volume of air through the microfluidic channels. The following template solutions were then prepared: 10 mM TRB97 in Buffer A, IOmM TRB98 in Buffer A, 13mM TRB-99 in Buffer A, and a mix of TRB97, TRB98, and TRB99 (10 mM each) in Buffer A. The PDMS substrate was removed using a razor blade from the microfluidic microarray device, the glass surface was then washed with 2X SSC buffer and dried with compressed air. Each of the four template solutions was spotted onto a different region having all three oligonucleotide probes on the glass surface. The template samples and the

oligonucleotide probes were placed in a closed box containing lab tissue saturated with water for humidity control, and incubated at 42°C for 2 hours in a hybridization oven. After hybridization, the glass surface was briefly washed with 2X SSC buffer, dried with compressed air and imaged using a PerkinElmer PROSCANARRAY™, a slide scanner typically used for traditional microarrays.

[0161] Results in FIGS. 4A-4D demonstrate successful synthesis of all three oligonucleotide probes, and successful detection of specific hybridization of the template samples to the oligonucleotide probes on the glass surface. In FIG. 4A, signal was detected from all three oligonucleotide probes as all three templates were added to this region of the glass surface.

FIGS. 4B-4D demonstrate specificity of the three oligonucleotide probes. For example, if there exists no specificity between probes and templates, when only the HIV template is added two or more probes would be illuminated. However, in FIG. 4B, no signal was detected from the HCV1 and HCV2 probes. Similarly, specific hybridization was observed in FIGS. 4C-4D.

Example 4: Synthesis of a single oligonucleotide probe in 5’-> 3’ direction

[0162] This example describes preparation of an exemplary microfluidic microarray device comprising 8 microfluidic channels each having a single type of oligonucleotide probe synthesized in the 5’ to 3’ direction.

[0163] The methodology and protocols for synthesizing a single type of oligonucleotide probe in the 5’- 3’ direction in a microfluidic device was very similar to those described in example 1

(3’ ->5’). However, for 5’- 3’ synthesis, the phosphoramidites had a 3’-photo-protecting group

( e.g ., NPPOC) and 5’-amidite instead of a 3’-amidite and 5’-photo-protecting group (e.g.,

NPPOC) as used in the 3’ ->5’ case. See, FIG. 5 showing the difference between the

phosphoramidites. The solutions used for coupling, capping, oxidation and deprotection for synthesis of the oligonucleotide probe in the 5’ ->3’ direction were the same as those described for the 3’ ->5’ synthesis described. The sequence input to the EXPEDITE ^® 8909 synthesizer was run in the reverse order as that in Example 1. Fluorescent labels could be attached to the 3’ end of the oligonucleotide probe.

Example 5: Synthesis of two or more different oligonucleotide probes in the 5’->3’ direction

[0164] In this example, two or more oligonucleotide probes having different sequences were synthesized in the 5’ ->3’ direction using the same method as in Example 2, but with nucleoside phosphoramidites having a 3’ NPPOC protecting group and a 5’ phosphoramidite group as described in Example 4. Four oligonucleotide probes were synthesized onto the microfluidic device in this example. The sequences of the oligonucleotide probes and their corresponding substrates are shown in Table 2 below. These probes were synthesized spatially to read“TTT,” “CCC,”“GGG,” and“AAA,” respectively, on the surface.

Table 2. Oligonucleotide probes and templates.

[0165] After synthesis of the entire probe sequences, the exocyclic groups were removed and the microfluidic device was washed in 4°C deionized water as described in Example 1.

Subsequently, all microfluidic channels were washed with 5X SSC buffer and air dried. The following template sample was added to all microfluidic channels: ImM TRB90-Cy3, ImM TRB9l-Cy3, ImM TRB92-Cy3, and ImM TRB93-Cy3 in Buffer A. The microfluidic device was incubated at 42°C for 2 hours. The template samples were removed from the microfluidic channels. One flowcell volume of 2X SSC buffer was added to each microfluidic channel, and the buffer was removed by pumping air through the microfluidic channels. The entire microfluidic device was then imaged through the glass surface. After the first imaging, the PDMS substrate was removed from the glass substrate. The glass surface alone was then imaged separately. [0166] As shown in FIGS. 6A-6B, hybridization of the template sample to all four

oligonucleotide probes can be detected either from the glass surface alone (FIG. 6A), or from both the glass surface and the PDMS surface.

[0167] In a second hybridization experiment, the PDMS was removed from the glass surface, as described above, and ImM TRB9l-Cy3 in Buffer A was spotted directly on the PDMS to demonstrate hybridization specificity. After hybridization, the PDMS substrate was washed with 0.2X SSC containing 0.1% Tween-20, dried and imaged. As shown in FIG. 7, specific hybridization between the TRB91 template and its complementary probe CCC was detected on the PDMS surface, although four probes (AAA, CCC, GGG and TTT) were synthesized on the PDMS surface in the 5’ to 3’ direction. The ability to detect signal from the PDMS surface provides opportunities for signal amplification, as the surface area of microarrays is usually limited to a relatively flat glass substrate.

Example 6: Biochemical assays on a microfluidic microarray device

[0168] This example demonstrates that the PDMS -based microfluidic microarray device is amenable for carrying out subsequent biochemical assays, such as hybridization and enzymatic DNA synthesis.

[0169] The microfluidic microarray device was prepared using a similar method as that described in Example 5. Two SNP probes, TRB18 and TRB19, were synthesized spatially to read“A” and“B” respectively on the surface. Each probe is 35 nucleotides long. Except for the 3’ terminal nucleotide, the sequences of TRB18 and TRB19 are identical. TRB18 has an adenosine, and TRB19 has a guanine at the 3’ end. The TRB40 template is 90 nucleotides long, and has a 5’ Cy55 label. TRB18 (probe A) has a perfectly complementary sequence to nucleotides 42-75 of the TRB40 template.

[0170] After synthesis of the entire probe sequences, the exocyclic groups were removed in the same manner as described in Example 5. The microfluidic device was prepared as described in example 1. Following these steps, 15m1 of ImM TRB40 in Buffer B was added to each microfluidic channel. The microfluidic device was incubated at 42°C for 2 hours in a closed box containing a laboratory tissue saturated with water for humidity control as described in example

3. The microfluidic channels were then washed with lO-flowcell volumes of 2X SSC buffer.

[0171] After this hybridization step, an extension mixture comprising DNA polymerase and dNTPs (including Cy3-labeled dUTP) was prepared. The extension mixture and the microfluidic device were held at 42°C for 10 minutes. 15m1 of the extension mixture was added to each microfluidic channel, and the microfluidic device was held at 42°C for 12 minutes.

Subsequently, the extension mixed was then pushed out of the microfluidic channels, which were then washed with 0.2X SSC buffer containing 0.l%Tween. The microfluidic device was then disassembled, and the glass substrate was imaged.

[0172] As shown in FIG. 8, TRB40 hybridized to both TRB18 and TRB19 ( i.e ., A and B in the right panel). However, only TRB18 (A probe) showed extension signal. This allele- specific extension demonstrates the ability of using the microfluidic microarray device to identify a single nucleotide polymorphism in a template DNA.

[0173] FIG. 9 shows a quantification of the hybridization and extension signals in FIG. 8. The “Cy5.5” bars reflect the mean fluorescence intensity of hybridization of template DNA to SNP oligonucleotide probes (A and B). The“Cy3” bars reflect the mean fluorescence intensity of products synthesized by DNA polymerase off of the probe sequences, using the hybridized DNA as a template and Cy3-lableled dUTP as a substrate. Error bars indicate the standard deviation of replicate spots within the letters shown in FIG. 8.

Example 7: Synthesis of two or more oligonucleotide probes in the 5’-> 3’ direction with subsequent in-channel hybridization and extension using Biotin dUTP

[0174] The fluorescence-based signal from the extension assay described in Example 6 may be amplified using a biotin-modified dUTP followed by labeling with Cy-3 labeled streptavidin, EXTRA VIDIN ^® or avidin.

[0175] The hybridization step in this example was carried out in the same manner as described in the previous examples. After hybridization, a pre-extension buffer was injected into the flowcell using a fluidic system and incubated on a hot plate at 42°C for 4 minutes. Following the incubation period, the pre-extension mix was removed using air and the extension master mix (including DNA polymerase, dATP, dCTP, dGTP, Biotin-labeled dUTP, and single-strand DNA-binding protein) was pre-heated to a specified temperature. Using the same fluidic system, the pre-heated extension mixture was injected into the flowcell, which was heated to the same specified temperature. The mixture was incubated at the specified temperature for a set period of time. After the extension incubation period, the flowcell was set back to room temperature and was washed with 2X SSC + 0.1% Tween20 followed by 0.2X SSC + 0.1% Tween20. A solution containing fluorescently labeled EXTRA VIDIN ^® was injected into the flowcell and incubated at room temperature for 6 minutes. The flowcell was then washed with deionized water prior to imaging. [0176] FIG. 10 shows extension of control probes and strand- specific probes hybridized to amplicons from Gyrase A of Neisseria gonorrhoeae.