[go: up one dir, main page]

WO2025224362A1 - Dispositifs et procédés d'amplification rapide d'acide nucléique avec détection facilitée de produits amplifiés - Google Patents

Dispositifs et procédés d'amplification rapide d'acide nucléique avec détection facilitée de produits amplifiés

Info

Publication number
WO2025224362A1
WO2025224362A1 PCT/EP2025/061585 EP2025061585W WO2025224362A1 WO 2025224362 A1 WO2025224362 A1 WO 2025224362A1 EP 2025061585 W EP2025061585 W EP 2025061585W WO 2025224362 A1 WO2025224362 A1 WO 2025224362A1
Authority
WO
WIPO (PCT)
Prior art keywords
heating zone
flow channel
sample
heating
amplification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/061585
Other languages
English (en)
Inventor
Jean-François BRUGERE
Stephen John MCCLEARY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Appolon Bioteck
Original Assignee
Appolon Bioteck
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Appolon Bioteck filed Critical Appolon Bioteck
Publication of WO2025224362A1 publication Critical patent/WO2025224362A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1883—Means for temperature control using thermal insulation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the present invention concerns a microfluidic device, configured for rapid amplification of target nucleic acids, the microfluidic device comprising (a) a microfluidic chip, comprising at least one flow channel configured to receive a sample and comprising a capture surface, and primers, that are grafted on the capture surface of the at least one flow channel, and (b) a heating plate, comprising two heating zones, including a first heating zone and a second heating zone, the heating plate being configured to heat the first heating zone at a first temperature that is constant, and the second heating zone at a second temperature that is constant and different to the first temperature, wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate.
  • PCR polymerase chain reaction
  • 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.
  • DNAs desoxyribonucleic acids
  • 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 pL to 50pL 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).
  • Microfluidics devices provide a potential solution to improve PCR speed as they can perform ultra-high ramping rates due to low system thermal capacity.
  • the sensitivity of the method is directly affected by the size of a PCR reactor, or the available nucleic acid templates. It has been tried to shorten the amplification and detection time by achieving a realtime (RT) detection. This implies either that a second reaction is added during each cycle of the PCR (e.g. hybridization onto a DNA array, as in US 2016/211698 or US 2011/0086361), and/or that the amplicons can be discriminated from native reagents in the reaction mix and differentially labeled to be distinguished from each other.
  • RT realtime
  • the detection rate is affected by the slow hybridization kinetics (e.g. about 2 min for a 50% yield of hybridization, according to the sequences, as shown in Zhang et al. (2017). Predicting DNA hybridization kinetics from sequence. Nature Chemistry, 10(1), 91-98. doi:10.1038/nchem.2877).
  • the differential labeling of amplicons requires not only expensive chemicals but also limits the multiplexing capability of testing, due to a limited number of fluorophores that can be efficiently discriminated (typically 5 with usual RT-PCR thermocyclers).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. Therefore, there is a current need for nucleic acid amplification techniques with both rapidity and a high sensitivity suitable for clinical tests.
  • the present invention meets these needs.
  • 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.
  • the present invention more preferably relates to microfluidic device, configured for rapid amplification of target nucleic acids, the microfluidic device comprising: a) a microfluidic chip, comprising: at least one flow channel configured to receive a sample and comprising a capture surface, and primers, that are grafted on the capture surface of the at least one flow channel, and b) a heating plate, comprising two heating zones, including a first heating zone and a second heating zone, the heating plate being configured to heat: the first heating zone at a first temperature that is constant, and the second heating zone (6) at a second temperature that is constant and different to the first 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.
  • the first temperature is higher than the second temperature.
  • the first heating zone is a denaturation zone and the second heating zone is an amplification zone.
  • the first heating zone is configurated to reach about 95°C and the second heating zone is configurated to 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.
  • the microfluidic chip further comprises in the flow channel an interspacing fluid or liquid, for example mineral oil.
  • the microfluidic chip further comprises in the flow channel an interspacing solid material or plug.
  • Interspacing fluid, liquid, solid material or plug are movable with the sample inside the flow channel, as this will be detailed herein.
  • the microfluidic device according to the invention further comprises at least one pump to repeatedly translate the sample from one heating zone to the other.
  • the microfluidic chip is configured to receive a sample having a volume smaller than about 20 pL, preferably smaller than about 10 pL.
  • the at least one flow channel comprises a plurality of flow channels, wherein each flow channel is configured to receive a sample and comprises primers, that are grafted on the capture surface of the considered flow channel, the primers being specific to certain target nucleic acids.
  • 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 goes back and forth between the two heating zones to effect thermal cycling.
  • the present invention more particularly 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 flow channel, c) aligning the sample with the first heating zone to effect denaturation, d) aligning the sample 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 goes back and forth between the two heating zones to effect thermal cycling.
  • the sample is introduced into the flow channel along with amplification reagents such as primers, deoxynucleotides (dNTPs), polymerase, buffers, and co-factors.
  • amplification reagents such as primers, deoxynucleotides (dNTPs), polymerase, buffers, and co-factors.
  • all or part of dNTPs are fluorescent.
  • the method according to the invention further comprises a step of detection of amplified products on the second heating zone after each thermal cycle.
  • said target nucleic acids are amplified simultaneously in a solid phase and a liquid phase.
  • 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.
  • the present invention more particularly relates to a kit for rapid amplification of target nucleic acids, the kit comprising: a) a microfluidic chip, wherein said microfluidic chip comprises: at least one flow channel configured to receive a sample and comprising a capture surface, and primers, that are grafted on the capture surface of the at least one flow channel; and b) a heating plate comprising two heating zones, including a first heating zone and a second heating zone, the heating plate being configured to heat: the first heating zone at a first temperature that is constant, and the second heating zone at a second temperature that is constant and different to the first temperature; and c) at least one vial.
  • 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 primers grafted on a capture surface of the flow channel.
  • 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.
  • the microfluidic device according to the invention is configured for rapid one-step amplification and real-time detection of nucleic acids.
  • the devices and methods presented herein facilitate the detection of amplified products by combining in a one-step process both the amplification and the detection of target nucleic acids (in real-time).
  • Primers grafted on the capture surface of the flow channel allows for a solid-phase PCR to be performed at the same time as a liquid-phase PCR.
  • the solid-phase amplified products are themselves detectable (in real time) as the liquid-phase is removed from this array at each cycle of polymerization, allowing the mix to contain detectable molecules (i.e. fluorescent primers, fluorescent dNTPs,...) without interfering with the reading of the array or the need of signals blockers, like quenchers of fluorochromes.
  • 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 pL), 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.
  • the present invention thus relates to a microfluidic device, configured for rapid amplification of target nucleic acids, the microfluidic device comprising: a) a microfluidic chip, comprising: at least one flow channel configured to receive a sample and comprising a capture surface, and primers, that are grafted on the capture surface of the at least one flow channel, and b) a heating plate, comprising two heating zones, including a first heating zone and a second heating zone, the heating plate being configured to heat: the first heating zone at a first temperature that is constant, and the second heating zone at a second temperature that is constant and different to the first temperature, wherein the capture surface is aligned with the second heating zone, and wherein the microfluidic chip is positioned on the heating plate.
  • the microfluidic device according to the invention is for rapid one-step amplification and real-time detection of target nucleic acids.
  • one step amplification and real-time detection it is meant that amplification and detection can be performed simultaneously in the same localisation or zone of the microfluidic device. This is in sharp contrast with other systems in which a first process of amplification such as a PCR reaction is performed before a second process of detection such as a microarray hybridization.
  • microfluidic device 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.
  • the microfluidic device comprises a microfluidic chip positioned on a heating plate.
  • positioned it is meant mounted, fixed or inserted.
  • the microfluidic chip is positioned above the heating plate.
  • the microfluidic device comprises at least one flow channel allowing for a continuous and cyclic flow of the sample back and forth between the two heating zones at constant temperature. This is in sharp contrast to conventional flowchannel 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.
  • the microfluidic chip is configured to receive a mobile phase comprising a sample, optionally at least one wash solution, and optionally a reading fluid, and a fixed phase comprising primers grafted on a capture surface of the flow channel.
  • 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.
  • the flow channel is a serpentine channel.
  • the flow channel is circular.
  • the flow channel is linear.
  • the diameter of said flow channel may vary or not.
  • the flow channel diameter or height is constant.
  • the flow channel diameter or height varies to form at least two reaction chambers separated by a thinner section of the flow channel.
  • 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).
  • the flow channel according to the invention is fixed, whereas the sample is mobile inside the flow channel.
  • the microfluidic device according to the invention comprises at least one flow channel.
  • 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.
  • 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 mobile phases of the two channels or sets.
  • 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, or cell(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 sample 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.).
  • the microfluidic device according to the invention comprises an inlet in fluid communication with the flow channel via which the sample can be introduced.
  • the microfluidic device according to the invention comprises an outlet in fluid communication with the flow channel via which the sample may exit.
  • the microfluidic chip is configured to receive at least one wash solution.
  • Said wash solution 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.
  • the wash solution may comprise water, wash buffer, detergent, salts, or any combination thereof.
  • the at least one wash solution according to the invention washes the capture surface before each reading.
  • the at least one wash solution according to the invention washes the capture surface at periodic intervals.
  • the at least one wash solution according to the invention is placed between the sample and the reading fluid.
  • the microfluidic chip according to the invention comprises more than one wash solution to improve the reading of an amplification signal.
  • nucleic acid 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.
  • 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.
  • target nucleic acid it is meant the nucleic acid one wish to amplify.
  • the microfluidic chip is configured to receive a reading fluid.
  • Said reading fluid may be any gas or buffer known in the art to not interfere with the used detection mean.
  • the reading fluid is water.
  • the reading fluid is air.
  • the reading fluid is adapted to improve signal detection.
  • the reading fluid is a buffer identical to the wash buffer.
  • the reading fluid is also a wash solution.
  • 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.
  • the microfluidic device according to the invention comprises at least one heating plate configured to heat a first heating zone at a first temperature that is constant and a second heating zone at a second temperature that is constant and different to the first temperature.
  • the microfluidic device according to the invention comprises a heating plate configured to heat a first heating zone at a first temperature that is constant and a second heating zone at a second temperature that is constant and different to the first temperature.
  • the microfluidic device comprises two heating plates, a first heating plate configured to heat a first heating zone at a first temperature that is constant and a second heating plate configured to heat a second heating zone at a second temperature that is constant and different to the first temperature.
  • a temperature controller regulates the temperature of each one of the heating zones. Said heating zones may be of different length.
  • said heating plate comprises two heating zones. In some embodiments, said heating plate comprises three heating zones.
  • the first heating zone is at a temperature that is constant for the amplification reaction, more particularly for the denaturation step of the amplification reaction.
  • the first heating zone could be at a constant temperature for a reverse transcriptase reaction taking place before the amplification reaction, in particular before the denaturation step of the amplification reaction, this constant temperature used for the reverse transcriptase reaction being the same or being different from the constant temperature used for the amplification reaction and in particular for the denaturation step.
  • 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.
  • temperature controller it is meant a device that adds heat to or removes heat from a sample.
  • 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.
  • a primer according to the invention is designed to prime polymerization of nucleotides, for example by solid-phase PCR or by primer extension. Said primer can base-pair to a second nucleic acid molecule that contains a complementary sequence (herein described as the “target nucleic acid”).
  • 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”.
  • target nucleic acid a complementary sequence
  • Said primers may be forward or reverse primers.
  • the primers are solid-phase, they are grafted on the capture surface.
  • the primers are liquid-phase primers. In some embodiments both solid-phase and liquid-phase primers are present.
  • 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.
  • primers are grafted on a capture surface of the flow channel and aligned with the second heating zone to facilitate amplification and detection reactions.
  • primers are grafted on the capture surface of the flow channel for priming polymerization of nucleotides and/or primer extension. Said primers may be grafted by any methods well-known in the art.
  • the primers are grafted covalently to the capture surface.
  • only reverse primers are grafted on the capture surface.
  • only forward primers are grafted on the capture surface.
  • only sensitivity defining primers are grafted on the capture surface.
  • 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.
  • capture surface it is meant the flow channel surface on which primers are grafted allowing for solid phase amplification.
  • 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.
  • the heating zone e.g. the second heating zone
  • the amplification zone in which annealing and extension can occur.
  • negative control primers and/or location markers such as a fluorescent dyes (e.g. fluorescein (FAM)) are also grafted on the capture surface.
  • FAM fluorescein
  • Said solid phase comprises the primers 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. It further allows the use of detectable molecules (i.e. fluorescent primers, fluorescent dNTPs, etc.) without interfering with the reading of the array or the need of signals blockers, like quenchers of fluorochromes.
  • the amplification products can also be detected in real-time.
  • primer length 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.
  • primer site or “primer binding site” refers to the segment of the target DNA to which a primer hybridizes.
  • 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.
  • primers are detectably-labeled, either radioactively, fluorescently, or non- radioactively, by methods well-known to those skilled in the art.
  • aligned it is meant that an element of the flow channel, e.g the sample or the wash solution, a capture surface or primers 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.
  • aligned it is also meant that a capture surface or primers 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.
  • 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.
  • 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.
  • 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.
  • the temperature of the first heating zone is higher than the temperature of the second heating zone.
  • the first heating zone is a denaturation zone and the second heating zone is an amplification zone.
  • the first heating zone is a denaturation zone and the second heating zone is an annealing and extension zone.
  • the first heating zone is a denaturation zone
  • the second heating zone is an annealing and extension zone.
  • 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.
  • the second heating zone is configured to reach 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.
  • the first heating zone is configured to reach about 94 to about 98°C, preferably about 95°C and the second heating zone is configured to 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.
  • the microfluidic chip further comprises an intercalating fluid that is non-miscible with the content of the flow channel such as an interspacing liquid, for example mineral oil.
  • the microfluidic chip further comprises a solid such as a valve, a tap, a piston or plug that is movable inside the flow channel along with the sample, the wash solution or buffer, and/or the reading solution. Said solid separates the different elements of the mobile phase inside the flow channel.
  • said intercalating fluid according to the invention washes the capture surface, preferably before each reading.
  • the microfluidic device according to the invention further comprises at least one pump to repeatedly translate the sample from one heating zone to another.
  • said at least one pump allows the sample solution to flow horizontally back and forth between the at least two heating zones while the flow channel remains fixed.
  • the microfluidic device according to the invention further comprises at least one piston to repeatedly translate the sample from one heating zone to another.
  • the microfluidic device according to the invention comprises one reversible pump to push and pump the sample inside the flow channel.
  • the microfluidic device according to the invention comprises a unidirectional pump in one extremity of the flow channel, which pushes the mobile phase(s) inside the flow channel. In such embodiments, the mobile phase(s) come back automatically to their original position when the pump is stopped.
  • 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 in one direction, the other pump being used to push the sample in the opposite direction. In some embodiments, no pumps or pistons are needed to move the sample inside the flow channel. In some embodiments, the sample circulates by convection flow.
  • the wash solution when the sample is aligned with the first heating zone, the wash solution is aligned with the second heating zone.
  • the wash solution is 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.
  • 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 pm to about 1 cm, or from about 1 pm to about 500pm, or from about 10 pm to about 100 pm, and is preferably about 100 pm. In some embodiments, the diameter or width is from about 0.1 pm to about 1 cm, or from about 1 pm to about 500pm, or from about 10 pm to about 200 pm, and is preferably about 200 pm. 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 pm to about 1 cm, and is preferably from 10 pm to about 100 pm.
  • 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.
  • the microfluidic chip is configured to receive a sample having a volume smaller than 20 pL, preferably smaller than 10 pL.
  • the microfluidic chip is configured to receive a sample having a volume comprised from 10 nL to 20 pL, in particular from 10 nL to 10 pL, more particularly from 10 nL to 2.5 pL or from 10 nL to 1 pL.
  • 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.
  • the device according to the invention may include a detection zone at which amplified products can be detected.
  • 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.
  • the detection zone is aligned with the second heating zone.
  • the detection zone is aligned with the amplification zone.
  • the detector is positioned to detect fluorescence from the amplified nucleic acids.
  • the detector can detect several fluorescence channels. In some embodiments, the detector can detect only one fluorescence channel.
  • the microfluidic device comprises a plurality of flow channels in order to amplify multiple target nucleic acids simultaneously in a multiplexing assay or serial of assays.
  • the at least one flow channel according to the invention comprises a plurality of flow channels, wherein each flow channel is configured to receive a sample and comprises primers, that are grafted on the capture surface of the considered flow channel, the primers being specific to a certain target.
  • the plurality of flow channels are on the same plane and/or parallel. Said primers may be complementary with different targets in separate flow channels.
  • each primer in each flow channel is detected by a different fluorescent dye.
  • each primer in each flow channel is detected by the same fluorescent dye.
  • each flow channel allows for the amplification of one specific target on the capture surface.
  • only one fluorescent channel may be necessary to co-detect multiple targets. The number of targets is therefore limited to the multiplexing of primers 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.
  • 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.
  • PCR polymerase chain reaction
  • 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 flow channel, c) aligning the sample with a first heating zone to effect denaturation, d) aligning the sample 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 goes back and forth between the at least two heating zones to effect thermal cycling.
  • 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.
  • the method according to the invention is a method of rapid real-time one-step amplification and detection of target nucleic acids.
  • the sample is introduced into the flow channel along with one or more reactants.
  • the sample is introduced along with amplification reagents selected from, but not limited to, primers, dNTPs, NTPs, polymerase, buffers, dsDNA dye, and co-factors.
  • amplification reagents further comprise a dye specific for dsDNA.
  • said primers are only forward primers. In some embodiments, said primers are only reverse primers.
  • the amplification reagents comprise NTPs (ATP, CTP, GTP, and UTP). Said dNTPs (or NTPs) may be modified dNTPs (or NTPs).
  • 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.
  • 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.
  • 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.
  • the method according to the invention effects extension of primers and/or amplification of the target nucleic acids simultaneously from the primers grafted on the capture surface and from free primers in the liquid phase.
  • 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.
  • the sample is repeatedly exposed to the at least two heating zones in a cyclic fashion whereas the flow channel remains fixed.
  • the flow channel remains fixed.
  • the method according to the invention further enables detection, and optionally quantification or semi-quantification, of target nucleic acids in a sample.
  • the method according to the invention further comprises a step of detection of amplified products on the second heating zone after each thermal cycle.
  • 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 cycles, 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.
  • the detection of amplified products is conducted in the second heating zone after the sample have been translated from the second heating zone to the first heating zone.
  • the detection step occurs when the sample 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.
  • the method according to the invention may allow for real-time amplification.
  • 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.
  • 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.
  • 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.
  • amplified products are detected by fluorescence.
  • the detection methods include, but are not limited to, light scattering, multichannel fluorescence detection, UV and visible wavelength 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).
  • 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).
  • dsDNA binding dyes dyes that fluoresce differentially when bound to doublestranded DNA than when bound to single-stranded DNA or free in solution, usually by fluorescing more strongly. Suitable dyes may be SYBR® Green.
  • Fluorescence detection is therefore indicative of an amplification of one (or more) target(s).
  • labeled primers and/or nucleotides are utilized.
  • 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.
  • labels can be utilized to label the primer and/or nucleotides.
  • the amplified product is detected using fluorescence-tagged dNTP for base extension.
  • said target nucleic acids are amplified simultaneously in a solid phase and a liquid phase.
  • 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).
  • a positive signal can be more quickly detected.
  • 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.
  • 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.
  • the amplification of target nucleic acids is multiplexed.
  • the method may be performed in a multiplexing format in which multiple target nucleic acids are simultaneously amplified.
  • each flow channel allows for the amplification of one specific target on the specific capture surface.
  • each primer, in each flow channel are detected by a different fluorescent dye.
  • each primer, in each flow channel are detected by the same fluorescent dye, which allows for the use of a single-channel detector.
  • the kit according to the invention comprises: a) a microfluidic chip, wherein said microfluidic chip comprises: at least one flow channel configured to receive a sample and comprising a capture surface , and
  • Primers that are grafted on the capture surface of the at least one flow channel; and b) a heating plate comprising two heating zones including a first heating zone and a second heating zone, the heating plate being configured to heat: the first heating zone at a first temperature that is constant, and the second heating zone at a second temperature that is constant and different to the first temperature; and c) at least one vial.
  • 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.
  • the kit according to the invention comprises: a) a microfluidic chip, wherein said microfluidic chip comprises (i) at least one flow channel configured to receive a sample, and primers grafted on a capture surface of the at least one flow channel; b) a heating plate comprising two heating zones including a first heating zone and a second heating zone, the heating plate being configured to heat: the first heating zone at a first temperature that is constant, and the second heating zone at a second temperature that is constant and different to the first temperature; and 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.
  • the reading buffer is identical to the wash buffer.
  • 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.
  • 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.
  • 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.
  • 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).
  • pathogens e.g., certain viruses, bacteria or fungi
  • identification purposes such as in paternity and forensic cases
  • specific nucleic acids that are correlated with infectious diseases, genetic disorders or cellular disorders (e.g., oncogenes associated with cancer).
  • 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.
  • Figure 4 Example of one microfluidic device for rapid specific detection of nucleic acids.
  • Figure 5 Illustration of the back and forth move of the sample across the different areas of the flow channel: the denaturation zone and the amplification zone.
  • FIG. 6 Schematic molecular description of the functioning of the invention in one of its embodiments.
  • A description of the principal molecular components used for rapid detection of a SARS-CoV 2 DNA target.
  • B illustration of results obtained on the capture surface.
  • Figure 7 Illustration of real-time results of the detection of the equivalent of 10 4 copies of SARS-CoV2 RNA genome with a flow channel encompassing an array of grafted primers.
  • the microfluidic device shown in Figure 1 is useful for conducting real-time PCR reactions. It comprises at least one flow channel 1 comprising a sample 2, and optionally a reading fluid 3; and a heating plate 4 including a first heating zone 5 and a second heating zone 6.
  • the sample and the reading fluid may be delimited by an interspacing liquid 8.
  • a wash solution 11 mays also be added, also delimited at each border by an interspacing liquid 8.
  • Primers 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 from one heating zone to the other.
  • Figure 2 represents nucleic acid amplification by thermal cycling using the method described herein.
  • the sample 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.
  • reagents necessary for nucleic acid amplification such as nucleic acid target, forward and revers primers, polymerase, and fluorescent dNTPs.
  • 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.
  • step N.1 of a thermal cycle N the sample 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 fluid 3 is aligned with a second heating zone 6 at a constant amplification temperature (e.g 60°C).
  • a constant denaturation temperature e.g 95°C
  • the reading fluid 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.
  • step N.2 of a thermal cycle N the sample is translated inside the flow channel 1 (e.g by the means of a pump 10) and aligned with the second heating zone 6 at a constant amplification temperature which allows for both liquidphase and solid-phase amplification of the nucleic acid target.
  • step 1 of the next cycle (i) the sample is moved back to the first heating zone 5 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 wash solution 11 to wash the capture surface from any unbound molecules, (iii) when the reading fluid 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.
  • 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.
  • 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.
  • step N.1 of a thermal cycle N the sample 2 is aligned with a first heating zone 5 at a constant denaturation temperature (e.g 95°C), and the reading fluid 3 is aligned with a second heating zone 6 at a constant amplification temperature (e.g 60°C).
  • Primers 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 and the reading fluid may be delimited by an interspacing liquid 8. In-between these two elements, a wash solution may also be added, also delimited at each border by an interspacing liquid.
  • step N.2 of a thermal cycle N the sample 2 is translated inside the serpentine part of the flow channel (e.g by the means of a pump 10) 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.
  • the device shown in Figures 4, 5 and 7 is one example dedicated to the rapid detection of SARS-CoV-2 virus, to illustrate some benefits of the present invention in a simple realization, here to rapidly detect SARS-CoV-2 virus with a NAAT (Nucleic Acid Amplification Testing).
  • the microfluidic chip was made by two pieces in polydimethylsiloxane (PDMS) 15 and 16, using molds fabricated by 3-dimensional (3D) printing using biocompatible material (Biomed, Formlabs). Then polydimethylsiloxane (PDMS) materials are used to make the replica structure.
  • the microfluidic flow channel has a first enlarged zone referred to as the upper chamber 17.
  • the upper chamber 17 is placed on a first heater made from a nichrome wire (NI80-012, OMEGA Engineering inc.) configured to heat a first heating zone 5, the wire being wrapped around a 3D-printed substrate that is attached to a customized aluminum block.
  • a first heater made from a nichrome wire (NI80-012, OMEGA Engineering inc.) configured to heat a first heating zone 5, the wire being wrapped around a 3D-printed substrate that is attached to a customized aluminum block.
  • the first heating zone 5 can be initially set at a temperature of 45°C to perform a reverse transcription reaction before beingraised and fixed at 95°C for the denaturation step of the PCR reaction.
  • the second heating zone 6 has a fixed temperature of 60°C.
  • an array of 3 x 3 spots (corresponding to the capture surface 7) has been designed with amino- 06 oligonucleotides attached to the surface via the amino linker. Attachment of the probes to the PDMS surface is performed via covalently binding. Briefly, the PDMS is subjected to oxygen plasma treatment and immersed in 5% (3-Aminopropyl) triethoxysilane solution. After washing, the surface is immersed in a 5% glutaraldehyde solution. Finally, the probes are spotted on their respective lanes.
  • oligonucleotide (Negative control (NegCTRL) primer, of sequence SEQ ID NO: 3: TCCAGCCTCATCTGCCAGGTCTACT with an amino- linker at its 5’ end: 5’ NH 2 -C6-TCCAGCCTCATCTGCCAGGTCTACT 3’).
  • NegCTRL Native control
  • the liquid reaction mix corresponds to a synthetic RNA control for SARS-CoV-2 (GenBank accession no. MN908947.3).
  • the liquid reaction mix encompasses a RT-PCR mix (here the SensiFASTTM Probe No-ROX One-Step kit from Meridian Bioscience) and primers specific to a part of the SARS-CoV-2 genome.
  • Reverse Transcription is performed in the upper chamber 17 at 45°C. Next, the temperature of the upper chamber 17 is increased to 95°C to allow polymerase activation for 1 min. Then, PCR amplification is performed at 95°C in the upper chamber 17 and then transferring the solution to the lower chamber 18 to perform annealing and extension at 60°C.
  • This back and forth move of the reaction mix is performed by regular mechanical pressure and release of a piston 19 placed above the upper chamber 17 ( Figure 5 at the upper left part).
  • This piston is lowered and enters in contact with the flexible upper layer part 16 of the microfluidic device. This exerts a pressure which pushes the liquid (the denatured PCR reaction mix) into the lower chamber 18 where primers are engrafted on the capture surface 7 ( Figure 5, right part).
  • DNA target SARS-CoV-2 cDNA
  • Figure 6 gives a schematic molecular description of the functioning of the invention in one of its embodiments.
  • Part A of Figure 6 highlights the principal molecular components used for rapid detection of a SARS-CoV 2 DNA target (with omission of enzyme and other components for clarity).
  • Part B illustrates the results of the reaction taking part in the lower chamber 18, and more specifically at the interface solid-liquid on the support, where SC2- Rv primers and NegCTRL primers are bound on the capture surface 7.
  • SC2- Rv primers and NegCTRL primers are bound on the capture surface 7.
  • This fluorescence gain on specific spots in the lower chamber 18 can be achieved for example using dsDNA binding dyes, or fluorescent dNTPs (upper right part of Figure 5B, resulting in much higher fluorescent gain at each new cycle) or, as illustrated lower part of Figure 5B and as shown on Figure 7, using a 5’ FAM- labelled SC2-Fw primer in the reaction mix, instead of a standard, non-fluorescent SC2-Fw primer.
  • Figure 7 is a concrete macro-illustration of the realization at various cycles during the reaction, instead of focusing solely on the 3 x 3 spots array corresponding to the capture surface 7.
  • the SensiFASTTM Probe No-ROX One-Step kit from Meridian Bioscience was used.
  • 16pl of RT-PCR master mix that includes 6pl of purified RNA (10,000 copies in total), is combined with 4pl of a solution of SC2-Fw primers of sequence SEQ ID NO: 4: GATCTCAATGGTAACTGGTATGATTTCGGTG labelled in 3’ with the fluorescent molecule FAM (FAM labelled SC2-FW: 5’ FAM- GATCTCAATGGTAACTGGTATGATTTCGGTG 3’) and of SC2-Rv primers of sequence SEQ ID NO: 2 (Seq: 5’ GCCCTGGTCAAGGTTAATATAGGCATTAAC 3’) mixed in equimolar concentrations (0.4pM).
  • SC2-Fw primers of sequence SEQ ID NO: 4: GATCTCAATGGTAACTGGTATGATTTCGGTG labelled in 3’ with the fluorescent molecule FAM (FAM labelled SC2-FW: 5’ FAM- GATCTCAATGGTAACTGGTATGATTTCGGTG 3’) and of SC2-Rv primers of sequence SEQ
  • primers are specific to the SARS-CoV-2 genome and should lead to a 119-bp amplicon of Orflab gene of SARS-CoV-2, in a standard RT- PCR assay.
  • Reverse Transcription was performed in the upper chamber 17 at 45°C.
  • the temperature of the upper was increased to 95°C to allow polymerase activation for 1 min.
  • PCR amplification was performed 5 seconds at 95°C in the upper chamber 17 and then transferring the solution to the lower chamber 18 to perform annealing and extension for 12 seconds at 60°C, by pressure of the piston. Cycles (lasting 17 seconds) were performed by back and forth move of the reaction mix caused by regular mechanical pressure and release of the piston 19 placed above the upper chamber17.
  • the 3 spots of control-signal probes are detected (corresponding to the lane 12 of three spots in Figures 4 and 5) using the FAM channel of a fluorescence microscope.
  • a picture is taken at cycles 5, 15, 25 and 35, when the piston 19 is raised.
  • the fluorescence is therefore detected at any cycle for these spots of FAM- control-signal probes in lane 12, but also in the denaturation zone (corresponding to the upper chamber 17) and around, corresponding to the zones where the liquid reaction mix stands at the time of picture acquisition, due to the presence of free FAM-labelled SC2-Fw primers and liquid-phase amplicons resulting from a FAM-labelled SC2-Fw / SC2-Rv PCR.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un dispositif microfluidique configuré pour l'amplification rapide d'acides nucléiques cibles, qui comprend : (a) une puce microfluidique, pourvue d'au moins un canal d'écoulement conçu pour recevoir un échantillon et comprenant une surface de capture, et des amorces, greffées sur la surface de capture du ou des canaux d'écoulement, et (b) une plaque chauffante, présentant deux zones de chauffage, à savoir une première zone de chauffage et une deuxième zone de chauffage, et qui est configurée pour chauffer la première zone de chauffage (5) à une première température constante, et la deuxième zone de chauffage à une deuxième température constante et différente de la première température, la surface de capture étant alignée avec la deuxième zone de chauffage, et la puce microfluidique étant positionnée sur la plaque de chauffage. L'invention concerne également des procédés les mettant en œuvre.
PCT/EP2025/061585 2024-04-26 2025-04-28 Dispositifs et procédés d'amplification rapide d'acide nucléique avec détection facilitée de produits amplifiés Pending WO2025224362A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP24305659.5A EP4640315A1 (fr) 2024-04-26 2024-04-26 Dispositifs et procédés d'amplification rapide d'acide nucléique
EP24305659.5 2024-04-26

Publications (1)

Publication Number Publication Date
WO2025224362A1 true WO2025224362A1 (fr) 2025-10-30

Family

ID=91073260

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/061585 Pending WO2025224362A1 (fr) 2024-04-26 2025-04-28 Dispositifs et procédés d'amplification rapide d'acide nucléique avec détection facilitée de produits amplifiés

Country Status (2)

Country Link
EP (1) EP4640315A1 (fr)
WO (1) WO2025224362A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110086361A1 (en) 2008-06-23 2011-04-14 Koninklijke Philips Electronics N.V. Amplification of nuceic acids using temperature zones
US20160211698A1 (en) 2015-01-21 2016-07-21 Eaton Corporation Battery Modules Having Detection Connectors and Related Systems
US20160251698A1 (en) * 2013-10-23 2016-09-01 Robert Bosch Gmbh Analysis Unit for Carrying Out a Polymerase Chain Reaction, Analysis Device, Method for Operating such an Analysis Unit, and Method for Producing such an Analysis Unit
US20200324292A1 (en) * 2016-03-29 2020-10-15 William Marsh Rice University Surface-based detection of nucleic acid in a convection flow fluidic device

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110086361A1 (en) 2008-06-23 2011-04-14 Koninklijke Philips Electronics N.V. Amplification of nuceic acids using temperature zones
US20160251698A1 (en) * 2013-10-23 2016-09-01 Robert Bosch Gmbh Analysis Unit for Carrying Out a Polymerase Chain Reaction, Analysis Device, Method for Operating such an Analysis Unit, and Method for Producing such an Analysis Unit
US20160211698A1 (en) 2015-01-21 2016-07-21 Eaton Corporation Battery Modules Having Detection Connectors and Related Systems
US20200324292A1 (en) * 2016-03-29 2020-10-15 William Marsh Rice University Surface-based detection of nucleic acid in a convection flow fluidic device

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG: "Predicting DNA hybridization kinetics from sequence", NATURE CHEMISTRY, vol. 10, no. 1, 2017, pages 91 - 98

Also Published As

Publication number Publication date
EP4640315A1 (fr) 2025-10-29

Similar Documents

Publication Publication Date Title
CA2682761C (fr) Procedes d'amplification multiplexe rapide d'acides nucleiques cibles
US20250050338A1 (en) Real-time polymerase chain reaction analysis plate
TWI539001B (zh) 快速多重擴增目標核酸的方法
EP2138587A1 (fr) Amplification d'acides nucléiques utilisant des zones de température
KR20120017033A (ko) 반응 카트리지에서의 다중 핵산 서열의 검출
JPH07506258A (ja) 微細加工装置を用いたポリヌクレオチド増幅分析
EP3006938B1 (fr) Procédé d'analyse quantitative et qualitative en temps réel d'une substance biologique
JP2023184748A (ja) 対流流体装置における核酸の表面ベースの検出方法
US20180135110A1 (en) Devices and methods to detect biomarkers using oligonucleotides
EP2231851A2 (fr) Système pour la détection d un pathogène biologique et utilisation de celui-ci
JP2006223309A (ja) 化学的な増幅反応のためのマクロ多孔質支持体及びその利用
JP2009034052A (ja) ハイブリダイゼーション方法および装置
US12186757B2 (en) Devices and methods for monitoring and quantifying nucleic acid amplification
EP2138232A1 (fr) Détection d'aliquotes taraudées d'acides nucléiques amplifiés
EP4640315A1 (fr) Dispositifs et procédés d'amplification rapide d'acide nucléique
JP2007043998A (ja) 改良されたマイクロ流体チップ
RU2423522C2 (ru) Способ гибридизации нк
WO2020225564A1 (fr) Amplification d'adn en phase solide et en phase soluble combinée
KR20250081630A (ko) 고감도 형광 다중분자진단용 미세유체칩
JP5258755B2 (ja) 捕捉プローブを含み、ｐｃｒ槽を開けることなしにハイブリダイゼーションによるｐｃｒ産物の検出を可能にするリアルタイムｐｃｒ用反応チェンバ
Seder et al. Fast and sensitive multiplexed diagnostic system enabled by real-time solid-phase PCR assay
JP2024536115A (ja) 核酸増幅生成物を試験する方法
WO2023046829A1 (fr) Détection d'acides nucléiques

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25721913

Country of ref document: EP

Kind code of ref document: A1