Nucleotide Sequencing Device with Increased Sensitivity and Improved Reliability

Abstract

A device for sequencing at least one nucleotide strand comprising a support (3), at least one moving part (4) relative to the support (3), means for vibrating the moving part (4) at a given frequency, means for measuring the vibration frequency of the moving part (4), a recognition probe (SR) mechanically connecting the support and the moving part (4), the recognition probe (SR) comprising at least one nucleotide sequence. Figure for the abstract: 1.

Description

Title of the invention: NUCLEOTIDE SEQUENCE SEQUENCING DEVICE WITH ENHANCED SENSITIVITY AND IMPROVED RELIABILITY

[0001] TECHNICAL FIELD AND PRIOR TECHNOLOGY

[0002] The present invention relates to a device for sequencing at least one nucleotide sequence and to a sequencing system comprising a large number of such devices.

[0003] A sequencing device or sequencer aims to determine the exact composition of a sequence of nucleic bases or nucleobases on a strand of DNA or RNA being analyzed. This determination can be performed by identifying the sequence through interaction with a probe formed by a strand. This interaction results in a hybridization reaction between the analyzed strand and the probe strand. The analyzed strand binds more readily to a probe whose sequence exactly matches the complementary sequence of the analyzed strand, and less readily to a probe whose sequence differs from its complementary sequence.

[0004] The methods of the state of the art exhibit a lack of sensitivity by causing a large number of false positives, i.e. causing the pairing of two strands when they are not complementary by their sequences.

[0005] One method to reduce the number of false positives is to reduce the length of the analyzed strands, but this reduction has the effect of increasing the time and cost of the analysis of the total sequence.

[0006] Another method consists of increasing the number of sensors equipped with the same recognition probe in order to perform the same analysis, thus statistically reducing the number of false positives. This method requires a large sample size.

[0007] Furthermore, there is a strong demand for DNA sequencing devices. In particular, for methods that are fast and have low error rates. Description of the invention

[0008] It is therefore an object of the present invention to offer a sequencing device for at least one fragment of DNA or RNA, which is more sensitive and more reliable than devices in the prior art.

[0009] The stated objective can be achieved by a sequencing device comprising at least one fixed part and one part configured to vibrate relative to the fixed part, an oligonucleotide probe mechanically linking the fixed part and the moving part, means for vibrating the moving part, and means to measure the vibration frequency of the moving part before and after hybridization of the analyzed strand on the probe.

[0010] The level of hybridization between the probe and the analyzed strand varies the stiffness of the probe and analyzed strand assembly, which has an influence on the mechanical resonance frequency of the vibrating part.

[0011] Depending on the variation of the mechanical resonance frequency, it can be determined whether the hybridization is total, partial or zero and thus deduce the exact sequence of nucleobases of the analyzed strand.

[0012] In other words, the mechanical stiffness of an assembly composed of an analyzed strand and a sequence of nucleotides is measured, this stiffness being characteristic of the level of pairing of the analyzed strand and the sequence, which makes it possible to deduce the structure of the strand in the case of complete pairing.

[0013] Advantageously the moving part is a resonant optomechanical ring.

[0014] The device can be integrated into a fluidic channel in which a fluid containing the analyzed strands circulates so as to come into contact with the probe.

[0015] Preferably, a system comprising a large number of sequencing devices is implemented, allowing, for example, the analysis of a complete genome in a single measurement.

[0016] According to one aspect, the present invention relates to a device for sequencing at least one strand of nucleotides comprising a support, at least one part movable relative to the support, means for vibrating the movable part at a given frequency, means for measuring the vibration frequency of the movable part, a recognition probe mechanically linking the support and the movable part, the recognition probe comprising at least one sequence of nucleotides.

[0017] Advantageously, the means for vibrating the moving part are configured to vibrate the moving part at its resonant frequency.

[0018] According to one possible implementation, the means for measuring the vibration frequency are opto-mechanical means.

[0019] According to one embodiment, the means for measuring the vibration frequency comprise at least one waveguide optically coupled with the moving part, a light source for injecting light into the waveguide and a unit for processing the light exiting the waveguide.

[0020] Advantageously, the movable part can be in the form of a disc, a ring, a racecourse or an ellipse suspended by at least one foot.

[0021] According to one possible implementation, the device may include several identical probes connecting the moving part to the support.

[0022] The support may comprise a first adapter precursor and a first adapter, one end of which is fixed to the first adapter precursor and the other end comprises a nucleotide sequence hybridized with one end of the recognition probe, and wherein the mobile part comprises a second adapter precursor and a second adapter comprising a probe one end of which is attached to the second adapter precursor and the other end comprises a nucleotide sequence hybridized with one end of the recognition probe.

[0023] The present invention also relates to a sequencing assembly comprising the device as defined above and a fluidic channel ensuring contact between a liquid containing said analyzed strand and the recognition probe.

[0024] The present invention also relates to a sequencing system comprising a plurality of sequencing devices as defined above.

[0025] Advantageously, the sequencing devices are divided into groups, each group comprising recognition probes having the same nucleotide structure.

[0026] Advantageously, the recognition probes are chosen so that in a single measurement a complete genome is analyzed.

[0027] For this, hundreds of thousands of sensors in parallel and / or several measurement cycles are typically planned.

[0028] The present invention also relates to a method for sequencing at least one nucleotide sequence comprising:

[0029] - bringing a liquid containing said sequence into contact with a device resonating device comprising a recognition probe connecting a fixed part and a moving part of the resonating device,

[0030] - the vibration of the moving part,

[0031] - the measurement of the variation in the vibration frequency of the moving part due to hybridization between the recognition probe and the nucleotide sequence.

[0032] Advantageously, the moving part is set into vibration at its resonant frequency.

[0033] Typically, the moving part can be set into vibration with an amplitude of the order of 0.1 nm.

[0034] According to one embodiment, the moving part is set into vibration at a frequency between 100 MHz and 1 GHz. Brief description of the drawings

[0035] The present invention will be better understood on the basis of the following description and the accompanying drawings in which:

[0036] [Fig-1] is a side view of an example of a sequencing device shown schematically mathematically,

[0037] [Fig.2] is a view of the device of [Fig.1] in a state in which a strand analyzed is hybridized on the probe,

[0038] [Fig.3B] is a detailed view of the device of [Fig.1],

[0039] [Fig.3A] is a detail view of the device of [Fig.1] without a latching probe,

[0040] [Fig.4] is a detail view of a variant of the device in [Fig.1],

[0041] [Fig.5] is a schematic representation of the device of the [Fig.1] integrated into a fluidic channel

[0042] [Fig.6] is a representation of a sequencing device implementing opto-mechanical detection means,

[0043] [Fig.7] is a top view of a system comprising a network of devices sequencing.

[0044] DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0045] Figure 1 shows an example of an embodiment of a genome sequencing device. The invention relates to the determination of DNA fragments and RNA fragments.

[0046] DNA or RNA sequencing consists of determining the order of the nucleotides for a given DNA fragment or a given RNA fragment respectively.

[0047] Figures 1, 2, 3A, and 3B show an example of a sequencing device comprising at least one fixed part 2, two in the example shown, for example attached to a support 3, and a part 4, designated the moving part, intended to be movable relative to the fixed part 2 in the plane of the device. The fixed part 2 is located near the moving part 4. The distance between the edge of the fixed part 2 and the opposite edge of the moving part 4 is, for example, between 100 nm and 200 nm. This distance is chosen so as to avoid any contact between the moving part and the fixed part under normal oscillation conditions of the moving part.

[0048] The plane of the device or sensor is the plane parallel to the support 3, generally formed by a substrate implemented in micro-electromechanics.

[0049] In the example shown, the moving part comprises a platform 6 suspended by a foot 8. The dimensions of the foot and of the platform 6 are such that the platform 6 can oscillate substantially in the plane.

[0050] The device is a resonant device and includes excitation means 10 for vibrating the movable part 4 in the plane. In the example schematically represented in [Fig. 1], the excitation means are electrostatic and comprise an electrode 10.1 on the fixed part and an electrode 10.2 on the movable part formed by the foot directly. Applying a potential difference between the electrodes generates electrostatic forces between the foot and the fixed part, causing the platform to vibrate. The implementation of excitation means at The foot level helps to limit the bulk.

[0051] Alternatively, optical excitation methods can also be used. In particular, radiation or thermo-optical forces are employed. In both cases, a laser with a wavelength within an optical peak is used, but its power is modulated at the frequency of interest, in this case, the mechanical resonance frequency of the device. This laser can be the same as the one used for optical reading, or another laser, for example, one that is superimposed on the first.

[0052] The device also includes means for measuring (not shown) the vibration frequency of the moving part. The moving part then forms a mechanical resonator. These means include, for example, one or more capacitive, piezoresistive, opto-mechanical, or acoustic wave transducers of the bulk acoustic wave (BAW) type or of the surface acoustic wave (SAW) type.

[0053] The moving part 4 can be a beam fixed at one end and set into vibration by electrostatic means and whose frequency is measured by piezoresistive gauges.

[0054] According to another example, the moving part 4 can be a vibrating plate in a Lame or breathing mode.

[0055] An example of opto-mechanical detection will be described in detail.

[0056] Preferably the moving part is excited at a resonance frequency allowing maximum amplification of the input signal and maximum sensitivity of the device's response to a disturbance (change in the environment).

[0057] The resonator is preferably chosen to exhibit reduced viscous damping. Indeed, the device is intended to be placed in a fluidic channel through which a liquid containing the analyzed strand(s) flows.

[0058] Figure 3B shows an enlarged view of the device of Figure 1. Figure 3A represents the device before the attachment of an SR reconnaissance probe.

[0059] The device includes a recognition probe SR designed to hybridize with the analyzed strand. The recognition probe is, for example, an oligonucleotide probe. The probe may contain, for example, between one hundred and several thousand nucleotides.

[0060] The device also includes nucleotide sequences 12, 14, one end of which is attached to the fixed part 2 and the other end of which is attached to the mobile part 4. The two free ends of the nucleotide sequences 12, 14 are designed to attach to the ends of the SR recognition probe by hybridization and allow the SR recognition probe to mechanically connect the fixed part 2 and the moving part 4.

[0061] Sequences 12 and 14 are designated "adapters" and comprise sequences complementary to the ends of the recognition probe, enabling hybridization of the two ends of the recognition probe onto the adapters. Once attached to the adapters 12 and 14, the SR recognition probe includes a sequence of free nucleotides designated as the "useful region," designed to cooperate by hybridization with the analyzed strand.

[0062] The fixed part has a layer 16 on its upper surface for attaching the adapter 12, and the upper surface of the mobile part has a layer 18 for attaching the adapter 14. Layers 16 and 18 are called "adapter precursors" and include, for example, silane or a thin metallic layer. A DNA-thiol adapter on a gold attachment layer may be provided. One or more thiols, polyethylene glycol (PEG), or epoxy may also be used for the adapter precursor. Furthermore, there is a strong affinity between biotin and streptavidin; thus, one of these two elements may serve as an adapter precursor, while the other may be provided at one end of the adapter 12 or 14, thereby "attaching" the adapter to the fixed or mobile part.

[0063] Preferably, prior to the deposition of the adapter precursors, the upper surfaces of the fixed and moving parts have different functionalizations to be selective with respect to the adapter precursors. The functionalization is obtained, for example, by forming a film 17 on the upper surface of the fixed part and a film 19 of a different material on the upper surface of the moving part. The films 17, 19 can, for example, be made of a polymer material or a metallic material such as gold. A particular embodiment provides:

[0064] - a SiO2 film 17, and a PLL-g-PEG or silane layer 16

[0065] - a gold film 19 with a thiol-based layer 18.

[0066] Typically, films 17, 19 have a thickness of around 100 nm.

[0067] Preferably, the functionalized areas are located at the most mechanically sensitive locations, i.e. those exhibiting the greatest displacement, for example the edge of the moving part and / or opposite the fixed part.

[0068] The adapters are therefore chosen according to the recognition probe to be placed between the fixed part and the moving part and therefore according to the analyzed strand.

[0069] Advantageously, the device comprises several fixed parts and several recognition probes connecting the fixed parts to the moving part, the probes having the same nucleotide sequences. The fixed parts may be a single piece and, for example, formed of a ring around the moving part. By increasing the number of recognition probes, the probability of binding to the strand(s) is increased. analyzed, which increases the sensitivity of the device.

[0070] Figure 5 shows a schematic representation of an assembly comprising the sequencing device of Figures 1 and 2 integrated into a fluidic channel C, for example formed by cooperation between a cover 21 and the support 3 of the device. The circulation of the liquid containing the strands to be analyzed is either open-circuit or continuous-circuit. In an open-circuit configuration, the channel is connected to a reservoir containing the liquid and the strands to be analyzed, and to a collection zone for the liquid after circulation in the channel. A circulation pump may be provided.

[0071] In a closed circuit, the liquid is, for example, injected into the channel by a syringe, and a pump ensures the recirculation of the liquid within the channel. Closed-loop operation allows for the recycling of products and the performance of multiple measurements. The sensors can be reset by thermally melting the paired DNA strands. It is also possible to accumulate measurement data without intermediate heating between consecutive measurement cycles by repeatedly circulating the fluid through the device(s).

[0072] The operation of this device will now be described.

[0073] The moving part 4, connected to the fixed part 2 by the "adapter-recognition probe-adapter" assembly, is set into vibration by the excitation means. The amplitude of the vibration is on the order of an interatomic distance, approximately 0.1 nm (1 Angstrom). The "adapter-recognition probe-adapter" assembly exhibits a certain stiffness and dissipates a certain amount of energy. The moving part oscillates in the plane at a given frequency, typically between 100 kHz and 1 GHz, preferably between 1 MHz and one or more tens of MHz, which results from the excitation means, the stiffness of the "adapter-recognition probe-adapter" assembly, and its dissipation.

[0074] A liquid containing the analyzed DNA or RNA strand circulates over the sequencing device. This device comes into contact with at least the portion of the SR recognition probe not hybridized with the adapters. The analyzed strand hybridizes more or less completely with the binding probe ([Fig. 2]). The higher the level of hybridization, the greater the stiffness of the "adapter-recognition probe-adapter" assembly and the analyzed strand, and the more the probe-strand assembly couples the fixed part to the moving part, which has the effect of modifying the oscillation frequency of the moving part. The measurement means measure this vibration frequency, which has changed relative to its vibration frequency before the hybridization of the analyzed strand. The frequency can vary from one to several hundred Hertz.

[0075] Indeed, when the analyzed strand hybridizes with the recognition probe, the two strands contract and the resulting assembly exhibits a determinate and known longitudinal stiffness, greater than that of the recognition probe alone. High-sensitivity measurement, down to the nucleotide level, of SNPs (single-nucleotide polymorphisms) can be offered in the implementation of kinetic competition such as strand displacement. The device therefore exhibits high reliability, high sensitivity, and a high detection rate.

[0076] The measurement of dissipated energy is a rich source of information on the hybridization process.

[0077] In the case where no hybridization takes place between the analyzed strand and the hooking probe, the vibration frequency of the moving part does not vary or does not vary significantly, in particular less than 0.001% variation, which corresponds for example to less than 300 Hz of variation for an initial resonance frequency of 30 MHz.

[0078] The high sensitivity of the detection means makes it possible to detect the level of hybridization between the strand and the recognition probe. The fact that the device makes it possible to detect and measure intermediate hybridization states (partial hybridization) nevertheless allows for obtaining selective information on the different sequences.

[0079] This device allows for high detection reliability.

[0080] Furthermore, the sequencer device allows the analysis of long nucleotide sequences, which reduces analysis time. For example, it is estimated that approximately 5*10⁶ base pairs are needed to analyze a bacterium.

[0081] Advantageously, mechanically stressing the hybridized strand allows for verification of the hybridization. If the strand is partially hybridized, the hybridization will be less resistant to axial mechanical stress. Axial stress thus significantly reduces the risk of false positives.

[0082] The sequencing process can then proceed according to the following steps:

[0083] - Vibrating the resonator at a given frequency, preferably its frequency mechanical resonance,

[0084] - Circulation of a solution containing the strand to be analyzed over the device,

[0085] - Measurement of the resonator's vibration frequency,

[0086] - Detection of perfect hybridization, partial hybridization or no hybridization.

[0087] - If there is perfect hybridization, the sequence of the analyzed strand is then known.

[0088] Preferably, during hybridization the liquid circulates.

[0089] The flow rate of the liquid is preferably limited, for example to a flow rate on the order of one or more ml / min. The hybridization can last on the order of several minutes, for example 5 minutes.

[0090] The flow rate, the repetition of measurements with cycles, and the use of rest periods can be adjusted. The liquid circulation is stopped, and the measurement is performed by data accumulation with a probability of capture controlled by the diffusion of the targets [Note: an additional option that allows different types of measurement without disrupting the flow].

[0091] Figure 6 shows an example of an embodiment in which the means of detection methods are opto-mechanical.

[0092] The device comprises an optical resonator 4, at least one waveguide 20 optically coupled to the resonator, the waveguide being supported by the substrate, a light source SL, and means for processing the light wave exiting the waveguide. The support, the waveguide, and the opto-mechanical resonator form a sensor structure.

[0093] The waveguide 20 has an input end 20.1 of a light wave connected to a light source via a coupling network 22.1 not shown in the figure, and an output end 20.2 connected to means for processing the light wave exiting the waveguide via a coupling network 20.2.

[0094] The resonator 4 is arranged near one side of the waveguide 20 so as to be optically coupled to it. The waveguide 20 is in the evanescent field of the resonator, so that the light wave from the source SL is injected into the optical resonator and the light wave that has traveled through the resonator is collected by the waveguide. The wave traveling through the resonator is symbolized by an arrow.

[0095] The width of the space between the side of the waveguide and the lateral edge of the resonator is for example between 10 nm and 50 nm.

[0096] In the example shown, the optomechanical resonator 4 has the form of a disk suspended from a foot 24 fixed to one face of the disk facing the substrate. The disk extends in a plane of the sensor. The resonator has two end faces 4.1, 4.2 substantially parallel to the plane of the sensor and a lateral face 6.3 ([Fig.6]).

[0097] Preferably the foot has a small diameter compared to the dimensions of the disk in the plane of the sensor, more particularly a small diameter compared to the diameter of the disk, preferably the foot has a diameter at least 10 times smaller than the diameter of the disk.

[0098] More generally, the diameter of the foot is ten times smaller than the smallest dimension of the resonator in the plane of the sensor, so the foot does little or no interference with the radial vibration of the resonator.

[0099] Alternatively, the resonator is suspended by in-plane springs or by radially extending nanometer-sized beams compressed and tensioned by the vibration of the disk. The springs or beams are then dimensioned to have a lower axial stiffness than that of the resonator.

[0100] Any other form of resonator may be suitable, for example, the top view of the D The sonorator can be in the shape of a ring, ellipse or racetrack. In the case of a ring, ellipse or racetrack shaped resonator, it is suspended from the substrate for example by means of a foot located in the center of the resonator, and beams extending between the inner edge of the resonator and the foot.

[0101] The resonator can be made of any material capable of confining an electromagnetic wave, such as GaAS, Ge or Si. The latter is particularly interesting for fabrication using microelectronic techniques offering a high level of integration on a substrate.

[0102] The functionalization layer is formed for example on face 4.2 of the resonator opposite that 4.1 with respect to the substrate.

[0103] The excitation means are, for example, electrostatic means (not shown) or optical means.

[0104] The operation of the sensor will now be described.

[0105] Preferably, the wavelength of the light wave to be injected into the resonator is chosen close to the optical resonance of the resonator, i.e., on the flank of the optical resonance peak. The light resonating inside the optical resonator is then very sensitive to the mechanical deformation of the mechanical resonator, in particular when the optical and mechanical resonators coincide.

[0106] The light wave at the chosen wavelength is injected into the waveguide by the light source, by optical coupling the light wave is injected into the optomechanical resonator 4.

[0107] When an analyzed strand hybridizes on the recognition probe, depending on the level of hybridization the stiffness of the link between the resonator and the fixed part is modified and the dissipated energy is also modified, which has an effect on the vibration frequency of the resonator, which generally increases.

[0108] Indeed, the mechanical frequency

[0109] f _ iraideur" I'm massing there

[0110] The mass of the resonator does not vary much during hybridization, therefore when the stiffness of the resonator increases, its frequency increases.

[0111] Measuring the variation in vibration frequency makes it possible to determine whether hybridization was complete or not, and if hybridization was complete, to determine the identity of the analyzed strand. Indeed, the optomechanical resonator is sensitive enough to distinguish between perfect and near-perfect pairing.

[0112] Alternatively, the measurement of the variation in the vibration frequency can be combined with a measurement of the variations in the optical properties of the resonator, allowing additional information to be acquired.

[0113] The optomechanical resonator, for example, has a vibration frequency between 100 MHz and 1 GHz with amplitude oscillations on the order of a few picometers. Operating at high frequencies increases detection sensitivity and the accuracy of the data collected, particularly through a level of temporal sampling never before achieved.

[0114] The optomechanical resonator as described above also offers the advantage of providing very good performance in liquid media.

[0115] Preferably, a system is implemented comprising a large number of D-sequencing devices ([Fig. 7]). For example, between 9 and on the order of 10,000 resonators may be used. An even higher number of resonators can be used, the limit being determined based on the manufacturing cost.

[0116] Each device or group of devices can be equipped with different recognition probes. Each detector delivers its own signal, which is processed by a processing unit. Only a portion of the devices is shown schematically.

[0117] The system includes, for example, a fluidic channel common to all devices or several parallel fluidic channels, each feeding a column of devices. The channel or channels are formed, for example, in a hood (not shown).

[0118] The implementation of optomechanical means facilitates such integration thanks to multiplexing techniques which are very well mastered in the field of optics.

[0119] In addition, the implementation of microelectronic manufacturing techniques allows the integration of a very large number of devices.

[0120] The recognition probe equipping each device is known since it depends on the adapters fixed to the fixed and moving parts, which are themselves determined by the functionalization of each part. The analyzed strand detectable by each device is therefore known.

[0121] By spatially locating the probes and therefore the responses given by each of the devices, it is possible to carry out usable redundant measurements, and to obtain low error rates.

[0122] The sequencing method described above applies to a sequencing system; however, such a system allows the detection of several different strands. The liquid then contains fragments of genomes, for example, a genome of several thousand bases. This is injected into the channel or channels of the system, and after a potential hybridization step of the strands on the appropriate recognition probes, the vibration frequencies of the different resonators are measured.

[0123] The frequencies can be measured continuously, allowing the hybridization process to be monitored.

[0124] The sequencing device and the sequencing system are reusable; after a sequencing cycle, they can be rinsed to remove the hybridized strands on the SR reconnaissance probes.

[0125] An example of a rinsing process uses a DNA-free buffer solution as the rinsing solution, for example, tris-acetate, tris-EDTA, PBS (Phosphate Buffered Saline), or HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)) with the addition of a surfactant, typically in small quantities, i.e., on the order of 0.1% or less. The surfactant may be SDS (Sodium dodecyl sulfate) or TWEEN (Polyethylene glycol sorbitan monolaurate). The rinsing solution is circulated to detach the DNA strands hybridized to the recognition probe. Preferably, the circulation should not be in a closed loop. This example is given for illustrative purposes only; those skilled in the art will be able to adapt the components of the rinsing solution according to the nature of the DNA strands and other reagents used.

[0126] It may also be envisaged to replace the recognition probes; these can be removed and replaced by other probes to modify the application of the device or system.

[0127] By raising the temperature to a range for example between 50 and 80°C, it is possible to detach the probes from the hooks and the hooks become free to put new probes.

[0128] It should be noted that the resonators can be set into vibration at different frequencies, for example they can have different mechanical resonance frequencies.

[0129] By way of example, using linear recognition probes of 50 micrometers, for a sequencing system comprising groups of 200 devices equipped with the same recognition probe containing 150 nucleotide pairs. The sequencing system comprises 4,104 optomechanical detectors / cm², i.e., approximately 200 groups of 200 devices having the same recognition probe and therefore capable of detecting 200 domains of different sequences. This system then makes it possible to analyze a complete viral genome of 30 kb, i.e., 30,000 bases or nucleotides, in a single series of measurements.

[0130] In another example, each device measures 40 pm on each side, which makes it possible to create sequencing systems with a device density of 62.5 x 03 cm².

[0131] As already mentioned above, the sequencing device and the sequencing system can advantageously be made at least in part by microelectronic techniques.

[0132] The moving part(s) and the fixed part(s), and optionally the waveguide(s), are produced by deposition of layers onto a substrate and lithography and etching. The electrical traces are also produced by deposition and etching.

[0133] Polymer layers and adapter precursors are, for example deposited and located by lithography and engraving.

[0134] The hood is for example made of glass and is structured to form with the substrate the fluidic channel or channels.

[0135] The cap is attached to the substrate and, for example, bonded to it. In a subsequent step, a liquid containing the adapters is injected into the channel or channels. The liquid is a physiological saline solution. The adapters then attach to the adapter precursors. During the attachment phase, the liquid is either at rest or circulating, generating a shear force on the fixed and moving parts.

[0136] Typically, if circulation is not implemented, sedimentation and inaccurate readings, with a risk of false positives, are likely to occur. However, if the liquid circulation is too rapid, poor adhesion, leading to a risk of false negatives, is likely to occur. A person skilled in the art will know how to adjust the liquid circulation rate. For example, a rate between 1 nl / min and several mL / min can be used.

[0137] In a subsequent step, the physiological fluid containing the recognition probes is injected into the duct or ducts. The probes are attached at one end to an adapter on the fixed part and at the other end to the moving part. During the probe attachment phase, the fluid is either at rest or circulating, generating a shear force on the fixed and moving parts.

[0138] It should be noted that the relative arrangements of two neighboring devices are such that there is no risk of a probe becoming fixed on a moving part of one device and on a fixed part of a fixed part of another device.

[0139] The sequencing device and the sequencing system have the advantage of using well-established materials and techniques. They exhibit good robustness.

[0140] In addition, the combination of a large number of sequencing devices enables many applications, such as parallel processing and profiling of coding sequences, gene selection and localization, parallel processing of multiple mutations, and selection and localization of viral variants.

Claims

Demands

1. A device for sequencing at least one strand of nucleotides comprising a support (3), at least one mobile part (4) relative to the support (3), means for vibrating the mobile part (4) at a given frequency, means for measuring the vibration frequency of the mobile part (4), a recognition probe (SR) mechanically linking a fixed part (2) attached to the support and the mobile part (4), the recognition probe (SR) comprising at least one nucleotide sequence.

2. Sequencing device according to claim 1, wherein the means for vibrating the moving part are configured to vibrate the moving part at its resonant frequency.

3. Sequencing device according to any one of claims 1 or 2, wherein the means for measuring the vibration frequency are opto-mechanical means.

4. Sequencing device according to claim 3, wherein the means for measuring the vibration frequency comprise at least one waveguide optically coupled with the moving part, a light source for injecting light into the waveguide and a light processing unit exiting the waveguide.

5. Sequencing device according to claim 4, wherein the moving part is a disc, a ring, a racetrack or an ellipse suspended by at least one foot.

6. Sequencing device according to any one of claims 1 to 5, comprising several identical probes connecting the moving part to the support.

7. A sequencing device according to any one of claims 1 to 6, wherein the fixed part comprises a first adapter precursor and a first adapter having one end attached to the first adapter precursor and the other end comprising a nucleotide sequence hybridized with one end of the recognition probe, and wherein the moving part comprises a second adapter precursor and a second adapter comprising a probe having one end attached to the second adapter precursor and the other end comprising a nucleotide sequence hybridized with one end of the recognition probe.

8. Sequencing device according to any one of claims 1 to 7, in in which the means for vibrating the moving part (4) at a given frequency are of the electrostatic type and comprise an electrode on the fixed part and an electrode on the moving part.

9. Sequencing assembly comprising the device according to any one of the preceding claims and a fluidic channel ensuring contact between a liquid containing said analyzed strand and the recognition probe.

10. Sequencing system comprising a plurality of sequencing devices according to any one of claims 1 to 8.

11. Sequencing system according to claim 10, wherein the sequencing devices are divided into groups, each group comprising recognition probes having the same nucleotide structure.

12. Sequencing system according to claim 11, wherein the recognition probes are chosen so that in a single measurement a complete genome is analyzed.

13. A method for sequencing at least one nucleotide sequence comprising: - bringing a liquid containing said sequence into contact with a resonant device comprising a recognition probe connecting a fixed part and a moving part of the resonant device, - vibrating the moving part, - measuring the variation in the vibration frequency of the moving part due to hybridization between the recognition probe and the nucleotide sequence.

14. A sequencing method according to the preceding claim, wherein the moving part is set into vibration at its resonant frequency.

15. A sequencing method according to any one of claims 13 or 14, wherein the moving part is vibrated with an amplitude of the order of 0.1 nm and / or vibrated at a frequency between 100 MHz and 1 GHz.

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