EP3645163A1

EP3645163A1 - Method for detecting nucleic acids

Info

Publication number: EP3645163A1
Application number: EP18752804.7A
Authority: EP
Inventors: Aurélien BANCAUD; Rémi MALBEC; Bayan CHAMI; Inga TIJUNELYTE
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 2017-06-30
Filing date: 2018-06-29
Publication date: 2020-05-06
Also published as: US20200114350A1; FR3068369A1; WO2019002800A1

Abstract

The present invention relates to a method for detecting a nucleic acid in a sample by separating and enriching nucleic acids using a bidirectional electrohydrodynamic stream in a non-Newtonian liquid medium, the method also comprising the injection of probes intended for hybridising with a target nucleic acid so as to introduce a modification of the molecular weight of the target nucleic acid upon hybridisation with the probes in order to allow the separation of the nucleic acid/probe complexes, of the nucleic acid alone or of the probes alone, when the sample is exposed to hydrodynamic and electric actuation to allow the detection of the target nucleic acid.

Description

Method for detecting nucleic acids

TECHNICAL AREA

The present invention relates to a method for detecting a nucleic acid, DNA or RNA, by nucleic acid separation and enrichment using a bidirectional electro-hydrodynamic flow in a non-Wonian liquid medium. STATE OF THE ART

The detection of nucleic acid sequences is fundamental in a wide range of biological applications such as in vitro diagnostics, clinical diagnosis, research ... In particular, the demonstration of the presence of a nucleic acid sequence specificity in a physiological sample is the first line of development of diagnostic methods.

Various methods and devices for detecting nucleic acids are known in the prior art, these techniques being based on the detection of a hybrid molecule consisting of the target nucleic acid molecule and a specific labeled probe.

For example, DNA recognition by molecular beacon, a conventional and validated method that makes it possible to detect point mutations on a fragment of 15-20 bases. However, this technique is insensitive because the beacon is never perfectly extinguished in the absence of a target so that the background noise can easily parasitize a signal when the target / beacon ratio is not correctly adjusted. Indeed, one of the crucial aspects in any method of nucleic acid detection is sensitivity. The most widely used methods for the detection of nucleic acids are based on the polymerase chain reaction (PCR). Real-time PCR, for example, is used to simultaneously amplify and quantify a targeted DNA molecule. PCR can also be applied to amplification of RNA, a process called reverse transcriptase PCR (RT-PCR). RT-PCR is similar to regular PCR, with the addition of an initial step in which DNA is synthesized from the RNA target using an enzyme called reverse transcriptase. A wide variety of RNA molecules have been used in RT-PCR, including ribosomal RNA, messenger RNA, and genomic viral RNA.

Amplification of the target sequence in these PCR-based techniques is time consuming, increases the likelihood of errors, and is highly susceptible to contamination. There is therefore a need to develop a method of detection of nucleic acids with high sensitivity, simple and quick to implement.

SUMMARY OF THE INVENTION Thus, the subject of the present invention is a method for detecting a nucleic acid in a sample by separation and enrichment of nucleic acids by using a bidirectional electro-hydrodynamic flow in a non-Newtonian liquid medium. method further comprising introducing probes for hybridizing to a target nucleic acid so as to introduce a change in the molecular weight of the target nucleic acid upon hybridization with the probes to allow separation of the acidic complexes nucleic / probes, nucleic acid alone or probes alone, when the sample is subjected to hydrodynamic and electrical actuation to allow detection of the target nucleic acid. The nucleic acid separation and enrichment technology using a bidirectional electro-hydrodynamic flow in a non-Newtonian liquid medium is described in particular in the article by Ranchon et al. : "DNA separation and enrichment using electro-hydrodynamic bidirectional flows in viscoelastic liquids".

The authors have indeed demonstrated that the nucleic acid separation and enrichment operations can be carried out by simultaneously using spatial modulations of the electric and flow fields via a constriction in a microfluidic channel or a capillary.

The technology involves the application of an electric field and a hydrodynamic flow in a non-Newtonian liquid medium contained in a channel. These forces increase the molecular weight of the nucleic acids and thus induce a gradual reduction in the nucleic acid migration rate, resulting in a size-dependent separation in one channel. The channel includes a constriction or funnel, for spatially modulating the hydrostatic and electric fields so as to stop the displacement of the nucleic acids at a predetermined location and to concentrate the nucleic acids at this determined position where they accumulate in a manner dependent on their molecular weights.

This method of concentration and separation by size is also described in the patent application WO 2016/016470.

Thus, the term "constriction" in the sense of the invention any section variation for spatially modulating the hydrostatic and electric fields so as to stop and concentrate the nucleic acids.

For a capillary, the constriction consists of a reduction of the diameter and is the ratio of the radii before and after the junction zone. Such constrictions are described on the following site: https://picometrics.com/piOduct/biabooster/. For a planar micro fluidic system with constant depth, it is the ratio of the widths at the beginning and at the end of the constriction. Such constrictions are described in the article by Ranchon et al., As well as in WO2016 / 016470. Examples of constrictions are shown in Figure 2 and its integration in a microfluidic system is shown in Figure 6. These examples are detailed below.

Other examples of constrictions, where the width and height of a pipe are modulated simultaneously, are also described in the article by Lettieri et al. : "A novel microfluidic concept for bioanalysis using freely moving beads trapped in recirculating flows".

The desired hydrodynamic flow profiles (characterized in particular by average flow rate and average speed values) are obtained by operating pressure control means, so as to generate a pressure difference between the inlet and the outlet of the channel. Together, an electric field is generated in the channel by means of electrodes. This electric field is adapted to apply an electrostatic force to the nucleic acids which tends to move them in the opposite direction to the applied hydrodynamic flow.

The applied electric field is from 10 V / m to 10000 V / m, preferably from 100 V / m to 5000 V / m, and more preferably from 200 V / m to 1000 V / m; and / or, the hydrodynamic flow is characterized by an average speed of 1 to 10,000 μητ / s, preferably from 5 to 5,000 μιη / s and more particularly preferably from 10 to 1,000 μιη / s.

The liquid is preferably non-Newtonian. In the present description, the term "Newtonian fluid" is intended to mean a fluid for which there is a linear relationship between the imposed mechanical stress (force exerted on the fluid per unit surface) and fluid shear (i.e., fluid velocity gradient). A "non-Newtonian fluid" is therefore a fluid that is not a Newtonian fluid.

For example, a non-Newtonian fluid according to the invention may have a shear-dependent viscosity coefficient; or he may have an elastic behavior. According to one embodiment, the fluid is viscoelastic.

Advantageously, and in the method according to the invention, the hybridization of the probe with the target nucleic acid introduces a modification of the molecular weight, by increasing the molecular weight of the target nucleic acid. This increase in molecular weight makes it possible to discriminate the target nucleic acid / probe complex of the free probe or free target nucleic acids subjected to hydrodynamic and electrical actuation. Modification of the molecular weight of the probe during hybridization with the target allows the selective enrichment of the signal with respect to the background of the solution.

Indeed, the nucleic acid / probe complex, according to its molecular weight, has a response in the flow such that there is a stopping point where its hydrodynamic speed is compensated for by its electrophoretic speed.

Thus, the probe, when it is not complexed, is not stopped at the same level as the acid / nucleic acid probe complex, thus making it possible to detect the nucleic acid of interest and to overcome the background noise. The present invention thus offers the possibility of selectively enriching the target nucleic acid and eliminating the background noise associated with the probe, as well as other non-specific nucleic acids to detect nucleic acids without fixing or washing steps . Thus, the method according to the present invention makes it possible to reach sensitivity levels for the detection of larger nucleic acids. DESCRIPTION OF THE FIGURES

Other advantages, aims and particular features of the present invention will emerge from the description which follows, made for an explanatory and non-limiting purpose, with reference to the appended drawings, in which:

Figure 1: Schematic representation of the hydrodynamic and electrophoretic force fields in the channel including a constriction for the detection of a target nucleic acid

Figure 2: Schematic representation of two constrictions

Figure 2A: Constriction with linear geometry

Figure 2B: Constriction with exponential geometry

FIG. 3: Schematic representation seen from above, of the geometry of two channels each comprising a constriction, one with a linear geometry, one with an exponential geometry, these two constrictions being opposite each other. FIG. 4: Graphical representation of Viscosity data as a function of the concentration of polyvinyl pyrrolidone (PVP)

FIG. 5: Graphical representation of viscosity data as a function of the concentration of polyvinyl pyrrolidone PVP or polyethylene glycol (PEG)

Figure 6: Schematic representation of a micro-fluidic chip having two channels, each channel comprising two constrictions vis-à-vis

Figure 7A: KRAS Target Nucleic Acid Sequence

Figure 7B: Structure of the KRAS Target Nucleic Acid FIG. 8A: sequence of the molecular beacon intended to hybridise with the KRAS nucleic acid

Figure 8B: Structure of the Molecular Beacon for Hybridizing with KRAS Nucleic Acid

Figure 9A: Sequence of the target miR21 nucleic acid

Figure 9B: Structure of the target miR21 nucleic acid

Figure 10A: sequence of the molecular beacon for hybridizing to the miR21 nucleic acid

Figure 10B: Structure of the Molecular Beacon for Hybridizing with miR21 Nucleic Acid

Figure 11: Representation of the constriction of the micro-fluidic chip 2.5D.

DETAILED DESCRIPTION

Thus, the present invention aims to provide a method for detecting a nucleic acid in a sample, by separation and enrichment of nucleic acids using a bidirectional electro-hydrodynamic flow in a non-Newtonian liquid medium, said method comprising :

introducing into a channel of said sample and a probe for hybridizing to a nucleic acid to form a nucleic acid / probe complex, said channel comprising a flow axis and at least one constriction;

the application of a hydrodynamic flow in the channel together with the application of an electric field in the channel to move the nucleic acids in the channel along the axis of flow and to stop and concentrate in an upstream zone said constriction the nucleic acid / probe complex to detect said complex. Alternatively, a mixture comprising a sample and a probe will be made and introduced into the channel.

Figure 1 is a schematic representation of the hydrodynamic and electrophoretic force fields in the channel 1 comprising a constriction 2.

The main axis of the cylinder is the flow axis 3 in the channel 1.

The nucleic acid / probe complex, according to its molecular weight has a response in the electro-hydrodynamic flow such that for each flow rate 20 Vo, there is an electrophoretic rate, 10, VE to induce its shutdown. In other words, if the torque (Vo, VE) is applied, the speed of the complex is zero. Indeed, with a constriction, one sweeps a range of hydrodynamic velocity and electrophoretic spatially: the flux of the two quantities is preserved. This configuration makes it possible to define a zone 4 where the speed of the complex is zero. Nucleic acids advance to upstream constriction and retreat downstream, as shown in Figure 1. Hybridization of the probe to the target nucleic acid introduces a change in the molecular weight of the target nucleic acid and thus allows for discriminating the target nucleic acid / probe complex of the free probe or free nucleic acids for its detection.

By "nucleic acid detection" is meant here the direct or indirect determination of the presence or absence of a specific nucleic acid sequence, including but not limited to, the detection of a nucleic acid. particular sequence in a nucleic acid molecule or the detection of a difference between the sequences of two different nucleic acid molecules, or the detection of a mutation on a nucleic acid. For the purpose of the present invention, the term "nucleic acid" is intended to mean single or double-stranded DNA molecules or RNA molecules.

Typically, the probe is an oligonucleotide probe, intended to hybridize specifically to the nucleic acid to be detected.

Alternatively, the probe may be chosen from a probe based on synthetic nucleic acids of the LNA (locked nucleic acid) or PNA (peptid nucleic acid) type.

Advantageously, these probes have the property of increasing the hybridization energy and may optionally improve the selectivity of the detection.

Typically, the probe comprises a single-stranded DNA or RNA sequence that is complementary and antiparallel to the fragment to be detected, which is preferentially labeled.

The labeling can be done with a radioisotope or by fluorescence. Alternatively, the probe may include an electrochemical probe. Preferably, a fluorophore will be grafted onto the oligonucleotide probe.

Typically, the fluorophore may be selected from FAM 6-carboxy-fluorescein, HEX ™, JOE ™, VIC®, CAL Fluor® Orange 560, Cy3, TetramethylRhodamine, Texas Red®, Cy5, Alexa series, Atto series.

Advantageously, the method according to the invention allows the detection of target nucleic acids by means of labeled linear probes, unsuited until then for detection in solution because of the background noise. Alternatively, the probe may be a molecular beacon or molecular beacon. The term molecular beacon means a probe formed of a strand of DNA pinned to hair, each end bearing a fluophore. One is said reporter and the other quencher (fire extinguisher).

Typically, the quencher or extinguisher may be selected from Black Hole Quencher®, such as BHQ-1, BHQ-2, BHQ-3, QXL® quenchers, DDQ-I, Dabcyl, Iowa Black® (FQ or RQ), QSY ® 21.

The sequence included in the oligonucleotide probe will be determined according to the target nucleic acid to be detected. In one embodiment, the oligonucleotide probe comprises a sequence of size between 30 and 120 bases.

These probes will be used when the detection of the nucleic acid must be specific.

In another embodiment, the oligonucleotide probe comprises a sequence of size between 15 and 30 bases.

In this embodiment, the probes will be used for detecting a mutation. Preferably, the oligonucleotide probe will have a size of 15 bases.

According to another embodiment, and for the detection of a mutation, the microfluidic system, at the constriction level, may be furthermore heated to a temperature of between 40 and 70 ° C., in order to reinforce the detection of a mutation. Indeed, a mutation induces a decrease in the T hybridization temperature,

In the same way, the methylated DNA can be detected by heating the microfluidic system at the constriction level.

Indeed, the DNA containing methylated cytosines have different structural and thermodynamic characteristics (Karberg et al., A Method for Quantifying DNA Methylation Percentage Without Chemical Modification). According to another embodiment, the probe further comprises polymers and / or nanoparticles, said polymers and / or nanoparticles being grafted onto said probe.

The grafting of polymer or nanoparticle chains makes it possible to increase the molecular weight of the probe in order to amplify the difference in molecular weight of the target / probe complex, and to increase the detection.

Typically, the polymer may be a polyethylene glycol (PEG). In one embodiment, the nanoparticles may be gold nanoparticles grafted to the probe by thiol grafting or nanoparticles of poly-styrene type polymer, grafted to the probe by a biotin-streptavidin bond.

Alternatively, the molecular weight of the probe can be increased by the addition of a biological molecule selected from a protein, an antibody.

As an illustration, antibodies specific for a double-stranded DNA could be added after the hybridization so as to significantly increase the molecular weight of the probe. In another embodiment, the probes may be designated by DNA engineering so as to induce a chain reaction associated with the formation of a nucleic acid / probe complex: the complex induces the recruitment of another DNA probe, which allows to amplify the difference in molecular weight with a 3-body complex.

In one embodiment, this engineering will be able to use the DNA origami technique by which the interaction of the target and the probe releases some free bases, which make it possible to trigger the attachment of an additional ssDNA (effect amplifier).

DNA origami is for example described in the article by Wang et al. : "The beauty and utility of DNA Origami". According to another preferred embodiment, the weight of complex nucleic acid target / probe will have a weight of between lOkDa 10 ⁶ kDa, 20kDa and 10 preferably between ⁴ kDa and even more preferably between 50 kDa and 10 ³ kDa.

In another embodiment, the weight of the nucleic acid / probe complex is greater than or equal to 1.5 times the molecular weight of the probe, preferably greater than or equal to 2 times the molecular weight of the probe, and even more preferably, greater than or equal to 4 times the molecular weight of the probe.

In one embodiment, the channel comprises at least one constriction, said constriction being formed by a first section of the channel of a width 1 and a second section of the channel of a width, the width of said second section of the channel being strictly less than the width 1 of said first section of the channel and corresponds to the width of the constriction. In one embodiment, the ratio of the width 1 of the first section of the channel to the width of the second section of the channel is greater than 5, preferably greater than 10, or at least greater than 20, or at least greater than 50 and even more preferably greater than 80.

Typically, the width 1 of the first section of the channel will be between 200 μιη and 5000 μιη, preferably between 600μιη and 2000μιη.

Preferably, the width 1 of the first section of the channel will be approximately 800μπι. Typically, the width of the second section of the channel will be between 2μιη and ΙΟΟμιη, preferably between 5 and 50μιη, and even more preferably between 5 and ΙΟμιη.

The walls of the channel at the constriction will form an angle with respect to the axis of flow of between 20 and 90 °, preferably 30 to 60 °, for a channel having a linear geometry.

The length of each constriction can vary between 500 and 2000μιη. The section of the mouth of the constriction is between 10 and 3000 μιη2, preferably 12 and 500 μιη2, and preferably between 2 and 100 μιη ² .

The shape of the constriction can be linear or of more complex form, for example parabolic or exponential.

According to one embodiment, the height of the detection channel of the constriction is between 1 and 6μιη, preferably between 2 and 4μιη.

Figure 2 schematically shows two channels, each having a constriction, one with linear geometry (Figure 2A), the other with exponential geometry (Figure 2B). In Figure 2A, the channel 1 has the shape of a hollow cylinder of rectangular section. The main axis of the cylinder is the flow axis 3 in the channel 1. Perpendicular to this flow axis 3, a first cross section la of the channel 1 is defined by a width of about ΙΟΟΟμιη and a second cross section 1b of the channel 1, a width of about 5μιη and forming the constriction 2. The constriction has a length of 550 μιη.

Thus, the ratio between the large section and the small section defining the constriction of the channel makes it possible to design a concentration factor of about 200. FIG. 2B represents a constriction having an exponential geometry. The elements shown in Figure 2B bearing the same references as those of Figures 1 and 2A represent the same objects, which are not described again below. The channel shown in Figure 2B has a first cross section la of a width of about 800μιη, and a second cross section lb of about 20μιη. The length of the constriction is about 800μιη.

Thus, the ratio between the large section and the small section defining the constriction of the channel makes it possible to design a concentration factor of about 40.

The constriction thus allows a "valve" effect allowing pass objects of low molecular weight. It thus makes it possible to stop the molecules in a small volume in order to obtain a high concentration factor, without being too astringent, with the risk of stopping the unhybridized probes under the same conditions. The height of the channel at the level of constriction is 2μηι.

The height of the channel at the constriction level plays a key role for the signal-to-noise ratio: the complexes accumulated at the wall become detectable if the signal they generate is greater than the voluminal signal linked to the probes present in volume. In other words, as the stuck molecules are pressed against the walls, increasing the height of the channel does not necessarily increase the signal, while it increases the background noise. Advantageously, the nucleic acid / target complex is stopped upstream of the constriction to avoid leakage associated with incomplete trapping.

Figure 3 generally shows two channels 1 each comprising a constriction 2 having a different geometric shape, the constrictions being vis-à-vis. Typically, the two different constrictions make it possible to compare their performance with respect to the separation, the concentration and the detection of the nucleic acids for the same experimental condition.

The constriction channel may be integrated into a microfluidic chip.

Different types of micro-fluidic chips can be used such as micro-fluidic chips with linear geometry (x shape), micro-fluidic chips power-law geometries (x 1 ^' 5, x 2, x 2 5, and x 3 ) as well as micro-fluidic chips "exponential-law geometries" (exp (3x), exp (4x), exp (5x) exp (6x) and exp (7x).

Alternatively, silicone chips made by lithography using grayscale masks may be used (2.5D chip). This manufacturing technique is described in the article "Grayscale lithography to fabricate varying nanochannels in a single step" by Naillon, Antoine & Massadi, Hajar & Courson, Rémi & Calmon, Pierre-François & Séveno, Lucie & Prat, Marc & Joseph, Pierre (2016). By way of example, the length of the concentration channel of the chip is 1.7 mm and has a height gradient of 5 μιη at 2 μιη, and the constriction width is 25 μιη. The geometry of the chip is shown in Figure 11.

Alternatively, the channel may be a light of a capillary tube. In this case, the constriction corresponds to the junction of two capillaries of different diameters.

The use of capillary tubes can allow easy multiplexing of the channels according to the invention, in the form of spindles of capillary tubes, for example such as those described in the article "Bundled capillary electrophoresis using micro structured fibers" of Rogers et al. , Electrophoresis, 32 (2): 223-229 (2011). In this case, the hydrodynamic flow application means and the electric field application means may be common for all the capillary tubes, or on the contrary be distinct for all the capillary tubes.

According to one embodiment, the liquid medium has a zero shear viscosity between 3cP and 40cP, preferably between 10 and 25cP (centipoise) at room temperature.

Typically, two methods may be used to measure the zero shear viscosity. It can be measured by dynamic light scattering (DLS), which measures the hydrodynamic radius of colloidal solutions. The measurement of DLS can be performed with a Malvern ZetaSizer type device. It is a question of using nanoparticles of given size R ₀ and measuring their apparent hydrodynamic size R _a in the solution of indeterminate viscosity. The viscosity is given by the ratio R _a / Ro _. The elasticity parameters can be measured by the use of fluorescent nanoparticles of calibrated size Ro of the order of 200 nm and the measurement of their Spatial fluctuations at room temperature by fluorescence video microscopy. Mean quadratic displacement (MSD) is measured as a function of time τ. These data are adapted according to the model described in (Zanten, PRE, 2000). The analytic expression is: t> x

where kT is the thermal agitation energy, m the mass of the particle, with μ the viscosity of the fluid, λ the relaxation time of the fluid which is μ / Ε where E is the elasticity of the fluid.

According to one embodiment, the liquid medium comprises uncharged polymers.

The use of a suitable dissolved polymer matrix allows the specific termination of nucleic acid complexes / probes of interest, and particularly those of small molecular weights for given pipe design and operating parameters limited by the materials used.

The term "unfilled" means that the polymers in question have a substantially total electrostatic charge in the aforementioned liquid medium. The presence of such polymers for example in an aqueous solution makes it possible to make the liquid medium non-Newtonian (for example viscoelastic).

According to one embodiment, the liquid medium comprises uncharged polymers, preferably chosen from polyvinylpyrrolidone (PVP), poly (ethylene glycol) polyacrylamide and / or their mixtures.

As an illustration, the liquid medium comprises a mixture of PVP and PEG. Preferably, the uncharged polymers will be chosen from polyvinylpyrrolidone 1.3MDa (PVP 1.3MDa), polyvinylpyrrolidone 360 KDa (PVP 360 Kda), polyvinylpyrrolidone 40kDa (PVP 40kDa), polyvinylpyrrolidone 10kDa (PVP 100Da) and polyvinylpyrrolidone. (ethylene glycol) 10 KDa (PEG 10 KDa).

In general, high concentrations of PVP are favorable to stop small molecules, but the viscosity becomes important, which requires imposing very important pressures not necessarily available to commercial devices. The electric field in the same way can not be modulated infinitely, because the risks of breakdown are strong with any type of device.

According to one embodiment, the uncharged polymers are present in a mass concentration of 0.5 to 30%, preferably 2 to 25%, and even more preferably 3 to 20%.

Figures 3 and 4 show the viscosity data as a function of the concentration of PVP or PEG.

Typically, for the detection of low molecular weight nucleic acids (less than 100 base pairs), high viscosity solutions will be used.

For example, the liquid medium comprises PVP 40kDa in a mass concentration of the order of 18%, or PVP 1.3MDa in a mass concentration of about 3%.

Separation is easier for DNAs in the 100-1000 base pair range where good performance is obtained for all conditions. Separation conditions for high molecular weight nucleic acids of the order of 10,000 base pairs or more are obtained with more dilute solutions. As an illustration, a liquid medium comprising 2.5 kDa PVP in a mass concentration of the order of 2% can be used.

Those skilled in the art will advantageously select the polymer and its concentration as a function of the target to be detected.

According to a preferred variant of the process according to the invention, the applied electric field is 0.1 kV / m at 10 kV / m, preferably 1 kV / m at 500 kV / m and even more preferably 100 kV / m. at 200 kV / m and / or the hydrodynamic flow is characterized by an average speed of 0.1 to 10 mm / s, preferably 1 to 100 mm / s, and even more preferably 5 to 10 mm / s. The desired hydrodynamic flow profiles (characterized in particular by average flow rate and average speed values) are obtained by operating pressure control means, so as to generate a pressure difference between the inlet and the outlet of the channel. For example, a voltage difference of less than 12 bar, preferably between 50mbar at 10 bar, preferably between 2 and 6 bar and more preferably between 0.1 and 3 bar, provides the desired hydrodynamic flow profiles.

Together, an electric field is generated in the channel by means of electrodes. This electric field is adapted to apply an electrostatic force to the electrically charged objects that tends to move them in the opposite direction to the applied hydrodynamic flow.

Typically, the voltage will be less than 400 V, and preferably between 10 to 300 V, and more preferably 100 to 200 V. In one embodiment, the introduction of the sample is performed in a channel introduction zone and the displacement of the electrically charged objects is effected from the introduction zone to a channel detection zone, the method comprising outraged ;

the detection of the nucleic acid / probe complex arriving in a detection zone.

Typically, the detection can be performed at the level of constriction. In another embodiment, the detection will be downstream of the constriction.

The concentration factor depends on the time, thus, the detection time is between 10 and 5000 seconds, preferably between 50 and 1000 seconds, and preferably between 100 and 500 seconds, in order to achieve sufficient enrichment.

Those skilled in the art will be able to determine the analysis time, while taking into account the dissociation of the nucleic acid / probe complex. EXAMPLES

In Examples 1 to 5 which follow the following materials and methods have been used: - micro-fluidic chip

In the following examples, a microfluidic chip with two channels manipulated with identical actuation parameters was used. This microfluidic chip is shown in FIG. 6 and comprises two channels each comprising two constrictions placed in view as represented in FIG. 3. This micro fluidic chip is also described in Malbec et al.

This chip has been used experimentally. Similar results can be obtained with a channel or a capillary comprising a constriction.

This system is used to simultaneously evaluate the signal in a channel where a sample is introduced into the channel at the top of Figure 6 and a sample into the channel at the bottom of Figure 6, the top channel serving as a control. Both channels are observed simultaneously by video fluorescence microscopy.

Analysis

The videos are analyzed with ImageJ, a program for tracing the fluorescence intensities and thus determining the positions of the nucleic acids in the channel.

The intensities are adjusted according to a Gaussian distribution and the resolution is calculated according to the ratio of the distance between the Gaussian consecutive peaks and the sum of their heights.

Example 1: Detection of the proto-oncogene KRAS

In this example, the invention is implemented to separate, concentrate and detect the proto-oncogene KRAS.

Thus, the target nucleic acid is the KRAS proto-oncogene comprising 111 bases (SEQ ID No. 1).

Its sequence is shown in Figure 7A and its structure is as shown in Figure 7B. The associated probe is a molecular beacon and corresponds to the KRAS probe, comprising 32 bases (SEQ ID No. 2) and a fluorophore: 6 FAM, and a fire extinguisher: Black Hole Quencher®: BHQ1. Its sequence is shown in Figure 8A and its structure is as shown in Figure 8B.

The interaction zone of the KRAS target nucleic acid with the probe is designated by the hook in Figure 7B.

The samples are diluted in a separation buffer comprising 1x TBE, and PVP 1.3MDa in a mass concentration of about 5%.

The viscosity of the non-Newtonian liquid medium is about 3cP at room temperature.

A sample comprising 100 nM of probe was introduced into the top channel and a sample comprising 100 nM of probe incubated for about 1 hour at 40 ° C. with ΙμΜ of target nucleic acid was introduced into the bottom channel.

An overall pressure difference of 1.7 bar and a voltage difference of 168V were implemented with cross-linked hydrodynamic and electrophoretic flows, that is, oriented along the axis of flow but along opposite axes. This pressure / tension pair enabled the enrichment and thus the detection of the nucleic acid complex KRAS and its probe.

This tension / pressure torque implies a fluid hydrodynamic velocity of 2cm / s and an electric field value prevailing within the constriction of 700k V / m. The pressure and voltage parameters have been modulated. An overall pressure difference of 1.7 bar and a voltage difference of 312V were implemented with hydrodynamic and electrophoretic cross flows. This pressure / voltage pair made it possible to enrich the probe and to detect it only.

This tension / pressure pair implies a fluid hydrodynamic velocity of 2cm / s and an electric field value prevailing within the constriction of 1300kV / m.

The method according to the invention thus makes it possible to detect the nucleic acid / probe complex at a given torque of pressure and of tension and to eliminate the background noise associated with the probe.

Example 2: Detection of miR 21

In this example, the invention is implemented to separate, focus and detect miR 21.

Thus, the target nucleic acid is 22 base miR21 (SEQ ID NO: 3).

Its sequence is shown in Figure 9A and its structure is as shown in Figure 9B.

The associated probe is the miR21 probe, comprising 32 bases (SEQ ID No. 4) and a fuorophore: 6 FAM, and a fire extinguisher: Black Hole Quencher®: BHQ1.

Its sequence is shown in Figure 10A and its structure is as shown in Figure 10B.

The samples are diluted in separation buffer comprising 1X TBE, and PVP 1.3MDa in a mass concentration of about 5%. The viscosity of the non-Newtonian liquid medium is about 3cP at room temperature. ΙΟΟηΜ of miR21 probe and 300nM of miR21 nucleic acid were used.

A sample comprising the probe was introduced into the top channel and a sample comprising the probe and the target nucleic acid was introduced into the bottom channel.

An overall pressure difference of 2 bar and a voltage difference of 235 V were implemented with cross-linked hydrodynamic and electrophoretic flows, ie oriented along the axis of flow but along opposite axes.

This pressure / voltage pair enabled the enrichment and therefore the detection of the miR21 / probe nucleic acid complex. The pressure / pressure torque was modulated and an overall pressure difference of 2 bar and a voltage difference of 314V were implemented with crossed hydrodynamic and electrophoretic flows. Under these conditions, the probe alone was concentrated and therefore detected. The method according to the invention thus makes it possible to detect the nucleic acid / probe complex at a given torque of pressure and of tension and to eliminate the background noise associated with the probe.

Example 3: Detection of nucleic acid using an excessively straight linear probe

In this example, the invention is implemented to separate, concentrate and detect the following nucleic acid:

5 'AAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AT AGCTTATC AG AC TGATGTTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3 '(SEQ ID NO: 5)

The probe is as follows: 5'-6FAM-TCAACATCAGTCTGATAAGCTA-3 '(SEQ ID NO: 6)

Samples are diluted in 1x TBE Separation Buffer, PVP 5% 1.3MDa

A sample comprising 100 nM probe and 1 nM target was injected into the top channel of the microfluidic chip. A sample comprising 100 nM probe and 10 nM target was injected into the bottom channel.

A pressure difference of 2 bars and a voltage of 230V were implemented with crossed hydrodynamic and electrophoretic flows. The method according to the invention thus made it possible to detect the only nucleic acid / target complexes for given voltage and pressure parameters.

A pressure difference of 2.5 bar and a voltage of 288V were implemented with crossed hydrodynamic and electrophoretic flows. Under these conditions, only the probe could be detected.

Advantageously, the method according to the invention makes it possible to detect low concentrations of nucleic acids. It is also possible to saturate the probe solution and efficiently detect the target nucleic acid because the method according to the invention allows the enrichment of the signal and to reduce the background noise. Example 4: Detection of a Target Sequence in a Sample Comprising Circulating DNA

1) Sample preparation

4 mL of blood plasmin were processed with a Norgenbiotek Plasma Kit / Serum Circulating DNA Purification Midi Kit.

Purified DNA was eluted in 50 μί TE IX.

50μΙ. of purified DNA were diluted with 450μί of TBE IX and PVP 1.3M 5%, and then filtered on a surface filter comprising 0.22μιη pores.

2) Preparation of target nucleic acids and probes

The target nucleic acid is as follows:

5'A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A AT AT AGCTTATCAG AT CTGATGTTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3 '(SEQ ID NO: 7)

The probe used comprises 22 bases, the fluorophore: 6 FAM and is the following: 5'-6FAM-TCAACATCAGTCTGATAAGCTA-3 '(SEQ ID No. 8).

A sample comprising 100 nM of probe was injected into the top channel of the microfluidic chip.

A sample comprising 10 nM of the nucleic acid to be detected as well as 100 nM of the probe was injected into the bottom channel. A pressure difference of 1.5 bar and a voltage difference of 280 V were implemented with cross-coupled hydrodynamic and electrophoretic flows, that is, oriented along the axis of flow but along opposite axes. This tension / pressure torque implies a fluid hydrodynamic velocity of 1.8cm / s and an electric field value prevailing within the constriction of 1170kV / m.

At these pressure and voltage differences, the probes alone were stopped in an area upstream of the constriction.

The pressure difference was changed and a pressure difference of 1.75 bar and a difference of 280 V were implemented with cross-linked hydrodynamic and electrophoretic flows as previously mentioned.

This tension / pressure torque implies a fluid hydrodynamic velocity of 2.1 cm / s and an electric field value prevailing within the constriction of 1170kV / m. This pressure and voltage pair makes it possible to stop the target nucleic acid / probe complex to be detected and to overcome the background noise constituted by the probes alone, which probes are not stopped at these parameters.

The method according to the invention thus makes it possible to detect target nucleic acids in complex samples at low concentrations.

Thus, and in the preceding examples, for a given pressure, it is possible to impose a weak electric field such that only the nucleic acid / probe complexes are stopped, while the probes circulate freely through the constriction. By increasing the electric field, the nucleic acid / probe complexes migrate backwards. It is thus possible, at a given threshold to observe an accumulation of the signal testifying to the detection of the nucleic acid / probe complex, and thus to isolate the nucleic acid / target complexes independently. Thus, and by properly adjusting the voltage and pressure parameters, and the constriction, it is possible to selectively enrich the nucleic acid / probe complex and eliminate the background noise associated with the probe.

The method according to the invention therefore makes it possible to reach higher levels of sensitivity than those obtained in volume measurement.

Example 5: Selective Enrichment of the Probe / Target Nucleic Acid Complex Vs. free probe The target nucleic acid is a 22 base ssDNA sequence, and the probe, a probe having a complementary sequence, labeled with a 6-FAM fluorophore.

A sample comprising ΙμΜ of probe and ΙμΜ of target nucleic acid was used.

The voltage / pressure pair was adjusted to allow formation of the nucleic acid / target complex.

The target nucleic acid complex could be detected after 1 second. After 10 seconds of enrichment according to the voltage / pressure parameters, all the nucleic acid / target complexes are stopped upstream of the construction.

The experiment was conducted using 1 μM probe and 1 μM target nucleic acid. Although the signal is weaker, it is possible to detect, thanks to the method according to the invention, the target nucleic acid / probe complex after 10 seconds of enrichment according to the pressure / voltage parameters. The enrichment factor was calculated by measuring the fluorescence intensity at the constriction as a function of the time applied for enrichment.

Similar enrichment factors were obtained for each of the experiments after 9 seconds. Advantageously, an enrichment factor of 160 was obtained for the experiment carried out on samples comprising ΙμΜ of probe and ΙμΜ of target nucleic acid, and an enrichment factor of 143 for the experiment carried out on samples comprising lnM. of probe and 1nM of target nucleic acid. Thus, the method according to the invention advantageously makes it possible to selectively enrich nucleic acids at low concentration, in order to allow their detection.

Example 6: Threshold of detection

The target nucleic acid is a ssDNA sequence of 99 bases, and the probe, a probe having a complementary sequence of 34 bases, labeled with a fluorophore 6- FAM (6-carboxyfluorescein) (Eurogentec, Liège, Belgium).

Thus, the target nucleic acid is as follows:

5'-

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAATGCTGGGCGATAAGAGG TTCCGTGTAGTCAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3 '

(SEQ ID NO: 9) The probe used is the following 5'-6FAM-

TTGACTACACGGAACCTCTAATCGCCCAGCATTT -3 '(SEQ ID NO: 10).

In this example, 2μΜ of probe were mixed with different concentrations of target nucleic acid from 2μM to 2μΜ in 0.2X PBS.

The solutions thus obtained were heated at 92 ° C for two minutes and then slowly cooled to room temperature. 20 μl of the probe / target nucleic acid mixture were diluted in 980 μl of freshly prepared buffer and filtered at 0.2 μιη containing 5% of 1.3 mM PVP in 0.2 × PBS.

The final solutions obtained contain constant concentrations of probes (20nM) and variable concentrations of target nucleic acids (from 20fM to 20nM).

For enrichment and detection of the nucleic acid / probe complex, lithographic silicone chips using grayscale masks were used.

The length of the concentration channel of the chip is 1.7 mm and has a height gradient of 5 μιη at 2 μιη.

The constriction width was set at 25 μιη. The geometry of the chip is shown in Figure 11

The channel was first filled with ethanol to remove air bubbles. It was then thoroughly rinsed with working buffer before adding 40 μΐ _^ solution containing various concentration ratios between target nucleic acids and probes (constant probe concentration (20nM) and varying concentrations of target nucleic acids (from 20fM to 20nM).

The viscosity of the non-Newtonian liquid medium is about 31cP at room temperature.

An overall pressure difference of 5 bar and a voltage difference of 190V were implemented with crossed hydrodynamic and electrophoretic flows. This pressure / voltage pair has made it possible to selectively enrich the nucleic acid / probe complex and thus its detection.

This tension / pressure torque implies a fluid hydrodynamic velocity of 10.18 cm / s and an electric field value prevailing within the constriction of 100 kV / m.

The method according to the invention thus makes it possible to detect the nucleic acid / probe complex at a given torque of pressure and of tension and to eliminate the background noise associated with the probe and also has a high sensitivity in that the process according to the invention The invention advantageously made it possible to detect the target nucleic acid at a concentration of 20 μM.

Claims

A method of detecting a nucleic acid in a sample by separating and enriching nucleic acids using a bidirectional electro-hydrodynamic flow in a non-Wontonian liquid medium, said method comprising: introducing into a channel of said nucleic acid; sample and a probe for hybridizing to a nucleic acid to form a nucleic acid / probe complex, said channel comprising a flow axis and at least one constriction;

the application of a hydrodynamic flow in the channel together with the application of an electric field in the channel to move the nucleic acids in the channel along the axis of flow and to stop and concentrate in an upstream zone said constriction the nucleic acid / probe complex to detect said complex.

2. Method according to claim 1 characterized in that the probe is an oligonucleotide probe.

3. Method according to claim 1 or 2 characterized in that the probe is an oligonucleotide probe comprising a sequence of size between 30 and 120 bases.

4. Method according to claim 1 or 2 characterized in that the probe is an oligonucleotide probe comprising a sequence of size between 15 and 30 bases.

5. Method according to any one of claims 1 to 4, characterized in that said probe further comprises polymers and / or nanoparticles, said polymers and / or nanoparticles being grafted onto said probe.

6. Method according to any one of claims 1 to 5 characterized in that the molecular weight of the nucleic acid / probe complex is greater than or equal to 2 times the molecular weight of the probe, preferably greater than or equal to 4 times the weight of the probe.

7. Method according to any one of claims 1 to 6, characterized in that the viscosity of the non-Newtonian liquid medium is between 3cP and 40cP, preferably between 10 and 25cP at room temperature.

8. Process according to any one of claims 1 to 7, characterized in that said non-Newtonian liquid medium comprises uncharged polymers, preferably chosen from polyvinylpyrrolidone, poly (ethylene glycol) and / or their mixtures .

9. Method according to any one of claims 1 to 8, characterized in that the detection time is between 10 and 5000 seconds, preferably between 50 and 1000 seconds, and even more preferably between 100 and 500 seconds.

The method of any one of claims 1 to 9, wherein:

the pressure difference applied will be less than 12 bars, and preferably between 50mbars and 10 bars, and / or

the applied voltage will be less than 400 V, and preferably between 10 to

300V and preferably between 100 and 200V,

so as to obtain the desired bi-directional electrodynamic flow.