WO2013177953A1 - Integrated system for real-time monitoring of microfluidic reactions - Google Patents

Abstract

A system for real-time monitoring of a microfluidic reaction and a method thereof are provided. The system comprises a platform for holding a microfluidic device; a temperature control module (4); a peripheral flow module; and a real-time imaging module. The method comprises providing a reagent to the microfluidic device; and monitoring the reaction using the real-time imaging module. Furthermore, methods for optimizing a reaction condition using the system are provided.

Description

INTEGRATED SYSTEM FOR REAL-TIME MONITORING OF MICROFLUIDIC

REACTIONS

Priority

[0001] The present application claims the priority benefit to Chinese Application No.

201210177086.6, filed on May 31, 2012, the content of which is incorporated by reference herein in its entirety for all purposes.

Technical Field

[0002] This invention relates to an integrated system for real-time monitoring of

microfluidic reactions. Background Art

[0003] A microarray chip can contain a substrate on which a number of probe molecules with known sequences are orderly fixed with high density by a micro-fabrication technology. The probe molecules can be DNA, RNA, protein and glycan, etc. Microarray chips can be used for the analysis of gene expression profiling, hereditary defect, protein distribution and reaction character by detecting the hybridization between the probes on the microarray and targets in a sample.

[0004] Standard analytical methods based on microarray chips include sample preparation, microarray chip manufacturing, microarray chip hybridization, microarray chip washing and drying and microarray chip detection, etc. Current microarray chip processing platforms include two types. One type of platform contains independent or integrated instalments for separated steps of hybridization, dyeing, washing, drying and detection, such as platforms from CapitalBio Corporation and from Affymetrix Incorporation, The platforms from CapitalBio Corporation, including BioMixer II hybridization station, SlideWasher slide clean-up station and LuxScan 10K microarray scanner, can complete these processing steps separately. The GeneTitan system is an integrated platform from Affymetrix Incorporation which can complete these processing steps by transferring the samples using robotic arms among functional modules. Another type of platform completes the steps of hybridization, washing and drying using a micro fluidic chip with micro fluidic channels associated with a peripheral flow system, and perform the step of detection on another instrument, such as the platform from Shanghai BioO Technology Co., Ltd., including the e-Hyb automated hybridization instrument and the BE-2.0 biochip reader. These two types of platforms cannot monitor the signals on a microarray in real-time during

microarray processing, thus require a series of control tests for the optimization of processing conditions.

[0005] For the control tests the same samples need to be treated by different processes under different conditions. Based on the results from final detection, an optimized condition for chip processing can be determined, such as hybridization time and dynamic, washing time, washing flow velocity, and conditions for drying, etc. It will take plenty of samples and time for the control tests if the signals on the chip cannot be monitored in real-time.

Summary of the Invention

[0006] in one aspect, the present in vention provides a system for real-time monitoring of a micro fluidic reaction, comprising a platform for holding a micro fluidic device; a temperature control module; a peripheral flow module; and a real-time imaging module.

[0007] In some embodiments, the real-time imaging module may comprise a light source and an optical detector. In some embodiments, the light source may be a photodiode, a fluorescent lamp, a filament lamp or a laser. In some embodiments, the optical detector may be a charge coupled device (CCD) or a photomultiplier tube (PMT). In some embodiments, the real-time imaging module may comprise a reflecting surface. In some embodiments, the light from the light source may be reflected to the optical detector by the reflecting surface. In some embodiments, the platform may comprise a microfluidic device. In some embodiments, the light from the light source may be reflected by the microfluidic device to the optical detector. In some embodiments, the microfluidic device may comprise a reaction chamber. In some embodiments, the microfluidic device may comprise an inlet and an outlet. In some

embodiments, the microfluidic device may comprise a pneumatic microvalve. In some embodiments, the pneumatic microvalve may be controlled by a control channel. In some embodiments, the peripheral flow module may comprise a pressure source connected to the microfluidic device. In some embodiments, the pressure source may control the pneumatic microvaive. In some embodiments, the pressure source may be a piston pump or a plunger pump. In some embodiments, the peripheral flow module may comprise a solution source connected to the microfluidic device through the inlet and/or outlet. In some embodiments, the peripheral flow module may comprise at least one pump providing air pressure and one valve controlling the fluid flow. In some embodiments, the valve may be an electromagnetic valve or an electric valve.

[0008] In a second aspect, the present invention provides a method for real-time monitoring of a micro fluidic reaction using the system disclosed herein, which method comprises: providing a reagent to the microfluidic device; and monitoring the reaction using the real-time imaging module,

[0009] in some embodiments, the optical detector may record an optical signal and transforms the optical signal to a pixel. In some embodiments, the optical detector may produce an image. In some embodiments, multiple images may be produced at multiple time intervals. In some embodiments, the multiple images may be transformed into a video recording. In some embodiments, the method may comprise providing a biological sample to the microfluidic device, In some embodiments, the reaction may comprise binding of a target in the biological sample to a probe, In some embodiments, the probe may be immobilized in the reaction chamber of the microfluidic device, In some embodiments, the optical detector may detect a label. In some embodiments, the method may comprise washing away non-targets in the biological sample. In some embodiments, the method may comprise mixing a reaction mixture,

[0010] In a third aspect, the present invention provides a method for optimizing a reaction time using the system disclosed herein, which method comprises: providing a reagent to the microfluidic device; recording images of the reaction using the real-time imaging module at multiple time intervals; and comparing the images to determine the optimal reaction time. [0011] In a fourth aspect, the present invention provides a method for optimizing a reagent using the system disclosed herein, which method comprises: providing multiple reagents to the microfluidic device; recording images of the reactions using the real-time imaging module with multiple reagents; and comparing the images to determine the optimal reagent. Brief Description of the Drawings

[0012] Figure 1 is a schematic diagram of an exemplary embodiment of a hybridization system incorporating real-time monitoring of microarray processing.

[0013] Figure 2 is a schematic diagram of an exemplar}' embodiment of a hybridization system incorporating real-time monitoring of microarray processing.

[0014] Figure 3 shows an exemplary microarray probe pattern.

[0015] Figure 4 shows exemplar}' images exported by the hybridization system during the hybridization step and after the washing step.

Detailed Description of the Invention [0016] In some embodiments, w^re provide an integrated system for real-time monitoring of microfluidic reactions and uses thereof. Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methodology, devices, solutions or apparatuses described, as such methods, devices, solutions or apparatuses can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention,

[0017] Unless defined otherwise or the context clearly dictates otherwise, ail technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the in vention, the preferred methods and materials are now described,

[0018] All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference I. Definitions

[0019] As used herein, the singular forms "a", "an", and "the" include plural references unless indicated otherwise. For example, "a" dimer includes one or more dimers,

[0020] As used herein, the term "microfluidic device" generally refers to a device through which materials, particularly fluid borne materials, such as liquids, can be transported, in some embodiments on a micro-scale, and in some embodiments on a nanoscale. Thus, the

microfluidic devices described by the presently disclosed subject matter can comprise

microsca!e features, nanoscale features, and combinations thereof.

[0021] Accordingly, an exemplary microfluidic device typically comprises structural or functional features dimensioned on the order of a millimeter-scale or less, which are capable of manipulating a fluid at a flow rate on the order of a p.L/min or less. Typically, such features include, but are not limited to channels, fluid reservoirs, reaction chambers, mixing chambers, and separation regions. In some examples, the channels include at least one cross-sectional dimension that is in a range of from about 0.1 μπι to about 500 μηι. The use of dimensions on this order al lows the incorporation of a greater number of channels in a smal ler area, and utilizes smaller volumes of fluids.

[0022] A microfluidic device can exist alone or can be a part of a microfluidic system which, for example and without limitation, can include: pumps for introducing fluids, e.g., samples, reagents, buffers and the like, into the system and/or through the system; detection equipment or systems; data storage systems; and control systems for controlling fluid transport and/or direction within the device, monitoring and controlling environmental conditions to which fluids in the device are subjected, e.g., temperature, current, and the like.

[0023] As used herein, the terms "channel," "micro-channel," "fiuidic channel," and

"microfluidic channel" are used interchangeably and can mean a recess or cavity formed in a material by imparting a pattern from a patterned substrate into a material or by any suitable material removing technique, or can mean a recess or cavity in combination with any suitable fluid-conducting structure mounted in the recess or cavity, such as a tube, capillary, or the like. In the present invention, channel size means the cross-sectional area of the microfluidic channel.

[0024] As used herein, the terms "flow channel" and "control channel" are used

interchangeably and can mean a channel in a microfluidic device in which a material, such as a fluid, e.g., a gas or a liquid, can flow through, More particularly, the term "flow channel" refers to a channel in which a material of interest, e.g., a solvent or a chemical reagent, can flow through. Further, the term, "control channel" refers to a flow channel in which a material, such as a fluid, e.g., a gas or a liquid, can flow through in such a way to actuate a valve or pump.

[0025] As used herein, "chip" refers to a solid substrate with a plurality of one-, two- or three-dimensional micro stnictures or micro-scale structures on which certain processes, such as physical, chemical, biological, biophysical or biochemical processes, etc., can be carried out. The micro structures or micro-scale structures such as, channels and wells, electrode elements, electromagnetic elements, are incorporated into, fabricated on or otherwise attached to the substrate for facilitating physical, biophysical, biological, biochemical, chemical reactions or processes on the chip. The chip may be thin in one dimension and may have various shapes in other dimensions, for example, a rectangle, a circle, an ellipse, or other irregular shapes. The size of the major surface of chips of the present invention can vary considerably, e.g., from about 1 mm² to about 0.25 m², Preferably, the size of the chips is from about 4 mm' to about 25 cm² with a characteristic dimension from about 1 mm to about 5 cm. The chip surfaces may be flat, or not flat. The chips with non-flat surfaces may include channels or wells fabricated on the surfaces.

[0026] A microfluidic chip can be made from any suitable materials, such as PDMS

(Polydimethylsiloxane), glass, PMMA (polymethylmethacrylate), PET (polyethylene terephthalate), PC (Polycarbonate), etc., or a combination thereof,

[0027] As used herein, "biological sample" refers to any sample obtained, from a living or viral source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid, or protein or other macromolecule can be obtained, The biological sample can be a sample obtained directly from a biological source or a sample that is processed, For example, isolated nucleic acids that are amplified constitute a biological sample. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom,

[0028] The term "molecule" is used herein to refer to any suitable molecule, such as nucleic acids, proteins, antibodies, small molecule compounds, peptides, carbohydrates, lipids, etc. [0029] The term "microarray" is used herein to refer to any suitable microarray, such as nucleic acid, protein and chemical microarrays. Specific nucleic acids, proteins, antibodies, small molecule compounds, peptides, and carbohydrates can be immobilized on solid surfaces to form microarrays. [0030] The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotid.es, analogs thereof, or mixtures thereof. This term refers only to the primary' structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double- and single-stranded ribonucleic acid ("RNA"). It also includes modified, for example by alkvlation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides

(containing D-ribose), including tRNA, rRNA, hRNA, and niRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids ("PNAs")) and polymorpholino (commercially available from the Anti-Virais, inc., Corvaliis, OR., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. Thus, these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' to P5' phosphoramidates, 2 -0- alkyl-substituted RNA, hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, "caps," substitution of one or more of the nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g. ,

animoalkylphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including enzymes (e.g. nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelates (of, e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide. The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" can comprise any suitable length, such as at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more

nucleotides.

[0031] it will be appreciated that, as used herein, the terms "nucleoside" and "nucleotide" will include those moieties which contain not only the known purine and pyrimidme bases, but also other heterocyclic bases which have been modified. Such modifications mclude methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxy! groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like. The term "nucleotidic unit" is intended to encompass nucleosides and nucleotides.

[0032] "Nucleic acid probe" refers to a structure comprising a polynucleotide, as defined above, that contains a nucleic acid sequence that can bind to a corresponding target. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.

[0033] As used herein, "complementary or matched" means that two nucleic acid sequences have at least 50% sequence identity. Preferably, the two nucleic acid sequences have at least 60°/», 70%, 80%, 90%, 95%, 96%, 97%, 98°/», 99% or 100% of sequence identity.

"Complementary or matched" also means that two nucleic acid sequences can hybridize under low, middle and/or high stringency condition(s).

[0034] As used herein, "substantially complementary or substantially matched" means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, "substantially complementary or substantially matched" means that two nucleic acid sequences can hybridize under high stringency condition(s).

[0035] In general , the stability of a hybrid is a function of the ion concentration and temperature, Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Moderately stringent hybridization refers to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule. The hybridized nucleic acid molecules generally have at least 60% identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions are conditions equivalent to hybridization in 50% forrnamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS, at 42°C. High stringency conditions can be provided, for example, by hybridization in 50% forrnamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in O.lx SSPE, and 0.1% SDS at 65°C. Low stringency hybridization refers to conditions equivalent to hybridization in 10% forrnamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS at 22°C, followed by washing in Ix SSPE, 0.2% SDS, at 37°C. Denhardt's solution contains 1% Ficoil, 1% polwinylpyrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025 M EDTA. Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et ai., Molecular Cloning: A

Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. ( 1989); and Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons (1999).

[0036] Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984).

[0037] The terms "homologous", "substantially homologous", and "substantial homology" as used herein denote a sequence of amino acids having at least 50%», 60%, 70%), 80% or 90% identity wherein one sequence is compared to a reference sequence of amino acids. The percentage of sequence identity or homology is calculated by comparing one to another when aligned to corresponding portions of the reference sequence.

[0038] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1 ,000 or more amino acids, The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non- amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc), as well as other modifications known in the art.

[0039] An "antibody" is an immunoglobulin molecule capable of specific binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, or a small molecule, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin mol ecule and can be an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD and IgE. IgY, which is the major antibody type in avian species such as chicken, is also included within the definition. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunogl obulin molecule that comprises an antigen recognition site of the required specificity.

[0040] The term "binding" is an attractive interaction between two molecules which results in a stable association in which the molecules are in close proximity to each other. Molecular binding can be classified into the following types: non-covalent, reversible covalent and irreversible covalent, Molecules that can participate in molecular binding include proteins, nucleic acids, carbohydrates, lipids, and small organic molecules such as pharmaceutical compounds, Proteins that form stable complexes with other molecules are often referred to as receptors while their binding partners are called ligands. Nucleic acids can also form stable complex with themselves or others, for example, DNA-protein complex, DNA-DNA complex, DNA-RNA complex.

[0041] As used herein, the term "specific binding" refers to the specificity of a binder, e.g., an antibody, such that it preferentially binds to a target, such as a polypeptide antigen.

Recognition by a binder or an antibody of a particular target in the presence of other potential interfering substances is one characteristic of such binding. Preferably, binders, antibodies or antibody fragments that are specific for or bind specifically to a target bind to the target with higher affinity than binding to other non-target substances. Also preferably, binders, antibodies or antibody fragments that are specific for or bind specifically to a target avoid binding to a significant percentage of non-target substances, e.g., non-target substances present in a testing sample. In some embodiments, binders, antibodies or antibody fragments of the present disclosure avoid binding greater than about 90% of non-target substances, although higher percentages are clearly contemplated and preferred. For example,binders, antibodies or antibody fragments of the present disclosure avoid binding about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%), about 98%), about 99%), and about 99%> or more of non- target substances. In other embodiments, binders, antibodies or antibody fragments of the present disclosure avoid binding greater than about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of non-target substances.

[0042] As used herein, the term "antigen" refers to a target molecule that is specifically bound by an antibody through its antigen recognition site. The antigen may be monovalent or polyvalent, i.e., it may have one or more epitopes recognized by one or more antibodies.

Examples of kinds of antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, or small molecules, etc.

[0043] As used herein, the term "epitope" refers to a peptide sequence of at least about 3 to 5, preferably about 5 to 10 or 15, and not more than about 1 ,000 amino acids (or any integer there between), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence. There is no critical upper limit to the length of the fragment, which may, for example, comprise nearly the full-length of the antigen sequence, or even a fusion protein comprising two or more epitopes from the target antigen. An epitope for use in the subject invention is not limited to a peptide having the exact sequence of the portion of the parent protein from which it is derived, but also encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions and substitutions (conservative in nature).

[0044] "Multiplexing" or "multiplex assay" herein refers to an assay or other analytical method in which the presence and/or amount of multiple targets, e.g., multiple nucleic acid target sequences, can be assayed simultaneously by using more than one capture probe conjugate, each of which has at least one different detection characteristic, e.g., fluorescence characteristic (for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime).

[0045] It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially of aspects and embodiments. [0046] Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to ha ve specifically disclosed all the possible sub-ranges as w^rell as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0047] Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

II. System for Real-Time Monitoring of a Microfluidic Reaction

[0048] In one aspect, the present disclosure pro vides a system for real-time monitoring of a microfluidic reaction, comprising: a platform for holding a microfluidic device: a temperature control module; a peripheral flow module; and a real-time imaging module. In some

embodiments, the system may comprise a microfluidic device on the platform.

[0049] Exemplary microfluidic devices may comprise a central body structure in which various microfluidic elements are disposed. The body structure includes an exterior portion or surface, as well as an interior portion which defines the various microscale channels and/or chambers of the overall microfluidic device. For example, the body structure of an exemplary microfluidic devices typically employs a solid or semi-solid substrate that may be planar in structure, i.e., substantially flat or having at least one flat surface. Suitable substrates may be fabricated from any one of a variety of materials, or combinations of materials. Often, the planar substrates are manufactured using solid substrates common in the fields of

micro fabrication, e.g., silica-based substrates, such as glass, quartz, silicon or polysilicon, as well as other known substrates, i.e., gallium arsenide. In the case of these substrates, common rmcroiabrication techniques, such as photolithographic techniques, wet chemical etching, micromachining, i.e., drilling, milling and the like, may be readily applied in the fabrication of microfluidic devices and substrates. Alternatively, polymeric substrate materials may be used to fabricate the devices of the present invention, including, e.g., polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), polyurethane, polyvinylchloride (PVC), polystyrene, polysulfone, polycarbonate and the like. In the case of such polymeric materials, injection molding or embossing methods may be used to form the substrates having the channel and reservoir geometries as described herein. In such cases, original molds may be fabricated using any of the abo ve described materials and methods.

[0050] The channels and chambers of an exemplary device are typically fabricated into one surface of a planar substrate, as grooves, wells or depressions in that surface. A second planar substrate, typically prepared from the same or similar material, is overlaid and bound to the first, thereby defining and sealing the channels and/or chambers of the device. Together, the upper surface of the first substrate, and the lower mated surface of the upper substrate, define the interior portion of the device, i.e., defining the channels and chambers of the device. In some embodiments, the upper layer may be reversibly bound to the lower layer.

[0051] In some embodiments, the real-time monitoring system disclosed herein may comprise a microfluidic device, such as a microarray, for the analysis of target molecules from a biological sample. The target molecules which can be analyzed by a microarray include nucleic acids, proteins, antibodies, small molecule compounds, peptides, carbohydrates and lipids, etc. The microarray may comprise probe molecules that bind to the target molecules. Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, microcontact printing, or electrochemistry on microelectrode arrays. In standard microarrays, the probe molecules are attached via surface engineering to a solid surface of supporting materials, which include glass, silicon, plastic, hydrogels, nitrocellulose and nylon.

[0052] In one embodiment, the array may be a "chip" composed, e.g., of one of the above- specified materials. Polynucleotide probes, e.g., RNA or DNA, such as cDNA, synthetic oligonucleotides, and the like, or binding proteins such as antibodies or antigen-binding fragments or derivatives thereof may be affixed to the chip in a logically ordered manner, i.e., in an array. Detailed discussions of methods for linking nucleic acids and proteins to a chip substrate, are found in, e.g., U.S. Pat. No. 5,143,854, U.S. Pat. No, 5,837,832, U.S. Pat. No. 6, 087,112, U.S. Pat. No. 5,215,882, U.S. Pat. No. 5,707,807, U.S. Pat. No. 5,807,522, U.S. Pat. No. 5,958,342, U.S. Pat. No, 5,994,076, U.S. Pat. No. 6,004,755, U.S. Pat. No. 6,048,695, U.S. Pat. No. 6,060,240, U.S. Pat. No. 6,090,556, and U.S. Pat. No. 6,040,138, each of which is hereby incorporated in its entirety.

[0053] For a DNA microarray, it may comprise an arrayed series of microscopic spots of DNA oligonucleotides, known as probes. This can be a short section of a gene or other DNA element that are used to hybridize a complementary nucleic acid sample (called target) under stringent conditions. Target molecules in a biological sample are usually detected and/or quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets hybridized on microarray. Since an array can contain several to tens of thousands of probes, a microarray experiment can accomplish many tests in parallel,

[0054] The systems described herein may comprise two or more probes that detect the same target nucleic acid, For example, in some embodiments where the system is a microarray, the probes may be present in multiple (such as any of 2, 3, 4, 5, 6, 7, or more) copies on the microarray. In some embodiments, the system comprises different probes that detect the same target nucleic acid. For example, these probes may bind to different (overlapping or

nonoverlapping) regions of the target nucleic acid.

[0055] Any probes that are capable of determining the levels of target nucleic acid can be used, in some embodiments, the probe may be an oligonucleotide, it is understood that, for detection of target nucleic acids, certain sequence variations are acceptable. Thus, the sequence of the oligon ucleotides (or their complementary sequences ) may be slightly different from those of the target nucleic acids described herein. Such sequence variations are understood by those of ordinary skill in the art to be variations in the sequence that do not significantly affect the ability of the oligonucleotide to determine target nucleic acid levels. For example, homologs and variants of these oligonucleotide molecules possess a relatively high degree of sequence identity when aligned using standard methods. Oligonucleotide sequences encompassed by the present invention have at least 40%, including for example at least about any of 50%, 60%, 70%, 80%, 90%, 95%, or more sequence identity to the sequence of the target nucleic acids described herein. In some embodiments, the oligonucleotide comprises a portion for detecting the target nucleic acids and another portion. Such other portion may be used, for example, for attaching the oligonucleotides to a substrate. In some embodiments, the other portion comprises a nonspecific sequence (such as poly-T or poly-dT) for increasing the distance between the

complementary sequence portion and the surface of the substrate.

[0056] The oligonucleotides for the systems described herein include, for example, DNA, RNA, PNA, ZNA, LNA, combinations thereof, and/or modified forms thereof. They may also include a modified oligonucleotide backbone. In some embodiments, the oligonucleotide comprises at least about any of 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more continuous oligonucleotides complementary or identical to all or part of target nucleic acids described herein, A single oligonucleotide may comprise two or more such complementary sequences. In some embodiments, there may be a reactive group (such as an amine) attached to the 5' or 3' end of the oligonucleotide for attaching the oligonucleotide to a substrate.

[0057] Several techniques are well-known in the art for attaching nucleic acids to a solid substrate such as a glass slide. One method is to incorporate modified bases or analogs that contain a moiety that is capable of attachment to a solid substrate, such as an amine group, a derivative of an amine group or another group with a positive charge, into the amplified nucleic acids. The amplified product is then contacted with a solid substrate, such as a glass slide, which may be coated with an aldehyde or another reactive group which can form a covalent link with the reactive group that is on the amplified product and become covalently attached to the glass slide. Microarrays comprising the amplified products can be fabricated using a Biodot (BioDot, Inc. Irvine, CA) spotting apparatus and aldehyde-coated glass slides (CEL Associates, Houston, TX). Amplification products can be spotted onto the aldehyde-coated slides, and processed according to published procedures (Schena et al, Proc. Natl. Acad. Sci. U.S.A. (1995), 93: 10614-10619). Arrays can also be printed by robotics onto glass, nylon (Ramsay, G., Nature Biotechnol. (1998), 16:40-44), polypropylene (Matson, et al., Anal Biochem, (1995),

224(1): 110-6), and silicone slides (Marshall and Hodgson, Nature Biotechnol. (1998), 16:27-31), Other approaches to array assembly include fine micropipetting within electric fields (Marshall, and Hodgson, Nature Biotechnol. (1998), 16:27-31), and spotting the polynucleotides directly onto positively coated plates. Methods such as those using amino propyl silicon surface chemistry are also known in the art, as disclosed at http://cmgm.stanford.edu/pbrown/.

[0058] In some embodiments, the real-time imaging module may comprise a light source and an optical detector which receives lights from the micro fluidic device that is being monitored, Any suitable light source may be used, such as natural light, chemical light, or electrical light. In some embodiments, the light source can be photodiode, fluorescent lamp, filament lamp or laser, and the optical detector can be charge coupled device (CCD) or photomultiplier tube (PMT). [0059] In some embodiments, the light from the light source may be reflected to the optical detector by the components of the microfluidic device, such as chemical or biological molecules involved in the microfluidic reaction. For example, the microfluidic reactions may comprise a biological molecule, e.g., DNA, RNA, protein, glycan, or a combination thereof, that are labeled with a fluorescent dye, which may be activated by the light from the light source and emit fluorescent light. The emitted fluorescent light may in turn be transmitted to the optical detector, in some embodiments, the chemical or biological molecule may be a fluorescent molecule, e.g., green fluorescent protein, that may be activated by the light from the light source and emit fluorescent light, In some embodiments, a lens may be located between the reflecting mirror and the optical detector, which may be used for focusing the light to the optical detector, [0060] in some embodiments, the optical detector of the real-time imaging module may record optical signals from a molecule of the microfluidic reaction, e.g., a PCR product, an antibody-antigen complex, a polynucleotide probe hybridized to a target molecule, or any other suitable reaction components, After recording the optical signals, the optical detector may produce an image from the optical signals, or multiple images from optical signals obtained at different time points, which may be further transformed into a video recording.

[0061] In some embodiments, the microfluidic channels in the microfluidic device may be connected to the peripheral flow module, through a conduit. The peripheral flow module may further comprises components, e.g., reservoirs, tubes, bottles, pumps, valves, pressure sources, etc., that control the fluidic flow in the microfluidic device. For example, the peripheral flow module may comprise a pressure source, such as a peristaltic pump, to pump solutions to and from the microfluidic device, so that a series of steps may be completed for the microfluidic reaction. The peripheral flow module may comprise a valve to control the fluidic flow among the connecting tubes or channels of the peripheral flow modul e,

[0062] In some embodiments, the pump can be a syringe pump or a plunger pump and may be connected with a step motor for the control of the fluid with micro-liter volume and thus providing accuracy control of reciprocating motion of the fluid (gas or liquid) in the channels, in certain embodiments, the valve can be a pneumatic valve, an electromagnetic valve or an electric valve, such as a pinch valve.

III. Methods of Using the System to Monitor Micro fluidic Reactions

[0063] in another aspect, the present disclosure provides methods for real-time monitoring of a micro fluidic reaction using the system disclosed herein, which method comprises providing a reagent to the microfluidic device, and monitoring the reaction using the real-time imaging module.

[0064] In some embodiments, the optical detector of the real-time imaging module may record optical signals from a molecule of the microfluidic reaction, e.g., a PCR product, an antibody-antigen complex, a polynucleotide probe hybridized to a target molecule, or any other suitable reaction components. After recording the optical signals, the optical detector may produce an image from the optical signals, or multiple images from optical signals obtained at different time points, which may be further transformed into a video recording. [0065] In some embodiments, the microfluidic reactions may be conducted by providing a biological sample to the microfluidic device, for example, in a reaction chamber for the reaction to occur. The biological sample may comprise a target molecule that reacts with a reagent that is immobilized in the reaction chamber. Any suitable biological sample may be used for the microfluidic reaction, e.g., body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples from animals and plants and processed samples derived therefrom.

[0066] The portion of the sample comprising or suspected of comprising the target molecules can be any source of biological material which comprises polynucleotides or polypeptides that can be obtained from a living organism directly or indirectly, including cells, tissue or fluid, and the deposits left by that organism, including viruses, mycoplasma, and fossils. The sample can also comprise a target molecule prepared through synthetic means, in whole or in part. Typically, the sample is obtained as or dispersed in a predominantly aqueous medium. Nonlimiting examples of the sample include blood, plasma, urine, semen, milk, sputum, mucus, a buccal swab, a vaginal swab, a rectal swab, an aspirate, a needle biopsy, a section of tissue obtained for example by surgery or autopsy, plasma, serum, spinal fluid, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, tumors, organs, samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components), and a recombinant source, e.g., a library, comprising polynucleotide sequences,

[0067] The sample can be a positive control sample which is known to contain the target molecule or a surrogate thereof, A negative control sample can also be used which, although not expected to contain the target molecule, is suspected of containing it, and is tested in order to confirm the lack of contamination by the target molecule of the reagents used in a given assay, as well as to determine whether a given set of assay conditions produces false positives (a positive signal even in the absence of target molecul e in the sample).

[0068] The sample can be diluted, dissolved, suspended, extracted or otherwise treated to solubilize and/or purify any target molecule present or to render it accessible to reagents which are used in an amplification scheme or to detection reagents. Where the sample contains ceils, the cells can be lysed or permeabilized to release the molecule within the cells.

Permeabiiization buffers can be used to lyse ceils which allow further steps to be performed directly after lysis, for example a polymerase chain reaction.

[0069] Any suitable physical, chemical or biological reactions may be monitored in realtime using the system disclosed herein. In certain embodiments, target molecules, e.g., nucleic acids, proteins, antibodies, small molecule compounds, peptides, and carbohydrates, from a biological sample may be used for the reactions. For example, the reactions may involve amplification of polynucleotides, e.g., polymerase chain reaction (PCR), reverse transcription PCR (RT-PCPv), asymmetric PCR, allele-specific (ASPCR); antibody binding assays, e.g., enzyme-linked immunosorbent assay (ELISA); hybridization of polynucleotides, or other suitable reactions known to one skilled in the art. The target molecules involved in the physical, chemical or biological reactions may be labeled for detection by the optical detector, for example, a fluorescent label, a radioactive label, a microparticle, a fluorophore, a silver-staining reagent, a ehemilumineseence reagent, an electrochemical reagent, or a nanoparticle, etc.

[0070] The nucleic acid target sequence (or "target polynucleotide") can be single-stranded, double-stranded, or higher order, and can be linear or circular. Exemplary single-stranded target polynucleotides include mRNA, rRNA, tRNA, hnRNA, ssRNA or ssDNA viral genomes and viroids, although these polynucleotides may contain internally complementary sequences and significant secondary structure. Exemplar}' double-stranded target polynucleotides include genomic DNA, mitochondrial DNA, chloroplast DNA, dsRNA or dsDNA viral genomes, plasmids, and phages. The target polynucleotide can be prepared synthetically or purified from a biological source. The target polynucleotide may be purified to remove or diminish one or more undesired components of the sample or to concentrate the target polynucleotide prior to amplification. Conversely, where the target polynucleotide is too concentrated for a particular assay, the target polynucleotide may first be diluted. [0071] Following sample collection and optional nucleic acid extraction and purification, the nucleic acid portion of the sample comprising the target polynucleotide can be subjected to one or more preparative treatments. These preparative treatments can include in vitro transcription (IVT), labeling, fragmentation, amplification and other reactions. mRNA can first be treated with reverse transcriptase and a primer, which can be the first primer comprising the target noncomplementary region, to create cDNA prior to detection and/or further amplification; this can be done in vitro with extracted or purified mRNA. or in situ , e.g,, in cells or tissues affixed to a slide. Nucleic acid amplification increases the copy number of sequences of interest and can be used to incorporate a label into an amplification product produced from the target polynucleotide using a labeled primer or labeled nucleotide. A variety of amplification methods are suitable for use, including the polymerase chain reaction method (PCR), transcription mediated amplification (TMA), the ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), the use of Q Beta replicase, reverse transcription, nick translation, and the like, particularly where a labeled amplification product can be produced and utilized in the methods taught herein.

[0072] Any nucleotides may be detected by the present devices and methods. Examples of such nucleotides include AMP, GMP, CMP, UMP, ADP, GDP, CDP, UDP, ATP, OTP, CTP, UTP, dAMP, dGMP, dCMP, dTMP, dADP, dGDP, dCDP, dTDP, clATP, dGTP, dCTP and dTTP. [0073] The target nucleic acid may be modified. In one embodiment, the modifications include a fluorescent label or a particle. The particle may be coated with a functional group. In another embodiment, the functional group is selected from the group consisting of streptavidin, neutravidin and avidin, In some embodiments, the target nucleic acid is coupled to the particle through an interaction between the modification and the functional group,

[0074] For the double-stranded target nucleic acid, they may be denatured by any suitable method, e.g., a chemical reaction, an enzymatic reaction or physical treatment such as heating, or a combination thereof. In some embodiments, the chemical reaction uses urea, formamide, methanol, ethanol, an enzyme, or sodium hydroxide (NaOH). In some embodiments, enzymatic methods include exonuclease and Uracil-N-giycosylase. in other embodiments, the double- stranded target nucleic acid is heat denatured at an appropriate temperature from about 30°C to about 95°C.

[0075] Employing PGR, RT-PCR (for RNA molecules) or other methods, nucleic acid molecules/agents of interest can be converted to nucleic acid fragments. For double-stranded nucleic acid fragments, they are denatured to single-stranded ones. Specific genes, S^'NPs or gene mutations, such as deletions, insertions, and indels, are thus identified. For

SNPs/niutations, they are valuable for biomedical research and for developing pharmaceutical compounds or medical diagnostics. SNPs are also evoiutionarily stable - not changing much from generation to generation - making them convenient to follow in population studies,

[0076] In some embodiments, the nucleic acid target sequence does not have a label directly incorporated in the sequence, When the nucleic acid target sequence is made with a directly incorporated label or so that a label can be directly bound to the nucleic acid target sequence, this label is one which does not interfere with detection of the capture probe conjugate substrate and/or the report moiety label.

[0077] Where the target polynucleotide is single-stranded, the first cycle of amplification forms a primer extension product complementary to the target polynucleotide, If the target polynucleotide is single-stranded RNA, a reverse transcriptase is used in the first amplification to reverse transcribe the RNA to DNA, and additional amplification cycles can be performed to copy the primer extension products. The primers for a PCR must, of course, be designed to hybridize to regions in their corresponding template that will produce an amplifiable segment: thus, each primer must hybridize so that its 3' nucleotide is base-paired with a nucleotide in its corresponding template strand that is located 3' from the 3' nucleotide of the primer used to prime the synthesis of the complementary template strand.

[0078] The target polynucleotide may be amplified by contacting one or more strands of the target polynucleotide with a primer and a polymerase having suitable activity to extend the primer and copy the target polynucleotide to produce a full-length complementary

polynucleotide or a smaller portion thereof. Any enzyme having a polymerase activity which can copy the target polynucleotide can be used, including DNA polymerases, RNA poiymerases, reverse transcriptases, enzymes having more than one type of polymerase activity. The polymerase can be thermolabile or thermostable. Mixtures of enzymes can also be used.

Exemplary enzymes include: DNA polymerases such as DN A Polymerase I ("Pol !"), the Klenow fragment of Pol I, T4, T7, Sequenase™ T7, Sequenase™ Version 2.0 T7, Tub, Taq, Tth, Pfx, Pfu, Tsp, Til, Tli and Pyrococcus sp GB-D DN A polymerases; RNA polymerases such as E. coli, SP6, T3 and T7 RNA polymerases; and reverse transcriptases such as AMV, M-MuLV, MMLV, RNAse H minus MMLV (Superscript™), Superscript™ II, ThermoScript™, HIV-1, and RAV2 reverse transcriptases. All of these enzymes are commercially available. Exemplary polymerases with multiple specificities include RAV2 and Tli (exo-) polymerases. Exemplary thermostable polymerases include Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli and Pyrococcus sp. GB- D DNA polymerases.

[0079] The reactions of the presen t invention may be implemented in a multipl ex format. Multiplex methods are provided employing 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000 or more different capture probes which can be used simultaneously to assay for amplification products from corresponding different target polynucleotides. In some embodiments, multiplex methods can also be used to assay for nucleic acid target sequences which have not undergone an amplification procedure. Methods amenable to multiplexing, such as those taught herein, allow acquisition of greater amounts of information from smaller specimens, The need for smaller specimens increases the ability of an investigator to obtain samples from a larger number of individuals in a population to validate a new assay or simply to acquire data, as less invasive techniques are needed.

[0080] Where different substrates are included in a multiplex assay as part of the capture probe conjugates, the different substrates can be encoded so that they can be distinguished. Any encoding scheme can be used; conveniently, the encoding scheme can employ one or more different fluorophores, which can be fluorescent semiconductor nanocrystals. High density spectral coding schemes can be used.

[0081] One or more different populations of spectrally encoded capture probe conjugates can be created, each population comprising one or more different capture probes attached to a substrate comprising a known or determinable spectral code comprising one or more

semiconductor nanocrystals or fluorescent nanoparticle. Different populations of the conjugates, and thus different assays, can be blended together, and the assay can be performed in the presence of the blended populations. The individual conjugates are scanned for their spectral properties, which allows the spectral code to be decoded and thus identifies the substrate, and therefore the capture probe(s) to which it is attached. Because of the large number of different semi conductor nanocrystals and fluorescent nanoparticles and combinations thereof which can be distinguished, large numbers of different capture probes and amplification products can be simultaneously interrogated.

[0082] In some embodiments, the reactions of the present disclosure involve the detection of target antibodies and/or antigens. The detection of antibodies and/or antigens may be achieved by immunoassays, including any immunoassay known in the art including, but not limited to, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich" assay, precipitin reaction, agglutination assay, fluorescent immunoassay, and chemiluminescence-based immunoassay. In some embodiments, the polypeptide-antibody complex may be assessed by a sandwich or competitive assay format, optionally with a binder or antibody. In some

embodiments, the binder or antibody may be attached to a surface and functions as a capture antibody, in some embodiments, the capture binder or antibody may be attached to the surface directly or indirectly. In some embodiments, the binder or antibody may be attached to the surface via a biotin-avidin (or streptavidin) linking pair. In some embodiments, at least one of the binders or antibodies may be labeled. In some embodiments, the polypeptide-antibody complex may be assessed by a format selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (1HA), complement fixation, indirect immunofluorescent assay (IF A), nephelometry, flow cytometry assay, plasmon resonance assay, chemi luminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay and avidity assay, In some embodiments, the polypeptide- antibody complex may be assessed in a homogeneous or a heterogeneous assay format.

[0083] In some embodiments, multiple reagents for detecting target antibodies and/or antigens may be included in the same assay, such as parallel immunoassay. A parallel immunoassay may include at least 2, 3, 4, 5, 10, 100, 1000 or more reagents, such as antibodies or antigenic polypeptides, in the same assay system.

[0084] Numerous technological platforms for performing parallel immunoassays are known. Generally, such methods involve a logical or physical array of either the subject samples, or the protein markers, or both. Common array formats include both liquid and solid phase arrays. For example, assays employing liquid phase arrays, e.g., for hybridization of nucleic acids, binding of antibodies or other receptors to ligand, etc., can be performed in multiwell or microti ter plates. Microtiter plates with 96, 384 or 1536 wells are widely available, and even higher numbers of wells, e.g., 3456 and 9600 can be used. In general, the choice of microtiter plates is determined by the methods and equipment, e.g., robotic handling and loading systems, used for sample preparation and analysis. Exemplary systems include, e.g., the ORCA^j;Vl system from Beckman- Couiter, Inc. (Fulierton, Calif.) and the Zymate systems from Zymark Corporation (Hopkinton, Mass.).

[0085] Alternatively, a variety of solid phase arrays can be employed for parallel

immunoassays in the context of the invention. Exemplary formats include membrane or filter arrays (e.g., nitrocellulose, nylon), pin arrays, and bead arrays (e.g., in a liquid "slurry").

Typically, probes corresponding to nucleic acid or protein reagents that specifically interact with (e.g., hybridize to or bind to) an expression product corresponding to a member of the candidate library, are immobilized, for example by direct or indirect cross- linking, to the solid support. Essentially any solid support capable of withstanding the reagents and conditions necessary for performing the particular expression assay can be utilized. For example, functionalized glass, silicon, silicon dioxide, modified silicon, any of a variety of polymers, such as

(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or

combinations thereof can all serve as the substrate for a solid phase array.

[0086] As used herein, "solid phase support" is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads and alumina gels. A suitable solid phase support may be selected on the basis of desired end use and suitability for various synthetic protocols, For example, for peptide synthesis, solid phase support may refer to resins such as polystyrene (e.g., PAM -resin obtained from Bachem Inc., Peninsula Laboratories, etc), PQLYHIPE^ resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) or

poly dimethylacryl amide resin (obtained from Milligen/Biosearch, California). In a preferred embodiment for peptide synthesis, solid phase support refers to polydimethylacrylamide resin. IV. Methods of Using the System for Optimizing Reaction Conditions

[0087] in a further aspect, the present disclosure provides methods of using the real-time monitoring system to optimize the reaction conditions of the hybridization reaction. For example, according to the changes of the hybridized target molecules at different time points of the reaction and/or under different hybridization conditions such as temperature, fluidic flow, pH, concentration of a reagent, composition of reaction reagents, the conditions for microarray hybridization can be optimized, including the hybridization time, hybridization temperature, hybridization dynamic, washing flow velocity, washing time and the conditions for drying, etc.

[0088] In some embodiments, the method may comprise providing a reagent to the micro fluidic device; recording images of the reaction using the real-time imaging module at multiple time intervals; and comparing the images to determine the optimal reaction time. In some embodiments, the method may comprise providing multiple reagents to the microfluidic device; recording images of the reactions using the real-time imaging module with multiple reagents; and comparing the images to determine the optimal reagent.

[0089] Those skilled in the art would recognize that the integrated real-time monitoring system enables continued monitoring of reaction results without interrupting the reaction. The integrated peripheral flow module and temperature control module may be used to provide and/or remove biological samples, solutions and reagents and control temperature continuously, so that reaction results with different reaction conditions may be monitored and compared to identify optimal reaction conditions. In some embodiments, the reactions may be controlled according to the data obtained from the real-time imaging module, so that desirable reaction results may be achieved.

[0090] For example, optimal reaction conditions may be chosen to permit amplification of the target molecule, including pH, buffer, ionic strength, presence and concentration of one or more salts, presence and concentration of reactants and cefaclors such as nucleotides and magnesium and/or other metal ions, optional cosolvents, temperature, thermal cycling profile for amplification schemes comprising a polymerase chain reaction, and may depend in part on the polymerase being used as well as the nature of the sample. Cosolvents include formamide (typically at from about 2 to about 10%), glycerol (typically at from about 5 to about 10%), and DMSO (typically at from about 0.9 to about 10%). Techniques may be used in the amplification scheme in order to minimize the production of false positives or artifacts produced during amplification, These include "touchdown" PCR, hot-start techniques, use of nested primers, or designing PCR primers so that they form stem-loop structures in the event of primer-dimer formation and thus are not amplified. Techniques to accelerate PCR can be used, for example centrifugal PCR, which allows for greater convection within the sample, and comprising infrared heating steps for rapid heating and cooling of the sample. One or more cycles of amplification can be performed. An excess of one primer can be used to produce an excess of one primer extension product during PCR: preferably, the primer extension product produced in excess is the amplification product to be detected. A plurality of different primers may be used to amplify different regions of a particular polynucleotide within the sample. Where the amplification reaction comprises multiple cycles of amplification with a polymerase, as in PCR, it is desirable to dissociate the primer extension product(s) formed in a given cycle from their tempiate(s). The reaction conditions are therefore altered between cycles to favor such dissociation; typically this is done by elevating the temperature of the reaction mixture, but other reaction conditions can be altered to favor dissociation, for example lowering the salt concentration and/or raising the pH of the solution in which the double-stranded polynucleotide is dissolved. Although it is preferable to perform the dissociation in the amplification reaction mixture, the polynucleotides may be first isolated using any effective technique and transferred to a different solution for dissociation, then reintroduced into an amplification reaction mixture for additional amplification cycles. Exemplary Embodiments

[0091] The following examples are offered to illustrate but not to limit the invention.

Example 1

System for real-time monitoring of a micro fluidic reaction [0092] Figure 1 shows an exemplar}' embodiment of a hybridization system provided by this invention. This hybridization system contains a hybridization chamber constructed by a microarray chip (1), a chamber enclosure (2) and a cover slip (3). A temperature control (4) device is connected with the hybridization chamber for the use of the temperature control of the chamber. The inlet of the hybridization chamber is connected with an air bottle (5), a sample bottle (6) and two washing solution bottles (7 and 8). A first pinch valve (91) is located between the hybridization chamber and the air bottle (5). A second pinch valve (92) is located between the hybridization chamber and the sample bottle (6). A third pinch valve (93) is located between the hybridization chamber and the washing solution bottle (7). A fourth pinch valve (94) is located between the hybridization chamber and the washing solution bottle (8). The pinch valves control the open/close of the channels. The outlet of the hybridization chamber is connected with a plunger pump (10) and a waste solution bottle (12) through a three-way valve (1 1 ). The hybridizations system contains a real-time imaging module constructed by the LED (13) and CCD (14). The light from the LED (13) is reflected by the surface of microarray (1) and subsequently by the reflecting mirror (15) which forms a 45° angel with the microarray chip, and then goes in parallel to the microarray chip through a focusing lens (16), and finally is projected onto the surface of a CCD ( 14) for transforming the light intensities to the pixel values of an image. Then, images collected at time intervals are encoded into a video file.

Example 2

System for real-time monitoring of a micro fluidic reaction with a four- way valve [0093] Figure 2 shows an exemplar}_'- embodiment of a hybridization system, which is similar to the embodiment shown in Figure 1, except that: there is an additional peristaltic pump (17) between a three-way valve (1 1) and a waste solution bottle (12) in order to continually pump the washing solution through the hybridization chamber for washing or continually pump the air through the hybridization chamber for drying; there is no sample bottle and the sample is injected to the hybridization chamber before the experiment; and the pinch valves are replaced by a four- way valve (18) for switching of the air and the washing solutions,

[0094] In the above exemplary hybridization system, the hybridization chamber (2) can be connected with several washing solution bottles (7 and 8), the pump (17) can be a syringe pump connected with a step motor, the light source (13) can be a fluorescent lamp, a filament lamp or a laser, and the optical detector (14) can be a photomultiplier tube (PMT).

Example 3

Real-time monitoring of multiplexed analysis of SNPs/mutations related to hereditary hearing loss [0095] The following experimental protocol is based on the exemplary hybridization system shown in Figure 1. The hybridization samples were prepared by the Nine Deafness Gene Mutations Detection Kit (Microarray) from CapitalBio Corporation, including the following steps.

L Genomic DNA extraction

[0096] Genomic DNA was extracted from whole blood or blood spot on filter paper. The genomic DNA with all wild-type alleles in this experiment came from the kit. The probe pattern is shown in Figure 3 and the probe information is shown in Table 1.

2. PGR amplification

[0097] PCR amplification was carried out using the Nine Deafness Gene Mutations

Detection Kit. The template is genomic DMA with all wild-type alleles in a concentration of 10 ng/fiL. PCR master mix components are shown in Tables 2 and 3.

Table 2 PCR master mix A

PCR thermal cycle protoco

3. Purification of nucleic acids

[0098] The magnetic beads were mixed using the vortex mixer. 60 \iL of binding buffer and 9 _uL of magnetic beads solution were added to a 200 μΤ tube located on a magnetic frame, incubated for 15 s, and then the buffer was removed. The tube was taken away from the magnetic frame, 48 \iL of binding buffer was added to this tube, and then the buffer was mixed using a vortex mixer.

[0099] 24 μ,Τ of PCR master mix A and 24 μΐ. of PCR master mix B were added to the tube, mixed and incubated for 10 min. The tube was put on a magnetic frame for 15 s. The solution was removed and the tube was taken away from the magnetic frame, 180 μΤ of denaturing solution (0. IN NaOH) was added, mixed and mcubated for 10 min. The tube was put on a magnetic frame for 15 s, the buffer was removed, and then the tube was taken away from the magnetic frame.

[0100] 120 μΤ of hybridization solution was added to the tube, mixed, and then the tube was put on a magnetic frame for 15 s. The solution was removed and the tube w^ras taken away from the magnetic frame. 60 μΐ, of hybridization solution was added to the tube and the solution was mixed.

4. Hybridization, washing and diving

[0101] 1) The washing solutions were prepared. The solution in washing solution bottle (7) included SSC (0.3x) and SDS (0.1%). The solution in washing solution bottle (8) included SSC (0.06*)·

[0102] 2) The LED light source (13) and CCD camera (14) were switched on for the monitoring during the steps of hybridization, washing and drying.

[0103] 3) The hybridization solution containing genomic DNA was added to the sample bottle (6). The target temperature was set to 50 °C. The pinch valve (92) was turned on and the pinch valves (91 , 93 and 94) were turned off. The plunger pump (10) and the hybridization chamber were connected by switching the three-way valve (11). The hybridization solution was pumped to the hybridization chamber by controlling the plunger to move downwards.

[0104] 4) Dynamic hybridization. The target temperature was set to 50 °C. The pinch valve (92) was turned on and the pinch valves (91 , 93 and 94) were turned off. The plunger pump (10) and the hybridization chamber were connected by switching the three-way valve (11). The plunger was set to move in a reciprocated motion, thus controlling the sample to move in a reciprocated motion in the hybridization chamber for the dynamic hybridization.

[0105] 5) The surface of the microarray chip was washed using the solution in bottle (7). The target temperature was set to 25 °C. The pinch valve (93) was turned on and the pinch valves (91, 92 and 94) were turned off. The plunger pump (10) and the hybridization chamber were connected by switching the three-way valve (1 1 ). The solution in the bottle (7) was pumped through the hybridization chamber to the tube region between the plunger pump (10) and the three-way valve (1 1 ) by controlling the plunger to move downwards. The plunger pump (10) and the w^raste solution bottle (12) were connected by switching the three-way valve (11). The washing solution was pumped to the waste solution bottle (12) by controlling the plunger to move upwards.

[0106] 6) The surface of the microarray chip was washed using the solution in bottle (8). The target temperature was set to 25 °C. The pinch valve (94) was turned on and the pinch valves (91, 92 and 93) were turned off. The plunger pump (10) and the hybridization chamber were connected by switching the three-way valve (1 1 ). The solution in the bottle (8) was pumped through the hybridization chamber to the tube region between the plunger pump (10) and the three-way valve (1 1 ) by controlling the plunger to move downwards. The plunger pump (10) and the waste solution bottle (12) were connected by switching the three-way valve (11). The washing solution was pumped to the waste solution bottle (12) by controlling the plunger pump (10) to move upwards.

[0107] 7) The surface of the microarray chip was dried. The target temperature was set to 25 °C. The pinch valve (91 ) was turned on and the pinch valves (92, 93 and 94) were turned off. The plunger pump (10) and the hybridization chamber were connected by switching the three- way valve (1 1 ). Air was pumped into the bottle (5) through the hybridization chamber by controlling the plunger to move downwards. The plunger pump (10) and the waste solution bottle (12) were connected by switching the three-way valve (1 1), The plunger pump (10) moved upwards for resetting,

[0108] 8) The LED light source (13) and CCD camera (14) were switched off. A series of image or a video files were saved.

[0109] A series of hybridization images during the process were monitored by the imaging module. Figure 4(A) shows the microarray image at the beginning of the hybridization, Figure 4(B) shows the microarray image after 15 min of hybridization, Figure 4(C) shows the microarray image after the washing,

[0110] The above examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Many variations to those described abo ve are possible.

Since modifications and variations to the examples described above will be apparent to those of skill in this art, it is intended that this in vention be limited only by the scope of the appended claims.

Claims

Claims What is claimed is:

1. A system for real-time monitoring of a micro iluidie reaction, comprising:

a platform for holding a micro fluidic device;

a temperature control module;

a peripheral flow module; and

a real-time imaging module.

2. The system of claim 1, wherein the real-time imaging module comprises a light source and an optical detector.

3. The system of claim 2, wherein the light source is a photodiode, a fluorescent lamp, a filament lamp or a laser.

4. The system of claim 2, wherein the optical detector is a charge coupled device (CCD) or a photomuitiplier tube (PMT).

5. The system according to any one of claims 1-4, wherein the real-time imaging module comprises a reflecting surface.

6. The system of claim 5, wherein the real-time imaging module comprises a lens between the reflecting surface and the optical detector.

7. The system according to claim 5 or 6, wherein the light from the light source is reflected to the optical detector by the reflecting surface.

8. The system according to any one of claims 1-7, wherein the platform comprises a microfiuidic device.

9. The system of claim 8, wherein the light from the light source is reflected by the microfiuidic device to the optical detector.

10. The system according to claim 8 or 9, wherein the microfluidic device comprises a reaction chamber.

11. The system according to any one of claims 8-10, wherein the microfluidic device comprises an inlet and an outlet.

12. The system according to any one of claims 8-1 1, wherein the microfluidic device comprises a pneumatic microvalve.

13. The system of claim 12, wherein the pneumatic microvalve is controlled by a control channel.

14. The system according to any one of claims 8-13, wherein the peripheral flow module comprises a pressure source connected to the microfluidic device.

15. The system of claim 14, wherein the pressure source controls the pneumatic microvalve.

16. The system of claim 15, wherein the pressure source is a piston pump or a plunger pump.

17. The system according to any one of claims 8-16, wherein the peripheral flow module comprises a solution source connected to the microfluidic device through the inlet and/or outlet.

18. The system of claim 17, wherein the peripheral flow module comprises at least one pump providing air pressure and one valve controlling the fluid flow.

19. The system of claim 18, wherein the valve is an electromagnetic valve or an

20. A method for real-time monitoring of a microfluidic reaction using the system according to any one of claims 8-19, which method comprises:

providing a reagent to the microfluidic device; and

monitoring the reaction using the real-time imaging module.

21 . The method of claim 20, wherein the optical detector records an optical signal and transforms the optical signal to a pixel.

22. The method of claim 21, wherein the optical detector produces an image.

23. The method of claim 22, wherein multiple images are produced at multiple time intervals.

24. The method of claim 23, wherein the multiple images are transformed into a video recording.

25. The method according to any one of claims 20-24, comprising providing a biological sample to the microfluidic device.

26. The method of claim 25, wherein the reaction comprises binding of a target in the biological sample to a probe.

27. The method of claim 26, wherein the probe is immobilized in the reaction chamber of the microfluidic device.

28. The method according to any one of claims 25-27, wherein the optical detector detects a label.

29. The method according to any one of claims 26-28, comprising washing away non-targets in the biological sample.

30. The method according to any one of claims 20-29, comprising mixing a reaction mixture.

31. A method for optimizing a reaction time using the system according to any one of claims 8-19, which method comprises:

providing a reagent to the rnicrofiuidic device;

recording images of the reaction using the real-time imaging module at multiple time intervals; and

comparing the images to determine the optimal reaction time.

32. A method for optimizing a reagent using the system according to any one of claims 8-19, which method comprises:

providing multiple reagents to the rnicrofiuidic device;

recording images of the reactions using the real -time imaging module with multiple reagents; and

comparing the images to determine the optimal reagent.