Device for detecting a link between two biological species

Abstract

The invention relates to a device for characterizing the affinity of a bond between a biological species of interest, for example an antibody, with a capture biological species, for example an antigen. The detection of the bond is carried out by a variation of a fluorescence light, using a detection device comprising nanowires, each nanowire acting as a transducer of a fluorescence light into an electrical signal. Figure 1E.

Description

Title of the invention: Device for detecting a link between two biological species Technical field

[0001] The technical field of the invention is the detection of a bond between two biological species by optical transduction carried out by nanowires. PREVIOUS ART

[0002] A hybridoma is a cell resulting from the artificial hybridization of mammalian lymphocyte cells and immortalized myeloma cells. Hybridomas are used for the production of monoclonal antibodies. These can be used in the prevention, diagnosis, or treatment of diseases. These are antibody-producing cells (ASC antibody secreting cells).

[0003] The characterization of hybridomas is a long and expensive process, aimed at selecting hybridomas producing monoclonal antibodies (Mab: Monoclonal antibody) with a high affinity with a predetermined target antigen. This involves a qualification of monoclonal antibodies, and more precisely an evaluation of their affinity with the target antigen.

[0004] Currently, the qualification of monoclonal antibodies is carried out using quartz microbalances or SPR (Surface Plasmon Resonance) sensors. These systems comprise a detection surface functionalized by a target biological species, generally an antigen. The formation of antibody-antigen bonds modifies the resonance frequency of the quartz balances or the phase or amplitude of a beam reflected on the surface of the SPR sensors. Thus, the temporal monitoring of these variables allows an evaluation of the antibody-antigen affinity constant. However, this is a global measurement, without information on the dispersion of the antibody-antigen affinity. In addition, the use of a quartz microbalance or an SPR sensor assumes a large quantity of antibodies.

[0005] Given the low yield of hybridomas (hybridization and production yields), this involves the characterization of a large number of hybridomas, in order to identify, among the latter, those exhibiting a good productive yield of an antibody exhibiting good affinity with a target antigen.

[0006] It is interesting to be able to have screening solutions requiring a lower quantification of antibodies, so as to limit the need for culturing hybridomas, and more generally ASCs.

[0007] The inventors propose a method for determining an affinity of a biological species of interest, in particular a monoclonal antibody, with a species biological capture agent, in particular an antigen, having a rapid response time, compact, and requiring a small quantity of the biological species of interest. Description of the invention

[0008] A first object of the invention is a device for characterizing a binding affinity between a biological species of interest and a capture biological species, the biological species of interest and the capture biological species being configured so as to induce, under the effect of the binding and of an exposure to an excitation light, a variation of a 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, comprising the second electrode; • an electrically insulating interface layer covering the second electrode, the second electrode being interposed between the interface layer and a nanowire, the interface layer being configured to be arranged between a sample, comprising the biological species of interest, and the second electrode, the interface layer being delimited by a functionalization surface, functionalized by the capture biological species, such that the functionalization surface forms an interface between the device and the sample; • the multilayer structure being such that the or each second electrode and the interface layer are transparent in the fluorescence spectral band;

[0009] 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 the band fluorescence spectral, the fluorescence light detected by each nanowire inducing an electrical detection signal in the detection circuit;

[0010] the device comprising a processing unit, programmed to: • acquire the detection signal; • determine a variation, over time, of a fluorescence light • depending on the variation, determination of a characteristic representative of an affinity between the biological species of interest and the biological species of capture.

[0011] The device may comprise a fluidic chamber, in contact with the functionalization surface.

[0012] According to one possibility, the functionalization surface is segmented into different capture sites, each capture site comprising the biological capture species, each capture site being configured to form a bond with the biological species of interest.

[0013] According to one possibility, the fluidic chamber comprises channels, each channel extending opposite several capture sites.

[0014] According to one possibility, each nanowire comprises: - a pn type homojunction; - or a heterojunction; - or a p-metal or n-metal Schottky junction.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] A second object of the invention is a method for characterizing an affinity between a biological species of interest with a biological capture species, using a device according to the first object of the invention, the method comprising: a. provision of a liquid sample, comprising the biological species of interest, in contact with the functionalization surface; b. establishment of a link between the biological species of interest and the biological capture species, carried by the functionalization surface, on a capture site located directly above at least one nanowire; c. excitation of the capture site by an excitation light, so as to cause a variation of a fluorescence light emitted at the capture site capture ; d. formation of a detection signal, by the detection circuit, the detection signal being representative of the variation in fluorescence; e. depending on the detection signal, determination of a characteristic of the affinity between the biological species of interest and the biological species of capture.

[0020] According to one possibility: - steps c) and d) are repeated; - during step e), the characteristic of the affinity between the biological species of interest and the biological capture species is determined based on the detection signal formed during several iterations of step d).

[0021] According to one possibility: - the binding between a biological species of interest and the capture biological species results in an increase or decrease in fluorescence light; - under the effect of the increase or decrease of the fluorescence light, the detection signal varies between a first level and a second

[0022]

[0023]

[0024]

[0025]

[0026] level ; - step e) comprises determining a duration during which the detection signal corresponds to the second level, assuming that at the capture site, the increase or decrease in fluorescence light is due to binding of a single biological species of interest to the capture biological species. According to one possibility: - the bonds between a plurality of identical biological species of interest and capture biological species result in an increase or decrease in fluorescence light; - following the first iterations of steps c) to d), steps c) and d) are repeated during second iterations; - prior to the second iterations of steps c) and d), a liquid sample, considered to be without the biological species of interest, is placed in contact with the functionalization surface; - during step e), the characteristic of the affinity between the biological species of interest and the capture biological species is determined based on detection signals formed during the first and second iterations of steps c) and d). - Method according to any one of claims 10 to 14 in which: - the biological species of interest is linked to a fluorescent marker; - binding with the capture biological species results in an increase in fluorescence light. According to one possibility: - the biological capture species is linked to a fluorescent marker; - the biological species of interest is linked to a fluorescent light extinguisher rescense; - binding with the biological capture species results in a decrease in fluorescence light. The biological species of interest may be a protein and the capture biological species may be an anti-protein. The biological species of interest may be an antibody and the capture biological species may be an antigen. The features of dependent claims will be added here before filing. 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

[0027] Figures 1A and 1B show the main components of a device according to the invention.

[0028] [Fig.lC] shows a spatial distribution of capture sites.

[0029] [Fig. 1D] represents an example of a device according to the invention, in which the electrodes are distributed according to a matrix arrangement.

[0030] [Fig. 1E] shows an example of implementation of the device.

[0031] [Fig. 1F] shows schematically an evolution, as a function of time, of a detection signal, according to a first embodiment of the invention.

[0032] [Fig. 1G] shows schematically an evolution, as a function of time, of a detection signal, according to a second embodiment of the invention.

[0033] [Fig.2A] shows a configuration in which the biological species of interest is labeled with a fluorophore, which corresponds to the example shown in [Fig.1E].

[0034] [Fig.2B] shows a configuration in which the biological species of interest is labeled by a fluorescence quencher, and the capture biological species is labeled by a fluorophore.

[0035] [Fig.2C] shows a configuration in which the biological species of interest is labeled by a first fluorophore, and the capture biological species is labeled by a second fluorophore. The excitation spectral band of the second fluorophore corresponds to the fluorescence spectral band of the first fluorophore.

[0036] Figures 3A to 3E diagram the steps of manufacturing nanowires according to a so-called bottom-up process.

[0037] [Fig.4] represents different possible arrangements of the nanowires.

[0038] Figures 5A and 5B show two different nanowire structures.

[0039] [Fig.6] shows different stages of implementation of the method. PRESENTATION OF SPECIAL METHODS OF IMPLEMENTATION

[0040] Figures 1A to 1E show a first example of an analysis device 1 enabling implementation of the invention. The analysis device 1 is configured to be placed in contact with a sample 2, comprising for example a liquid medium capable of containing a biological species of interest 28, the affinity of which with a capture biological species 26, which may also be designated a target biological species, is to be characterized. In this example, the biological species of interest are monoclonal antibodies and the capture biological species (or target biological species) are antigens. The device is intended to characterize the affinity of the binding of the monoclonal antibody to the antigen forming the capture biological species.

[0041] The device comprises a substrate 10, forming or comprising at least a first electrode 11c. 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 first surface 11, comprising a first electrode 11c. 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 deposit of a conductive layer, for example graphene, forming all or part of the first surface 11.

[0042] Nanowires 30 are formed on the substrate 10, and more precisely 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 are intersecting, and preferably 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 may be conductive.

[0043] 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.

[0044] 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.

[0045] The nanowires 30 may be synthesized directly on the substrate 10, as described in connection with FIGS. 3A to 3E. 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.

[0046] The nanowires 30 extend, from the first surface 11, to a second surface 21 delimiting a multilayer structure 20. Like the first surface 11, the second surface 21 is conductive at least at the intersection with each nanowire 30. In the example shown in [Fig. 1B], the second surface 21 is formed from a layer 22 of a conductive material transparent in a detection spectral band described below. It may for example be ITO (indium tin oxide).

[0047] 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 two different dopings respectively: thus, the first part 31 and the second part 32 are respectively formed from a same semiconductor material respectively 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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 that at the level of the second surface 21, several second electrodes 21c are electrically insulated from each other. Thus, the conductive layer can comprise openings or insulating materials delimiting the electrodes 21c. This allows differentiated detection at the level of each nanowire.

[0052] 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.

[0053] The interface layer 23 is intended to form an interface between the layer 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.

[0054] 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.

[0055] The functionalization surface 25, which forms an interface between the multi-layer structure 20 and the sample 2 to be analyzed, is functionalized by capture species 26.

[0056] 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 functionalized at the level of each nanowell 27, the spaces between each well not being functionalized. The formation of the nanowells may be obtained by local thinning of the interface layer 23.

[0057] 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 the biological species of interest 28.

[0058] 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.

[0059] 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. Capture species 26 can be grafted onto the functionalization surface by covalent bonding.

[0060] When the functionalization of the functionalization surface 25 is carried out according to a spatial pattern, the parts of the functionalization surface outside the spatial pattern can be coated with an anti-biofouling coating so as to avoid non-specific adsorption of the biological species of interest. An example of an anti-biofouling material is a polyethylene glycol or a fluorinated polymer.

[0061] 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.

[0062] As described in connection with Figures 1A to 1C, each nanowire extends between a first end, on a first surface 11 of the substrate 10 and a second end, on a second surface 21 of the multilayer structure 20. The first surface 11 and the second surface 21 are conductive at least at each intersection with a nanowire. Thus, at each intersection with a nanowire, the first surface comprises a first electrode 11c and the second surface comprises a second electrode 21c. In the example shown in Figures 1A to 1B, the first surface 11 and the second surface 21 are formed from a conductive material. They are conductive over their entire area.

[0063] The first and second surfaces may be structured, and comprise different electrodes 11c, 21c isolated from each other. In [Fig.1D], each electrode is represented by a dotted line. On the first surface 11, each first electrode 11c describes a line, parallel to the longitudinal axis X. On the second surface 21, each second electrode 21c describes a column, parallel to the lateral axis Y. Each nanowire is functional when the electrodes 11c, 21c, between which it extends, are polarized. The structuring of the electrodes in rows / columns makes it possible to select the functional nanowires, the latter extending between two polarized electrodes: according to this arrangement, the nanowires connected to a polarized row and column are functional. By functional nanowire, we mean a nanowire polarized to perform fluorescence photon detection.

[0064] Several lines 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.

[0065] The functionalized parts of the functionalization surface form capture sites for the biological species of interest.

[0066] As previously indicated, each capture biological species 26 is configured to capture a biological species of interest, for example an antibody. By capture, we mean the establishment of a bond between the capture biological species 26 and the biological species of interest 28.

[0067] In [Fig. 1E], a fluidic chamber 4 is shown, in which a liquid sample 2 comprising biological species of interest 28 is placed. The fluidic chamber is placed above the functionalization surface, so that the functionalization surface forms a wall of the fluidic chamber. The fluidic chamber 4 may comprise channels, so as to direct the sample in front of all or part of the capture sites, according to a predefined path.

[0068] On the functionalization surface, three capture sites 25i, 252 and 253 are represented. At each capture site, the functionalization surface 25 is functionalized. In this example, the monoclonal antibodies, forming the biological species of interest 28, are functionalized by a fluorescent marker symbolized by the symbol *. Each capture site is functionalized by the same antigen 26.

[0069] The light source is configured to emit excitation light in a short duration, of the order of a few ps to la 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 quantity of antibodies 28 bound to the antigens 26.

[0070] An important aspect of the invention, described below, consists in translating the fluorescence signal into an electrical signal dependent on the fluorescent marker, which makes it possible to obtain, at each capture site, an electrical signal dependent on the quantity of antibodies bound to the antigen 26.

[0071] 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 antigen-antibody bonds inducing an electrical response of the device.

[0072] 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 *, 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 fluorescent photons rescence 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, which results in an increase in the detection signal.

[0073] 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 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. Optionally, an absorbing layer can be added between the functionalization surface 25 and the conductive layer 22 to reduce the spectral width of the response of the nanowires, for example a layer of zinc oxide deposited by sputtering. When the semiconductor material is GaAs, the fluorescent marker can be Cy3 (Cyanine): excitation wavelength 540nm and emission wavelength between 555 and 600nm.

[0074] Preferably, the diameter of the nanowires is controlled so as to provide sensitivity in a narrow spectral band. By narrow spectral band is meant a bandwidth typically of a few tens of nm, and preferably less than 100 nm. The bandwidth corresponds to the width at half-height of the absorption peak on the absorption spectrum. The detection bandwidth is for example of the order of 50 nm. It is thus possible to adjust the detection spectral band so that it includes the fluorescence wavelength of the fluorescent marker and does not include the excitation wavelength of the fluorescent marker. The ability of nanowires to form a wavelength-selective photodetector has been described in Mokkapati et al., “Optical design of nanowire absorbers for wavelength selective photodetectors”, Sci. Rep. 2015, 5 15339.

[0075] 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.

[0076] Each nanowire thus forms a nanophotodetector.

[0077] In this example, the antigen-antibody binding results in a variation in the fluorescence light. In this case, it is an increase in the light of fluorescence. Preferably, the antibody concentration in the fluidic chamber is adjusted so as to graft a single or a small number of antibodies, typically less than twenty, directly above each nanowire.

[0078] The fluorescence light detected by the nanowire placed directly above a capture site passes from a first level, in the absence of grafted antibodies, to a second level, representative of the quantity of antibodies captured.

[0079] S denotes a detection signal, resulting from the detection circuit 40, formed from the charges collected at the level of each nanowire. The device comprises a processing unit 41, allowing characterization of the detection signal. The processing unit 41 may comprise a microprocessor, connected to a memory 42 in which instructions are stored. The processing unit 41 may be programmed to determine a duration during which the detection signal occupies a high level.

[0080] The device may comprise a non-functionalized capture site, which makes it possible to establish the first level of the detection signal, in the absence of antibody-antigen binding.

[0081] The device makes it possible to characterize the affinity of the antibody with respect to an antigen. When the antibody concentration is sufficiently low, a single antibody-antigen bond can be detected on the surface of a nanowire. This corresponds to a first embodiment of the invention, described in connection with [Fig. 1F]. According to this embodiment, each grafting can be detected and its duration determined. Having several capture sites makes it possible to obtain measurement statistics.

[0082] The concentration of antibody and / or antigen is then determined by taking into account the number of nanowires, the surface area 25 and the volume of the fluidic chamber. Assuming that the height of the fluidic chamber is 100 pm, and that the surface area 25 measures 1 cm2, with a nanowire distributed in a matrix manner according to a spatial pitch of 1 pm, i.e. 108 nanowires, the concentration of antibody is at least 1 ng / ml.

[0083] In [Fig.lF], an evolution of a detection signal (y-axis) as a function of time (x-axis) is shown diagrammatically. The detection signal reflects the intensity of the fluorescence light detected by a nanowire. At a time t1, an antibody is bound to an antigen located on a capture site, directly above a nanowire. The binding extends over a duration Ata, up to a time t2. At a time t3, after t2, an antibody is bound to an antigen located on the same capture site. The binding extends over a duration Atb, up to a time t4. Between times t2 and t3, no antibody is bound to an antigen at the capture site.

[0084] According to this embodiment, an average duration of each antibody-antigen bond can be determined at the level of the same nanowire. The large number of nanowires makes it possible to obtain correct measurement statistics to characterize the affinity by a average duration of attachment between antibodies and antigens.

[0085] The embodiment described in connection with [Fig. 1F] is particularly interesting, because it allows an individual characterization of the affinity of the antibodies, by a measurement of the duration of antibody-antigen grafting. It does not assume that the concentration of the antibodies is known, nor the surface density of the biological capture species.

[0086] According to a second embodiment, several identical antibodies are grafted onto the same capture site, directly above a nanowire, or several nanowires arranged directly above the same capture site. In this case, the device makes it possible to establish characteristics of the affinity of the antibody with respect to the antigen.

[0087] In [Fig.lG], an evolution of a detection signal (y-axis) is shown diagrammatically as a function of time (x-axis). Under the effect of the progressive grafting of antibodies onto the capture site, the intensity of the fluorescence signal increases, until it reaches a plateau. On the plateau, some antibodies then detach, and are replaced by others. An equilibrium is thus reached between the association of antibodies and the dissociation of antibodies. At a time ts, the fluidic chamber 4 is supplied with a buffer, without antibodies. The antibodies gradually detach from the capture site, which leads to a decrease in the fluorescence signal. The affinity can be characterized by a ratio between a duration of the plateau (Atmax) and a duration corresponding to half the height of the plateau (Ath / 2). h corresponds to the height of the plateau. It is possible to calculate the rising and falling slopes and to calculate their ratio.

[0088] Regardless of the embodiment, a characteristic of the affinity is determined by exciting each nanowire at a predetermined excitation frequency, and by analyzing the detection signal obtained following each excitation. At each capture site, the detection signal is acquired during a detection time period subsequent to the excitation. This makes it possible to determine a number of capture sites at which at least one antigen-antibody bond has been produced.

[0089] The fact of providing different capture sites, implementing the same antigen-antibody pair, makes it possible to establish statistics relative to each site: this makes it possible to take into account variability in the antigen-antibody binding. The detection signals resulting from the different capture sites are acquired sequentially, by playing on the addressing of the electrodes.

[0090] [Fig.2A] corresponds to the configuration described in connection with [Fig.1E]. The labeling of the antibody can be carried out via a secondary antibody.

[0091] In [Fig.2B], a configuration is shown in which the biological capture species 26 is marked by a fluorescent marker, represented by the symbol *. The biological species of interest 28, in this case the antibody 28, is linked to a quencher (fluorescence deactivator) represented by the symbol +. The quencher induces a reduction or extinction of an emission of fluorescence photons. In the absence of capture, fluorescence photons are emitted, which causes an electric current to flow in the electrical circuit of the detection unit 40. Following capture, the emission of fluorescence photons is reduced, or stopped, by the quencher, which causes a variation in the electric current. According to this embodiment, under the effect of the establishment of antibody-antigen bonds, the detection current decreases.

[0092] In [Fig.2C], a configuration is shown in which the capture biological species 26 is marked by a first fluorophore, materialized by the symbol H, and the biological species of interest is marked by a second fluorophore, materialized by the symbol *. The first fluorophore H emits fluorescence light in a first fluorescence spectral band under the effect of illumination in a first excitation spectral band. The second fluorophore * emits fluorescence light in a second fluorescence spectral band under the effect of illumination in a second excitation spectral band. The second excitation spectral band corresponds to the first fluorescence spectral band.When the first fluorophore and the second fluorophore are close to each other, by FRET (Fôrster Resonance Energy Transfer) effect, under the effect of illumination in the first excitation spectral band, fluorescence light is emitted in the second fluorescence spectral band.

[0093] Thus, according to this embodiment, during illumination in the first excitation spectral band: - in the absence of antigen-antibody binding, fluorescence light is emitted, at the level of the functionalization surface 25, in the first fluorescence spectral band; - in the presence of an antigen-antibody bond, a fluorescence light is emitted, at the level of the functionalization surface 25, in the second fluorescence spectral band;

[0094] Preferably, the detection spectral band of the nanowire comprises the second fluorescence spectral band and does not comprise the first fluorescence spectral band. Thus, the nanowire is blind in the first fluorescence spectral band. In the absence of antigen-antibody binding, no fluorescence signal is detected. Establishment of an antigen-antibody binding results in the detection of fluorescence light, which results in an increase in the detection signal.

[0095] The use of nanowires is particularly advantageous. This makes it possible to obtain an exploitable signal using a small number of monoclonal antibodies.

[0096] The excitation spectral band is preferably between 350nm and 550nm. The detection spectral band of each nanowire is preferably sized to enable detection of fluorescence light while masking the excitation light. Alternatively, the detection currents, respectively formed at each nanowire during excitation and emission of fluorescence light, can be temporally separated. Advantage is taken of the fast response time of nanowires, on 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.

[0097] We will now describe different design aspects of the device according to the invention. Formation of nanowires

[0098] Figures 3A to 3E diagram steps for forming nanowires from a silicon substrate 10. Figures 3A to 3E 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 PMMA with a thickness of 45nm. The layers 10i and 102 are etched, so as to form nanowells isolated from each other, according to a predetermined pattern. See [Fig.3A]. The nanowells open onto the Si substrate. A thin layer of metal 103, for example gold, with a thickness of 5 to 10nm is deposited on the SiO2 layer. The added metal, in this case gold, acts as a catalyst. See [Fig. 3B]. The excess gold between the nanowells is removed by lifting (removing) the PMMA layer 102. See [Fig. 3C].We thus obtain islands of gold 103 isolated from each other, at positions corresponding to the position of the previously formed nanowells.

[0099] 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 jets towards the substrate to achieve epitaxial growth. See [Fig.3D]. The vapor-phase molecular jets contain the chemical species composing the semiconductor nanowire as well as the doping species. The molecular species collide and diffuse into the gold droplets. When the latter reach saturation, the nucleation of the nanowires occurs first at the droplet / substrate interfaces, then at the droplet / nanowire interfaces being formed.

[0100] The method is not limited to the use of gold as a catalyst. Other catalysts may be used, for example Ga. When the catalyst is Ga and Ga also constitutes an element constituting the semiconductor, this is referred to as self-catalyzed nanowire growth.

[0101] This method allows for example a growth of nanowires using As, Ga as well than C and Si respectively as p and n dopant, the processing temperature being 610°C. The process continues until the nanowires reach a predetermined height. Cf. [Fig.3E].

[0102] Following the step shown in [Fig.3E], 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. 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.

[0103] 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.

[0104] In the embodiment described in FIGS. 3A to 3E, the formation of the nanowells on the substrate 10 makes it possible to control the position of the nanowires.

[0105] Another advantage of the ascending approach, described in connection with Figures 3A to 3E, is that it allows more precise control of the crystalline structure of the nanowires. The ascending approach allows controlled incorporation of quantum dots or quantum wells, by controlling their position, in particular along the Z axis.

[0106] Figure 4 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

[0107] When the nanowires are distributed into clusters, as described in connection with [Fig. 4], 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 are thus simultaneously functional. The nanowires not connected to the detection circuit are not functional.

[0108] 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.lB]. In this case, each nanowell 27 extends facing the nanowires belonging to the same cluster 35.

[0109] The arrangement of the nanowires in a predefined arrangement on the substrate 10 is not necessary. According to one possibility, drops of metal catalyst are distributed randomly on the substrate 10. Following the addition, in the vapor phase, of the molecules constituting the nanowire, for example Ga, As and dopants, for example C, Si, the nanowires grow from the positions initially occupied, on the substrate, by the catalyst drops.

[0110] 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. Functionalization - fluorescence

[0111] The functionalization of the functionalization surface 25 is ensured by the grafting of biological capture probes 26 onto the functionalization surface 25. The biological capture probes 26 are intended to selectively capture a biological species of interest, the affinity of which with the biological capture probe is to be characterized.

[0112] According to one embodiment, in multiplexing, different biological capture probes 26 are separated on the functionalization surface 25. With reference to [Fig.4], the part of the functionalization surface located opposite the same cluster 35 can be functionalized by the same biological capture probe 26, so as to address the same biological species of interest. Advantage is then taken of the potential detection, by several adjacent nanowires, of the fluorescence resulting from a bond between the biological capture species and the biological species of interest. This makes it possible to characterize the affinity between the biological capture species and the biological species of interest from several capture sites, and this in a sequential manner.

[0113] Two different capture sites 251 252 of the functionalization surface 25, respectively arranged opposite two different clusters, can be functionalized by two different biological capture species, so as to address different biological capture species / biological species of interest pairs.

[0114] The fluidic chamber 4 may comprise channels, so as to simultaneously direct the same antibody towards different lines, each line being formed of capture sites functionalized with the same biological species of interest. Two different channels are isolated from each other so as to be able to test the affinity of the same antibody to different capture sites respectively functionalized by different capture species.

[0115] 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 pair of biological species to be captured / biological species of interest. It is thus possible to arrange a large number of clusters, respectively addressing identical pairs, or different pairs, in a single compact device. This results in the use of a reduced volume of reagents. The sensitivity of the nanowires makes it possible to detect and characterize antibodies with low affinity.

[0116] Another advantage of the invention is that the affinity can be characterized by time measurements, regardless of the maximum intensity of the detection signal. It does not assume knowledge of the density of antigens or the concentration of antibodies in the fluidic chamber. Structure of nanowires

[0117] Figures 5A and 5B represent other nanowire structures that can be implemented in a device according to the invention. [Fig.5A] 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.5A], a passivation sheath 34 bypasses the nanowire.

[0118] In the example of [Fig.5B], 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.

[0119] 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.

[0120] 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.5B] comprises an annular sheath 34 as described in connection with [Fig.5A].

[0121] [Fig.6] diagrams the main steps of a method for implementing the invention.

[0122] Step 100: arrangement of the microfluidic chamber on the functionalization surface, the latter having been functionalized, so as to obtain capture sites, each capture site being functionalized by an antigen.

[0123] Step 110: flow of the sample, comprising the antibodies, into the fluidic chamber.

[0124] Step 120: excitation of one or more capture sites by an excitation light.

[0125] Step 130: following each excitation, detection of a variation in the fluorescent light. rescense.

[0126] Step 140: acquisition of a detection signal, representative of the variation of the fluorescence light at each capture site.

[0127] Step 150: depending on the detection signal, determination of a characteristic of the affinity between at least one antigen, grafted at a capture site, and an antibody contained in the sample.

[0128] Steps 120 and 140 are repeated, so as to obtain a plurality of detection signals as a function of time. Step 150 is implemented from the detection signals respectively acquired during each step 140.

[0129] According to one possibility, described in connection with [Fig. 1F], following first iterations of steps 120 to 140, the method comprises a step 110', during which a sample, considered to be devoid of biological species of interest, flows into the fluidic chamber 4. Steps 120 to 140 are the subject of second iterations. Step 150 Step 150 is implemented from the detection signals respectively acquired during the first and second iterations of steps 120 to 140.

[0130] The device benefits from a fast response time, typically of the order of ns. 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.

[0131] Finally, since the device is 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.

[0132] In addition, the device is sensitive, which makes it possible to be used to characterize the affinity of a small number of monoclonal antibodies. This limits the need to cultivate and clone a large number of hybridomas, and this makes it possible to reduce the time required to determine the quality of production of an ASC. The antibodies can be collected from the supernatant of a culture medium comprising at least one ASC-type cell. The objective is to rapidly identify hybridomas, and more generally ASCs, producing antibodies exhibiting a high affinity with predetermined antigens. The invention allows the implementation of a functional screening requiring a small quantity of antibodies.

[0133] The invention can also be applied to the characterization of antibodies generated in-silico, for example on the basis of algorithms implementing artificial intelligence.

[0134] Although described in connection with a determination of antibody-antigen affinity, the invention can be applied, more generally, to characterize an affinity of protein-antiprotein type interactions, or the affinity of antibody fragments with antigens.

Claims

Claims

1. Device (1) for characterizing a binding affinity between a biological species of interest (28) and a capture biological species (26), the biological species of interest and the capture biological species being configured so as to induce, under the effect of the binding and of exposure to an excitation light, a variation of a 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), comprising the second electrode; • an electrically insulating interface layer (23) covering each second electrode (21c), the second electrode being interposed between the interface layer and a nanowire, the interface layer being configured to be arranged between a sample, comprising the biological species of interest, and the second electrode, the interface layer being delimited by a functionalization surface (25), functionalized by the capture biological species, 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 the fluorescence spectral band, the fluorescence 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; • determine a variation, over time, of a fluorescence light • as a function of the variation, determination of a characteristic representative of an affinity between the biological species of interest and the biological capture species.

2. Device according to claim 1, comprising a fluidic chamber (4), in contact with the functionalization surface.

3. Device according to any one of the preceding claims, wherein the functionalization surface (25) is segmented into different capture sites, each capture site comprising the biological capture species, each capture site being configured to form a bond with the biological species of interest.

4. A dispenser according to any one of claims 2 and 3, wherein the fluidic chamber comprises channels, each channel extending opposite several capture sites.

5. 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.

6. A device according to any preceding claim in which: - 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.

7. 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.

8. Device according to claim 7, 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.

9. 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 current detection induced by each nanowire extending between the selected first electrode and second electrode.

10. A method of characterizing an affinity between a biological species of interest (28) and a capture biological species (26) using a device according to any one of the preceding claims, the method comprising: a. provision of a liquid sample, comprising the biological species of interest, in contact with the functionalization surface; b. establishment of a bond between the biological species of interest and the biological capture species, carried by the functionalization surface, on a capture site located directly above at least one nanowire; c. excitation of the capture site by an excitation light, so as to cause a variation of a fluorescence light emitted at the capture site; d. formation of a detection signal, by the detection circuit, the detection signal being representative of the variation in fluorescence; e. depending on the detection signal, determination of a characteristic of the affinity between the biological species of interest and the biological species of capture.

11. The method of claim 10, wherein - steps c) and d) are repeated; - during step e), the characteristic of the affinity between the biological species of interest and the biological capture species is determined based on the detection signal formed during several iterations of step d).

12. The method of claim 11, wherein - the binding between a biological species of interest and the capture biological species results in an increase or decrease in fluorescence light; - under the effect of the increase or decrease of the fluorescence light, the detection signal varies between a first level and a second level; - step e) comprises determining a duration during which the detection signal corresponds to the second level, assuming that at the capture site, the increase or decrease in fluorescence light is due to binding of a single biological species of interest to the capture biological species.

13. Method according to claim 11, wherein: - the bonds between a plurality of identical biological species of interest and capture biological species result in an increase or a decrease in the fluorescence light; - following first iterations of steps c) to d), steps c) and d) are repeated during second iterations; - prior to the second iterations of steps c) and d), a liquid sample, considered to be without the biological species of interest, is placed in contact with the functionalization surface; - during step e), the characteristic of the affinity between the biological species of interest and the capture biological species is determined as a function of detection signals formed during the first and second iterations of steps c) and d).

14. Method according to any one of claims 10 to 14 in which: - the biological species of interest is linked to a fluorescent marker; - the linking with the capture biological species results in an increase in the fluorescence light.

15. Method according to any one of claims 10 to 14, in which - the biological capture species is linked to a fluorescent marker; - the biological species of interest is linked to a fluorescence light quencher; - binding with the biological capture species results in a decrease in fluorescence light.

16. A method according to any one of claims 10 to 15, wherein the biological species of interest is an antibody and the capture biological species is an antigen.

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