Devices and methods for rapid nucleic acid amplification

Abstract

The present invention concerns a microfluidic device for rapid amplification of target nucleic acids comprising (a) a microfluidic chip comprising at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the flow channel, (b) a heating plate comprising two heating zones at constant temperature, wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate. It also concerns methods using the same.

Description

[0001] The present invention concerns a microfluidic device for rapid amplification of target nucleic acids comprising (a) a microfluidic chip comprising at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the flow channel (b) a heating plate comprising two heating zones at constant temperature, wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate.

[0002] The polymerase chain reaction (PCR) is the most common technic for nucleic acid amplification in clinical practice and is widely used in many applications such as infectious disease detection, tumor screening, forensic identification, genomic programs, food safety testing, and environmental monitoring. However, most PCR devices have inefficient heating and cooling ramp rates for the solution, which significantly limits their application.

[0003] PCR may be divided into 3 reactions (denaturation, annealing, and extension) occurring at 3 temperatures over 3 time periods each cycle. The PCR reaction requires repeated thermal cycles at different temperatures to perform denaturation and amplification (annealing and extension) of desoxyribonucleic acids (DNAs) for the number of target DNA sequences to increase exponentially. Typically, the PCR reaction will execute 30-40 cycles and will take between 1 and 4 hours, making it increasingly difficult to meet the demand for rapid clinical testing. Conventional PCR is performed in a volume from 20 µL to 50µL and cannot achieve ultra-fast ramping rate because of both the big reaction size and the large thermal resistance between the heating block and the reaction tube (with high thermal capacity).

[0004] Since the invention of PCR, many efforts have been made to reduce the required amplification time. Microfluidics devices provide a potential solution to improve PCR speed as they can perform ultra-high ramping rates due to low system thermal capacity. However, the sensitivity of the method is directly affected by the size of a PCR reactor, or the available nucleic acid templates.

[0005] To date, various microfluidic techniques have been applied for PCR. Two design methods have been reported for microfluidic PCR: one is to make the sample position fixed, and the temperature changed in a single heating zone; the other is to move the sample between multiple heating zones, and the temperature of each heating zone is fixed.

[0006] The microfluidic PCR with a fixed sample position cannot achieve rapid PCR because temperature changes take a long time. The second method consists in moving the fluid to circulate in multiple heating zones each with a fixed temperature and can be divided into two main types: convective PCR and flow-channel PCR.

[0007] With convective PCR, the heating source is placed under the PCR sample, and fluid heat conduction and convection are used to create the temperature gradient and fluid cycle, respectively. The fluid will thermally circulate in the temperature gradient area in the capillary to achieve PCR. However, the amplification efficiency is low as the solution cannot maintain a stable and prolonged constant temperature, and the PCR speed is limited by the speed of fluid convection.

[0008] The flow-channel PCR is where the sample flows inside a long (often serpentine) microchannel that is directly placed on multiple heaters with fixed temperatures. Because of the high surface to volume ratio of long microchannels, PCR efficiency is deteriorated due to an increased number of biomolecules adsorbed on the channel surface, resulting in reduced detection sensitivity.

[0009] The current amplification methods have the following problems: (i) most devices have slow solution temperature ramp rates, resulting in long amplification times, (ii) microfluidic devices have a reduced sensitivity, which limits potential in clinical applications.

[0010] Therefore, there is a current need for nucleic acid amplification techniques with both rapidity and a high sensitivity suitable for clinical tests.

[0011] The present invention meets these needs.SUMMARY OF THE INVENTION

[0012] The present invention relates to a microfluidic device for rapid amplification of target nucleic acids comprising: a) a microfluidic chip comprising at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the flow channel, b) a heating plate comprising two heating zones at constant temperature, wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate.

[0013] In some embodiments, the first heating zone is hotter, or can be heated at a temperature that is higher, than the second heating zone.

[0014] In some embodiments, the first heating zone is a denaturation zone and the second heating zone is an amplification zone.

[0015] In some embodiments, the temperature of the first heating zone is or can reach about 95°C and the temperature of the second heating zone is or can reach about 45C° to 72°C, for example about 50°C to 65°C, for example 52, 55, 57 or 59°C, and preferably about 60°C.

[0016] In some embodiments, the compartments of the microfluidic chip are delimited or separated by an interspacing fluid or liquid, for example mineral oil.

[0017] In some embodiments, the compartments of the microfluidic chip are delimited or separated by an interspacing solid material or plug.

[0018] Interspacing fluid, liquid, solid material or plug are movable with the sample inside the channel, as this will be detailed herein.

[0019] In some embodiments, the microfluidic device according to the invention further comprises at least one pump to repeatedly translate the sample compartment from one heating zone to the other.

[0020] In some embodiments, the sample compartment has a volume smaller than about 20 µL, preferably smaller than about 10 µL.

[0021] In some embodiments, the microfluidic device according to the invention comprises a plurality of flow channels, wherein each flow channel comprises (i) a sample compartment, optionally a washing compartment, optionally a reading compartment, (ii) oligonucleotides grafted on a capture surface that are specific to a certain target and (iii) at least two heating zones.

[0022] The present invention further relates to a method of rapid amplification of target nucleic acids comprising the steps of: a) providing the microfluidic device according to the invention, b) introducing a sample into the sample compartment, c) aligning the sample compartment with the first heating zone to effect denaturation, d) aligning the sample compartment with the second heating zone and the capture surface to effect amplification of the target nucleic acids, and e) repeating step c and step d so that the sample compartment go back and forth between the two heating zones to effect thermal cycling.

[0023] In some embodiments, the sample is introduced along with amplification reagents such as primers, deoxynucleotides (dNTPs), polymerase, buffers, and co-factors.

[0024] In some embodiments, all or part of dNTPs are fluorescent.

[0025] In some embodiments, the method according to the invention, further comprises a step of detection of amplified products on the second heating zone after each thermal cycle.

[0026] In some embodiments, said target nucleic acids are amplified simultaneously in a solid phase and a liquid phase.

[0027] In some embodiments, the amplification of target nucleic acids is multiplexed. The present invention further relates to a kit for rapid amplification of target nucleic acids comprising: a) a microfluidic chip, wherein said microfluidic chip comprises at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the at least one flow channel; b) a heating plate comprising at least two heating zones at constant temperature; and c) at least one vial. DETAILED DESCRIPTION OF THE INVENTION

[0028] The inventors designed a microfluidic device for rapid amplification of nucleic acids in which the sample is moved back and forth between two constant-temperature zones, and further comprises oligonucleotides grafted on a capture surface of the flow channel. Thus, the microfluidic device according to the invention combines the advantages of a solid-phase amplification and flow channel thermocyclers, and provides ultrafast, accurate, and stable temperature conditions for PCR. This is in sharp contrast with most microfluidic devices that enable liquid-phase amplification only, and comprise three to more temperature regions.

[0029] The heat transfer time of the sample on this device is very short. The sample size is also very small (preferably less than about 20 µL), which can reduce the reagent volume and reduce the cost per analysis. The microfluidic device described herein aims to achieve rapid nucleic acid amplification suitable for practical application, for example point-of-care tests.

[0030] Most rapid PCR methods have focused on shortening thermal cycle time. The methods described herein judiciously combine a solid phase and a liquid phase amplification resulting in an increased sensitivity. Moreover, the combination with labeled or fluorescent dNTPs leads to a rapid signal enhancement. Thus, a lower number of cycles are necessary for the detection of a target nucleic acid.Definitions

[0031] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art.

[0032] It is also understood that various implementations described herein can be utilized in combination with any other implementation described or disclosed, without departing from the scope of the present disclosure. Therefore, devices, elements, methods and / or processes according to certain implementations of the present disclosure can include, incorporate, or otherwise comprise properties, features, components, elements, steps, and / or the like described in other disclosed herein without departing from the scope of the present disclosure. Thus, reference to a specific feature in relation to one implementation should not be construed as being limited to applications only within that implementation.

[0033] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 5%.Microfluidic device

[0034] The present invention thus relates to a microfluidic device for rapid amplification of target nucleic acids comprising: a) a microfluidic chip comprising at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the flow channel, b) a heating plate comprising two heating zones at constant temperature, and wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate.

[0035] By "microfluidic device", it is meant an instrument that uses very small amounts of fluid on a microchip with small channel(s), generally microscale channel(s), to do certain laboratory tests. According to the invention, the microfluidic device comprises a microfluidic chip positioned on a heating plate. By "positioned" it is meant mounted, fixed or inserted. In some embodiments, the microfluidic chip is positioned above the heating plate.

[0036] The microfluidic device according to the invention comprises at least one flow channel allowing for a continuous and cyclic flow of the sample compartment back and forth between the two heating zones at constant temperature. This is in sharp contrast to conventional flow-channel microfluidic devices in which the sample flows in one direction inside a long and often serpentine channel, or to rotary (or circular) microfluidic devices in which the flow channel forms a loop.

[0037] According to the invention, the microfluidic chip comprises a mobile phase comprising a sample compartment, optionally at least one washing compartment and optionally a reading compartment, and a fixed phase comprising oligonucleotides grafted on a capture surface of the flow channel.

[0038] By "flow channel", it is meant a flow path through which a solution can flow. The at least one flow channel may be present in various sizes and forms, linear or not. In some embodiments, the flow channel is a serpentine channel. Preferably, the flow channel is linear. The diameter of said flow channel may vary or not. In some embodiments, the flow channel diameter or height is constant. In some embodiments, the flow channel diameter or height varies to form at least two reaction chambers separated by a thinner section of the flow channel. By "reaction chamber", it is meant a widen zone of the flow channel, wherein a reaction can occur (e.g a denaturation reaction or amplification reaction). Preferably, the flow channel according to the invention is fixed, whereas the sample compartment and the at least one washing compartment are mobile inside the flow channel. In some embodiments, the microfluidic device according to the invention comprises at least one flow channel. In some embodiments, the microfluidic device according to the invention comprises a plurality of flow channels, preferably in order to detect multiple targets. Said plurality of flow channels may be on the same plane and / or parallel. In some embodiments, two channels or two sets of channels each located in the same plane and in parallel, are superimposed with the heating plate interposed between the two channels or the two sets, the heating plate having at least two heating zones in regard with the corresponding compartments of the two channels or sets.

[0039] By "sample compartment", it is meant a space inside the flow channel capable to collect the sample and / or reagents necessary for the amplification reaction. In some embodiments, the sample compartment is a reaction chamber wherein a nucleic acid amplification can occur. In some embodiments, the microfluidic device according to the invention comprises an inlet in fluid communication with the flow channel via which the sample can be introduced in the sample compartment. In some embodiments, the microfluidic device according to the invention comprises an outlet in fluid communication with the flow channel via which the sample may exit.

[0040] By "sample", it is meant a nucleic acid extract of a cell(s), tissue, or organ taken directly from a biological specimen, human or animal subject, orcell(s) maintained in culture or from a cultured cell line, a cell lysate or cell extract, a solution containing one or more molecules derived from a cell, cellular material, or viral material (e.g. a polypeptide or nucleic acid). A sample may also be a nucleic acid extract of any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile, or cerebrospinal fluid) that may or may not contain host or pathogen cells, cell components, or nucleic acids. Samples may also include a nucleic acid extract of an environmental samples such as, but not limited to, soil, water (fresh water, wastewater, etc.), air monitoring system samples (e.g., material captured in an air filter medium), surface swabs, and vectors (e.g., mosquitos, ticks, fleas, etc.).

[0041] By "washing compartment", it is meant a space inside the flow channel comprising a wash solution. Said wash solution (or wash buffer) may be any solution or buffer which are well known in the art to remove contaminants from the capture surface such as enzymes, deoxyribonucleosides triphosphate (dNTPs), nucleosides triphosphate (NTPs), and unbound nucleic acids. Accordingly, the wash solution may comprise water, wash buffer, detergent, salts, or any combination thereof. In some embodiments, the at least one washing compartment according to the invention washes the capture surface before each reading. In some embodiments, the at least one washing compartment according to the invention washes the capture surface at periodic intervals. In some embodiments, the at least one washing compartment according to the invention is placed between the sample compartment and the reading compartment. In some embodiments, the microfluidic chip according to the invention comprises more than one washing compartment to improve the reading of an amplification signal.

[0042] By "nucleic acid", it is meant a naturally occurring or synthetic polynucleotide, or polynucleotide chain comprising individual nucleic acid residues. Said nucleic acid may be DNA, ribonucleic acid (RNA) or DNA-RNA hybrid, and can include triple-, double-, and single-stranded molecules. In particular, nucleic acids can include, without limitation, DNA, RNA, messenger RNA (mRNA), ribosomal RNA (rRNA), complementary DNA (cDNA), genomic DNA (gDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), or any combination thereof. By "target nucleic acid", it is meant the nucleic acid one wish to amplify.

[0043] By "reading compartment", it is meant a space inside the flow channel comprising a reading fluid. Said reading fluid may be any gas or buffer known in the art to not interfere with the used detection mean. In some embodiments, the reading fluid is water. In some embodiments, the reading fluid is air. Preferably, the reading fluid is adapted to improve signal detection. In some embodiments, the reading fluid is a buffer identical to the wash buffer. In some embodiments, the reading compartment is also a washing compartment.

[0044] The microfluidic device according to the invention comprises a heating plate comprising at least two heating zones at constant temperature spaced along the flow channel. Preferably, a temperature controller regulates the temperature of each one of the heating zones. Said heating zones may be of different length. In some embodiments, said heating plate comprises two heating zones. In some embodiments, said heating plate comprises three heating zones.

[0045] By "heating plate", it is meant any device or element that can regulate temperature of the heating zones. The temperature of the heating zones may be controlled by any temperature controller known in the art including, but not limited to, a Peltier device, a heat exchanger, a resistance heater, an induction heater, an electromagnetic heater, a thin film heater, and combinations thereof. By "temperature controller", it is meant a device that adds heat to or removes heat from a sample.

[0046] According to the invention, oligonucleotides are grafted on a capture surface of the flow channel and aligned with the second heating zone to facilitate amplification reactions. By "oligonucleotides", it is meant a short nucleic acid, usually about 15 to 30 nucleotides long.

[0047] Said oligonucleotides may be grafted by any methods well-known in the art. In some embodiments, the oligonucleotides are grafted covalently to the capture surface. Said oligonucleotides may be primers. In one embodiment, only reverse primers are grafted on the capture surface. In another embodiment, only forward primers are grafted on the capture surface. In one embodiment, only sensitivity defining primers are grafted on the capture surface. By "sensitivity defining primers", it is meant oligonucleotides designed to increase the sensitivity of the PCR, i.e the probability of a positive detection of a nucleic acid target.

[0048] By "capture surface", it is meant the flow channel surface on which oligonucleotides, for example primers, are grafted allowing for solid phase amplification. According to the invention, the capture surface is aligned with the heating zone (e.g. the second heating zone), which is the amplification zone in which annealing and extension can occur. With such an approach, when the sample compartment is aligned on the second heating zone it is in the flow channel part comprising the capture surface, that annealing and extension can occur in both a liquid phase and a solid phase.

[0049] Said solid phase comprises the oligonucleotides grafted in the capture surface. Said liquid phase may further be a sample solution comprising free oligonucleotides. The combination of a liquid phase and a solid phase amplification accelerates and facilitates the solid-phase reaction and fewer amplification cycles are necessary for detection.

[0050] By "primer", it is meant a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA or RNA synthesis under appropriate conditions (i.e., in the presence of four different nucleosides triphosphates or deoxyribonucleosides triphosphate, and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. Said primer can base-pair to a second nucleic acid molecule that contains a complementary sequence (herein described as the "target nucleic acid"). In some embodiments, said primer can base-pair to a second nucleic acid molecule that contains a complementary sequence (herein described as the "target nucleic acid") by its 3' part, and its 5' part corresponds to a defined and unique sequence per target nucleic acid thereafter referred to as "unique tag". By "complementary", it is meant that one nucleic acid hybridizes selectively to, another nucleic acid molecule. Said primers may be forward or reverse primers. In some embodiments, the primers are solid-phase, they are grafted on the capture surface. In some embodiments, the primers are liquid-phase primers. In some embodiments both solid-phase and liquid-phase primers are present. In some embodiments, the primers grafted on the capture surface are reverse primers. In some embodiments, the primers grafted on the capture surface are forward primers. In some embodiments, the primers grafted on the capture surface are complementary to the unique tags. In some embodiments, the primers on the capture surface are complementary to internal parts of amplified targets in liquid-phase, allowing solid-phase / grafted extension and amplification with a second level of specificity. In some embodiments, the primers may comprise modified NTPs, or dNTPs. In some embodiments, the primers may comprise fluorescent NTPs, or dNTPs.

[0051] The appropriate length of a primer depends on the intended use of the primer but typically is at least 7 nucleotides long and, more typically range from 10 to 30 nucleotides in length. Other primers can be somewhat longer such as 30 to 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer needs not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term "primer site" or "primer binding site" refers to the segment of the target DNA to which a primer hybridizes. The term "primer pair" means a set of primers including a 5' upstream primer that hybridizes with the complement of the 5' end of the DNA sequence to be amplified and a 3' downstream primer that hybridizes with the 3' end of the sequence to be amplified. In some embodiments, primers are detectably-labeled, either radioactively, fluorescently, or non-radioactively, by methods well-known to those skilled in the art.

[0052] By "aligned", it is meant that a compartment, e.g the sample compartment or the washing compartment, a capture surface or oligonucleotides is / are placed inside the flow channel so that it is located in regard with a certain heating zone and can be heated or cooled to reach the temperature of said heating zone. Said flow channel can be placed above or under the heating plate, preferably above. By "aligned", it is also meant that a capture surface or oligonucleotides is / are placed inside the flow channel so that it is located in regard with the heating zone allowing annealing and / or elongation / extension, or amplification.

[0053] Traditional thermocycling devices comprise a heater that raises and lowers the temperature of a sample to perform a number of cycles of annealing, elongation (also described herein as extension), and denaturation. In contrast, the device according to the invention can achieve high speed thermal cycling because there is no need to ramp the temperature to the desired level as the temperature of the two heating zones is constant.

[0054] The temperature within the at least two heating zone is selected to promote the major processes involved in nucleic acids amplification, namely denaturation of the nucleic acids, annealing of primers to the target nucleic acid, and extension of the primers.

[0055] In some embodiments, the first heating zone is hotter than the second heating zone. In some embodiments, the first heating zone is a denaturation zone and the second heating zone is an amplification zone. In some embodiments, the first heating zone is a denaturation zone and the second heating zone is an annealing and extension zone. By "the first heating zone is a denaturation zone", it is meant that the first heating zone is set to a temperature that is optimal for denaturation of the nucleic acids. By "the second heating zone is an amplification zone" or "the second heating zone is an annealing and extension zone", it is meant that the second heating zone is set to a temperature that is optimal for annealing (or hybridization) and extension processes associated with the amplification reaction. In some embodiments, the second heating zone has a limited range of temperature from about 45°C to about 72°C and can be set to an annealing temperature from about 45°C to 72°C, for example from about 55°C to 70°C, for example about 56, 58, 60, 62, 64, 66, 68 or 70°C, and preferably about 60°C before being raised, or not, to an extension temperature from about 60°C to 72°C, for example about 60, 65, 70 or 72°C, and preferably about 72°C. It is understood that the annealing temperature cannot be above the extension temperature. In some embodiments, the temperature of the second heating zone is fixed at about 45°C to about 72°C, for example from about 55°C to 72°C, for example about 56, 58, 60, 62, 64, 66, 68, 70°C, and preferably about 60°C. In some embodiments, the temperature of the first heating zone is about 94 to about 98°C, preferably about 95°C and the temperature of the second heating zone is about 45°C to 72°C, for example about 50°C to 65°C, for example 52, 55, 57 or 59°C, and preferably about 60°C.

[0056] In some embodiments, the different compartments according to the invention are delimited by an intercalating fluid that is non-miscible with the content of the compartments such as an interspacing liquid, for example mineral oil. In some embodiments, the compartments are delimited by a solid such as a piston or plug that is movable inside the channel along with the sample, the wash solution or buffer, and / or the reading solution. In some embodiments, said intercalating fluid according to the invention washes the capture surface, preferably before each reading.

[0057] In some embodiments, the microfluidic device according to the invention further comprises at least one pump to repeatedly translate the sample compartment from one heating zone to another. By increasing or decreasing pressure inside the flow channel, said at least one pump allows the sample solution in the sample compartment to flow horizontally back and forth between the at least two heating zones while the flow channel remains fixed. However, a wide variety of other techniques can be utilized to move the sample compartment between the at least two heating zones. In some embodiments, the microfluidic device according to the invention comprises one reversible pump to push and pump the sample compartment inside the flow channel. In some embodiments, the microfluidic device according to the invention comprises a unidirectional pump in one extremity of the flow channel, which pushes the compartment(s) inside the flow channel. In such embodiments, the compartment(s) come back automatically to their original position when the pump is stopped. In some embodiments, the microfluidic device according to the invention comprises two pumps at each extremity of the flow channel, one pump being used to push the sample compartment in one direction, the other pump being used to push the sample compartment in the opposite direction.

[0058] In some embodiments, when the sample compartment is aligned with the first heating zone, the washing compartment is aligned with the second heating zone. When the sample compartment is translated to the second heating zone, the washing compartment moved away from the second heating zone allowing to wash the capture surface from any unbound molecules. It is possible to regulate how long the sample is exposed to each heating zones.

[0059] The device according to the invention allows reactions to be conducted with very small reaction volume. The flow channel according to the invention may vary in size and shape. In some embodiments, the flow channel is rectangular. In a preferred embodiment, the flow channel is cylindrical. In some embodiments, the diameter or height of the flow channel is constant. In some embodiments, the diameter or height of the flow channel is from about 0.1 µm to about 1 cm, or from about 1 µm to about 500µm, or from about 10 µm to about 100 µm, and is preferably about 100 µm. In some embodiments, the diameter or width is from about 0.1 µm to about 1 cm, or from about 1 µm to about 500µm, or from about 10 µm to about 200 µm, and is preferably about 200 µm. In some embodiments, some part of the flow channel is enlarged to form reaction chambers. The diameter or height of said reaction chambers may be from about 1 µm to about 1 cm, and is preferably from 10 µm to about 100 µm. In some embodiments, the two heating zones according to the invention have a length from about 0.1 cm to about 2 cm, preferably about 0.5 cm. In some embodiments, the sample compartment has a volume smaller than 20 µL, preferably smaller than 10 µL. In some embodiments, the sample compartment has a volume comprised from 10 nL to 20 µL, in particular from 10 nL to 10 µL, more particularly from 10 nL to 2.5 µL or from 10 nL to 1 µL.

[0060] In some embodiments, the device according to the invention is about the size of a credit card. In some embodiments, the device according to the invention has a thickness of less than 10 mm, less than 5 mm or less than 1 mm. Preferably, the device according to the invention has a thickness of less than 5 mm.

[0061] The device according to the invention may include a detection zone at which amplified products can be detected. By "detection zone", it is meant the portion of the microfluidic device at which detection occurs. This detection zone may include detectors that are incorporated into the device. As a variant, detectors are not incorporated into the device. In some embodiments, the detection zone is aligned with the second heating zone. In some embodiments, the detection zone is aligned with the amplification zone. In some embodiments, the detector is positioned to detect fluorescence from the amplified nucleic acids. In some embodiments, the detector can detect several fluorescence channels. In some embodiments, the detector can detect only one fluorescence channel.

[0062] In some embodiments, the microfluidic device according to the invention comprises a plurality of flow channels in order to amplify multiple target nucleic acids simultaneously in a multiplexing assay or serial of assays. In some embodiments, the microfluidic device according to the invention comprises a plurality of flow channels, wherein each flow channel comprises (i) a sample compartment, and optionally at least one washing compartment, optionally a reading compartment, (ii) oligonucleotides grafted on a capture surface that are specific to a certain target and (iii) at least two heating zones. In some embodiments, the plurality of flow channels are on the same plane and / or parallel. Said oligonucleotides may be complementary with different targets in separate flow channels. In some embodiments, each oligonucleotides in each flow channels are detected by a different fluorescent dye. In some embodiments, each oligonucleotides in each flow channels are detected by the same fluorescent dye. In some embodiments, each flow channel allows for the amplification of one specific target on the capture surface. Thus, as the location of each amplified target is known, only one fluorescent channel may be necessary to co-detect multiple targets. The number of targets are therefore limited to the multiplexing of oligonucleotides in solution, therefore allowing a dozen to thousands targets to be discriminated. This is very different from actual techniques that are limited to the simultaneously detection of only 4 or 5 targets as they need several fluorescent channels.

[0063] Amplification reactions that can be performed with the microfluidic device according to the invention include, but are not limited to, polymerase chain reaction (PCR), and other nucleic acid-based sequence amplification.Method of rapid amplification

[0064] The present invention further relates to a method of rapid amplification of target nucleic acids comprising the steps of: a) providing the microfluidic device according to the invention, b) introducing a sample into the sample compartment, c) aligning the sample compartment with a first heating zone to effect denaturation, d) aligning the sample compartment with a second heating zone and the capture surface to effect amplification of the target nucleic acids, and e) repeating step c) and step d) so that the sample compartment go back and forth between the at least two heating zones to effect thermal cycling.

[0065] While PCR is the amplification method described herein, it is understood that any amplification method that uses a primer may be suitable, in particular thermocycling amplification methods.

[0066] In some embodiments, the sample is introduced into the sample compartment along with one or more reactants. In some embodiments, the sample is introduced along with amplification reagents selected from, but not limited to, primers, dNTPs, NTPs, polymerase, buffers, dsDNA dye, and co-factors. In some embodiments, amplification reagents further comprise a dye specific for dsDNA. In some embodiments, said primers are only forward primers. In some embodiments, said primers are only reverse primers. There are four dNTP typically used for PCR reactions: dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate). In some embodiments, when the sample comprises RNA, the amplification reagents comprises NTPs (ATP, CTP, GTP, and UTP). Said dNTPs (or NTPs) may be modified dNTPs (or NTPs). The term "modified dNTPs" or "modified NTPs" refer to modification with respect to the four dNTPs (dATP, dGTP, dCTP, and dTTP) or NTPs (ATP, GTP, CTP, and UTP). Said modifications can include, for example, backbone modifications, sugar modifications or base modifications. Modified nucleotides can be synthesized by any useful method known in the art, such as chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleotides.

[0067] In some embodiments, at least one, at least two, or at least three dNTPs are labeled, especially fluorescent. In some embodiments, all dNTPs are labeled, especially fluorescent. In some embodiments, all dNTPs are linked to the same fluorochrome. In some embodiments, forward primers are fluorescent. In some embodiments, reverse primers are fluorescent.

[0068] Once the sample is introduced into the sample compartment of the microfluidic device, the sample is repeatedly move from one heating zone to the other at fixed temperature chosen to promote reaction between the sample and the one or more reactants within the sample compartment leading to the formation of an amplified product. The sample is exposed to several thermal cycle until an amplified product is formed and can be detected. By "thermal cycle" or "thermal cycling", it is meant the completion of the three phases involved in PCR reactions: denaturation, annealing and extension. Each thermal cycle may be completed by repeating step c and d of the method according to the invention.

[0069] In some embodiments, the amplified products can be detected after a lower number of thermal cycles than a conventional PCR. In some embodiments, the amplified products can be detected after less than 25 cycles. In some embodiments, the amplified products can be detected after only 10 to 20 thermal cycles. Reducing the number of thermal cycles to effectively detect a specific target significantly lowers the necessary amplification reaction time.

[0070] The sample is repeatedly exposed to the at least two heating zones in a cyclic fashion whereas the flow channel remains fixed. Thus, it allows for a high ramping rate as only the sample needs to be heated. This is very different from microfluidic devices in which the heater is translated between different reaction zones and which require the flow channel to heat up or cool down.

[0071] The method according to the invention further enables detection, and optionally quantification or semi-quantification, of target nucleic acids in a sample. In some embodiments, the method according to the invention further comprises a step of detection of amplified products on the second heating zone after each thermal cycle. In some embodiments, the method according to the invention further comprises a step of detection of amplified products on the second heating zone after a certain number of thermal cycle, for example from the first, from the second, from the third or more thermal cycle. The resulting amplified product can be detected according to the methods described herein. In some embodiments, the detection of amplified products is conducted in the second heating zone after the sample compartment have been translated from the second heating zone to the first heating zone. In particular, the detection step occurs when the sample compartment is not aligned with the second heating zone, allowing the detection to occur during the cycling process without affecting the duration of each step, and therefore accelerating the overall process compared to other traditional systems. Thus, the method according to the invention may allow for real-time amplification. By "real-time amplification", it is meant that the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each thermal cycle.

[0072] A wide variety of detection means can be used to detect the amplified product depending on the nature of the reactant and / or product being detected. The detection of amplified products can be performed by any physical, chemical, electromagnetic and other analytical techniques described in the art. In some embodiments, the method according to the invention uses labels such as radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, or molecules that emit chemiluminescence. Preferably, amplified products are detected by fluorescence. The detection methods include, but are not limited to, light scattering, multichannel fluorescence detection, UV and visible wave length absorption, luminescence, differential reflectivity, and confocal laser scanning. Applications can also utilize scintillation proximity assay techniques, radiochemical detection, fluorescence polarization, fluorescence correlation spectroscopy (FCS), time-resolved energy transfer (TRET), fluorescence resonance energy transfer (FRET) and variations such as bioluminescence resonance energy transfer (BRET), electrical resistance, resistivity, impedance, voltage sensing and surface plasmonic resonance (SPR).

[0073] One method for detecting the amplified product is the use of dsDNA binding dyes. By "dsDNA binding dyes", it is meant dyes that fluoresce differentially when bound to double-stranded DNA than when bound to single-stranded DNA or free in solution, usually by fluorescing more strongly. Suitable dyes may be SYBR ®< Green. Thus, as the amplification reaction progresses, an increasing amount of dye becomes bound and is accompanied by a concomitant increase in signal. Fluorescence detection is therefore indicative of an amplification of one (or more) target(s). When the sample compartment is aligned with the first heating zone, remaining fluorescence and its position(s) is indicative of the identity of the detected target(s) In some embodiments, labeled primers and / or nucleotides are utilized. Hence, product formed as the result of primer extension is labeled because of the labeled primer and / or the labeled nucleotides that are incorporated into the extension products. A wide variety of labels can be utilized to label the primer and / or nucleotides. In some embodiments, the amplified product is detected using fluorescence-tagged dNTP for base extension.

[0074] In some embodiments, said target nucleic acids are amplified simultaneously in a solid phase and a liquid phase. When the sample compartment is aligned with the second heating zone, in particular the amplification zone, and the capture surface, the steps of annealing and extension of the target nucleic acids can occur with the primers in the sample solution (liquid-phase amplification) as well as with the primers grafted on the capture surface (solid phase amplification). Thus, a positive signal can be more quickly detected.

[0075] In some embodiments, the signal enhancement acquired at each thermal cycle by the method according to the invention is significantly superior to the signal enhancement acquired by a PCR. In some embodiments, the signal enhancement acquired at each thermal cycle by the method according to the invention is superior by at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 200-fold than the signal enhancement acquired by a PCR, depending on the size of the extended targets. Thanks to the rapid signal enhancement, the method according to the invention allows for a faster detection of the presence of a target nucleic acid with a fewer number of thermal cycles.

[0076] In some embodiments, the method according to the invention is used to detect an amplified product and is completed in 25 minutes or less, 15 minutes or less, 5 minutes or less, or 1 minute or less. In some embodiments, each thermal cycle is completed in 10 seconds or less, or 5 seconds or less.

[0077] In some embodiments, the amplification of target nucleic acids is multiplexed. The method may be performed in a multiplexing format in which multiple target nucleic acids are simulteanously amplified. In some embodiments, each flow channel allows for the amplification of one specific target on the specific capture surface. In some embodiments, each oligonucleotide, for example each primer, in each flow channel are detected by a different fluorescent dye. In some embodiments, each oligonucleotide, for example each primer, in each flow channel are detected by the same fluorescent dye, which allows for the use of a single-channel detector.

[0078] In some embodiments, the kit according to the invention comprises: a) a microfluidic chip, wherein said microfluidic chip comprises at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and primers grafted on a capture surface of the at least one flow channel; b) a heating plate comprising two heating zones at constant temperature; c) at least one vial.

[0079] Said at least one vial may be chosen from a wash buffer, a reading buffer, amplification reagents, or an interspacing fluid or liquid. Said amplification reagents may be selected from, but not limited to, primers, dNTPs, NTPs, polymerase, buffers, co-factors, dsDNA dye, and any combinations thereof.

[0080] In some embodiments, the kit according to the invention comprises: a) a microfluidic chip, wherein said microfluidic chip comprises (i) at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, andprimers grafted on a capture surface of the at least one flow channel; b) a heating plate comprising two heating zones at constant temperature; c) a vial comprising a wash buffer; d) a vial comprising a reading buffer; e) a vial comprising amplification reagents; and f) a vial comprising an interspacing liquid.

[0081] In some embodiments, the reading buffer is identical to the wash buffer.Applications

[0082] The microfluidic device according to the invention and methods using the same can be used as an analytical tool to amplify a target nucleic acid potentially present in a sample and then to detect the amplified product to determine whether the target nucleic acid is present or absent in the sample. Amplification serves to enhance the ability to detect target nucleic acids present at low levels. Thus, the microfluidic device can be used in various diagnostic applications that involve a determination of whether a particular nucleic acid is present in a sample.

[0083] The microfluidic device and the method according to the invention may be used to detect and determine a wide variety of nucleic acid sequences from a wide variety of samples, including, human, veterinary, industrial, and environmental. Hence, samples can be tested for the presence of a particular nucleic acid associated with particular pathogens (e.g., certain viruses, bacteria or fungi), for identification purposes, such as in paternity and forensic cases, or to detect specific nucleic acids that are correlated with infectious diseases, genetic disorders or cellular disorders (e.g., oncogenes associated with cancer).FIGURES

[0084] Figure 1. Schematic representation of an exemplary microfluidic device with linear flow channel(s) for rapid amplification of target nucleic acids. Figure 2. Schematic representation of thermal cycling performed with the microfluidic device with linear flow channel(s). Figure 3. Schematic representation of thermal cycling performed with the microfluidic device with reaction chambers separated by a flow channel built as a serpentine in order to save space on the microfluidic chip. EXAMPLES

[0085] The figures and examples presented herein as only exemplary and demonstrative embodiments without limiting the scope of the present invention.

[0086] The device shown in Figure 1 is useful for conducting real-time PCR reactions. It comprises at least one flow channel 1 comprising a sample compartment 2, and optionally a reading compartment 3; and a heating plate 4 including a first heating zone 5 and a second heating zone 6. The sample compartment and the reading compartment may be delimited by an interspacing liquid 8. In-between these two compartments, a washing compartment 11 mays also be added, also delimited at each border by an interspacing liquid 8. Oligonucleotides are grafted on a capture surface 7 of the flow channel and aligned with the second heating zone. The device may comprise a detection device 9 aligned with the second heating zone and at least one pump 10 to repeatedly translate the sample compartment from one heating zone to the other.

[0087] Figure 2 represents nucleic acid amplification by thermal cycling using the method described herein. The sample compartment 2 is charged with all the reagents necessary for nucleic acid amplification such as nucleic acid target, forward and revers primers, polymerase, and fluorescent dNTPs. To improve the yield of solid-phase amplification, if reverse primers are grafted onto the capture surface 7 than a lower concentration of free / liquid-phase reverse primers are used compared to forward free / liquid-phase primers. During step n.1 of a thermal cycle n, the sample compartment is aligned with a first heating zone 5 at a constant denaturation temperature (e.g 95°C) that allows denaturation of the nucleic acid target, and the reading compartment 3 is aligned with a second heating zone 6 at a constant amplification temperature (e.g 60°C). Reverse primers are grafted on the capture surface 7 of the flow channel that is aligned with the second heating zone. A light excitation is emitted by the fluorescence detector 9 on the second heating zone. During the first cycle, no signal is detected as nucleic acid amplification has not yet occurred. During step n.2 of a thermal cycle n, the sample compartment is translated inside the flow channel 1 (e.g by the means of a pump 10) and aligned with the second heating zone at a constant amplification temperature which allows for both liquid-phase and solid-phase amplification of the nucleic acid target. During step 1 of the next cycle, (i) the sample compartment is moved back to the first heating zone to allow for denaturation of the nucleic acid target as well as the liquid-phase amplified products, (ii) the second heating zone is washed by the motion of the washing compartment to wash the capture surface from any unbound molecules, (iii) when the reading compartment is moved back to the second heating zone a light excitation is emitted by the fluorescence detector on the second heating zone to detect solid-phase amplified products. At each cycle, the signal is exponentially increased and thermal cycling is conducted until a significant signal is detected to determine the presence of a target nucleic acid.

[0088] The device shown in Figure 3 represents an alternative microfluidic chip according to the invention comprising at least one flow channel 1 comprising two reaction chambers separated by a serpentine.

[0089] During step N.1 of a thermal cycle N, the sample compartment 2 is aligned with a first heating zone 5 at a constant denaturation temperature (e.g 95°C), and the reading compartment 3 is aligned with a second heating zone 6 at a constant amplification temperature (e.g 60°C). Oligonucleotides are grafted on the capture surface 7 of the second reaction chamber of the flow channel that is aligned with the second heating zone 6. The sample compartment and the reading compartment may be delimited by an interspacing liquid 8. In-between these two compartments, a washing compartment may also be added, also delimited at each border by an interspacing liquid.

[0090] During step N.2 of a thermal cycle N , the sample compartment 2 is translated inside the serpentine part of the flow channel (e.g by the means of a pump) to the second reaction chamber and aligned with the second heating zone 6 at a constant amplification temperature which allows for both liquid-phase and solid-phase amplification of the nucleic acid target.

Examples

examples

EXAMPLES

[0085]The figures and examples presented herein as only exemplary and demonstrative embodiments without limiting the scope of the present invention.

[0086]The device shown in Figure 1 is useful for conducting real-time PCR reactions. It comprises at least one flow channel 1 comprising a sample compartment 2, and optionally a reading compartment 3; and a heating plate 4 including a first heating zone 5 and a second heating zone 6. The sample compartment and the reading compartment may be delimited by an interspacing liquid 8. In-between these two compartments, a washing compartment 11 mays also be added, also delimited at each border by an interspacing liquid 8. Oligonucleotides are grafted on a capture surface 7 of the flow channel and aligned with the second heating zone. The device may comprise a detection device 9 aligned with the second heating zone and at least one pump 10 to repeatedly translate the sample compartment from one heating zone to the other.

[0087]Figure 2 re...

[0001] The present invention concerns a microfluidic device for rapid amplification of target nucleic acids comprising (a) a microfluidic chip comprising at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the flow channel (b) a heating plate comprising two heating zones at constant temperature, wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate.

[0002] The polymerase chain reaction (PCR) is the most common technic for nucleic acid amplification in clinical practice and is widely used in many applications such as infectious disease detection, tumor screening, forensic identification, genomic programs, food safety testing, and environmental monitoring. However, most PCR devices have inefficient heating and cooling ramp rates for the solution, which significantly limits their application.

[0003] PCR may be divided into 3 reactions (denaturation, annealing, and extension) occurring at 3 temperatures over 3 time periods each cycle. The PCR reaction requires repeated thermal cycles at different temperatures to perform denaturation and amplification (annealing and extension) of desoxyribonucleic acids (DNAs) for the number of target DNA sequences to increase exponentially. Typically, the PCR reaction will execute 30-40 cycles and will take between 1 and 4 hours, making it increasingly difficult to meet the demand for rapid clinical testing. Conventional PCR is performed in a volume from 20 µL to 50µL and cannot achieve ultra-fast ramping rate because of both the big reaction size and the large thermal resistance between the heating block and the reaction tube (with high thermal capacity).

[0004] Since the invention of PCR, many efforts have been made to reduce the required amplification time. Microfluidics devices provide a potential solution to improve PCR speed as they can perform ultra-high ramping rates due to low system thermal capacity. However, the sensitivity of the method is directly affected by the size of a PCR reactor, or the available nucleic acid templates.

[0005] To date, various microfluidic techniques have been applied for PCR. Two design methods have been reported for microfluidic PCR: one is to make the sample position fixed, and the temperature changed in a single heating zone; the other is to move the sample between multiple heating zones, and the temperature of each heating zone is fixed.

[0006] The microfluidic PCR with a fixed sample position cannot achieve rapid PCR because temperature changes take a long time. The second method consists in moving the fluid to circulate in multiple heating zones each with a fixed temperature and can be divided into two main types: convective PCR and flow-channel PCR.

[0007] With convective PCR, the heating source is placed under the PCR sample, and fluid heat conduction and convection are used to create the temperature gradient and fluid cycle, respectively. The fluid will thermally circulate in the temperature gradient area in the capillary to achieve PCR. However, the amplification efficiency is low as the solution cannot maintain a stable and prolonged constant temperature, and the PCR speed is limited by the speed of fluid convection.

[0008] The flow-channel PCR is where the sample flows inside a long (often serpentine) microchannel that is directly placed on multiple heaters with fixed temperatures. Because of the high surface to volume ratio of long microchannels, PCR efficiency is deteriorated due to an increased number of biomolecules adsorbed on the channel surface, resulting in reduced detection sensitivity.

[0009] The current amplification methods have the following problems: (i) most devices have slow solution temperature ramp rates, resulting in long amplification times, (ii) microfluidic devices have a reduced sensitivity, which limits potential in clinical applications.

[0010] Therefore, there is a current need for nucleic acid amplification techniques with both rapidity and a high sensitivity suitable for clinical tests.

[0011] The present invention meets these needs.SUMMARY OF THE INVENTION

[0012] The present invention relates to a microfluidic device for rapid amplification of target nucleic acids comprising: a) a microfluidic chip comprising at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the flow channel, b) a heating plate comprising two heating zones at constant temperature, wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate.

[0013] In some embodiments, the first heating zone is hotter, or can be heated at a temperature that is higher, than the second heating zone.

[0014] In some embodiments, the first heating zone is a denaturation zone and the second heating zone is an amplification zone.

[0015] In some embodiments, the temperature of the first heating zone is or can reach about 95°C and the temperature of the second heating zone is or can reach about 45C° to 72°C, for example about 50°C to 65°C, for example 52, 55, 57 or 59°C, and preferably about 60°C.

[0016] In some embodiments, the compartments of the microfluidic chip are delimited or separated by an interspacing fluid or liquid, for example mineral oil.

[0017] In some embodiments, the compartments of the microfluidic chip are delimited or separated by an interspacing solid material or plug.

[0018] Interspacing fluid, liquid, solid material or plug are movable with the sample inside the channel, as this will be detailed herein.

[0019] In some embodiments, the microfluidic device according to the invention further comprises at least one pump to repeatedly translate the sample compartment from one heating zone to the other.

[0020] In some embodiments, the sample compartment has a volume smaller than about 20 µL, preferably smaller than about 10 µL.

[0021] In some embodiments, the microfluidic device according to the invention comprises a plurality of flow channels, wherein each flow channel comprises (i) a sample compartment, optionally a washing compartment, optionally a reading compartment, (ii) oligonucleotides grafted on a capture surface that are specific to a certain target and (iii) at least two heating zones.

[0022] The present invention further relates to a method of rapid amplification of target nucleic acids comprising the steps of: a) providing the microfluidic device according to the invention, b) introducing a sample into the sample compartment, c) aligning the sample compartment with the first heating zone to effect denaturation, d) aligning the sample compartment with the second heating zone and the capture surface to effect amplification of the target nucleic acids, and e) repeating step c and step d so that the sample compartment go back and forth between the two heating zones to effect thermal cycling.

[0023] In some embodiments, the sample is introduced along with amplification reagents such as primers, deoxynucleotides (dNTPs), polymerase, buffers, and co-factors.

[0024] In some embodiments, all or part of dNTPs are fluorescent.

[0025] In some embodiments, the method according to the invention, further comprises a step of detection of amplified products on the second heating zone after each thermal cycle.

[0026] In some embodiments, said target nucleic acids are amplified simultaneously in a solid phase and a liquid phase.

[0027] In some embodiments, the amplification of target nucleic acids is multiplexed. The present invention further relates to a kit for rapid amplification of target nucleic acids comprising: a) a microfluidic chip, wherein said microfluidic chip comprises at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the at least one flow channel; b) a heating plate comprising at least two heating zones at constant temperature; and c) at least one vial. DETAILED DESCRIPTION OF THE INVENTION

[0028] The inventors designed a microfluidic device for rapid amplification of nucleic acids in which the sample is moved back and forth between two constant-temperature zones, and further comprises oligonucleotides grafted on a capture surface of the flow channel. Thus, the microfluidic device according to the invention combines the advantages of a solid-phase amplification and flow channel thermocyclers, and provides ultrafast, accurate, and stable temperature conditions for PCR. This is in sharp contrast with most microfluidic devices that enable liquid-phase amplification only, and comprise three to more temperature regions.

[0029] The heat transfer time of the sample on this device is very short. The sample size is also very small (preferably less than about 20 µL), which can reduce the reagent volume and reduce the cost per analysis. The microfluidic device described herein aims to achieve rapid nucleic acid amplification suitable for practical application, for example point-of-care tests.

[0030] Most rapid PCR methods have focused on shortening thermal cycle time. The methods described herein judiciously combine a solid phase and a liquid phase amplification resulting in an increased sensitivity. Moreover, the combination with labeled or fluorescent dNTPs leads to a rapid signal enhancement. Thus, a lower number of cycles are necessary for the detection of a target nucleic acid.Definitions

[0031] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art.

[0032] It is also understood that various implementations described herein can be utilized in combination with any other implementation described or disclosed, without departing from the scope of the present disclosure. Therefore, devices, elements, methods and / or processes according to certain implementations of the present disclosure can include, incorporate, or otherwise comprise properties, features, components, elements, steps, and / or the like described in other disclosed herein without departing from the scope of the present disclosure. Thus, reference to a specific feature in relation to one implementation should not be construed as being limited to applications only within that implementation.

[0033] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 5%.Microfluidic device

[0034] The present invention thus relates to a microfluidic device for rapid amplification of target nucleic acids comprising: a) a microfluidic chip comprising at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and oligonucleotides grafted on a capture surface of the flow channel, b) a heating plate comprising two heating zones at constant temperature, and wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate.

[0035] By "microfluidic device", it is meant an instrument that uses very small amounts of fluid on a microchip with small channel(s), generally microscale channel(s), to do certain laboratory tests. According to the invention, the microfluidic device comprises a microfluidic chip positioned on a heating plate. By "positioned" it is meant mounted, fixed or inserted. In some embodiments, the microfluidic chip is positioned above the heating plate.

[0036] The microfluidic device according to the invention comprises at least one flow channel allowing for a continuous and cyclic flow of the sample compartment back and forth between the two heating zones at constant temperature. This is in sharp contrast to conventional flow-channel microfluidic devices in which the sample flows in one direction inside a long and often serpentine channel, or to rotary (or circular) microfluidic devices in which the flow channel forms a loop.

[0037] According to the invention, the microfluidic chip comprises a mobile phase comprising a sample compartment, optionally at least one washing compartment and optionally a reading compartment, and a fixed phase comprising oligonucleotides grafted on a capture surface of the flow channel.

[0038] By "flow channel", it is meant a flow path through which a solution can flow. The at least one flow channel may be present in various sizes and forms, linear or not. In some embodiments, the flow channel is a serpentine channel. Preferably, the flow channel is linear. The diameter of said flow channel may vary or not. In some embodiments, the flow channel diameter or height is constant. In some embodiments, the flow channel diameter or height varies to form at least two reaction chambers separated by a thinner section of the flow channel. By "reaction chamber", it is meant a widen zone of the flow channel, wherein a reaction can occur (e.g a denaturation reaction or amplification reaction). Preferably, the flow channel according to the invention is fixed, whereas the sample compartment and the at least one washing compartment are mobile inside the flow channel. In some embodiments, the microfluidic device according to the invention comprises at least one flow channel. In some embodiments, the microfluidic device according to the invention comprises a plurality of flow channels, preferably in order to detect multiple targets. Said plurality of flow channels may be on the same plane and / or parallel. In some embodiments, two channels or two sets of channels each located in the same plane and in parallel, are superimposed with the heating plate interposed between the two channels or the two sets, the heating plate having at least two heating zones in regard with the corresponding compartments of the two channels or sets.

[0039] By "sample compartment", it is meant a space inside the flow channel capable to collect the sample and / or reagents necessary for the amplification reaction. In some embodiments, the sample compartment is a reaction chamber wherein a nucleic acid amplification can occur. In some embodiments, the microfluidic device according to the invention comprises an inlet in fluid communication with the flow channel via which the sample can be introduced in the sample compartment. In some embodiments, the microfluidic device according to the invention comprises an outlet in fluid communication with the flow channel via which the sample may exit.

[0040] By "sample", it is meant a nucleic acid extract of a cell(s), tissue, or organ taken directly from a biological specimen, human or animal subject, orcell(s) maintained in culture or from a cultured cell line, a cell lysate or cell extract, a solution containing one or more molecules derived from a cell, cellular material, or viral material (e.g. a polypeptide or nucleic acid). A sample may also be a nucleic acid extract of any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile, or cerebrospinal fluid) that may or may not contain host or pathogen cells, cell components, or nucleic acids. Samples may also include a nucleic acid extract of an environmental samples such as, but not limited to, soil, water (fresh water, wastewater, etc.), air monitoring system samples (e.g., material captured in an air filter medium), surface swabs, and vectors (e.g., mosquitos, ticks, fleas, etc.).

[0041] By "washing compartment", it is meant a space inside the flow channel comprising a wash solution. Said wash solution (or wash buffer) may be any solution or buffer which are well known in the art to remove contaminants from the capture surface such as enzymes, deoxyribonucleosides triphosphate (dNTPs), nucleosides triphosphate (NTPs), and unbound nucleic acids. Accordingly, the wash solution may comprise water, wash buffer, detergent, salts, or any combination thereof. In some embodiments, the at least one washing compartment according to the invention washes the capture surface before each reading. In some embodiments, the at least one washing compartment according to the invention washes the capture surface at periodic intervals. In some embodiments, the at least one washing compartment according to the invention is placed between the sample compartment and the reading compartment. In some embodiments, the microfluidic chip according to the invention comprises more than one washing compartment to improve the reading of an amplification signal.

[0042] By "nucleic acid", it is meant a naturally occurring or synthetic polynucleotide, or polynucleotide chain comprising individual nucleic acid residues. Said nucleic acid may be DNA, ribonucleic acid (RNA) or DNA-RNA hybrid, and can include triple-, double-, and single-stranded molecules. In particular, nucleic acids can include, without limitation, DNA, RNA, messenger RNA (mRNA), ribosomal RNA (rRNA), complementary DNA (cDNA), genomic DNA (gDNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), or any combination thereof. By "target nucleic acid", it is meant the nucleic acid one wish to amplify.

[0043] By "reading compartment", it is meant a space inside the flow channel comprising a reading fluid. Said reading fluid may be any gas or buffer known in the art to not interfere with the used detection mean. In some embodiments, the reading fluid is water. In some embodiments, the reading fluid is air. Preferably, the reading fluid is adapted to improve signal detection. In some embodiments, the reading fluid is a buffer identical to the wash buffer. In some embodiments, the reading compartment is also a washing compartment.

[0044] The microfluidic device according to the invention comprises a heating plate comprising at least two heating zones at constant temperature spaced along the flow channel. Preferably, a temperature controller regulates the temperature of each one of the heating zones. Said heating zones may be of different length. In some embodiments, said heating plate comprises two heating zones. In some embodiments, said heating plate comprises three heating zones.

[0045] By "heating plate", it is meant any device or element that can regulate temperature of the heating zones. The temperature of the heating zones may be controlled by any temperature controller known in the art including, but not limited to, a Peltier device, a heat exchanger, a resistance heater, an induction heater, an electromagnetic heater, a thin film heater, and combinations thereof. By "temperature controller", it is meant a device that adds heat to or removes heat from a sample.

[0046] According to the invention, oligonucleotides are grafted on a capture surface of the flow channel and aligned with the second heating zone to facilitate amplification reactions. By "oligonucleotides", it is meant a short nucleic acid, usually about 15 to 30 nucleotides long.

[0047] Said oligonucleotides may be grafted by any methods well-known in the art. In some embodiments, the oligonucleotides are grafted covalently to the capture surface. Said oligonucleotides may be primers. In one embodiment, only reverse primers are grafted on the capture surface. In another embodiment, only forward primers are grafted on the capture surface. In one embodiment, only sensitivity defining primers are grafted on the capture surface. By "sensitivity defining primers", it is meant oligonucleotides designed to increase the sensitivity of the PCR, i.e the probability of a positive detection of a nucleic acid target.

[0048] By "capture surface", it is meant the flow channel surface on which oligonucleotides, for example primers, are grafted allowing for solid phase amplification. According to the invention, the capture surface is aligned with the heating zone (e.g. the second heating zone), which is the amplification zone in which annealing and extension can occur. With such an approach, when the sample compartment is aligned on the second heating zone it is in the flow channel part comprising the capture surface, that annealing and extension can occur in both a liquid phase and a solid phase.

[0049] Said solid phase comprises the oligonucleotides grafted in the capture surface. Said liquid phase may further be a sample solution comprising free oligonucleotides. The combination of a liquid phase and a solid phase amplification accelerates and facilitates the solid-phase reaction and fewer amplification cycles are necessary for detection.

[0050] By "primer", it is meant a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA or RNA synthesis under appropriate conditions (i.e., in the presence of four different nucleosides triphosphates or deoxyribonucleosides triphosphate, and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. Said primer can base-pair to a second nucleic acid molecule that contains a complementary sequence (herein described as the "target nucleic acid"). In some embodiments, said primer can base-pair to a second nucleic acid molecule that contains a complementary sequence (herein described as the "target nucleic acid") by its 3' part, and its 5' part corresponds to a defined and unique sequence per target nucleic acid thereafter referred to as "unique tag". By "complementary", it is meant that one nucleic acid hybridizes selectively to, another nucleic acid molecule. Said primers may be forward or reverse primers. In some embodiments, the primers are solid-phase, they are grafted on the capture surface. In some embodiments, the primers are liquid-phase primers. In some embodiments both solid-phase and liquid-phase primers are present. In some embodiments, the primers grafted on the capture surface are reverse primers. In some embodiments, the primers grafted on the capture surface are forward primers. In some embodiments, the primers grafted on the capture surface are complementary to the unique tags. In some embodiments, the primers on the capture surface are complementary to internal parts of amplified targets in liquid-phase, allowing solid-phase / grafted extension and amplification with a second level of specificity. In some embodiments, the primers may comprise modified NTPs, or dNTPs. In some embodiments, the primers may comprise fluorescent NTPs, or dNTPs.

[0051] The appropriate length of a primer depends on the intended use of the primer but typically is at least 7 nucleotides long and, more typically range from 10 to 30 nucleotides in length. Other primers can be somewhat longer such as 30 to 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer needs not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term "primer site" or "primer binding site" refers to the segment of the target DNA to which a primer hybridizes. The term "primer pair" means a set of primers including a 5' upstream primer that hybridizes with the complement of the 5' end of the DNA sequence to be amplified and a 3' downstream primer that hybridizes with the 3' end of the sequence to be amplified. In some embodiments, primers are detectably-labeled, either radioactively, fluorescently, or non-radioactively, by methods well-known to those skilled in the art.

[0052] By "aligned", it is meant that a compartment, e.g the sample compartment or the washing compartment, a capture surface or oligonucleotides is / are placed inside the flow channel so that it is located in regard with a certain heating zone and can be heated or cooled to reach the temperature of said heating zone. Said flow channel can be placed above or under the heating plate, preferably above. By "aligned", it is also meant that a capture surface or oligonucleotides is / are placed inside the flow channel so that it is located in regard with the heating zone allowing annealing and / or elongation / extension, or amplification.

[0053] Traditional thermocycling devices comprise a heater that raises and lowers the temperature of a sample to perform a number of cycles of annealing, elongation (also described herein as extension), and denaturation. In contrast, the device according to the invention can achieve high speed thermal cycling because there is no need to ramp the temperature to the desired level as the temperature of the two heating zones is constant.

[0054] The temperature within the at least two heating zone is selected to promote the major processes involved in nucleic acids amplification, namely denaturation of the nucleic acids, annealing of primers to the target nucleic acid, and extension of the primers.

[0055] In some embodiments, the first heating zone is hotter than the second heating zone. In some embodiments, the first heating zone is a denaturation zone and the second heating zone is an amplification zone. In some embodiments, the first heating zone is a denaturation zone and the second heating zone is an annealing and extension zone. By "the first heating zone is a denaturation zone", it is meant that the first heating zone is set to a temperature that is optimal for denaturation of the nucleic acids. By "the second heating zone is an amplification zone" or "the second heating zone is an annealing and extension zone", it is meant that the second heating zone is set to a temperature that is optimal for annealing (or hybridization) and extension processes associated with the amplification reaction. In some embodiments, the second heating zone has a limited range of temperature from about 45°C to about 72°C and can be set to an annealing temperature from about 45°C to 72°C, for example from about 55°C to 70°C, for example about 56, 58, 60, 62, 64, 66, 68 or 70°C, and preferably about 60°C before being raised, or not, to an extension temperature from about 60°C to 72°C, for example about 60, 65, 70 or 72°C, and preferably about 72°C. It is understood that the annealing temperature cannot be above the extension temperature. In some embodiments, the temperature of the second heating zone is fixed at about 45°C to about 72°C, for example from about 55°C to 72°C, for example about 56, 58, 60, 62, 64, 66, 68, 70°C, and preferably about 60°C. In some embodiments, the temperature of the first heating zone is about 94 to about 98°C, preferably about 95°C and the temperature of the second heating zone is about 45°C to 72°C, for example about 50°C to 65°C, for example 52, 55, 57 or 59°C, and preferably about 60°C.

[0056] In some embodiments, the different compartments according to the invention are delimited by an intercalating fluid that is non-miscible with the content of the compartments such as an interspacing liquid, for example mineral oil. In some embodiments, the compartments are delimited by a solid such as a piston or plug that is movable inside the channel along with the sample, the wash solution or buffer, and / or the reading solution. In some embodiments, said intercalating fluid according to the invention washes the capture surface, preferably before each reading.

[0057] In some embodiments, the microfluidic device according to the invention further comprises at least one pump to repeatedly translate the sample compartment from one heating zone to another. By increasing or decreasing pressure inside the flow channel, said at least one pump allows the sample solution in the sample compartment to flow horizontally back and forth between the at least two heating zones while the flow channel remains fixed. However, a wide variety of other techniques can be utilized to move the sample compartment between the at least two heating zones. In some embodiments, the microfluidic device according to the invention comprises one reversible pump to push and pump the sample compartment inside the flow channel. In some embodiments, the microfluidic device according to the invention comprises a unidirectional pump in one extremity of the flow channel, which pushes the compartment(s) inside the flow channel. In such embodiments, the compartment(s) come back automatically to their original position when the pump is stopped. In some embodiments, the microfluidic device according to the invention comprises two pumps at each extremity of the flow channel, one pump being used to push the sample compartment in one direction, the other pump being used to push the sample compartment in the opposite direction.

[0058] In some embodiments, when the sample compartment is aligned with the first heating zone, the washing compartment is aligned with the second heating zone. When the sample compartment is translated to the second heating zone, the washing compartment moved away from the second heating zone allowing to wash the capture surface from any unbound molecules. It is possible to regulate how long the sample is exposed to each heating zones.

[0059] The device according to the invention allows reactions to be conducted with very small reaction volume. The flow channel according to the invention may vary in size and shape. In some embodiments, the flow channel is rectangular. In a preferred embodiment, the flow channel is cylindrical. In some embodiments, the diameter or height of the flow channel is constant. In some embodiments, the diameter or height of the flow channel is from about 0.1 µm to about 1 cm, or from about 1 µm to about 500µm, or from about 10 µm to about 100 µm, and is preferably about 100 µm. In some embodiments, the diameter or width is from about 0.1 µm to about 1 cm, or from about 1 µm to about 500µm, or from about 10 µm to about 200 µm, and is preferably about 200 µm. In some embodiments, some part of the flow channel is enlarged to form reaction chambers. The diameter or height of said reaction chambers may be from about 1 µm to about 1 cm, and is preferably from 10 µm to about 100 µm. In some embodiments, the two heating zones according to the invention have a length from about 0.1 cm to about 2 cm, preferably about 0.5 cm. In some embodiments, the sample compartment has a volume smaller than 20 µL, preferably smaller than 10 µL. In some embodiments, the sample compartment has a volume comprised from 10 nL to 20 µL, in particular from 10 nL to 10 µL, more particularly from 10 nL to 2.5 µL or from 10 nL to 1 µL.

[0060] In some embodiments, the device according to the invention is about the size of a credit card. In some embodiments, the device according to the invention has a thickness of less than 10 mm, less than 5 mm or less than 1 mm. Preferably, the device according to the invention has a thickness of less than 5 mm.

[0061] The device according to the invention may include a detection zone at which amplified products can be detected. By "detection zone", it is meant the portion of the microfluidic device at which detection occurs. This detection zone may include detectors that are incorporated into the device. As a variant, detectors are not incorporated into the device. In some embodiments, the detection zone is aligned with the second heating zone. In some embodiments, the detection zone is aligned with the amplification zone. In some embodiments, the detector is positioned to detect fluorescence from the amplified nucleic acids. In some embodiments, the detector can detect several fluorescence channels. In some embodiments, the detector can detect only one fluorescence channel.

[0062] In some embodiments, the microfluidic device according to the invention comprises a plurality of flow channels in order to amplify multiple target nucleic acids simultaneously in a multiplexing assay or serial of assays. In some embodiments, the microfluidic device according to the invention comprises a plurality of flow channels, wherein each flow channel comprises (i) a sample compartment, and optionally at least one washing compartment, optionally a reading compartment, (ii) oligonucleotides grafted on a capture surface that are specific to a certain target and (iii) at least two heating zones. In some embodiments, the plurality of flow channels are on the same plane and / or parallel. Said oligonucleotides may be complementary with different targets in separate flow channels. In some embodiments, each oligonucleotides in each flow channels are detected by a different fluorescent dye. In some embodiments, each oligonucleotides in each flow channels are detected by the same fluorescent dye. In some embodiments, each flow channel allows for the amplification of one specific target on the capture surface. Thus, as the location of each amplified target is known, only one fluorescent channel may be necessary to co-detect multiple targets. The number of targets are therefore limited to the multiplexing of oligonucleotides in solution, therefore allowing a dozen to thousands targets to be discriminated. This is very different from actual techniques that are limited to the simultaneously detection of only 4 or 5 targets as they need several fluorescent channels.

[0063] Amplification reactions that can be performed with the microfluidic device according to the invention include, but are not limited to, polymerase chain reaction (PCR), and other nucleic acid-based sequence amplification.Method of rapid amplification

[0064] The present invention further relates to a method of rapid amplification of target nucleic acids comprising the steps of: a) providing the microfluidic device according to the invention, b) introducing a sample into the sample compartment, c) aligning the sample compartment with a first heating zone to effect denaturation, d) aligning the sample compartment with a second heating zone and the capture surface to effect amplification of the target nucleic acids, and e) repeating step c) and step d) so that the sample compartment go back and forth between the at least two heating zones to effect thermal cycling.

[0065] While PCR is the amplification method described herein, it is understood that any amplification method that uses a primer may be suitable, in particular thermocycling amplification methods.

[0066] In some embodiments, the sample is introduced into the sample compartment along with one or more reactants. In some embodiments, the sample is introduced along with amplification reagents selected from, but not limited to, primers, dNTPs, NTPs, polymerase, buffers, dsDNA dye, and co-factors. In some embodiments, amplification reagents further comprise a dye specific for dsDNA. In some embodiments, said primers are only forward primers. In some embodiments, said primers are only reverse primers. There are four dNTP typically used for PCR reactions: dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate). In some embodiments, when the sample comprises RNA, the amplification reagents comprises NTPs (ATP, CTP, GTP, and UTP). Said dNTPs (or NTPs) may be modified dNTPs (or NTPs). The term "modified dNTPs" or "modified NTPs" refer to modification with respect to the four dNTPs (dATP, dGTP, dCTP, and dTTP) or NTPs (ATP, GTP, CTP, and UTP). Said modifications can include, for example, backbone modifications, sugar modifications or base modifications. Modified nucleotides can be synthesized by any useful method known in the art, such as chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleotides.

[0067] In some embodiments, at least one, at least two, or at least three dNTPs are labeled, especially fluorescent. In some embodiments, all dNTPs are labeled, especially fluorescent. In some embodiments, all dNTPs are linked to the same fluorochrome. In some embodiments, forward primers are fluorescent. In some embodiments, reverse primers are fluorescent.

[0068] Once the sample is introduced into the sample compartment of the microfluidic device, the sample is repeatedly move from one heating zone to the other at fixed temperature chosen to promote reaction between the sample and the one or more reactants within the sample compartment leading to the formation of an amplified product. The sample is exposed to several thermal cycle until an amplified product is formed and can be detected. By "thermal cycle" or "thermal cycling", it is meant the completion of the three phases involved in PCR reactions: denaturation, annealing and extension. Each thermal cycle may be completed by repeating step c and d of the method according to the invention.

[0069] In some embodiments, the amplified products can be detected after a lower number of thermal cycles than a conventional PCR. In some embodiments, the amplified products can be detected after less than 25 cycles. In some embodiments, the amplified products can be detected after only 10 to 20 thermal cycles. Reducing the number of thermal cycles to effectively detect a specific target significantly lowers the necessary amplification reaction time.

[0070] The sample is repeatedly exposed to the at least two heating zones in a cyclic fashion whereas the flow channel remains fixed. Thus, it allows for a high ramping rate as only the sample needs to be heated. This is very different from microfluidic devices in which the heater is translated between different reaction zones and which require the flow channel to heat up or cool down.

[0071] The method according to the invention further enables detection, and optionally quantification or semi-quantification, of target nucleic acids in a sample. In some embodiments, the method according to the invention further comprises a step of detection of amplified products on the second heating zone after each thermal cycle. In some embodiments, the method according to the invention further comprises a step of detection of amplified products on the second heating zone after a certain number of thermal cycle, for example from the first, from the second, from the third or more thermal cycle. The resulting amplified product can be detected according to the methods described herein. In some embodiments, the detection of amplified products is conducted in the second heating zone after the sample compartment have been translated from the second heating zone to the first heating zone. In particular, the detection step occurs when the sample compartment is not aligned with the second heating zone, allowing the detection to occur during the cycling process without affecting the duration of each step, and therefore accelerating the overall process compared to other traditional systems. Thus, the method according to the invention may allow for real-time amplification. By "real-time amplification", it is meant that the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each thermal cycle.

[0072] A wide variety of detection means can be used to detect the amplified product depending on the nature of the reactant and / or product being detected. The detection of amplified products can be performed by any physical, chemical, electromagnetic and other analytical techniques described in the art. In some embodiments, the method according to the invention uses labels such as radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, or molecules that emit chemiluminescence. Preferably, amplified products are detected by fluorescence. The detection methods include, but are not limited to, light scattering, multichannel fluorescence detection, UV and visible wave length absorption, luminescence, differential reflectivity, and confocal laser scanning. Applications can also utilize scintillation proximity assay techniques, radiochemical detection, fluorescence polarization, fluorescence correlation spectroscopy (FCS), time-resolved energy transfer (TRET), fluorescence resonance energy transfer (FRET) and variations such as bioluminescence resonance energy transfer (BRET), electrical resistance, resistivity, impedance, voltage sensing and surface plasmonic resonance (SPR).

[0073] One method for detecting the amplified product is the use of dsDNA binding dyes. By "dsDNA binding dyes", it is meant dyes that fluoresce differentially when bound to double-stranded DNA than when bound to single-stranded DNA or free in solution, usually by fluorescing more strongly. Suitable dyes may be SYBR ®< Green. Thus, as the amplification reaction progresses, an increasing amount of dye becomes bound and is accompanied by a concomitant increase in signal. Fluorescence detection is therefore indicative of an amplification of one (or more) target(s). When the sample compartment is aligned with the first heating zone, remaining fluorescence and its position(s) is indicative of the identity of the detected target(s) In some embodiments, labeled primers and / or nucleotides are utilized. Hence, product formed as the result of primer extension is labeled because of the labeled primer and / or the labeled nucleotides that are incorporated into the extension products. A wide variety of labels can be utilized to label the primer and / or nucleotides. In some embodiments, the amplified product is detected using fluorescence-tagged dNTP for base extension.

[0074] In some embodiments, said target nucleic acids are amplified simultaneously in a solid phase and a liquid phase. When the sample compartment is aligned with the second heating zone, in particular the amplification zone, and the capture surface, the steps of annealing and extension of the target nucleic acids can occur with the primers in the sample solution (liquid-phase amplification) as well as with the primers grafted on the capture surface (solid phase amplification). Thus, a positive signal can be more quickly detected.

[0075] In some embodiments, the signal enhancement acquired at each thermal cycle by the method according to the invention is significantly superior to the signal enhancement acquired by a PCR. In some embodiments, the signal enhancement acquired at each thermal cycle by the method according to the invention is superior by at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 200-fold than the signal enhancement acquired by a PCR, depending on the size of the extended targets. Thanks to the rapid signal enhancement, the method according to the invention allows for a faster detection of the presence of a target nucleic acid with a fewer number of thermal cycles.

[0076] In some embodiments, the method according to the invention is used to detect an amplified product and is completed in 25 minutes or less, 15 minutes or less, 5 minutes or less, or 1 minute or less. In some embodiments, each thermal cycle is completed in 10 seconds or less, or 5 seconds or less.

[0077] In some embodiments, the amplification of target nucleic acids is multiplexed. The method may be performed in a multiplexing format in which multiple target nucleic acids are simulteanously amplified. In some embodiments, each flow channel allows for the amplification of one specific target on the specific capture surface. In some embodiments, each oligonucleotide, for example each primer, in each flow channel are detected by a different fluorescent dye. In some embodiments, each oligonucleotide, for example each primer, in each flow channel are detected by the same fluorescent dye, which allows for the use of a single-channel detector.

[0078] In some embodiments, the kit according to the invention comprises: a) a microfluidic chip, wherein said microfluidic chip comprises at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, and primers grafted on a capture surface of the at least one flow channel; b) a heating plate comprising two heating zones at constant temperature; c) at least one vial.

[0079] Said at least one vial may be chosen from a wash buffer, a reading buffer, amplification reagents, or an interspacing fluid or liquid. Said amplification reagents may be selected from, but not limited to, primers, dNTPs, NTPs, polymerase, buffers, co-factors, dsDNA dye, and any combinations thereof.

[0080] In some embodiments, the kit according to the invention comprises: a) a microfluidic chip, wherein said microfluidic chip comprises (i) at least one flow channel comprising a sample compartment, optionally at least one washing compartment, optionally a reading compartment, andprimers grafted on a capture surface of the at least one flow channel; b) a heating plate comprising two heating zones at constant temperature; c) a vial comprising a wash buffer; d) a vial comprising a reading buffer; e) a vial comprising amplification reagents; and f) a vial comprising an interspacing liquid.

[0081] In some embodiments, the reading buffer is identical to the wash buffer.Applications

[0082] The microfluidic device according to the invention and methods using the same can be used as an analytical tool to amplify a target nucleic acid potentially present in a sample and then to detect the amplified product to determine whether the target nucleic acid is present or absent in the sample. Amplification serves to enhance the ability to detect target nucleic acids present at low levels. Thus, the microfluidic device can be used in various diagnostic applications that involve a determination of whether a particular nucleic acid is present in a sample.

[0083] The microfluidic device and the method according to the invention may be used to detect and determine a wide variety of nucleic acid sequences from a wide variety of samples, including, human, veterinary, industrial, and environmental. Hence, samples can be tested for the presence of a particular nucleic acid associated with particular pathogens (e.g., certain viruses, bacteria or fungi), for identification purposes, such as in paternity and forensic cases, or to detect specific nucleic acids that are correlated with infectious diseases, genetic disorders or cellular disorders (e.g., oncogenes associated with cancer).FIGURES

[0084] Figure 1. Schematic representation of an exemplary microfluidic device with linear flow channel(s) for rapid amplification of target nucleic acids. Figure 2. Schematic representation of thermal cycling performed with the microfluidic device with linear flow channel(s). Figure 3. Schematic representation of thermal cycling performed with the microfluidic device with reaction chambers separated by a flow channel built as a serpentine in order to save space on the microfluidic chip. EXAMPLES

[0085] The figures and examples presented herein as only exemplary and demonstrative embodiments without limiting the scope of the present invention.

[0086] The device shown in Figure 1 is useful for conducting real-time PCR reactions. It comprises at least one flow channel 1 comprising a sample compartment 2, and optionally a reading compartment 3; and a heating plate 4 including a first heating zone 5 and a second heating zone 6. The sample compartment and the reading compartment may be delimited by an interspacing liquid 8. In-between these two compartments, a washing compartment 11 mays also be added, also delimited at each border by an interspacing liquid 8. Oligonucleotides are grafted on a capture surface 7 of the flow channel and aligned with the second heating zone. The device may comprise a detection device 9 aligned with the second heating zone and at least one pump 10 to repeatedly translate the sample compartment from one heating zone to the other.

[0087] Figure 2 represents nucleic acid amplification by thermal cycling using the method described herein. The sample compartment 2 is charged with all the reagents necessary for nucleic acid amplification such as nucleic acid target, forward and revers primers, polymerase, and fluorescent dNTPs. To improve the yield of solid-phase amplification, if reverse primers are grafted onto the capture surface 7 than a lower concentration of free / liquid-phase reverse primers are used compared to forward free / liquid-phase primers. During step n.1 of a thermal cycle n, the sample compartment is aligned with a first heating zone 5 at a constant denaturation temperature (e.g 95°C) that allows denaturation of the nucleic acid target, and the reading compartment 3 is aligned with a second heating zone 6 at a constant amplification temperature (e.g 60°C). Reverse primers are grafted on the capture surface 7 of the flow channel that is aligned with the second heating zone. A light excitation is emitted by the fluorescence detector 9 on the second heating zone. During the first cycle, no signal is detected as nucleic acid amplification has not yet occurred. During step n.2 of a thermal cycle n, the sample compartment is translated inside the flow channel 1 (e.g by the means of a pump 10) and aligned with the second heating zone at a constant amplification temperature which allows for both liquid-phase and solid-phase amplification of the nucleic acid target. During step 1 of the next cycle, (i) the sample compartment is moved back to the first heating zone to allow for denaturation of the nucleic acid target as well as the liquid-phase amplified products, (ii) the second heating zone is washed by the motion of the washing compartment to wash the capture surface from any unbound molecules, (iii) when the reading compartment is moved back to the second heating zone a light excitation is emitted by the fluorescence detector on the second heating zone to detect solid-phase amplified products. At each cycle, the signal is exponentially increased and thermal cycling is conducted until a significant signal is detected to determine the presence of a target nucleic acid.

[0088] The device shown in Figure 3 represents an alternative microfluidic chip according to the invention comprising at least one flow channel 1 comprising two reaction chambers separated by a serpentine.

[0089] During step N.1 of a thermal cycle N, the sample compartment 2 is aligned with a first heating zone 5 at a constant denaturation temperature (e.g 95°C), and the reading compartment 3 is aligned with a second heating zone 6 at a constant amplification temperature (e.g 60°C). Oligonucleotides are grafted on the capture surface 7 of the second reaction chamber of the flow channel that is aligned with the second heating zone 6. The sample compartment and the reading compartment may be delimited by an interspacing liquid 8. In-between these two compartments, a washing compartment may also be added, also delimited at each border by an interspacing liquid.

[0090] During step N.2 of a thermal cycle N , the sample compartment 2 is translated inside the serpentine part of the flow channel (e.g by the means of a pump) to the second reaction chamber and aligned with the second heating zone 6 at a constant amplification temperature which allows for both liquid-phase and solid-phase amplification of the nucleic acid target.

Examples

examples

EXAMPLES

[0085]The figures and examples presented herein as only exemplary and demonstrative embodiments without limiting the scope of the present invention.

[0086]The device shown in Figure 1 is useful for conducting real-time PCR reactions. It comprises at least one flow channel 1 comprising a sample compartment 2, and optionally a reading compartment 3; and a heating plate 4 including a first heating zone 5 and a second heating zone 6. The sample compartment and the reading compartment may be delimited by an interspacing liquid 8. In-between these two compartments, a washing compartment 11 mays also be added, also delimited at each border by an interspacing liquid 8. Oligonucleotides are grafted on a capture surface 7 of the flow channel and aligned with the second heating zone. The device may comprise a detection device 9 aligned with the second heating zone and at least one pump 10 to repeatedly translate the sample compartment from one heating zone to the other.

[0087]Figure 2 re...

Claims

1. A microfluidic device for rapid amplification of target nucleic acids comprising: a) a microfluidic chip comprising at least one flow channel (1) comprising a sample compartment (2), optionally at least one washing compartment (11), optionally a reading compartment (3), and oligonucleotides grafted on a capture surface (7) of the flow channel, b) a heating plate (4) comprising two heating zones (5) and (6) at constant temperature, wherein the capture surface (7) is aligned with the second heating zone (6), and wherein the microfluidic chip is positioned on the heating plate.

2. The microfluidic device according to claim 1, wherein the first heating zone (5) is hotter, or can be heated at a temperature that is higher, than the second heating zone (6).

3. The microfluidic device according to claim 1 or 2, wherein the first heating zone is a denaturation zone and the second heating zone is an amplification zone.

4. The microfluidic device according to any one of the preceding claims, wherein the temperature of the first heating zone is or can reach about 95°C and the temperature of the second heating zone is or can reach about 45°C to 72°C, for example about 50°C to 65°C, for example 52, 55, 57 or 59°C, and preferably about 60°C.

5. The microfluidic device according to any one of the preceding claims, wherein the compartments of the microfluidic chip are delimited or separated by (i) an interspacing fluid or liquid (8), for example mineral oil, or (ii) an interspacing solid material or plug.

6. The microfluidic device according to any one of the preceding claims, further comprising at least one pump (10) to repeatedly translate the sample compartment from one heating zone to the other.

7. The microfluidic device according to any one of the preceding claims, wherein the sample compartment has a volume smaller than about 20 µL, preferably smaller than about 10 µL.

8. The microfluidic device according to any one of the preceding claims comprising a plurality of flow channels, wherein each flow channel comprises (i) a sample compartment, optionally at least one washing compartment, and optionally one reading compartment, (ii) oligonucleotides grafted on a capture surface that are specific to a certain target in each channel and (iii) two heating zones.

9. A method of rapid amplification of target nucleic acids comprising the steps of: a) providing the microfluidic device according to any one of claims 1-8, b) introducing a sample into the sample compartment (2), c) aligning the sample compartment with the first heating zone (5) to effect denaturation, d) aligning the sample compartment with the second heating zone (6) and the capture surface (7) to effect amplification of the target nucleic acids, and e) repeating step c and step d so that the sample compartment go back and forth between the two heating zones to effect thermal cycling.

10. The method according to claim 9, wherein the sample is introduced along with amplification reagents such as primers, deoxynucleotides (dNTPs), polymerase, buffers, and co-factors.

11. The method according to claim 10, wherein all or part of dNTPs are fluorescent.

12. The method according to any one of claims 9 to 11, further comprising a step of detection of amplified products on the second heating zone after each thermal cycle.

13. The method according to any one of claims 9 to 12, wherein target nucleic acids are amplified simultaneously in a solid phase and a liquid phase.

14. The method according to any one of claims 9 to 13, wherein the amplification of target nucleic acids is multiplexed.

15. A kit for rapid amplification of target nucleic acids comprising: a) a microfluidic chip, wherein said microfluidic chip comprises at least one flow channel (1) comprising a sample compartment (2),optionally at least one washing compartment (11), optionally a reading compartment (3), and oligonucleotides grafted on a capture surface (7) of the at least one flow channel; b) a heating plate (4) comprising two heating zones (5) and (6) at constant temperature; and c) at least one vial.

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