Fluorescence detection device using nanophotodetectors

Abstract

The invention relates to a device for identifying a fluorescent marker. The detection device comprises nanowires, each nanowire acting as a transducer of fluorescence light into an electrical signal. One intended application is nucleic acid sequencing. Figure 1E.

Description

Title of the invention: Fluorescence detection device using nanophotodetectors Technical field

[0001] The technical field of the invention is the detection of biomolecules by optical transduction carried out by nanowires. PREVIOUS ART

[0002] Devices for detecting biomolecules may be based on optical detection. The biomolecules to be detected are previously labeled using a luminescent marker, such as a fluorophore or quantum dot. Detection is carried out by a matrix photodetector, such as a CCD, CMOS imager or avalanche photodetector arrays (APD: Avalanche Photodetectors).

[0003] A widespread application of fluorescent labeling is sequencing, in particular so-called SBS sequencing (Sequencing by Synthesis). According to this method, a single-stranded nucleotide sequence, arranged downstream of a primer, is amplified so as to form a cluster. During sequencing, a complementary sequence is progressively synthesized, by successive addition of nucleic bases complementary to those forming the sequence studied. The complementary bases are added one by one, respectively during cycles. After each addition of a complementary base, an image of the cluster is produced. All or part of the complementary bases is marked by a fluorescent label. The observation of the fluorescence during each cycle makes it possible to identify the complementary base added during said cycle. Thus, by producing a succession of fluorescence images, it is possible to decode the nucleotide sequence examined.

[0004] Fluorescence detection is performed by a photodetector, usually a matrix photodetector. However, photodetectors have a detection matrix whose spatial pitch is generally greater than 1 pm. By spatial pitch is meant the distance between two adjacent pixels. Such a spatial pitch is considered too high to obtain a device capable of addressing a large number of detections in parallel. These devices also have limited sensitivity: it is necessary to have sufficient amplification to obtain a sufficient number of identical sequences at the level of the cluster studied, so as to obtain a usable signal.

[0005] Some biosensors are based on the detection of charges carried by the target biomolecules to be detected. These are, for example, field effect transistors. This type of device allows for rapid detection with good sensitivity. In addition, CMOS (complementary metal oxide semiconductor) technology allows the fabrication of devices with a large number of sensors. This makes it possible to perform a large number of analyses in parallel. However, this type of sensor can be sensitive to environmental parameters affecting the sample, for example pH or temperature or interactions with non-target biomolecules or ions inducing detection errors. The use of this type of biosensor in DNA (deoxyribonucleic acid) sequencing applications requires a certain redundancy of measurements, in order to increase the robustness of the measurements. DNA sequencers based on nanopores have the same disadvantages.

[0006] Photonic sensors based on nanowires have been developed for the purpose of detecting DNA fragments. This is for example the case of the publication Sing, et al., “Silicon nanowire optical rectangular waveguide biosensor for DNA Hybridization”, IEEE Photonics Technology Letters, 2018, 30(12) 1123-1126. In this publication, DNA hybridization is detected by detecting a variation in refractive index during hybridization

[0007] The publication Irrera, et al., “New generation of ultrasensitive label-free optical Si nanowire-based biosensors”, ACS Photonics 2018, 5(2) 471-479 describes a nanowire-based biosensor used to detect CRP (C-reactive protein) in human serum.

[0008] Applications related to DNA sequencing assume fluorescence coding, so as to differentiate the added bases. In applications using a sensor, the coding is carried out either by the emission wavelength or by a light intensity at the level of the image acquired by the photodetector.

[0009] The inventors have designed a compact and sensitive sample analysis device, making it possible to take advantage of the fluorescence detection capabilities of nanowires. The device can allow simultaneous or sequential analysis of different biomolecules. The device makes it possible to benefit from the detection sensitivity and compactness conferred by nanowires, while allowing differentiated detection of nucleic bases labeled by fluorescent markers. Statement of the invention

[0010] A first object of the invention is a device for identifying a fluorescent marker, the fluorescent marker being configured to emit fluorescence light in a fluorescence spectral band, the device comprising: - a substrate, comprising at least one first electrode; - a multilayer structure, comprising at least one second electrode; - nanowires, extending between the first electrode and the second electrode, parallel to a transverse axis; - an encapsulation layer extending around the nanowires, between the substrate and the multilayer structure, the encapsulation layer being formed of an insulating material; - the multi-layer structure comprising: • a conductive layer, forming each second electrode; • an electrically insulating interface layer covering each second electrode, each second electrode being interposed between the interface layer and a nanowire, the interface layer being delimited by a functionalization surface, the interface layer being configured to be arranged between a sample, comprising the fluorescent marker, and the second electrode, such that the functionalization surface forms an interface between the device and the sample; • the multilayer structure being such that the second electrode and the interface layer are transparent in a detection spectral band comprising the fluorescence spectral band;

[0011] the device being such that: - each nanowire comprises a homojunction type junction, or a heterojunction, or a Schottky junction between the first electrode and the second electrode; - the first electrode and the second electrode are configured to be connected to a detection circuit; - such that each nanowire forms a nanophotodetector of the fluorescence light when the fluorescent marker is bound to the functionalization surface, the light detected by each nanowire inducing an electrical detection signal in the detection circuit;

[0012] the device comprising a processing unit, programmed to: • acquire the detection signal during a detection time period; • determine a characteristic of the detection signal during the detection time period; • identify the fluorescent marker based on the characteristic.

[0013] According to one possibility, the device comprises a light source, configured to emit excitation light in the excitation spectral band of the fluorescent marker.

[0014] According to one possibility: - the light source is configured to emit the excitation light during an excitation time period;

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[0023] - the detection time period is later than the excitation time period. According to one possibility: - following the excitation time period, the intensity of the fluorescence light increases and then decreases; - the characteristic of the detection signal is representative of the decrease in the intensity of the fluorescence light. According to one possibility the functionalization surface is configured to capture a strand forming an oligonucleotide chain. According to one possibility, each nanowire comprises: - a pn type homojunction; - or a heterojunction; - or a p-metal or n-metal Schottky junction. According to one possibility at least one nanowire comprises a first part and a second part, separated by the junction, the first part being formed of an n-doped semiconductor, the second part being formed of a p-doped semiconductor, the junction forming a homojunction or a heterojunction. According to one possibility at least one nanowire comprises a first part and a second part, separated by the junction, the first part being formed of a semiconductor, the second part, adjacent to an electrode, being formed of a metal, the junction forming a Schottky junction. According to one possibility, the first part and the second part extend, along the transverse axis, on either side of the junction. According to one possibility the second part surrounds the first part, around the transverse axis. According to one possibility the functionalization surface is segmented into different capture sites, each capture site being configured to capture a strand forming a chain of oligonucleotides. According to one possibility: - the interface layer comprises two sub-layers stacked on top of each other, forming a lower sub-layer and an upper sub-layer, the lower sub-layer being interposed between the conductive layer and the upper sub-layer; - the upper sub-layer comprises wells, opening into the lower sub-layer, each well being arranged facing a nanowire, each well forming part of the functionalization surface; - the functionalization surface is segmented at the level of each well, so that each well forms a capture site.

[0024] According to one possibility, the device comprises several nanowires, extending between the same first electrode and the same second electrode, the nanowires forming a cluster of nanowires.

[0025] According to one possibility, the device comprises several clusters of nanowires (35), distant from each other, such that a nanowire of a cluster is closer to another nanowire of said cluster than to another nanowire of another cluster, the nanowires of the same cluster extending between the same first electrode and the same second electrode.

[0026] According to one possibility, the device comprises several nanowires, the device being such that: - several first electrodes are formed on the substrate, and several second electrodes are formed on the multilayer structure, each nanowire extending between a first electrode and a second electrode; - each first electrode is connected to a first addressing unit, configured to select at least one first electrode; - each second electrode is connected to a second addressing unit, configured to select at least one second electrode; - such that the detection circuit detects a detection current induced by each nanowire extending between the selected first electrode and second electrode.

[0027] A second subject of the invention is a method for identifying a fluorescent marker using a device according to the first subject of the invention, the fluorescent marker being capable of emitting fluorescence light, in the detection spectral band, when it is illuminated by the excitation light, the functionalization surface being configured to capture a strand of nucleic acid, the device comprising: a. arrangement of a sample, comprising nucleic acids, in contact with the functionalization surface; b. capture of at least one nucleic acid strand on the functionalization surface; c. adding nucleic bases to the sample, at least two different nucleic bases being labeled with two different fluorescent markers, the sample comprising active ingredients configured to allow hybridization of a nucleic base to the nucleic acid strand captured on the functionalization surface; d. exposing the functionalization surface to an excitation light, in an excitation spectral band of at least one fluorescent marker; e. following step d), detection of a detection signal at the terminals of the circuit of

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[0035] detection, during a detection time period; f. depending on the detection signal detected during step e), identification of the fluorescent marker; g. repeating steps c) to f) so as to progressively hybridize nucleic bases along the nucleic acid strand. Step b) may involve amplification of each captured nucleic acid strand. According to one possibility, steps b) to g) are implemented on different capture sites distributed on the functionalization surface. According to one possibility, step f) comprises: - determination of a characteristic of the detection signal during the detection time period; - identification of the fluorescent marker according to the characteristic. According to one possibility: - the fluorescent marker is chosen from several candidate fluorescent markers; - step f) comprises a selection of the fluorescent marker from among the candidate fluorescent markers based on the characteristic of the detection signal. According to one possibility, step f) comprises an estimation of a time derivative of the detection signal. According to one possibility, step f) comprises a detection of an intensity level or an integration of the detection signal during at least a predetermined time period. According to one possibility: - prior to step a), each nucleotide sequence is linked to a known calibration sequence; - steps c) to f) are implemented so as to hybridize the bases of the calibration sequence, steps c) to f) forming a calibration phase; - the detection signal obtained during each step e) of the calibration phase is used to calibrate a response of the device to the bases of the calibration sequence. According to one possibility: - each candidate fluorescent marker emits fluorescence light with increasing then decreasing fluorescence intensity; - the decrease in the fluorescence intensity of each fluorescent marker is characterized by a decrease constant; - the decay constants of two different fluorescence markers, are different.

[0036] According to one possibility, following step g), the method comprises a step h) of identifying the hybridized nucleic acid base.

[0037] According to one possibility, - following step h), the fluorescent marker is cleaved and the sample is rinsed; - following rinsing, steps c) and h) are repeated, so as to identify a sequence of nucleotides forming the captured nucleic acid strand.

[0038] The invention will be better understood upon reading the description of the exemplary embodiments presented in the remainder of the description, in conjunction with the figures listed below. FIGURES

[0039] Figures 1A and 1B show the main components of a device according to the invention.

[0040] [Fig.lC] shows a spatial distribution of capture sites.

[0041] [Fig. 1D] represents an example of a device according to the invention, in which the electrodes are distributed according to a matrix arrangement.

[0042] [Fig.1E] shows an example of sequencing three different sequences, respectively on three capture sites.

[0043] [Fig.2A] shows temporal distributions of fluorescence light emission of different fluorescent markers.

[0044] Figures 2B and 2C show examples of characterization of the temporal distributions of fluorescence light emission, the characterization being carried out during a detection time period.

[0045] [Fig.2D] shows schematically different time periods of detection during the decay of the emission of a fluorescence light.

[0046] [Fig.3A] illustrates a calibration sequence.

[0047] [Fig.3B] shows different intensity levels detected during a calibration phase.

[0048] [Fig.3C] shows diagrammatically successive characterization signals, respectively during sequencing of a TTCG sequence, with cleavage of the fluorescent markers during each cycle.

[0049] [Fig.3D] shows schematically successive characterization signals, respectively during sequencing of a TTCG sequence, without cleavage of the fluorescent markers during each cycle.

[0050] Figures 4A to 4E show diagrams of steps in manufacturing nanowires using a so-called bottom-up process.

[0051] [Fig.5] represents different possible arrangements of the nanowires.

[0052] Figures 6A and 6B show two different nanowire structures.

[0053] [Fig.7] shows different stages of implementation of the method. PRESENTATION OF SPECIAL EMBODIMENTS

[0054] Figures 1A to 1E show a first example of an analysis device 1 enabling implementation of the invention. The analysis device 1 is configured to be placed in contact with a sample 2. The sample comprises, for example, a liquid medium capable of containing strands of nucleic acids that it is desired to sequence. Each strand is derived from a fragmentation of genetic material, forming a library of strands. Each strand may comprise an adapter at the 3' end and the 5' end. In a known manner, the adapter may comprise a primer binding site and possibly an index comprising a sample identification code. One of the adapters may comprise a so-called calibration sequence, described below.

[0055] The device comprises a substrate 10, forming or comprising at least a first electrode 111c. In the example shown in [Fig. 1A], the substrate is a crystalline silicon substrate, for example a Si substrate with a (111) crystal orientation. The substrate 10 is delimited by a surface, called the first surface 11, comprising a first electrode 111c. In the example of [Fig. 1A], the first surface 11 is formed of Si comprising conductive regions. According to another possibility, the substrate 10 is subject to a deposition of a conductive layer, for example graphene, forming all or part of the first surface 11.

[0056] Nanowires 30 are formed on the substrate 10, and more precisely from the first surface 11. The first surface 11 extends along a plane PXY. The plane PXY is defined by a longitudinal axis X and a lateral axis Y. The axes X and Y are intersecting, and preferably perpendicular to each other. The nanowires extend parallel to a transverse axis Z intersecting the plane PXY. In the embodiments described below, the transverse axis Z is perpendicular to the plane PXY. The first surface 11 is conductive at least at the intersection with each nanowire 30. The entire first surface 11 may be conductive.

[0057] According to other configurations, the nanowires can be inclined and not perpendicular to the PXY plane. For example, if the crystalline orientation of the material forming the substrate 10 is (001), the nanowires can grow in a (111) direction, therefore at an angle relative to the PXY plane.

[0058] The nanowires 30 preferably have a diameter of between 1 nm and 500 nm and a height, along the transverse axis Z, of between 300 and 1000 nm, or even 10000 nm.

[0059] The nanowires 30 may be synthesized directly on the substrate 10, as described in connection with FIGS. 4A to 4E. The nanowires may be formed on another substrate and then transferred to the substrate 10. The transfer may be performed as described in the publication Valente, et al., “Light-Emitting GaAs Nanowires on a Flexible Substrate”, Nano Lett 2018, 18(7) 4206-4213.

[0060] The nanowires 30 extend, from the first surface 11, to a second surface 21 delimiting a multilayer structure 20. Like the first surface 11, the second surface 21 is conductive at least at the intersection with each nanowire 30. In the example shown in [Fig. 1B], the second surface 21 is formed from a layer 22 of a conductive material transparent in a detection spectral band described below. It may for example be ITO (indium tin oxide).

[0061] Each nanowire is formed from one or more semiconductor materials, and possibly from a metallic material. Each nanowire comprises a junction 33. In the example shown, the junction 33 is a homojunction: each nanowire comprises a first part 31, adjacent to the first surface 11, and a second part 32, adjacent to the second surface 21. The first and second parts are formed from the same semiconductor material, with respectively two different dopings: thus, the first part 31 and the second part 32 are respectively formed from the same semiconductor material respectively doped n and p or p and n. In the example shown, the first part 31 is formed from p-doped GaAs (gallium arsenide) and the second part is formed from n-doped GaAs. The interface between the two parts forms the pn junction 33.

[0062] Alternatively, the junction 33 can be made at a metal disposed on or in contact with the surface 21 so as to establish a Schottky-type semiconductor-metal junction.

[0063] Each nanowire comprises at least one semiconductor chosen from materials of columns III and V, usually designated by the term III-V materials, for example, GaAs. It may preferably be a material of column III and arsenic, for example InAs (indium arsenide). In the example shown, the nanowires 30 are formed from GaAs. Other semiconductor materials may be envisaged, for example, and in a non-limiting manner, Si, InGaAs, AlGaAs, InGaP, InGaN, GaN, ZnSe, ZnS, ZnO, ZnCdO, ZnTe, CdSe, Ge, GeSn.

[0064] Between the first surface 11 and the second surface 21, the nanowires 30 are embedded in an encapsulation layer 15 formed of an insulating material. The encapsulation layer 15 may be formed of a material of the PMMA (Polymethylmethacrylate), BCB (Benzocyclobuthene) or SOG (Spin-On Glass) type materials consisting mainly of silicon oxides and other chemical additives to provide specific properties such as adhesion and thermal stability. The encapsulation layer 15 may be deposited by spin-coating.

[0065] The encapsulation layer 15 is preferably formed following the growth of the nanowires, prior to the deposition of a second conductive layer 22, delimited by the second surface 21, and intended to form second electrodes 21c. The second electrodes 21c are formed, at the second surface 21, from the conductive layer 22. The conductive layer 22 may be structured so that at the second surface 21, several second electrodes 21c are electrically insulated from each other. Thus, the conductive layer may comprise openings or insulating materials delimiting the electrodes 21c. This allows differentiated detection at the level of each nanowire.

[0066] In addition to the conductive layer 22, the multilayer structure 20 comprises an interface layer 23, adjacent to the conductive layer 22. The interface layer 23 is transparent in the detection spectral band. The interface layer 23 may for example be formed from a layer of a polymer, for example PMMA (Polymethylmethacrylate). The interface layer 23 is electrically insulating, in particular at the interface with the conductive layer 22. The interface layer 23 is preferably planar.

[0067] The interface layer 23 is intended to form an interface between the conductive layer 22, forming the electrodes 21c, and the sample 2. Thus, the interface layer 23 extends between the conductive layer 22 and the sample 2. An important aspect of the device is that the sample is not in direct contact with the nanowires. It is isolated from the latter by the interface layer 23.

[0068] The interface layer 23 is for example a thin layer formed of SiO2 or PMMA. The surface of the interface layer 23, intended to be in contact with the sample, is a surface, called the functionalization surface 25, functionalized by biological capture species 26. It is important that the interface layer 23 is formed of a material having as low an autofluorescence as possible in the detection spectral band.

[0069] The functionalization surface 25, which forms an interface between the multilayer structure 20 and the sample 2 to be analyzed, is intended to be functionalized by capture species 26. By capture species, we mean a species configured to capture a biomolecule of interest, and more precisely a sequence of nucleotides to be analyzed.

[0070] According to one possibility, the capture species is formed of oligonucleotides configured to graft onto the nucleic acid to be analyzed. It may in particular comprise a base chain of nucleotides complementary to all or part of the adapters connected to the strands to be characterized. The connection between the capture species 26 and each strand of oligonucleotides to be analyzed, or more precisely an adapter of each strand of oligonucleotides to be analyzed, is then carried out by hybridization. The interface layer 23 may be nanostructured, so that nanowells 27 are formed at the interface between the multilayer structure 20 and the sample to be analyzed. The diameter or the most large diagonal of the nanowells can be between 70 nm and 700 nm. The nanowells 27 can for example be arranged in a matrix manner, or, more generally, according to a predetermined pattern. The functionalization surface is then functionalized at the level of each nanowell 27, the spaces between each well not being functionalized. The formation of the nanowells can be obtained by local thinning of the interface layer 23.

[0071] According to one possibility, shown in [Fig.1C], the interface layer 23 comprises two superimposed sub-layers 231 and 232. The interface layer comprises a lower sub-layer 231 interposed between the conductive layer 22 and an upper sub-layer 232. The nanowells are formed by local thinning of the upper sub-layer 232, so that the nanowells open into the lower sub-layer 231. The upper sub-layer 232 may be formed of a non-functionalizable material, for example an anti-biofouling material (anti-biological fouling), for example a hydrophobic material. Such a structuring allows the functionalization of the functionalization surface 25 to be carried out only at the level of the nanowells 27, on the lower sub-layer 231. Each nanowell 27 thus forms a capture site for a DNA strand.

[0072] More generally, the functionalization surface 25 can be functionalized according to a predetermined functionalization pattern. Apart from the functionalization pattern, the functionalization surface is not functionalized.

[0073] The functionalization can be carried out by a treatment of the functionalization surface 25, for example a plasma / oxygen surface treatment. When the interface layer 23 is formed from PMMA, a plasma / oxygen treatment makes it possible to form carboxyl functions. The capture species 26 can be grafted onto the functionalization surface by covalent bonding. To this end, the capture species comprise a function, for example an amine function, so as to form a covalent bond with the functions of the functionalization surface 25. It can for example be a bond obtained by grafting a Thiol function, at the capture species, onto an Amine function present at the functionalization surface.

[0074] According to one possibility, each sequence to be analyzed comprises a Thiol function, for example at the 3' end, so as to form a covalent bond with the functionalization surface. In this case, the functionalization surface acts as a capture species.

[0075] As shown in Figures 1B and 1E, the device comprises a detection circuit 40, a first terminal of which is connected to a first electrode 11c, on the substrate 10, and a second terminal of which is connected to a second electrode 21c, on the multilayer structure 20. The detection circuit 40 makes it possible to measure the difference of potential, or an electric current, between the first electrode 1 lc and the second electrode 21c.

[0076] As described in connection with Figures 1A to 1C, each nanowire extends between a first end, on a first surface 11 of the substrate 10 and a second end, on a second surface 21 of the multilayer structure 20. The first surface 11 and the second surface 21 are conductive at least at each intersection with a nanowire. Thus, at each intersection with a nanowire, the first surface comprises a first electrode 11c and the second surface comprises a second electrode 21c. In the example shown in Figures 1A to 1B, the first surface 11 and the second surface 21 are formed from a conductive material. They are conductive over their entire area.

[0077] The first and second surfaces can be structured, and comprise different electrodes 11c, 21c isolated from each other. In [Fig.1D], each electrode is represented by a dotted line. On the first surface 11, each first electrode 11 describes a line, parallel to the longitudinal axis X. On the second surface 21, each second electrode 21c describes a column, parallel to the lateral axis Y. Each nanowire is functional when the electrodes 11c, 21c, between which it extends, are polarized. The structuring of the electrodes in rows / columns makes it possible to select the functional nanowires, the latter extending between two polarized electrodes: according to this arrangement, the nanowires connected to a polarized row and column are functional. By functional nanowire, we mean a nanowire polarized to perform fluorescence photon detection.

[0078] Several rows and / or several columns can be polarized simultaneously or successively. The detection circuit 40 comprises: - a 40x addressing unit, intended to polarize all or part of the first llc electrodes, parallel to the X axis, - and an addressing unit 40Y intended to polarize all or part of the second electrodes 21c parallel to the Y axis.

[0079] The functionalized parts of the functionalization surface form capture sites for nucleotide sequences to be characterized.

[0080] As previously indicated, each biological capture species 26 is configured to capture a biological species of interest, in particular a sequence of nucleotides to be analyzed.

[0081] Preferably, after a nucleotide sequence has been captured at a capture site, the latter is amplified, so as to obtain, at the same capture site, a plurality of identical replicas, apart from amplification errors, of the captured nucleotide sequence.

[0082] The amplification of each captured sequence can be of the “amplification by bridge”, usually referred to as “bridge amplification”, and usually implemented in devices offered by the company Illumina. When implementing bridge amplification, the functionalization surface carries capture species configured to graft onto the adapters of the captured sequences.

[0083] In [Fig. 1E], three Sb sequences S2 and S3 are shown, respectively captured, then amplified, on three capture sites 251, 252 and 253. Each nucleotide sequence to be characterized is for example obtained according to a method for preparing a library of short DNA fragments, the length of which is typically 300 bases or 150 bases.

[0084] Generally speaking, sequencing involves several cycles, during which complementary bases are progressively hybridized along each sequence to be characterized. In a known manner, during each cycle, the sequences to be characterized are immersed in a reaction medium comprising bases labeled with a fluorescent marker and a hybridase enzyme. Hybridization is carried out progressively along each strand, base by base, in a predetermined direction, for example from the free end to the end linked to the capture species.

[0085] According to one possibility, the fluorescent markers are coupled to a terminator, which prevents the hybridization of two consecutive bases during the same cycle. The reaction medium then comprises a mixture of bases, each base of the same type being labeled with the same fluorescent marker. Two bases of different types are respectively labeled with different fluorescent markers. Following each hybridization, detection of the fluorescent marker is carried out after rinsing, which makes it possible to identify the base that has hybridized during the cycle. Following detection, at least the terminator undergoes cleavage. The fluorescent marker may also undergo cleavage. The medium is then rinsed and another cycle is initiated. The cycles are repeated until the sequences grafted at the different capture sites are completely sequencing. Among the four different bases used, one base may not be labeled.

[0086] According to another possibility, during each cycle, the sequences are bathed successively in baths, each bath comprising a single type of base. Each base of the same type is labeled with the same fluorescent marker. According to this possibility, it is possible, but not necessary, for two bases of different types to be respectively labeled with different markers. Indeed, the identification of each type of base is carried out by each bath. Between each bath, rinsing is carried out and detection of fluorescence at each capture site is carried out. According to this possibility, the fluorescent markers do not necessarily comprise terminators. Thus, a plurality of identical successive bases can be hybridized by complementary bases of the reaction medium. This results in a fluorescent signal rescence whose intensity increases according to the number of successive bases hybridized.

[0087] Following detection of a fluorescence signal, the fluorescent markers labeling each hybridized base during the cycle can be cleaved. As described below, cleavage is not necessary.

[0088] Thus, at each capture site, each cycle comprises: - the addition of a reaction medium containing bases and a hybridase enzyme (polymerase enzyme): • either in the form of a mixture of bases of different types, in which case the fluorescent markers include a terminator; • either in the form of successive baths, each bath containing bases of the same type, in which case the fluorescent markers may not contain a terminator; - a rinse; - detection, by nanowires, of fluorescence (or absence of fluorescence) - following the detection of fluorescence, possible cleavage of any terminators and possibly fluorescent markers, followed by rinsing.

[0089] Between each cycle, the reaction medium is washed and renewed. Each cluster is excited by a light source 5, in an excitation band of the fluorescent marker or of each fluorescent marker. The light source is configured to emit excitation light in a short duration, of the order of ns. It may for example be a laser light source. This makes it possible to generate, at the level of each cluster, a fluorescence signal, which depends on the base added (A, C, T or G in the case of a DNA strand).

[0090] An important aspect of the invention, described below, consists in translating the fluorescence signal into an electrical signal dependent on the fluorescent label, which makes it possible to obtain, during a cycle, an electrical signal dependent on the base added during the cycle.

[0091] This is a significantly different approach from the sequencing approach based on forming an image of the sample, with each base being identified by a color code and / or by an intensity level.

[0092] The invention takes advantage of the ability of a nanowire to detect an optical signal, in a predefined spectral band, and to convert the optical signal into an electrical detection signal. Thus, the device 1 is based on an optical detection of the hybridization of a base on a sequence to be analyzed, inducing an electrical response of the device.

[0093] In [Fig.1E], a cross-sectional view of the device is shown, after amplification of sequences. It is assumed that at each capture site, a single sequence was captured (SI sequence at site 251, S2 sequence at capture site 252). Each captured sequence was then amplified by bridging. After amplification, at each capture site, identical sequences extend, formed at their 3' end from a PI adapter. The sequences have, at their 5' end, a P2 adapter. The PI adapter is part of the capture species 26 grafted, by covalent bond, onto the functionalization surface. The P2 adapter of each sequence is free.

[0094] The reaction medium 2 is placed in a fluidic chamber 4, in contact with the interface layer 23. The sequences to be characterized and the reaction medium are isolated from the nanowires 30 by the interface layer 23. In this example, we are in the configuration according to which the reaction medium comprises different types of bases marked by fluorescent markers.

[0095] During each cycle, a light source 5 illuminates the functionalization surface 25, in an excitation spectral band centered on a fluorescence excitation wavelength of a fluorescent marker.

[0096] We refer to the sequence Si, captured and amplified on the capture site 25i. During a first cycle, a base T was hybridized. [Fig.lE] represents a second cycle, during which a base C labeled by a fluorescent marker C*, represented by the symbol *, hybridizes to a base G of the sequence. The objective of the device is to detect the fluorescent marker C*, by means of transduction of the fluorescence light.

[0097] The light source 5 generates an excitation light 7, which propagates through the sample 2 to the functionalization surface 25. Under the effect of the illumination at the excitation wavelength, a fluorescence light 8 is emitted by the fluorescent marker C*, in a fluorescence wavelength, higher than the excitation wavelength. Fluorescence photons are emitted, forming the fluorescence light 8. Due to the transparency of the interface layer 23, or the material 22 constituting the electrode 21c, certain fluorescence photons propagate through a nanowire 30. When a fluorescence photon is absorbed at the junction 33, electron / hole pairs are formed.Under the effect of the potential difference between the first part 31 and the second part 32, the electrons propagate through the n-doped portion, towards the higher potential while the holes propagate in the opposite direction through the p-doped portion. This results in an increase in the electric current flowing in the detection circuit 40.

[0098] The choice of the semiconductor material forming the nanowire 30 depends on the absorption spectral band of said material, the latter having to contain both the fluorescence wavelength, and, preferably, not contain the excitation wavelength of the fluorescent marker. The nanowire thus acts as a nanophoto fluorescence light detector, while not being sensitive to the excitation wavelength. Each nanowire thus forms a filter with respect to the excitation wavelength. Optionally, an absorbing layer can be added between the functionalization surface 25 and the conductive layer 22 to reduce the spectral width of the response of the nanowires, for example a layer of zinc oxide deposited by sputtering. When the semiconductor material is GaAs, the fluorescent marker can be Cy3 (Cyanine): excitation wavelength 540nm and emission wavelength between 555 and 600nm.

[0099] Preferably, the diameter of the nanowires is controlled so as to provide sensitivity in a narrow spectral band. By narrow spectral band is meant a bandwidth typically of a few tens of nm, and preferably less than 100 nm. The bandwidth corresponds to the width at half-height of the absorption peak on the absorption spectrum. The detection bandwidth is for example of the order of 50 nm. It is thus possible to adjust the detection spectral band so that it includes the fluorescence wavelength of the fluorescent marker and does not include the excitation wavelength of the fluorescent marker. The ability of nanowires to form a wavelength-selective photodetector has been described in Mokkapati et al., “Optical design of nanowire absorbers for wavelength selective photodetectors”, Sci. Rep. 2015, 5 15339.

[0100] The spectral detection sensitivity can be adjusted by incorporating quantum wells or quantum dots at the junction 33. This makes it possible to obtain a junction 33 whose composition is different from the rest of the nanowire. The detected wavelength then corresponds to the wavelength of the gap defined by the quantum well or dot.

[0101] Each nanowire thus forms a nanophotodetector. During each cycle, it is necessary not only to detect a fluorescence light, but also to identify the fluorescent label that generated the fluorescence light, in order to identify the base to which it was bound. In this example, it is assumed that the nucleic bases contained in the reaction medium are respectively labeled by a fluorescent label A*, G*, C*. It is assumed that the T bases are not labeled by a fluorescent label. During each cycle, the following are determined: - either the absence of fluorescence light, which results in an absence of variation in the current resulting from the nanowires: this corresponds to a hybridization of a T base; - either a detection of a fluorescence light, by discriminating the markers A*, G* and C*.

[0102] An important aspect of the invention is that the discrimination is carried out by a characterization of the detection electric current, resulting from the nanowires.

[0103] In order to allow discrimination of the fluorescence light respectively emitted by each fluorescent marker, each fluorescence marker used has one or more emission parameters different from one another. By emission parameter is meant a parameter characterizing a temporal distribution of the fluorescence intensity emitted by the fluorescent marker. It may for example be the maximum intensity Imax of the distribution and / or the decay time of the fluorescence light. The decay time of the fluorescence light corresponds to a duration during which the fluorescence intensity decreases between two predetermined levels, for example between the maximum intensity Imax and a certain percentage of the maximum intensity, for example 50% or 80%. The decay of the fluorescence intensity may follow a variation of type gr , where t corresponds to the time and T is a decay constant.In this case, the decay time can be characterized by the decay constant T. .

[0104] In Figure 2A, three temporal distributions of the fluorescence intensity of the three fluorescent markers G*, C* and A* are shown diagrammatically, marking the bases G, C and A respectively. In this example, the maximum intensity Imax is considered to be identical for these three fluorescent markers. However, the decrease is faster for A* compared to C*, and for C* compared to G*. Thus, denotes the decay constant of A* and similarly for c* and G

[0105] S denotes a detection signal, resulting from the detection circuit 40, formed from the charges collected at the level of each nanowire. The device comprises a processing unit 41, allowing characterization of the detection signal, so as to identify the fluorescent marker having generated the fluorescence light. The processing unit 41 may comprise a microprocessor 41, connected to a memory 42 in which instructions are stored. The processing unit forms a characterization signal S* from the detection signal.

[0106] In [Fig.2A], we have represented: - an Atex excitation time period, which corresponds to the light pulse emitted by the light source 5. The Atex excitation time period can be, for example, a few tens of ps or of the order of ns. - a detection time period AL, during which the detection signal S is characterized, so as to identify the fluorescent marker having generated fluorescence light, or the absence of fluorescent marker. The detection time period AL may be for example 100 ps to 1 ns or a few ns. The detection period preferably extends during the detection growth of the fluorescence light emitted by the fluorescent marker.

[0107] The emission of fluorescence light makes it possible to obtain a detection signal which can be used for a fluorescence duration of the order of one or more ns.

[0108] Figure 2B illustrates a first exemplary embodiment, in which the characterization signal corresponds to an absolute value of the time derivative of the intensity of the _ I I. We observe that: | dt | the characterization signal is maximal when the fluorescent marker at the origin of the fluorescence is A*; the characterization signal is minimal when the fluorescent marker at the origin of the fluorescence is G*; the characterization signal is between the maximum value and the minimum value when the fluorescent marker at the origin of the fluorescence is C*. electric current resulting from each nanowire. Thus,

[0109] An absence of variation in the detection signal allows us to conclude that no fluorescence has been detected. This indicates hybridization of a T base on the sequence to be characterized.

[0110] The embodiment described in connection with [Fig.2B], based on a different measurement relative to the fluorescence intensity, has the advantage of being relatively insensitive to the reliability of the number of sequences respectively amplified at each capture site.

[0111] In a second example, shown in Figure 2C, the processing unit is programmed to determine a characterization signal S* which corresponds to a integral of the detection signal the characterization signal is maximal when the fluorescent marker at the origin of the fluorescence is G*; the characterization signal is minimal when the fluorescent marker causing the fluorescence is A*; the characterization signal is between the maximum value and the minimum value when the fluorescent marker at the origin of the fluorescence is C*.

[0112] When the characterization signal corresponds to an integration of the detection signal, the level reached by the characterization signal allows discrimination between the fluorescent markers G*, A* and C*. The same is true when the characterization signal is an average or a median or resulting from another statistical indicator representative of the intensity of the detection signal during the detection period.

[0113] However, the number of sequences to be characterized immobilized on the functionalization surface, directly above a nanowire or the same capture site can be variable, as well as the number of amplification errors. Thus, the number of identical sequences replicated by amplification at each capture site can vary from one capture site to another capture site. However, the intensity of the photoinduced current depends on the number of fluorescent markers marking hybridized bases during the same cycle. To address this difficulty, a calibration sequence Sc can be grafted onto each sequence to be characterized, so as to establish the intensity levels respectively associated with each fluorescent marker.

[0114] The calibration sequence Sc corresponds to a series of bases arranged in a known order, and whose location in the adapter is known. For example, the calibration sequence is arranged at a known location in the adapter P2 intended to remain free after amplification. The use of a calibration sequence is particularly useful when the characterization signal corresponds to the detection signal at a given instant, or comprises an integral of the detection signal.

[0115] [Fig.3A] illustrates an example of calibration sequences, composed of a succession of 4 T bases, 4 G bases and 4 C bases. In this example, the calibration sequence is part of the adapter P2. The characterization signal is an integral of the detection signal during a predetermined time period. When sequencing the calibration sequence: - during a first period Atb which corresponds to a period during which complementary bases A are successively hybridized to the bases T of the calibration sequence, the intensity of the characterization signal corresponds to a first level S*i. The fluorescent marker A* is symbolized by *1 in [Fig.3A]. - during a second period At2, which corresponds to a period during which complementary bases C are successively hybridized to the bases G of the calibration sequence, the intensity of the characterization signal corresponds to a second level S*2. The fluorescent marker C* is symbolized by *2 in [Fig.3A]. - during a third period At3, which corresponds to a period during which complementary bases G are successively hybridized to the bases C of the calibration sequence, the intensity of the characterization signal corresponds to a third level S*3. The fluorescent marker G* is symbolized by *3 in [Fig.3A].

[0116] [Fig.3B] shows the evolution of the characterization signal during the periods Atb At2 and At3. [Fig.3B] corresponds to a configuration according to which each fluorescent marker has a terminator. During the period Atb, the signal S*i is measured over four successive cycles. During the period At2, the signal S*2 is measured over four successive cycles. During the period At3, the signal S*1 is measured over four successive cycles. four successive cycles, the signal S*3.

[0117] The use of the calibration sequence allows an experimental determination of the levels S*b S*2 and S*3, and this on each capture site, knowing that the number of identical sequences can vary from one site to another. The levels thus established are used to interpret the detection signals detected during the cycles carried out on each sequence to be characterized.

[0118] According to a variant, illustrated in [Fig.2D], the detection signal is characterized during several successive detection periods Atd-1, Atd-2 and Atd-3. In such an embodiment, the use of a calibration sequence is also preferable.

[0119] During each cycle, the processing unit 41 establishes a characterization signal S*, which characterizes the detection signal S measured at the terminals of the detection circuit 40, the detection signal resulting from an electric current formed at the level of at least one nanowire. The characterization signal S* may comprise an integration or a derivative of the intensity measured by each nanowire taken into account. The processing unit compares the characterization to stored values ​​or to the values ​​established during the calibration period, each value being assigned to a fluorescent marker.

[0120] The characterization signal makes it possible to identify a fluorescent marker from among several so-called candidate fluorescent markers (in this example A*, C* and G*), the characteristics of which are known, in terms of emission intensity or decay, and this in the or each detection period. The characteristics can be stored in the memory 42. This makes it possible to identify the hybridized nucleic base during the cycle.

[0121] When three labeled bases are used, fluorescent labels suitable for implementing the invention are: DAPI (half-life time 1.88 ns), Hoechst33342 (half-life time 1.05 ns) and Hoeschst 33258 (half-life time 1.22 ns). The wavelengths conferring maximum excitation are respectively 358 nm, 350 nm and 349 nm.

[0122] The embodiment according to which the different types of complementary bases are implemented simultaneously is particularly suitable for implementing the invention. The fluorescent markers linked to each base comprise a terminator, the latter being eliminated by cleavage following the formation of the characterization signal. However, the invention can be implemented by exposing the sequences to be characterized to known bases by implementing successive baths.

[0123] It is noted that the fluorescent markers may be cleaved during each cycle, following the formation of the characterization signal. This is not, however, imperative.

[0124] In [Fig.3C], the characterization signal resulting from the sequencing of a TTCG sequence is shown schematically. In this example, the characterization signal corresponds to a integral of the detection signal during a predetermined detection period. Each base can be identified from the S*i, S*2 and S*3 signals established during calibration. In this example, it is assumed that following each detection, the fluorescent label undergoes cleavage. The x-axis designates the detected base. The y-axis corresponds to the characterization signal.

[0125] In [Fig.3D], the characterization signal resulting from the same sequence is shown schematically. In this example, the characterization signal corresponds to an integral of the detection signal during a predetermined detection period. Each base can be identified from the signals S*b S*2 and S*3 established during the calibration and stored in the memory 42. In this example, there is no cleavage of the fluorescent marker. The intensity of the fluorescence signal increases progressively, under the effect of the accumulation of fluorescent markers linked to bases hybridized on the sequence to be characterized.

[0126] The use of a calibration sequence can also be useful when the characterization signal is a differential signal, of the time derivative type. This can make it possible to calibrate the detection signal for the different bases. When the characterization signal is of the time derivative type, it is preferable for the fluorescent markers to be cleaved following the detection of the hybridization.

[0127] Generally speaking, the use of the calibration sequence makes it possible to establish a response of the device on each capture site. This makes it possible to take into account the fluorescence emission characteristics of each fluorescent marker, the variability of the number of sequences amplified at each capture site, but also the possible variabilities of the detection performance of each nanowire.

[0128] Preferably, the fluorescent markers are excited in the same excitation spectral band, which allows the use of the same light source. According to an alternative, the fluorescent markers are excited in different excitation spectral bands, which may require the use of different light sources.

[0129] The use of nanowires is particularly advantageous. This makes it possible to obtain an exploitable signal using a small number of sequences replicated on the same capture site. This makes it possible to reduce the size of the capture sites, relative to the prior art. It is thus possible to characterize, on the same functionalization surface, a larger number of sequences. The cycles can be implemented in parallel on each capture site defined on the functionalization surface. The reduction in the number of amplifications makes it possible to limit the disadvantages linked to the sequence amplification process: lower volume of reagents required, lower number of replicates, hence a lower number of amplification errors.

[0130] Indeed, the detection circuit allows measurement of very low currents, of the order of a few tens of pA to a few nA. In addition, the nanowires can be closely spaced from each other, typically with a spatial pitch of less than 500 nm. (compared to typically 1.1-1.7 pm for a CMOS optical detector). This makes it possible to have a device with a high useful surface area ratio (i.e. the functionalization surface area) to fluid volume.

[0131] The excitation spectral band is preferably between 350 nm and 550 nm. The detection spectral band of each nanowire is preferably sized so as to allow detection of the fluorescence light while masking the excitation light. Alternatively, the detection currents, respectively formed at each nanowire during the excitation and emission of the fluorescence light, can be temporally separated. Advantage is taken of the fast response time of the nanowires, of the order of ps, as described in the literature Gallo, et al., “Picosecond response times in GaAs / AlGaAs core / shell nanowire-based photo-detectors” Appl. Phys. Lett. 2011, 98(24) 241113.

[0132] We will now describe different design aspects of the device according to the invention. Formation of nanowires

[0133] Figures 4A to 4E diagram steps for forming nanowires from a silicon substrate 10. Figures 4A to 4E correspond to a so-called bottom-up approach. The substrate 10 is formed of Si, with an orientation (111) and comprises a surface layer 10i of SiO2, intended to form a barrier layer, with a thickness of 10nm - 15nm. The SiO2 layer is covered with a layer 102 of electrosensitive PMMA with a thickness of 45-80nm. The layers 10i and 102 are subjected to photolithography and etching respectively, so as to form nanowells isolated from each other, according to a predetermined pattern. See [Fig.4A]. The nanowells open onto the Si substrate. A thin layer of metal 103, for example gold, with a thickness of 5 to 10 nm is deposited on the SiO2 layer. The added metal, in this case gold, acts as a catalyst. See [Fig.4B]. The excess gold between the nanowells is removed by lifting (removing) the PMMA layer 102. See [Fig.4C].We thus obtain islands of gold 103 isolated from each other, at positions corresponding to the position of the previously formed nanowells.

[0134] After heating to a temperature above 450°C, the islands form drops. The gold drops act as a catalyst. The semiconductor nanowires are then formed by molecular beam epitaxy (MBE). This involves sending one or more molecular beams towards the substrate to achieve epitaxial growth. See [Fig.4D]. The vapor phase molecular beams contain the chemical species composing the semiconductor nanowire as well as the species dopants (e.g. Ga and As to form GaAs). The atomic species adsorb and diffuse to the surface of the Si substrate in the form of adatoms. The adatoms are incorporated into the gold droplets. When the latter saturate, the nucleation of the nanowires occurs first at the droplet / substrate interface, then at the droplet / forming nanowire interface.

[0135] The method is not limited to the use of gold as a catalyst. Other catalysts may be used, for example Ga or Sn. When the catalyst is Ga and Ga is also a constituent element of the semiconductor, this is referred to as self-catalyzed nanowire growth.

[0136] This method allows for example a growth of nanowires using As, Ga as well as C and Si respectively as p and n dopant, the implementation temperature being 600°C-610°C. The method continues until the nanowires reach a predetermined height. Cf. [Fig.4E].

[0137] Following the step shown in [Fig.4E], the encapsulation layer 15 is formed between the nanowires, for example by spin-coating. The encapsulation layer makes it possible to electrically insulate the nanowires from each other, and gives better mechanical strength to the assembly. Chemical, plasma etching and / or polishing can be carried out at the end of the nanowires opposite the substrate, so as to remove the catalyst residues and homogenize the height of the nanowires and the encapsulation layer 15.

[0138] The conductive layer 22, then the interface layer 23, are then successively deposited on the assembly formed by the nanowires and the encapsulation layer 15.

[0139] In the embodiment described in FIGS. 4A to 4E, the formation of the nanowells on the substrate 10 makes it possible to control the position of the nanowires. Thus, the nanowires can be arranged regularly, for example according to respectively square or hexagonal mesh patterns. The pitch between two adjacent nanowires can be small, of the order of twice the diameter, or be higher, for example a few to several hundred nm.

[0140] Another advantage of the bottom-up approach, described in connection with Figures 4A to 4E, is that it allows for more precise control of the crystalline structure of the nanowires. The bottom-up approach allows for controlled incorporation of quantum dots or quantum wells, by controlling their position and composition, in particular along the Z axis.

[0141] Figure 5 illustrates a configuration in which the nanowires are arranged to form clusters 35, or clusters. The nanowires of the same cluster are brought close to each other, the distance d between two adjacent nanowires of the same cluster preferably being greater than or equal to the diameter of the nanowires. The distance between two adjacent clusters may be equal to or greater than twice the distance d.

[0142] When the nanowires are distributed into clusters, (or bunches) as described in connection with [Fig. 5], the nanowires of the same cluster are preferably connected to the same electrode, both on the substrate 10 and on the multilayer structure 20. The nanowires of the same cluster address the same capture site. The nanowires of the same cluster are thus simultaneously functional. The nanowires not connected to the detection circuit are not functional. The distribution of the nanowires into clusters makes it possible to take advantage of the potential detection, by several adjacent nanowires, of the fluorescence resulting from the hybridization of bases of the same type on sequences of the same type.

[0143] Due to the high sensitivity of each nanowire, the number of nanowires making up a single cluster can be relatively small. Thus, the surface area, in the PXY plane, of each cluster is small, each cluster selectively addressing a different nucleotide sequence from another cluster. It is thus possible to arrange a large number of clusters, respectively addressing different nucleotide sequences, in a single compact device.

[0144] The arrangement of the nanowires in clusters 35 can be combined with a structuring of the functionalization surface in nanowells 27, as described in connection with [Fig. 1C]. In this case, each nanowell 27 extends opposite the nanowires belonging to the same cluster 35.

[0145] Alternatively, the nanowires are obtained by etching, using a so-called top-down approach. The top-down approach is described in patent application FR2114563 filed on 12 / 27 / 2021. Structure of the nanowires

[0146] Figures 6A and 6B represent other nanowire structures that can be implemented in a device according to the invention. [Fig.6A] represents a nanowire similar to the nanowires previously described. The junction 33 is arranged axially, extending between two parts 31, 32, of different doping, spaced from each other along the transverse axis Z. In the example of [Fig.6A], a passivation sheath 34 bypasses the nanowire.

[0147] In the example of [Fig.6B], the junction 33 extends radially between two different doping zones. Thus, the junction 33 extends around the transverse axis Z, parallel to the latter. The first part 31 and the second part 32 are separated radially, the separation between the two parts corresponding to a separation radius. The first part extends between the axis of the nanowire and the junction 33, while the second part extends around the junction 33.

[0148] An axial structure is considered advantageous because it favors an incorporation of quantum wells or quantum dots inside the nanowires in order to adjust the absorption spectrum.

[0149] A radial structure makes it possible to have a junction 33 extending along a significant height along the Z axis, which makes it possible to increase the detection sensitivity. Optionally, the radial structure shown in [Fig.6B] comprises an annular sheath 34 as described in connection with [Fig.6A].

[0150] [Fig.7] shows schematically the main steps of a method for implementing the invention.

[0151] Step 100: arrangement of a sample comprising nucleic acid sequences from a library previously prepared from the DNA strand to be sequenced, brought into contact with the functionalization surface 25.

[0152] Step 110: capture of nucleic acid sequences by the functionalization surface, preferably at capture sites arranged directly above the nanowires.

[0153] Step 115: amplification, for example by bridging, of the nucleic acid sequence captured in each site. Step 115 is optional, but it makes it possible to obtain, during each cycle, a detection signal of higher intensity, due to the increase in fluorescence light.

[0154] Step 120: addition of a reaction medium, comprising nucleic bases labeled with fluorescent markers and a hybridase. Preferably, only one nucleic base is capable of being hybridized on each sequence. Each type of base is labeled with a fluorescent marker called a predetermined candidate fluorescent marker, coupled to a terminator. Bases of the same type are labeled with the same candidate fluorescent marker.

[0155] Step 130: rinsing the sample;

[0156] Step 140: illumination of the functionalization surface, so as to cause fluorescence of hybridized fluorescent markers following step 120;

[0157] Step 150: detection of a detection signal at the terminals of the detection circuit.

[0158] Step 160: characterization of the detection signal, so as to identify the marker fluorescent among the candidate fluorescent markers.

[0159] Step 170: cleavage of the terminator, possible cleavage of the fluorescent marker, and rinsing of the sample;

[0160] Step 180: repeating steps 120 to 170, until each nucleotide sequence captured at a capture site is sequencing.

[0161] In the method described in connection with [Fig.7], during step 120, the reaction medium comprises a mixture of bases. As previously indicated, during each cycle, 4 successive baths can be provided, respectively comprising the 4 types of bases marked by fluorescent markers. These can then be identical or different from each other.

[0162] The device benefits from a fast response time, typically of the order of ns.

[0163] It is observed that the device does not require the use of optical components. bulky. In addition, the device's response is stable and not very sensitive to environmental variations: pH of the sample, temperature, presence of molecules or ions different from the biomolecule of interest. This is due to the fact that the nanowires are not in contact with the sample, but isolated, physically and electrically, from the latter by the interface layer 23.

[0164] Finally, since the device is based on nanophotodetectors, it allows for a compact analysis platform. The device can be obtained by implementing a collective manufacturing process, which allows for lowering the cost.

Claims

Claims

1. Device (1) for identifying a fluorescent marker, the fluorescent marker being configured to emit fluorescence light in a fluorescence spectral band, the device comprising: - a substrate (10), comprising at least one first electrode (lie); - a multilayer structure (20), comprising at least one second electrode (21c); - nanowires (30), extending between the first electrode (11c) and the second electrode (21c), parallel to a transverse axis (Z); - an encapsulation layer (15) extending around the nanowires, between the substrate and the multilayer structure (20), the encapsulation layer being formed from an insulating material; - the multi-layer structure comprising: • a conductive layer (22), forming each second electrode; • an electrically insulating interface layer (23) covering each second electrode (21c), each second electrode being interposed between the interface layer and a nanowire, the interface layer being delimited by a functionalization surface (25), the interface layer being configured to be arranged between a sample, comprising the fluorescent marker, and the second electrode, such that the functionalization surface forms an interface between the device and the sample; • the multilayer structure being such that the second electrode and the interface layer are transparent in a detection spectral band comprising the fluorescence spectral band; the device being such that: - each nanowire (30) comprises a junction (33) of homojunction type, or a heterojunction, or a Schottky junction between the first electrode and the second electrode; - the first electrode and the second electrode are configured to be connected to a detection circuit (40); - such that each nanowire forms a nanophotodetector of the fluorescence light when the fluorescent marker is bound to the functionalization surface, the light detected by each nanowire inducing an electrical detection signal in the detection circuit; the device comprising a processing unit, programmed to: • acquire the detection signal during a detection time period; • determine a characteristic of the detection signal during the detection time period; • identify the fluorescent marker as a function of the characteristic.

2. Device according to claim 1, comprising a light source, configured to emit excitation light in the excitation spectral band of the fluorescent marker.

3. Device according to any one of the preceding claims, wherein: - the light source is configured to emit the excitation light during an excitation time period; - the detection time period is subsequent to the excitation time period.

4. Device according to claim 3, in which: - following the excitation time period, the intensity of the fluorescence light follows an increase then a decrease; - the characteristic of the detection signal is representative of the decrease in the intensity of the fluorescence light.

5. A device according to any preceding claim, wherein the functionalization surface is configured to capture a strand forming a chain of oligonucleotides.

6. Device according to any one of the preceding claims, in which the functionalization surface (25) is segmented into different capture sites, each capture site being configured to capture a strand forming a chain of oligonucleotides.

7. Device according to claim 6 wherein: - the interface layer comprises two sub-layers (23b 232), stacked on top of each other, forming a lower sub-layer and an upper sub-layer, the lower sub-layer being interposed between the conductive layer (22) and the upper sub-layer; - the upper sub-layer comprises wells, opening into the lower sub-layer, each well being arranged facing a nanowire, each well forming a part of the functionalization surface; - the functionalization surface is segmented at the level of each well, so that each well forms a capture site.

8. Device according to any one of the preceding claims, comprising several nanowires, extending between the same first electrode and the same second electrode, the nanowires forming a cluster of nanowires.

9. Device according to claim 8, comprising several clusters of nanowires (35), spaced apart from each other, such that a nanowire of a cluster is closer to another nanowire of said cluster than to another nanowire of another cluster, the nanowires of the same cluster extending between the same first electrode and the same second electrode.

10. Device according to any one of the preceding claims, comprising several nanowires, the device being such that: - several first electrodes are formed on the substrate, and several second electrodes are formed on the multilayer structure, each nanowire extending between a first electrode and a second electrode; - each first electrode is connected to a first addressing unit (40x), configured to select at least one first electrode; - each second electrode is connected to a second addressing unit (40Y), configured to select at least one second electrode; - such that the detection circuit (40) detects a detection current induced by each nanowire extending between the selected first electrode and second electrode.

11. A method of identifying a fluorescent marker using a device according to any one of the preceding claims, the fluorescent marker being capable of emitting fluorescence light, in the detection spectral band, when illuminated by the excitation light, the functionalization surface being configured to capture a nucleic acid strand, the device comprising: a. arrangement of a sample, comprising nucleic acids, in contact with the functionalization surface; b. capture of at least one nucleic acid strand on the functionalization surface; c. adding nucleic bases to the sample, at least two different nucleic bases being labeled with two different fluorescent markers, the sample comprising active ingredients configured to allow hybridization of a nucleic base to the nucleic acid strand captured on the functionalization surface; d. exposing the functionalization surface to an excitation light, in an excitation spectral band of at least one fluorescent marker; e. following step d), detection of a detection signal at the terminals of the detection circuit, during a detection time period; f. depending on the detection signal detected during step e), identification of the fluorescent marker; g. repeating steps c) to f) so as to progressively hybridize nucleic bases along the nucleic acid strand.

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13.

14.

15.

16.

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18. The method of claim 11, wherein step b) comprises amplification of each captured nucleic acid strand. Method according to any one of claims 11 or 12, in which steps b) to g) are carried out on different capture sites distributed over the functionalization surface. The method of claim 13, wherein step f) comprises: - determination of a characteristic of the detection signal during the detection time period; - identification of the fluorescent marker according to the characteristic. The method of claim 14 wherein: - the fluorescent marker is chosen from several candidate fluorescent markers; - step f) comprises a selection of the fluorescent marker from among the candidate fluorescent markers based on the characteristic of the detection signal. Method according to any one of claims 11 to 15, in which step f) comprises an estimation of a time derivative of the detection signal. A method according to any one of claims 11 to 16, wherein step f) comprises detecting an intensity level or integrating the detection signal over at least a predetermined time period. A method according to any one of claims 11 to 17, wherein: - prior to step a), each nucleotide sequence is linked to a known calibration sequence; - steps c) to f) are implemented so as to hybridize the bases of the calibration sequence, steps c) to f) forming a calibration phase; - the detection signal obtained during each step e) of the calibration phase is used to calibrate a response of the device to the bases of the calibration sequence.

19. Method according to any one of claims 11 to 18, wherein: - each candidate fluorescent marker emits fluorescence light with an increasing then decreasing fluorescence intensity; - the decrease in the fluorescence intensity of each fluorescent marker is characterized by a decay constant; - the decay constants of two different fluorescence markers are different.

20. Method according to any one of claims 11 to 19, comprising, following step g), a step h) of identifying the hybridized nucleic acid base.

21. Method according to claim 20, wherein - following step h), the fluorescent marker is cleaved and the sample is rinsed; - following rinsing, steps c) and h) are repeated, so as to identify a sequence of nucleotides forming the captured nucleic acid strand.

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