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. EARLIER ART

[0002] Devices for detecting biomolecules can be based on optical detection. The biomolecules to be detected are first labeled with a luminescent marker, such as a fluorophore or quantum dot. Detection is performed by a matrix photodetector, such as a CCD imager, CMOS, or avalanche photodetector array (APD).

[0003] A widespread application of fluorescent labeling is sequencing, particularly sequencing by synthesis (SBS). According to this method, a single-stranded nucleotide sequence, located downstream of a primer, is amplified to form a cluster. During sequencing, a complementary sequence is progressively synthesized by successively adding nucleic bases complementary to those forming the sequence under study. The complementary bases are added one by one, respectively, during cycles. After each addition of a complementary base, an image of the cluster is acquired. All or part of the complementary bases are labeled with a fluorescent tag. Observing the fluorescence during each cycle allows the identification of the complementary base added during that cycle. Thus, by acquiring a succession of fluorescence images, the examined nucleotide sequence can be decoded.

[0004] Fluorescence detection is performed by a photodetector, usually a matrix photodetector. However, photodetectors have a detection matrix with a spatial pitch that is generally greater than 1 pm. A spatial pitch is defined as the distance between two adjacent pixels. Such a spatial pitch is considered too large for a device capable of addressing a large number of detections in parallel. These devices also have limited sensitivity: sufficient amplification is required to obtain a sufficient number of identical sequences at the cluster under study in order to obtain a usable signal.

[0005] Some biosensors are based on the detection of charges carried by the target biomolecules to be detected. Examples include field-effect transistors. This type of device allows for rapid detection with good sensitivity. Furthermore, CMOS (complementary metal oxide semiconductor) technology enables the fabrication of devices with a large number of sensors. This allows for the parallel performance of numerous analyses. However, this type of sensor can be sensitive to environmental parameters affecting the sample, such as pH or temperature, or interactions with non-target biomolecules or ions, leading to detection errors. The use of this type of biosensor in DNA (deoxyribonucleic acid) sequencing applications requires a degree of measurement redundancy to increase robustness. Nanopore-based DNA sequencers present the same drawbacks.

[0006] Nanowire-based photonic sensors have been developed for the purpose of detecting DNA fragments. For example, see the publication by 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 change in the 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 require fluorescence coding to differentiate the added bases. In sensor-based applications, coding is performed either by the emission wavelength or by the light intensity in the image acquired by the photodetector.

[0009] The inventors have designed a compact and sensitive sample analysis device that takes advantage of the fluorescence detection capabilities of nanowires. The device can perform simultaneous or sequential analysis of different biomolecules. The device leverages the detection sensitivity and compactness provided by nanowires, while also enabling the differentiated detection of nucleic bases labeled with fluorescent markers. Description 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 a first electrode; - a multilayer structure, comprising at least a 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 multilayer 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 disposed between a sample, containing 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 has a homojunction, heterojunction, or 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 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 includes 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 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 temporal period of excitation, the intensity of the fluorescence light follows a growth and then a decrease; - 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 a chain of oligonucleotides. According to one possibility, each nanowire contains: - a pn type homojunction; - or a heterojunction; - or a p-metal or n-metal Schottky junction. According to at least one possibility, a 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 at least one possibility, a 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 has two sublayers stacked one on top of the other, forming a lower sublayer and an upper sublayer, the lower sublayer being interposed between the conductive layer and the upper sublayer; - the upper sublayer has wells opening into the lower sublayer, each well being positioned opposite 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), spaced apart from each other, such that a nanowire of one 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 first and second selected electrodes.

[0027] A second object of the invention is a method for identifying a fluorescent marker using a device according to the first object of the invention, the fluorescent marker being capable of emitting fluorescence light, in the detection spectral band, when illuminated by excitation light, the functionalization surface being configured to capture a strand of nucleic acid, the device comprising: a. disposition of a sample, comprising nucleic acids, in contact with the functionalization surface; b. capture of at least one strand of nucleic acid on the functionalization surface; c. addition of nucleic bases into the sample, at least two different nucleic bases being labeled by two different fluorescent markers, the sample comprising active ingredients configured to allow hybridization of a nucleic base onto the nucleic acid strand captured on the functionalization surface; d. exposure of the functionalization surface to 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

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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. reiteration of 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 at different capture sites distributed over the functionalization area. According to one possibility, step f) includes: - determination of a characteristic of the detection signal during the detection time period; - identification of the fluorescent marker based on the characteristic. According to one possibility: - the fluorescent marker is chosen from several candidate fluorescent markers; - step f) involves selecting the fluorescent marker from among the candidate fluorescent markers based on the characteristic of the detection signal. According to one possibility, step f) involves an estimation of a time derivative of the detection signal. According to one possibility, step f) involves detecting an intensity level or integrating the detection signal over 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 in such a way 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 an increasing then decreasing fluorescence intensity; - the decrease in fluorescence intensity of each fluorescent marker is characterized by a decay constant; - the decay constants of two different fluorescence markers, are different.

[0036] According to one possibility, following step g), the process includes a step h) of identification of 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 later in this description, in connection with the figures listed below. FIGURES

[0039] Figures IA and IB show the main components of a device according to the invention.

[0040] Fig. 1C 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 from 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 schematically illustrates different time periods of detection during the decay of the emission of fluorescence light.

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

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

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

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

[0050] Figures 4A to 4E schematically illustrate steps in the manufacture of nanowires according to a so-called bottom-up process.

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

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

[0053] Figure 7 shows different implementation steps of the process. PRESENTATION OF SPECIFIC IMPLEMENTATION METHODS

[0054] Figures IA to 1E show a first example of an analysis device 1 for implementing 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 one wishes to sequence. Each strand originates from a fragmentation of genetic material, forming a strand library. Each strand may include an adapter at the 3' end and the 5' end. As is known, the adapter may include a primer binding site and optionally an index containing a sample identification code. One of the adapters may include a so-called calibration sequence, described below.

[0055] The device comprises a substrate 10, forming or comprising at least one first electrode llc. In the example shown in [Fig. 1A], the substrate is a crystalline silicon substrate, for example, a Si substrate with a crystalline orientation (111). The substrate 10 is delimited by a surface, called the first surface 11, comprising a first electrode llc. In the example in [Fig. 1A], the first surface 11 is formed of Si comprising conductive regions. According to another possibility, the substrate 10 is coated with 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 specifically from the first surface 11. The first surface 11 extends along a PXY plane. The PXY plane is defined by a longitudinal axis X and a lateral axis Y. The X and Y axes intersect, and preferably are perpendicular to each other. The nanowires extend parallel to a transverse axis Z intersecting the PXY plane. In the embodiments described below, the transverse axis Z is perpendicular to the PXY plane. The first surface 11 is conductive at least at the intersection with each nanowire 30. The entire first surface 11 can 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 direction (111), therefore at an angle to the PXY plane.

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

[0059] The nanowires 30 can be synthesized directly on the substrate 10, as described in connection with Figures 4A to 4E. The nanowires can be formed on another substrate and then transferred to the substrate 10. The transfer can be carried out as described in the publication by 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. This could, for example, be ITO (indium tin oxide).

[0061] Each nanowire is formed of one or more semiconductor materials, and optionally of a metallic material. Each nanowire has a junction 33. In the example shown, the junction 33 is a homojunction: each nanowire has 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 of the same semiconductor material, with two different doping levels respectively: thus, the first part 31 and the second part 32 are respectively formed of the same semiconductor material doped n and p or p and n. In the example shown, the first part 31 is formed of p-doped GaAs (gallium arsenide) and the second part is formed of n-doped GaAs. The interface between the two parts forms the pn junction 33.

[0062] Alternatively, the junction 33 can be made at the level of 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 selected from materials in columns III and V, usually referred to as III-V materials, for example, GaAs. Preferably, it may be a material from column III and arsenic, for example, InAs (indium arsenide). In the example shown, the nanowires 30 are made of GaAs. Other semiconductor materials may be considered, for example, but not limited to, 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 can be made of a material such as PMMA (polymethyl methacrylate), BCB (benzocyclobuthene), or SOG (spin-on glass) materials consisting mainly of silicon oxides and other chemical additives to impart specific properties such as adhesion and thermal stability. The encapsulation layer 15 can 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 is designed to form second electrodes 21c. These second electrodes 21c are formed at the second surface 21 from the conductive layer 22. The conductive layer 22 can be structured so that, at the second surface 21, several second electrodes 21c are electrically isolated from each other. Thus, the conductive layer can have openings or insulating materials delimiting the electrodes 21c. This allows for 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 (Polymethyl methacrylate). The interface layer 23 is electrically insulating, particularly 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 them by the interface layer 23.

[0068] The interface layer 23 is, for example, a thin film made 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 be made of a material exhibiting the lowest possible autofluorescence 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 specifically 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 adaptors linked to the strands to be characterized. The binding between the capture species 26 and each oligonucleotide strand to be analyzed, or more precisely an adaptor of each oligonucleotide strand to be analyzed, is then achieved 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 The long diagonal of the nanowells can range from 70 nm to 700 nm. The nanowells 27 can, for example, be arranged in a matrix pattern, or, more generally, according to a predetermined pattern. The functionalization surface is then functionalized at the level of each nanowell 27, while the spaces between each well remain unfunctionalized. The formation of the nanowells can be achieved by local thinning of the interface layer 23.

[0071] According to one possibility, shown in [Fig. 1C], the interface layer 23 comprises two superimposed sublayers 231 and 232. The interface layer has a lower sublayer 231 interposed between the conductive layer 22 and an upper sublayer 232. The nanowells are formed by local thinning of the upper sublayer 232, such that the nanowells open into the lower sublayer 231. The upper sublayer 232 can be made of a non-functionalizable material, for example, an anti-biofouling material, such as a hydrophobic material. This structure allows the functionalization of the functionalization surface 25 to be carried out only at the nanowells 27, on the lower sublayer 231. Each nanowell 27 thus forms a DNA strand capture site.

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

[0073] Functionalization can be carried out by treating the functionalization surface 25, for example, by plasma / oxygen surface treatment. When the interface layer 23 is made of PMMA, plasma / oxygen treatment allows the formation of carboxyl groups. The capture species 26 can be grafted onto the functionalization surface by covalent bonding. To this end, the capture species have a functional group, for example, an amine group, so as to form a covalent bond with the functional groups on the functionalization surface 25. This could, for example, be a bond obtained by grafting a thiol group, at the level of the capture species, onto an amine group present at the level of the functionalization surface.

[0074] According to one possibility, each sequence to be analyzed has 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 IB and IE, the device comprises a detection circuit 40, a first terminal of which is connected to a first electrode 1lc on the substrate 10, and a second terminal is connected to a second electrode 21c on the multilayer structure 20. The detection circuit 40 makes it possible to measure the difference potential, or an electric current, between the first electrode 1 lc and the second electrode 21c.

[0076] As described in relation to Figures IA to IC, each nanowire extends from a first end, on a first surface 11 of the substrate 10, to 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 has a first electrode 1lc and the second surface has a second electrode 21c. In the example shown in Figures IA to IB, the first surface 11 and the second surface 21 are made of a conductive material. They are conductive over their entire area.

[0077] The first and second surfaces can be structured and comprise different electrodes llc, 21c isolated from each other. In [Fig. 1D], each electrode is represented by a dashed line. On the first surface 11, each first electrode 1 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 llc, 21c between which it extends are polarized. The structuring of the electrodes into rows / columns allows for the selection of functional nanowires, which extend between two polarized electrodes: according to this arrangement, nanowires connected to a polarized row and column are functional. A functional nanowire is defined as 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 sites for capturing nucleotide sequences to be characterized.

[0080] As previously stated, each biological capture species 26 is configured to capture a biological species of interest, in particular a nucleotide sequence 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, up to amplification errors, of the captured nucleotide sequence.

[0082] The amplification of each captured sequence can be of the "amplification by" type "Bridge amplification," usually referred to as "bridge amplification," is typically implemented in devices offered by Illumina. When bridge amplification is used, the functionalization surface incorporates capture devices configured to connect to the adapters of the captured sequences.

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

[0084] Generally, sequencing involves several cycles, during which complementary bases are progressively hybridized along each sequence to be characterized. As is known, during each cycle, the sequences to be characterized are immersed in a reaction medium containing bases labeled with a fluorescent marker and a hybridase enzyme. Hybridization occurs progressively along each strand, base by base, in a predetermined direction, for example, from the free end to the end bound 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, the fluorescent marker is detected after rinsing, which allows identification of the base that 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 complete sequencing of the grafted sequences at the different capture sites. Among the four different bases used, one base may not be labeled.

[0086] According to another possibility, during each cycle, the sequences are successively bathed in baths, each bath containing only one 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 labeled with different markers. Indeed, the identification of each type of base is performed by each bath. Between each bath, a rinse is performed, and fluorescence is detected at each capture site. According to this possibility, the fluorescent markers do not necessarily have terminators. Thus, a plurality of identical successive bases can be hybridized by complementary bases in the reaction medium. This results in a fluorescence signal. recession whose intensity increases according to the number of successive bases hybridized.

[0087] Following the detection of a fluorescence signal, the fluorescent markers labeling each base hybridized 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 the nanowires, of fluorescence (or the 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, within an excitation band of the fluorescent marker(s). The light source is configured to emit excitation light for a short duration, on the order of nanoseconds. For example, it could be a laser light source. This allows the generation, at each cluster, of a fluorescence signal that depends on the added base (A, C, T, or G in the case of a DNA strand).

[0090] An important aspect of the invention, described below, consists of translating the fluorescence signal into an electrical signal dependent on the fluorescent marker, 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, 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, within a predefined spectral band, and to convert the optical signal into an electrical detection signal. Thus, device 1 is based on optical detection of the hybridization of a base on a sequence to be analyzed, inducing an electrical response from the device.

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

[0094] The reaction medium 2 is arranged 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 in which the reaction medium comprises different types of bases labeled 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] Reference is made to the Si sequence, captured and amplified at capture site 25i. During a first cycle, a T base was hybridized. Figure 1E represents a second cycle, during which a C base labeled with a fluorescent marker C*, represented by the symbol *, hybridizes to a G base of the sequence. The objective of the device is to detect the fluorescent marker C* by means of fluorescence light transduction.

[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 illumination at the excitation wavelength, a fluorescence light 8 is emitted by the fluorescent marker C*, at 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 of the material 22 constituting the electrode 21c, some fluorescence photons propagate through a nanowire 30. When a fluorescence photon is absorbed at the junction 33, electron / hole pairs are formed.Due to the potential difference between the first part 31 and the second part 32, electrons propagate through the n-doped portion towards the higher potential, while 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 both contain the fluorescence wavelength and, preferably, not contain the excitation wavelength of the fluorescent marker. The nanowire thus acts as a nanophoto The detector detects fluorescence light while being insensitive to the excitation wavelength. Each nanowire thus acts as 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 nanowire response; 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 540 nm and emission wavelength between 555 and 600 nm.

[0099] Preferably, the diameter of the nanowires is controlled to confer sensitivity in a narrow spectral band. By narrow spectral band, we mean a bandwidth typically of a few tens of nm, and preferably less than 100 nm. The bandwidth corresponds to the full width at half maximum (FWHM) of the absorption peak in the absorption spectrum. The detection bandwidth is, for example, on 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 excludes the excitation wavelength of the fluorescent marker. The ability of nanowires to form a wavelength-selective photodetector was described in Mokkapati et al., “Optical design of nanowire absorbers for wavelength-selective photodetectors,” Sci. Rep. 2015, 5 15339.

[0100] The spectral sensitivity of detection can be adjusted by incorporating quantum wells or quantum dots at junction 33. This allows for a junction 33 with a different composition compared to the rest of the nanowire. The detected wavelength then corresponds to the wavelength of the band gap defined by the well or quantum dot.

[0101] Each nanowire thus forms a nanophotodetector. During each cycle, it is necessary not only to detect fluorescence light, but also to identify the fluorescent marker 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 with a fluorescent marker A*, G*, C*. It is assumed that the T bases are not labeled with a fluorescent marker. During each cycle, the following is 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 discrimination is performed by characterizing the detection electric current resulting from the nanowires.

[0103] In order to allow discrimination of the fluorescence light emitted by each fluorescent marker, each fluorescence marker used has one or more emission parameters that differ from one another. An emission parameter is defined as a parameter characterizing a temporal distribution of the fluorescence intensity emitted by the fluorescent marker. This could, 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 can follow a variation of the type gr, where t corresponds to time and T is a decay constant.In this case, the decay time can be characterized by the decay constant T.

[0104] Figure 2A schematically represents three temporal distributions of the fluorescence intensity of the three fluorescent markers G*, C*, and A*, labeling the bases G, C, and A, respectively. In this example, the maximum intensity Imax is considered identical for these three fluorescent markers. However, the decay 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 each nanowire. The device includes a processing unit 41, enabling characterization of the detection signal so as to identify the fluorescent marker that generated the fluorescence light. The processing unit 41 may include 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] Figure 2A shows: - a time period of excitation Atex, which corresponds to the light pulse emitted by the light source 5. The time period of excitation Atex can be, for example, a few tens of ps or on the order of ns. - a detection time period AL, during which the detection signal S is characterized in order to identify the fluorescent marker that generated fluorescence light, or the absence of a fluorescent marker. The detection time period AL can be, for example, from 100 ps to 1 ns or a few ns. The detection period preferably extends over the duration of the detection. growth of fluorescence light emitted by the fluorescent marker.

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

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

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

[0110] The embodiment described in relation to [Fig. 2B], based on a different measurement The relative intensity of fluorescence has the advantage of being relatively insensitive to va 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 causing 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 causing 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 a mean 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 is 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. However, the intensity of the photoinduced current depends on the number of fluorescent markers labeling bases hybridized during the same cycle. To address this difficulty, a Sc calibration sequence can be grafted onto each sequence to be characterized, in order 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 located at a known position in the adapter P2, which is 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 time, or includes an integral of the detection signal.

[0115] Figure 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 P2 adapter. The characterization signal is an integral of the detection signal over a predetermined time period. During the sequencing of 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 on [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 on [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 on [Fig.3A].

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

[0117] The use of the calibration sequence allows for the experimental determination of the S*b, S*2, and S*3 levels at each capture site, bearing in mind that the number of identical sequences may vary from one site to another. The levels thus established are used to interpret the detection signals detected during the cycles performed on each sequence to be characterized.

[0118] According to one 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 across 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 include an integration or a derivative of the intensity measured by each nanowire considered. The processing unit compares the characterization to stored values ​​or to 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 among several so-called candidate fluorescent markers (in this example A*, C*, and G*), whose characteristics are known in terms of emission intensity or decay, and this in the detection period(s). The characteristics can be stored in memory 42. This makes it possible to identify the nucleic base hybridized during the cycle.

[0121] When using three labeled bases, suitable fluorescent markers for an embodiment of the invention are: DAPI (half-life 1.88 ns), Hoechst 33342 (half-life 1.05 ns), and Hoechst 33258 (half-life 1.22 ns). The wavelengths conferring maximum excitation are 358 nm, 350 nm, and 349 nm, respectively.

[0122] The embodiment in which the different types of complementary bases are used simultaneously is particularly well-suited to implementing the invention. The fluorescent markers associated with each base include a terminator, which is eliminated by cleavage following the formation of the characterization signal. However, the invention can also be implemented by exposing the sequences to be characterized to known bases using successive baths.

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

[0124] Figure 3C schematically represents the characterization signal resulting from the sequencing of a TTCG sequence. In this example, the characterization signal corresponds to a The integral of the detection signal during a predetermined detection period. Each base can be identified from the S*1, S*2, and S*3 signals established during calibration. In this example, it is assumed that following each detection, the fluorescent marker undergoes cleavage. The x-axis represents the detected base. The y-axis represents the characterization signal.

[0125] Figure 3D shows a schematic representation of the characterization signal resulting from the same sequence. In this example, the characterization signal corresponds to an integral of the detection signal over a predetermined detection period. Each base can be identified from the S*b, S*2, and S*3 signals established during calibration and stored in memory 42. In this example, there is no cleavage of fluorescent markers. The intensity of the fluorescence signal increases progressively due to the accumulation of fluorescent markers bound to bases hybridized to 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-dependent type. This can allow the detection signal to be calibrated for the different bases. When the characterization signal is of the time-dependent type, it is preferable for the fluorescent markers to undergo cleavage following the detection of hybridization.

[0127] In general, the use of the calibration sequence makes it possible to establish a response of the device at 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 variability in the detection performance of each nanowire.

[0128] Preferably, the fluorescent markers are excited in the same spectral excitation band, thus allowing the use of a single light source. Alternatively, the fluorescent markers are excited in different spectral excitation bands, which may require the use of different light sources.

[0129] The use of nanowires is particularly advantageous. It allows for obtaining a usable signal using a small number of replicated sequences at the same capture site. This reduces the size of the capture sites compared to the prior art. A greater number of sequences can thus be characterized on the same functionalization surface. The cycles can be implemented in parallel at each defined capture site on the functionalization surface. Reducing the number of amplifications limits the drawbacks associated with the sequence amplification process: a smaller volume of reagents required, a smaller number of replicates, and therefore fewer amplification errors.

[0130] Indeed, the detection circuit allows for the measurement of very low currents, on the order of a few tens of pA to a few nA. Furthermore, the nanowires can be spaced close together, typically with a spatial pitch of less than 500 nm (compared to typically 1.1–1.7 pm for a CMOS optical detector). This allows for a device with a high ratio of effective surface area (i.e., functional 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 to allow detection of fluorescence light while masking the excitation light. Alternatively, the detection currents, respectively formed at each nanowire during excitation and fluorescence light emission, can be temporally separated. The fast response time of nanowires, on the order of ps, is taken advantage of, 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 aspects of the design of the device according to the invention. Nanowire formation

[0133] Figures 4A to 4E schematically illustrate the steps involved in forming nanowires from a silicon substrate 10. Figures 4A to 4E correspond to a bottom-up approach. The substrate 10 is made of (111) oriented Si and has a surface layer 10i of SiO2, intended to form a barrier layer, 10m - 15 nm thick. The SiO2 layer is covered with a layer 102 of electrosensitive PMMA, 45-80 nm thick. The layers 10i and 102 are photolithographed and etched, respectively, 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, 5 to 10 nm thick, is deposited on the SiO2 layer. The added metal, in this case gold, acts as a catalyst. See [Fig. 4B]. Excess gold between the nanowells is removed by lifting (removing) the PMMA 102 layer. See [Fig. 4C].This results in isolated islands of gold 103, in positions corresponding to the position of the previously formed nanowells.

[0134] After heating to a temperature above 450°C, the islands form droplets. The gold droplets act as a catalyst. The semiconductor nanowires are then formed by molecular beam epitaxy (MBE). This involves sending one or more molecular beams toward 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 Doping agents (e.g., Ga and As to form GaAs) are used. The atomic species adsorb and diffuse onto the surface of the Si substrate as adatoms. The adatoms are incorporated into the gold droplets. When these droplets become saturated, nanowire nucleation occurs first at the droplet / substrate interface, and then at the droplet / nanowire interface as they form.

[0135] The process is not limited to the use of gold as a catalyst. Other catalysts can 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 process allows, for example, the growth of nanowires using As, Ga, and C and Si respectively as p and n dopants, with an operating temperature of 600°C-610°C. The process continues until the nanowires reach a predetermined height. See [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 electrically insulates the nanowires from each other and provides improved mechanical strength to the assembly. Chemical etching, plasma etching, and / or polishing can be performed on the end of the nanowires opposite the substrate to remove 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 Figures 4A to 4E, the formation of nanowells on the substrate 10 allows for precise control of the nanowire positions. Thus, the nanowires can be arranged regularly, for example, according to square or hexagonal mesh patterns. The spacing between two adjacent nanowires can be small, on the order of twice the diameter, or larger, for example, from a few to several hundred nanometers.

[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, particularly along the Z-axis.

[0141] Figure 5 illustrates a configuration in which the nanowires are arranged to form clusters. The nanowires in the same cluster are placed close together, the distance d between two adjacent nanowires in 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 arranged in clusters (or bunches) as described in relation to [Fig. 5], the nanowires in the same cluster are preferably connected to the same electrode, both on the substrate 10 and on the multilayer structure 20. The nanowires in the same cluster address the same capture site. The nanowires in the same cluster are thus simultaneously functional. Nanowires not connected to the detection circuit are not functional. The clustering of the nanowires 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 composing a single cluster can be relatively small. Thus, the surface area, in the PXY plane, of each cluster is small, with each cluster selectively addressing a nucleotide sequence different from that of any other cluster. It is therefore possible to arrange a large number of clusters, each addressing different nucleotide sequences, in the same 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 relation to [Fig. 1C]. In this case, each nanowell 27 extends opposite the nanowires belonging to the same cluster 35.

[0145] According to another possibility, 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 27 / 12 / 2021. Structure of the nanowires

[0146] Figures 6A and 6B show other nanowire structures that can be implemented in a device according to the invention. Figure 6A shows a nanowire similar to the nanowires described above. The junction 33 is arranged axially, extending between two portions 31, 32, with different doping levels, spaced apart along the transverse axis Z. In the example of Figure 6A, a passivation sheath 34 surrounds 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 it. 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 promotes the incorporation of quantum wells or quantum dots inside the nanowires in order to adjust the absorption spectrum.

[0149] A radial structure allows for a junction 33 extending along a significant height along the Z-axis, which increases detection sensitivity. Optionally, the radial structure shown in [Fig.6B] includes an annular sheath 34 as described in connection with [Fig.6A].

[0150] Figure 7 schematically illustrates the main steps of a method for implementing the invention.

[0151] Step 100: Disposal of a sample containing nucleic acid sequences from a library previously prepared from the DNA strand to be sequenced, placed in contact with the functionalization surface 25.

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

[0153] Step 115: Amplification, for example by bridging, of the nucleic acid sequence captured at each site. Step 115 is optional, but it allows for obtaining a higher intensity detection signal during each cycle, 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 hybridizing to each sequence. Each type of base is labeled with 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 induce fluorescence of fluorescent markers hybridized 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: Reiteration of steps 120 to 170, until sequencing of each nucleotide sequence captured at a capture site.

[0161] In the process described in relation to [Fig. 7], during step 120, the reaction medium comprises a mixture of bases. As previously indicated, during each cycle, four successive baths can be used, each containing one of the four types of bases labeled with fluorescent markers. These markers may be identical or different from one another.

[0162] The device takes advantage of a fast response time, typically on the order of ns.

[0163] It is observed that the device does not require the use of optical components Furthermore, the device's response is stable and relatively insensitive to environmental variations such as sample pH, temperature, and the presence of molecules or ions other than the biomolecule of interest. This is because the nanowires are not in contact with the sample but are physically and electrically isolated from it by the interface layer 23.

[0164] Finally, since the device is based on nanophotodetectors, it provides a compact analysis platform. The device can be produced using a collective manufacturing process, which reduces costs.

Claims

Demands

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 (link); - a multilayer structure (20), comprising at least a second electrode (21c); - nanowires (30), extending between the first electrode (1 lc) 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 of an insulating material; - the multilayer 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 disposed 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) has a homojunction (33), 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 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 according to 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, wherein: - following the time period of excitation, the intensity of the fluorescence light follows a growth and 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 one of the preceding claims, wherein the functionalization surface is configured to capture a strand forming a chain of oligonucleotides.

6. A device according to any one of the preceding claims, wherein 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 sublayers (23b 232), stacked one on top of the other, forming a lower sublayer and an upper sublayer, the lower sublayer being interposed between the conductive layer (22) and the upper sublayer; - the upper sublayer comprises wells, opening into the lower sublayer, each well being disposed opposite a nanowire, each well forming a part of the functionalization surface; - the functionalization surface is segmented at each well, so that each well forms a capture site.

8. A 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 one 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. A 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 first and second selected electrodes.

11. A method for 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 excitation light, the functionalization surface being configured to capture a strand of nucleic acid, the device comprising: a. disposition of a sample, comprising nucleic acids, in contact with the functionalization surface; b. capture of at least one strand of nucleic acid on the functionalization surface; c. addition of nucleic bases into the sample, at least two different nucleic bases being labeled by two different fluorescent markers, the sample comprising active ingredients configured to allow hybridization of a nucleic base onto the nucleic acid strand captured on the functionalization surface; d. exposure of the functionalization surface to 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. reiteration of steps c) to f) so as to progressively hybridize nucleic bases along the nucleic acid strand.

12.

13.

14.

15.

16.

17.

18. A method according to claim 11, wherein step b) comprises an amplification of each captured nucleic acid strand. A method according to any one of claims 11 or 12, wherein steps b) to g) are carried out at different capture sites distributed over the functionalization surface. A method according to claim 13, wherein step f) comprises: - determination of a characteristic of the detection signal during the detection time period; - identification of the fluorescent marker based on the characteristic. Method according to claim 14, wherein: - the fluorescent marker is chosen from several candidate fluorescent markers; - step f) involves selecting the fluorescent marker from among the candidate fluorescent markers based on the characteristic of the detection signal. A method according to any one of claims 11 to 15, wherein 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 in such a way 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. A 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. A method according to any one of claims 11 to 19, comprising, following step g), a step h) of identification of the hybridized nucleic acid base.

21. A 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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