Fluorescence detection device using nanophotodetectors

Abstract

The invention relates to a device for identifying a fluorescent marker or for determining an amount of 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 the sequencing of a nucleic acid. 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 marked using a luminescent marker, such as a fluorescent marker or quantum dot. Detection is carried out by a matrix photodetector, such as a CCD, CMOS imager or avalanche photodetector array (APD: Avalanche Photo-detectors).

[0003] A widespread application of fluorescent labeling is sequencing, in particular so-called SBS (Sequencing by Synthesis) sequencing. 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. Between the addition of each cycle, an image of the cluster is produced. All or part of the complementary bases are marked with a fluorescent label. Observing 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] Usually, fluorescence detection is performed by a photodetector, usually a matrix photodetector. However, usual matrix photodetectors have a detection matrix whose spatial pitch is generally greater than 1 pm. By spatial pitch, we mean 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 biomolecules of interest to be detected. These are, for example, field effect transistors. This This type of device allows for rapid detection with good sensitivity. In addition, CMOS (complementary metal oxide semiconductor) technology allows for 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 leading to 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 drawbacks.

[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 “New generation of ultrasensitive label-free optical Si nanowire-based biosensors”, ACS Photonics 2018,5,471-479 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. It can also allow 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 or for determining a quantity of 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 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 in a detection spectral band comprising the fluorescence spectral band 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 detection signal intensity level during the detection time period; • identify the fluorescent marker or determine a quantity of 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 marker

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[0023] fluorescent. Depending on the possibility, the intensity level is average intensity or median intensity or total intensity or maximum intensity. Alternatively, the detection band extends over a spectral width greater than 150 nm, so as to address the fluorescence spectral bands of several different fluorescent markers. According to one possibility, the functionalization surface is configured to capture a strand forming an oligonucleotide chain. According to one possibility, one or 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 an oligonucleotide chain. 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 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. According to one possibility, several nanowires extend between a single 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, 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 or for determining a quantity of 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 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, the sample comprising active ingredients configured to allow hybridization of a nucleic base to the nucleic acid strand captured on the functionalization surface, at least one added nucleic base being labeled with a fluorescent marker; 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;

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[0034] f. determining an intensity level of the detection signal detected in step e), and identifying the fluorescent marker or determining an amount of the fluorescent marker, depending on the intensity level detected; the process being such that - the nucleic acid comprises a calibration sequence, formed of a known number of known bases; - steps c) to f) are repeated so as to identify each nucleic acid base hybridized to the calibration sequence, each iteration of steps c) and f) forming a calibration cycle; - the detection signals respectively detected during each calibration cycle are used to define intensity levels corresponding respectively to each fluorescent marker, the defined levels being taken into account during steps f) of the sequencing cycles different from the calibration cycles. The intensity level can be an average intensity or a median intensity or a total intensity or a maximum intensity during the detection time period. Preferably, step b) comprises an amplification of each captured nucleic acid strand. According to one possibility: - the fluorescent marker is chosen from several candidate fluorescent markers; - each candidate fluorescent marker emits fluorescence light in a fluorescence spectral band; - the fluorescence spectral bands of the different candidate fluorescent markers are centered around fluorescence wavelengths that are respectively different from each other; - step f) comprises a selection of the fluorescent marker from among the candidate fluorescent markers according to the determined intensity level. According to one possibility, the method comprises, following step f), a step g) of identifying the hybridized nucleic acid base. According to one possibility: - following step g), the fluorescent marker is cleaved and the sample is rinsed; - following rinsing, steps c) and g) are repeated, so as to identify a sequence of nucleotides forming the captured nucleic acid strand, each iteration of steps c) and g) forming a sequencing cycle. The invention will be better understood by reading the description of the exemplary embodiments presented in the remainder of the description, in conjunction with the figures listed below. FIGURES

[0035] Figures 1A and 1B show the main components of a device according to the invention.

[0036] [Fig.lC] shows a spatial distribution of capture sites.

[0037] [Fig. 1D] represents an example of a device according to the invention, in which the electrodes are distributed according to a matrix arrangement.

[0038] [Fig.1E] shows an example of sequencing three different sequences, respectively at three capture sites.

[0039] [Fig.2A] shows a time distribution of an intensity of a fluorescence light.

[0040] [Fig.2B] schematizes a spectral band for detecting a fluorescence marker common to different bases.

[0041] [Fig.2C] illustrates a calibration sequence.

[0042] [Fig.2D] shows different intensity levels detected during a calibration phase with cleavage of the fluorescent markers during each cycle.

[0043] [Fig.2E] shows different intensity levels detected during a calibration phase without cleavage of the fluorescent markers during each cycle.

[0044] [Fig.3A] schematizes a spectral band for detecting different fluorescence markers, each marker marking the same type of base.

[0045] [Fig.3B] shows detection signals resulting from the use of the fluorescent markers described in connection with [Fig.3A].

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

[0047] [Fig.3D] shows different characterization signals detected during a calibration phase.

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

[0049] [Fig.3F] 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 diagram steps in the manufacture of 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 METHODS OF IMPLEMENTATION

[0054] Figures 1A to 1E show a first example of an analysis device. 1 allowing an implementation of the invention. The analysis device 1 is configured to be placed in contact with a sample 2, comprising for example a liquid medium likely to contain 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 the subject of 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. As well as 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.lB], 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 level of the second surface 21, from the conductive layer 22. The conductive layer 22 can be structured so as to that at the level of 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 probes 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 largest diagonal of the nanowells may be between 70 nm and 700 nm. The nanowells 27 may for example be arranged in a matrix manner, or, more generally, according to a predetermined pattern. The functionalization surface is then dark 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.lC], the interface layer 23 comprises two superimposed sub-layers 23i 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 23b. The upper sub-layer 232 may be formed of a non-functionalizable material, for example an anti-biofouling material, 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 23b. 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 biological probes comprise a function, for example an amine function, so as to form a covalent bond with the functions of the functionalization surface 25. This 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 potential difference, or an electric current, between the first electrode 11c and the second electrode 21c.

[0076] As previously indicated, each biological capture species 26 is configured to capture a biological species of interest, in particular a nucleotide sequence to be analyzed. By capture is meant the establishment of a bond, in particular a hybridization, between the capture probe 26 and the single-stranded fragment of nucleic acid to be characterized.

[0077] In [Fig. 1E], three Sb sequences S2 and S3 are represented, 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.

[0078] The captured sequences are amplified so as to form several replicas of each sequence, so as to form clusters of identical sequences. Thus, 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.

[0079] The amplification of each captured sequence can be of the “bridge amplification” type, usually referred to as “bridge amplification”, and usually implemented in devices proposed by the company Illumina. When bridge amplification is implemented, the functionalization surface carries capture species configured to graft onto the adapters of the captured sequences.

[0080] 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. 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 functionalization surface.

[0081] According to one possibility, during each cycle, the sequences bathe successively in baths, each bath comprising a single type of base, each base of the same type being labeled by the same fluorescent marker. According to this possibility, it is possible, but not necessary, for two bases of different types to be respectively labeled by 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. A plurality of identical successive bases can be hybridized by complementary bases of the reaction medium. This results in a fluorescence signal whose intensity increases as a function of the number of identical successive bases hybridized.

[0082] Following the detection of a fluorescence signal, the fluorescent markers marking Each base hybridized during the cycle can be cleaved. As described later, cleavage is not necessary.

[0083] According to another possibility, the fluorescent markers are coupled to a terminator, which prevents the hybridization of two consecutive bases during the same cycle. The reaction medium can then comprise a mixture of bases, each base of the same nature being labeled by the same fluorescent marker. Two bases of different natures are respectively labeled by different fluorescent markers. Following each hybridization, detection of the fluorescent marker is carried out after rinsing, which makes it possible to identify the base which has hybridized during the cycle. Following detection, the terminator undergoes cleavage, and 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.

[0084] Thus, at each capture site each cycle comprises: - the addition of a reaction medium comprising bases and a 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 comprising 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 the fluorescent markers and / or possible terminators, followed by rinsing.

[0085] 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).

[0086] An important aspect of the invention, described below, consists in translating the fluorescence signal into an electrical signal dependent on the fluorescence, which makes it possible to obtain, during a cycle, an electrical signal dependent on the base added during the cycle.

[0087] This is a significantly different approach from the sequencing approach based on imaging the sample, with each base being identified by a color code and / or an intensity level.

[0088] 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.

[0089] The device comprises a processing unit 41, allowing characterization of the detection signal, so as to identify an occurrence of fluorescence, and quantify it. The processing unit 41 may comprise a microprocessor 41, connected to a memory 42 in which instructions are stored. The processing unit 41 is configured to determine an intensity level of the detection signal. The term intensity level designates a quantitative value representative of the detected intensity. It may be a maximum, or average, or median, or total intensity level, or any other quantitative statistical indicator, for example of the fractile type. During sequencing, the processing unit compares the detected signals with the stored signals or the signals established during the calibration period, which makes it possible to quantify the number of identical bases hybridized during the same cycle.

[0090] In [Fig.lE], a cross-sectional view of the device is shown, after sequence amplification. It is assumed that at each capture site, only one sequence has been captured (sequence S1 at site 25b, sequence S2 at capture site 252, etc.). Each captured sequence was then amplified by bridging. After amplification, at each capture site, identical sequences extend, formed at their 3' end from a Pb adapter. The sequences comprise, at their 5' end, a P2 adapter. The Pi adapter is part of the capture species 26 grafted, by covalent bonding, onto the functionalization surface. The P2 adapter of each sequence is free.

[0091] 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.

[0092] 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.

[0093] 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 G base of the sequence. The objective of the device is to detect the fluorescent marker C*, through transduction of fluorescence light.

[0094] 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 of 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.

[0095] 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 nanophotodetector of the fluorescence light, while not being sensitive to the excitation wavelength. Each nanowire thus forms a filter with respect to the excitation wavelength. When the semiconductor material is GaAs, the fluorescent marker can be Cy3 (Cyanine): excitation wavelength 540nm and emission wavelength between 555 and 600nm.

[0096] Preferably, the diameter of the nanowires is controlled so as to provide sensitivity in a predetermined detection spectral band. Preferably, the detection spectral band is sufficiently wide to be able to detect the fluorescence lights respectively emitted by different fluorescent labels. Preferably, the detection spectral band extends with a width greater than 50 nm or 100 nm or 200 nm. The bandwidth corresponds to the half-maximum width of the absorption peak on the absorption spectrum. The detection spectral band can be adjusted so that it includes the fluorescence wavelength of the fluorescent label and does not include the excitation wavelength of the fluorescent label. The fluorescence spectral band can be adjusted by providing an optical filter in the multilayer structure 20. The ability of nanowires to form a wavelength-selective photodetector has been described in Mokkapati.S., et al “Optical design of nanowire absorbers for wavelength selective photodetectors”, Sci. Rep. 5, . 15339.

[0097] 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.

[0098] 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 marker having generated the fluorescence light, in order to identify the base to which it was bound.

[0099] In [Fig.2A], the variation of the fluorescence intensity as a function of time of a fluorescent marker excited during a time Atex, which corresponds to the light pulse emitted by the light source 5, is shown diagrammatically. Following excitation, the fluorescence intensity increases up to a maximum intensity Imax and then decreases.

[0100] In [Fig.2A], we have represented: - the Atex excitation time period, which 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 a fluorescence light, or the absence of fluorescent marker. The detection time period Atd may be for example 500 ps or a few nanoseconds

[0101] [Fig.2B] shows an example in which the bases G, C and A are labeled with the same fluorescent label F*. The bases T are not labeled. In this example, it is assumed that during each cycle, the amplified sequences are successively immersed in different media, each medium being specific to a base. As previously described, following each bath comprising identical labeled bases, detection of the fluorescence light is carried out at each capture site. In [Fig.2B], the spectral emission band of the F* label is represented. The emission of a fluorescence signal causes the formation of an electric current by a nanowire. This results in a detection signal at the terminals of the detection circuit 40. The detection signal is integrated, during a detection time period, by the processing unit 40, so as to form a characterization signal.The characterization signal makes it possible to detect hybridization, and possibly the number of identical bases hybridized during the cycle.

[0102] 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. Indeed, 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.

[0103] 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.

[0104] [Fig.2C] illustrates an example of calibration sequences, composed of a succession of 1 T base, 2 G bases and 3 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. In this example, after each fluorescence detection, the fluorescent markers are cleaved. During the sequencing of the calibration sequence, - during a first period Atb which corresponds to a period during which a complementary base A is hybridized to the calibration sequence, the intensity of the detection signal corresponds to a first level Sp - 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 detection signal corresponds to a second level S2, which is substantially double the first level Si because the bases are labeled with the same fluorescent marker, the latter being materialized by the reference * 1; - 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 detection signal corresponds to a third level S3, which is substantially three times the first level Si.

[0105] [Fig.2D] shows the evolution of the detection signal during the calibration periods Atb At2 and At3.

[0106] The use of the calibration sequence allows an experimental determination of the levels S1, S2 and S3, and this on each capture site, knowing that the number of monoclonal (or amplified) sequences can 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. This makes it possible in particular to detect and quantify several hybridizations during the same cycle.

[0107] When the fluorescent markers are not cleaved after detection of a fluorescence signal, the intensity of the detection signal is increasing, due to the presence of the fluorescent markers linked to the previously hybridized bases. [Fig.2E] shows the evolution of the signal during the periods Atb At2 and At3, without cleavage of the fluorescent markers. The detection signals corresponding to the hybridization of several successive bases are deduced from the intensity jumps between each calibration period.

[0108] According to one embodiment, the bases of different types are labeled with different fluorescent markers. Each fluorescent marker comprises a terminator, so that during each cycle, only one base is hybridized. An interesting aspect of the device is that it makes it possible to identify each marker by characterizing the detection electric current resulting from the nanowires.

[0109] In order to allow discrimination of the fluorescence light respectively emitted by each fluorescent marker, each fluorescence marker used has a fluorescence spectral band centered around a different wavelength compared to the other markers considered. Preferably, the respective fluorescence spectral bands of the fluorescence markers do not overlap, or do so insignificantly.

[0110] In [Fig.3A], the detection spectral band AXd of a nanowire is shown. In [Fig.3A], the abscissa axis corresponds to the wavelength and the ordinate axis corresponds to the intensity of current photoinduced by the light propagating through the nanowire. Also shown are: - a first fluorescence spectral band AXi, which corresponds to the spectral band of a fluorescent marker G* used to mark nucleic bases G. The first fluorescence spectral band extends around a first central wavelength Xi. - a second fluorescence spectral band AX2, which corresponds to the spectral band of a fluorescent marker C* used to label C nucleic bases. The second fluorescence spectral band extends around a second central wavelength X2; - a third fluorescence spectral band AX3, which corresponds to the spectral band of a fluorescent marker A* used to label nucleic bases A. The third fluorescence spectral band extends around a third central wavelength X3;

[0111] According to this embodiment, the detection spectral band comprises the bands respective fluorescence spectral bands of the fluorescent markers used. This is why the detection spectral band extends by at least 50 nm, and preferably at least 100 nm or even at least 150 nm or 200 nm.

[0112] Another particularity of the detection spectral band is that the sensitivity of the nanowire is different with respect to the fluorescence lights respectively emitted by the different fluorescent markers. Thus, the sensitivity of the nanowire is different for each fluorescence spectral band, and more precisely at each central fluorescence wavelength. By sensitivity, we mean the number of charge carriers created relative to a quantity of photons incident on the nanowire. It can also be the intensity of the photoinduced current in the nanowire relative to a flux of photons incident on the nanowire.

[0113] In the example shown in [Fig.3A], it is shown that for the same illumination of the nanowire, the intensity of the induced photocurrent is: - maximum when the illumination of the nanowire is due to fluorescence light emitted by the fluorescent marker G* marking a base G; - minimal when the illumination of the nanowire is due to fluorescence light emitted by the fluorescent marker A* marking a base A; - intermediate when the illumination of the nanowire is due to fluorescence light emitted by the fluorescent marker C* marking a base C;

[0114] The particular configuration of the detection spectral band makes it possible to identify each fluorescent marker by a level of intensity of the photoinduced current.

[0115] The processing unit is for example programmed to determine a characterization signal S* which corresponds to an integral of the detection signal, ç* _ f ° Jtd [Fig.3B] shows different characterization signals corresponding to the hybridization of different bases.

[0116] We observe that: - 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 at the origin of 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*.

[0117] Thus, 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 indicator sta characteristic representing the intensity of the detection signal during the detection period.

[0118] [Fig.3C] illustrates an example of a calibration sequence, 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 over a predetermined time period. When sequencing the calibration sequence:.

[0119] 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 detection signal corresponds to a first level Si. The fluorescent marker A* is symbolized by *1 in [Fig.3C]. - 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 detection signal corresponds to a second level S2. The fluorescent marker C* is symbolized by *2 in [Fig.3C]. - 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 detection signal corresponds to a third level S3. The fluorescent marker G* is symbolized by *3 in [Fig.3C].

[0120] [Fig.3D] shows the evolution of the detection signal during the calibration periods Atl, At2 and At3.

[0121] The use of the calibration sequence allows an experimental determination of the Sb S2 and S3 levels, and this on each capture site, knowing that the number of monoclonal (or amplified) sequences can vary from one site to another. The levels thus established are used to interpret the detection signals detected during the other cycles.

[0122] The calibration sequence may involve hybridization of unlabeled bases, so as to determine a dark current for each nanowire.

[0123] Generally, the calibration sequence may comprise a number of identical bases of between 1 and a few dozen. Preferably, in the calibration sequence, the identical bases are adjacent. This makes it possible to detect a detection signal representative of each base during a sufficiently high number of successive cycles.

[0124] In [Fig.3E], the characterization signal resulting from the sequencing is shown diagrammatically. of a TTCG sequence. 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 S 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.3F], we have schematized the characterization signal resulting from the same sequence. 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 S1, S2 and S3 signals established during calibration. In this example, there is no fluorescent label cleavage. The intensity of the fluorescence signal increases progressively, due to the accumulation of fluorescent labels linked to bases hybridized on the sequence to be characterized.

[0126] In the preceding examples, it is assumed that the nucleic bases A, G and C are respectively labeled by a fluorescent marker A*, G*, C*. During each cycle, the following are determined: - either the absence of a 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 on a nucleotide sequence; - either a detection of a fluorescence light, by discriminating the fluorescent markers A*, G* and C*.

[0127] Following measurement and characterization of the detection signal, the fluorescence markers can be removed, but this is not necessary. The sample is rinsed and a new cycle can be performed. This characterizes the detection signal during each cycle, allowing the hybridized base to be identified during each cycle.

[0128] The device 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. This makes it possible to identify the nucleic base hybridized during the cycle.

[0129] 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.

[0130] The embodiment described in connection with Figures 3A to 3F can be implemented using three fluorescent markers whose emission characteristics are mentioned in the table below, and using an excitation wavelength of 532nm (YAG Neodymium laser). These fluorescent markers are available from the supplier ATTO GmbH under the references ATTO 532, ATTO 550 or ATTO Rhol2. Maximum excitation wavelength (nm) Maximum emission wavelength (nm) Quantum yield (%) Fluorescence decay time (ns) ATTO 532 532 552 90 3.8 ATTO 550 554 576 80 3.6 ATTORhol2 577 600 80 4.0

[0131] [Table 1]

[0132] In Table 1, the quantum yield is given at the maximum excitation wavelength. Excitation at 532 nm allows to reduce the quantum yield of the fluorescent markers ATTO 550 and ATTO Rhol2.

[0133] 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 excitation spectral bands, which may require the use of different light sources. Such an alternative is not preferred, since this assumes that no emission spectral band of the light sources is located in the detection spectral band of the nanowires.

[0134] 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 greater 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.

[0135] 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.

[0136] 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.

[0137] We will now describe different design aspects of the device according to the invention. Formation of nanowires

[0138] 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.

[0139] 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 doping species (for example, Ga and As to form GaAs). The atomic species adsorb and diffuse onto 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 / nanowire interface being formed.

[0140] The method is not limited to the use of gold as a catalyst. Other ca 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 called self-catalyzed nanowire growth.

[0141] 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].

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] Figure 5 illustrates a configuration in which the nanowires are arranged to form clusters 35. 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.

[0147] 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.

[0148] 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.

[0149] 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 facing the nanowires belonging to the same cluster 35.

[0150] According to another possibility, the nanowires are obtained by etching, according to a so-called top-down approach. The top-down approach is described in patent application FR2114563 filed on 12 / 27 / 2021. Structure of nanowires

[0151] 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.

[0152] 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.

[0153] 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.

[0154] A radial structure makes it possible to have a junction 33 extending over 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].

[0155] [Fig.7] shows schematically the main steps of a method for implementing the invention.

[0156] Step 100: arrangement of a sample comprising sequences of nu acids cleics from a library previously prepared from the DNA strand to be sequenced, placed in contact with the functionalization surface 25.

[0157] Step 110: capture of nucleic acid sequences by the functionalization surface, preferably at capture sites arranged directly above the nanowires.

[0158] Step 115: amplification, for example by bridging, of the nucleic acid sequence captured in each site, so as to maximize the number of identical sequences replicated on each capture 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.

[0159] 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, comprising a terminator. Bases of the same type are labeled with the same candidate fluorescent marker.

[0160] Step 130: rinsing the sample;

[0161] Step 140: illumination of the functionalization surface, so as to cause fluorescence of hybridized fluorescent markers following step 120;

[0162] Step 150: detection of a detection signal at the terminals of the detection circuit.

[0163] Step 160: characterization of the detection signal, so as to identify the marker fluorescent among the candidate fluorescent markers.

[0164] Step 170: cleavage of the terminator, possible cleavage of the fluorescent marker, and rinsing of the sample;

[0165] Step 180: repeating steps 120 to 170, until each nucleotide sequence captured at a capture site is sequencing.

[0166] 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. The latter can be identical or different from each other. In this case, the fluorescent markers are not linked to a terminator.

[0167] Preferably, steps 120 to 170 are implemented during the decoding of a calibration sequence Sc, as previously mentioned. This makes it possible to have a calibration, at the level of each capture site or at the level of each nanowire, making it possible to define the intensity levels corresponding respectively to each fluorescent marker.

[0168] The device benefits from a fast response time, typically of the order of ns.

[0169] It is observed that the device does not require the use of bulky optical components. In addition, the response of the device 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.

[0170] Finally, the device being based on nanophotodetectors, it makes it possible to obtain a compact analysis platform. The device can be obtained by implementing a collective manufacturing process, which makes it possible to lower the cost.

Claims

Claims

1. Device (1) for identifying a fluorescent marker or determining an amount of 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 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 in a detection spectral band comprising the fluorescence spectral band 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 an intensity level of the detection signal during the detection time period; • identify the fluorescent marker or determine a quantity of 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. A device according to any preceding claim, wherein the intensity level is an average intensity or a median intensity or a total intensity or a maximum intensity.

4. A device according to any preceding claim, wherein the detection band extends over a spectral width greater than 150 nm, so as to address the fluorescence spectral bands of several different fluorescent markers.

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

6. Device according to any one of the preceding claims, in which each nanowire comprises: - a pn-type homojunction; - or a heterojunction; - or a p-metal or n-metal Schottky junction.

7. 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.

8. Device according to claim 7 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.

9. 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.

10. Device according to claim 9, 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.

11. 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.

12. A method of identifying a fluorescent marker or determining an amount of 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, the sample comprising active ingredients configured to allow hybridization of a nucleic base to the nucleic acid strand captured on the functionalization surface, at least one added nucleic base being labeled with a fluorescent marker; 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. determining an intensity level of the detection signal detected in step e), and identifying the fluorescent marker, or determining an amount of the fluorescent marker, as a function of the intensity level detected; the process being such that - the nucleic acid comprises a calibration sequence, formed of a known number of known bases; - steps c) to f) are repeated so as to identify each nucleic acid base hybridized to the calibration sequence, each iteration of steps c) and f) forming a calibration cycle; - the detection signals respectively detected during each calibration cycle are used to define intensity levels corresponding respectively to each fluorescent marker, or to a predetermined quantity of the fluorescent marker, the defined levels being taken into account during steps f) of the sequencing cycles different from the calibration cycles.

13. The method of claim 12, wherein the intensity level is an average intensity or a median intensity or a total intensity or a maximum intensity during the detection time period.

14. A method according to any one of claims 12 or 13, wherein step b) comprises an amplification of each captured nucleic acid strand.

15. Method according to any one of claims 12 to 14, wherein: - the fluorescent marker is chosen from several candidate fluorescent markers; - each candidate fluorescent marker emits fluorescence light in a fluorescence spectral band; - the fluorescence spectral bands of the different candidate fluorescent markers are centered around fluorescence wavelengths respectively different from each other; - step f) comprises a selection of the fluorescent marker from among the candidate fluorescent markers according to the determined intensity level.

16. Method according to any one of claims 12 to 15, comprising, following step f), a step g) of identifying the acid base hybridized nucleic acid.

17. The method of claim 16, wherein - following step g), the fluorescent marker is cleaved and the sample is rinsed; - following rinsing, steps c) and g) are repeated, so as to identify a sequence of nucleotides forming the captured nucleic acid strand, each iteration of steps c) and g) forming a sequencing cycle.

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