Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
  • G01N21/6454—Individual samples arranged in a regular 2D-array, e.g. multiwell plates using an integrated detector array
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides

Definitions

• the present invention aims to improve the detection of chromophoric or chromogenic signals linked directly or indirectly to cells, molecules or macromolecules, chemical or biological, attached to supports.
• chromophore signal or "chromogenic signal” means any light signal emitted directly or indirectly after excitation by an appropriate light or after enzymatic transformation of a chromogenic substrate.
• chromophoric or chromogenic signals the colorimetric, photoluminescent, fluorescent, chemiluminescent, bioluminescent and similar signals. These signals are emitted either directly by the molecules of interest, or by elements which are added to them and / or grafted, such as, for example, dye molecules or certain types of semiconductor nanostructures such as wires or quantum dots. .
• molecule here is meant any type of molecule or macromolecule, isolated or linked to a structure or to another molecule.
• the term “molecule” can also be understood to mean chemical molecules having a particular affinity for a specific ligand.
• the accessibility of biological material and the search for the presence of a particular molecule in a complex mixture have led to the development of miniaturized techniques which use "chips", “micro-networks” or “macro- networks ", these last two elements being better known under the English names” micro-arrays "and” macro-arrays ".
• the molecules of which one wishes to search for the presence or which one wishes to quantify are placed in the presence of known specific ligands (the probes) fixed on a support or a substrate which, today, is generally made of glass, made of silica, nylon or even metal.
• the detection of a target is then ensured by its coupling to a fixed probe (or specific ligand), because the target carries a chromophore (it may happen that it is the probe which carries the chromophore).
• support or “substrate” here is meant any means enabling chromophoric elements to be supported. Consequently, it may be a monolayer structure or else a multilayer structure.
• the monolayer or one of the layers of the multilayer may be a commercially available and possibly modified substrate.
• the fixing of the probes on their supports can be carried out in different ways. Mention will be made, for example, of addressing by chemical means, addressing by electrochemical means, or addressing using a technology similar to the technology of ink jets implemented in printers.
• the sensitivity of the detection means used today requires the presence of at least 10 4 molecules of the same category to obtain a significant response.
• the probes becoming more numerous on very small supports, it becomes particularly difficult to detect the origin of luminescent or colorimetric signals generated after coupling or probe / target hybridization.
• Document US-A-6,008,892 discloses a device for supporting chromophoric elements which comprises a rigid base with a reflecting surface, in particular made of metal, covered with a transparent layer, for example of silica, on which are fixed chromophoric elements.
• the optical path between the reflecting surface of the base and the upper face of the transparent layer is an odd multiple of a quarter of the wavelength of the excitation light of the chromophoric elements, so that the standing wave of the incident excitation light presents a belly at the level of the chromophoric elements.
• the excitation of the chromophores is then maximum.
• the reflecting base also makes it possible to increase the collection of the fluorescence emitted in response to the excitation, by almost doubling the solid angle of collection of the fluorescence by a microscope system.
• the object of the present invention is in particular to further improve the detection of the light emitted by the chromophoric elements in response to a light excitement.
• a device for supporting chromophoric elements of the aforementioned type which makes it possible to concentrate at least part of the light emitted by the chromophoric elements in a selected part of the space, so that it can be received.
• the term “concentrating” is understood here to mean, first of all, recovering the light which is emitted by the chromophoric elements in a part of the region which surrounds them, then, forcing this recovered light to gain one or more zones of collection.
• the invention provides for this purpose a device for supporting at least one chromophore element, comprising a substrate having an upper surface on which said chromophore element is fixed and means for increasing the amount of light emitted by the chromophore element towards a collection device, these means belonging to a group comprising reflecting means placed in the substrate at a distance from its upper surface, characterized in that this group also comprises:
• microlenses each associated with a chromophore element and operating in transmission or in reflection
• - diffraction means placed at a distance from the chromophoric element or elements and operating in transmission or in reflection
• first reflecting means and second reflecting means mutually parallel and placed on either side of the chromophoric element or elements to define an asymmetrical resonant cavity
• a planar waveguide formed in the substrate under the upper surface carrying the chromophores, to capture a part of the light emitted by the chromophores in the substrate and / or to bring about an excitation light
• the means increasing the quantity of light emitted by the chromophoric element or elements comprise at least one of the means belonging to the aforementioned group.
• the device according to the invention therefore provides for modifying the optical environment of each chromophore element so that a greater proportion of the light which it emits is collected.
• the yield is increased, as is the sensitivity of the molecules detection tests.
• the invention can allow a reduction in the excitation intensity.
• This invention can also allow, in certain embodiments, an improvement in the rejection of the exciting light collected in the detector, and consequently an improvement in the signal / noise ratio (S / N).
• the invention may also allow, in certain embodiments, a strengthening of the optical excitation of the chromophores in addition to the strengthening of the collection of the light emitted by said chromophores.
• a first so-called “distant” mirror secured to (or formed in) a substrate at a distance (d) from the chromophores, large in front of the wavelength emission ( ⁇ ), i.e. verifying the criterion d> n ⁇ / 2NA 2 (where “NA” is the numerical aperture of the collection objective and “n” the index of the environment between the mirror and the chromophores), and intended to reflect the light emitted by the chromophoric elements towards the chosen part of the space.
• the first far mirror can be produced in the form of a stack of dielectric layers;
• microlenses each associated with a chromophore element and capable of operating in transmission or in reflection, that is to say by focusing forwards or backwards, as is the case, in particular, of a Fresnel mirror;
• the chromophores are emitted in a planar waveguide (the chromophores are not necessarily placed in the guide), grooves defining a blazed network, so as to collect the light emitted by the chromophoric elements in the direction means for supporting and guiding this light towards the collection device;
• a planar waveguide intended to convey an excitation light of the chromophores while minimizing the diffusion of this light in the surrounding environments; • in the absence of a collection waveguide, it is possible to provide a matrix of light detection elements by charge coupling (CCD), associated with the rear face of the substrate, some at least of the detection elements being addressed electronically in correspondence of at least one chromophore element.
• CCD charge coupling
• CCD either at sites carrying active or empty or non-active chromophoric elements, or at sites having a reference activity signal, so as to obtain by a differential measurement on the pixels of the CCD detector the signal of the active chromophore with an increased S / N ratio, by subtracting the background noise and the parasitic excitation signal.
• a first reflector or mirror
• close similar to that of geometric optics but placed at a distance (d) from the chromophores such that said reflector provides an interference effect allowing an amplification of the collection of light emitted in the support (d checking the relation: d ⁇ n ⁇ / 2NA 2 ), and coupled to excitation waves arriving on the chromophores under a non-zero incidence compared to the normal to the support.
• This incidence ensures in addition a reinforcement of the excitation when there is a double resonance, that is to say a coincidence of the bellies of the field for the two wavelengths of excitation ( ⁇ exc) and emission ( ⁇ issued). Detection remains centered on normal to the support;
• this first mirror, placed under the chromophoric elements, can be combined with a second semi-reflecting mirror placed above these chromophoric elements, substantially parallel to the first mirror and at a chosen distance from it so as to define an asymmetrical resonant cavity, in particular of the Fabry-Perot type, delivering the light collected through the second mirror.
• the second mirror or output mirror may be below and the mirror most reflecting the wavelength emitted above, the latter possibly being largely transparent at the excitation wavelength.
• a condition of double resonance similar to that of the near mirror can be satisfied, taking as the distance from that which separates the chromophores from the most reflecting mirror.
• the second mirror can be deposited directly either on the substrate, or on the entry face of a collecting optic, or even be constituted by the diopter of an existing entry face;
• a planar resonator formed in the substrate around each chromophore element, intended to store the electromagnetic energy of the field which it induces and arranged so that the associated chromophore element is substantially placed on a belly of the electromagnetic field.
• They may be concentric circular grooves or grooves (at least two) in the center of which is placed a chromophore element. It may also be rectangular or parallel grooves or grooves defining a lamellar network; • a two-dimensional (2D) or three-dimensional (3D) network of holes or columns, defining a photonic crystal, preferably with a photonic band prohibited so as to control the propagation of light in its region, which can go as far as 'to prohibit ;
• nanometric structures for example islets
• regular or not or nanometric holes, produced in selected metals, in particular in silver (Ag).
• nanometric islands or holes are capable of increasing the emission, and incidentally the excitation, as in Raman scattering of increased surface, in particular of the SERS type;
• light concentration means comprising an asymmetrical resonant cavity, in particular of the Fabry-Pérot type, integrated into a substrate below its upper layer, the latter being at least partially permeable, vertically and / or laterally, so to allow the migration of chromophoric elements to selected sites relative to the resonant cavity.
• This cavity is preferably defined by first and second mirrors, and the upper layer is preferably made of a porous material, for example Silicagel.
• the upper layer may have holes in selected locations, so as to encourage the migration of the chromophoric elements towards the sites.
• the invention relates to all fields in which the device presented above can be used, and in particular in the fields of biology, biochemistry and chemistry.
• a device of the type presented above for the implementation of biochips intended to allow, in particular, the detection of pairings of target and probe oligonucleotides, such as strands of DNA, the detection of interaction between proteins or target polypeptide sequences, labeled with a molecule capable of directly or indirectly emitting chromophoric or chromogenic signals, in particular luminescent, fluorescent or colorimetric signals, and proteins or sequences probe polypeptides, and detection of interaction between probe chemical molecules and target chemical molecules.
• FIG. 1 is a very schematic sectional view of a device according to the invention, with a far mirror formed only of dielectric layers, or of hybrids with dielectric and metallic layers,
• FIGS. 2A and 2B are very schematic sectional views of devices according to the invention, with mirror cups,
• FIGS. 3A and 3C are very schematic sectional views of a device according to the invention, with a "close” mirror coupled to an excitation under non-zero incidence, the curves of FIGS. 3B and 3C respectively illustrating the luminescence intensity curves (1 Efl U0 (Z)
• FIGS. 4A and Ab are very schematic sectional views of devices according to the invention, with a resonant cavity
• FIG. 5 is a very schematic sectional view of a device according to the invention, with planar resonator in grooves,
• FIG. 6 is a very schematic sectional view of a device according to the invention, with planar resonator and waveguide,
• FIG. 7 is a very schematic sectional view of a device according to the invention, with groove structure, coupled to CCD type detection elements.
• FIGS. 8 to 14 are very schematic sectional views of different alternative embodiments of the device according to the invention.
• FIG. 15 is a very schematic top view of the device of Figure 14.
• the attached drawings are, for the most part, certain. Consequently, they can not only serve to complete the invention, but also contribute to its definition, if necessary.
• reference will be made to devices for supporting chromophoric elements comprising at least one support on which is arranged a multiplicity of zones suitable for receiving probes which it is desired to couple to targets.
• the targets and the probes are oligonucleotides such as DNA strands.
• the invention is not limited to this application, the targets and the probes possibly being proteins or polypeptide sequences, or more generally any type of chemical, biochemical or biological molecule.
• the device according to the invention is intended to recover at least part of the photons of light which are emitted by chromophoric elements in a part of the region which surrounds them, so that they are delivered in a selected part of space where a collection device is located, such as a microscope objective coupled to a photodetector, such as for example a photomultiplier or a matrix of photodetector elements, for example of the CCD (Charge Coupled Device) type.
• the support device comprises means for concentrating light which can be classified into two different categories according to whether they function according to the laws of wave optics or geometric optics.
• Figure 1 We first refer to Figure 1 to describe an embodiment of a device according to the invention operating according to the laws of geometric optics.
• the device comprises a support 1 formed of a substrate 2 in which a reflecting mirror 3 is formed at a distance (d) from its upper face 24.
• the mirror can be formed by depositing a layer highly reflecting at wavelengths working (emission wavelengths chromophores).
• the mirror consists of a multiplicity of dielectric layers 3.
• the substrate 2 and the dielectric layers 3 can be made of semiconductor materials or oxides or glasses. For example, for an emission wavelength ( ⁇ emiss) of the order of 600 nm, it is possible to choose layers of
• nitrides Si 3 N
• organic or organometallic polymers these polymers being either amorphous (isotropic material), or “oriented” and birefringent.
• the mirror is said to be “distant” so as to operate according to the laws of geometric optics.
• the term “distant mirror” is understood to mean any type of reflective structure operating, by virtue of its distance from the chromophores, according to the laws of geometric optics and not according to the laws of wave optics (by interference phenomena).
• the mirror is distant from the chromophores 5 by a distance d satisfying the criterion: d> n ⁇ / 2NA 2 , where “NA” is the numerical aperture of the objective of collecting the light emitted by the chromophores according to the wavelength ( ⁇ ), and "n” is the index of the medium between the mirror and the chromophores.
• Such a “distant” reflecting mirror makes it possible to multiply by about two the number of photons collected substantially around the direction normal N to the upper face 24 of the support 1, the angle of incidence of the excitation light on the face 24 being arbitrary.
• the support 1 receives, on its upper face 24, the various probes at selected locations.
• the fixing of the probes on the support 1 can be carried out according to addressing techniques known to those skilled in the art, which vary according to the nature of the probes and of the upper surface 24 of the support 1. It can be, for example, example, a chemical addressing technique, or electrochemical addressing, or a technique similar to the ink jet technique used in the field of printers.
• the supports 1 equipped with their probes are then brought into contact with targets, for example by immersion, so that certain targets interact with certain probes.
• targets for example by immersion
• interaction is understood here to mean any type of coupling or hybridization or even pairing which makes it possible to form target / probe pairs in a relatively durable manner.
• the targets are, for example, strands of DNA provided with luminescent markers.
• luminescence is understood here to mean any type of light emission, induced or spontaneous, such as for example photoluminescence, fluorescence, or phosphorescence.
• the luminescence is ensured locally by the structures on which the target / probe pairs are fixed. This is particularly the case for certain semiconductor structures such as quantum wires and dots (see in particular the document Bruchez et al., Science 281, p.2013, 25 Sept. 1998), or dyes for which the couple which s fixed there acts as a destroyer or an amplifier of luminescence.
• the substrate may be produced in semiconductor materials.
• chromophore element therefore means any element capable of emitting a chromophore signal or a signal chromogenic, directly or indirectly after excitation by an appropriate light or after enzymatic transformation of a chromogenic substrate. It could therefore be either a target / probe couple secured to the upper surface 24 of the support 1 and capable of spontaneously or indirectly emitting photons of light, or of a three-dimensional structure which, once associated with a target / probe pair, emits light spontaneously or in an induced manner, or even an element secured to the substrate and which once associated with a target / probe pair changes behavior, emitting much more or much less than in the absence of the couple, whether spontaneous or induced emission.
• the invention therefore aims to promote the collection of photons emitted by the chromophores, and in particular all or part of the light which would be emitted in the direction of the support 1. It is recalled here that approximately 70% of the light emitted by the chromophores get lost in the substrate and the remaining 30% are not necessarily picked up by the collection device, for reasons of limitation of the collection opening.
• the support 1 of the device may comprise, substantially below each chromophore 5, microlenses, structured or not, operating in transmission or in reflection (micromirror), that is to say by focusing forward or towards the back, as is the case, in particular, of Fresnel mirrors.
• Diffractive devices can also be used placed at sufficiently large distances from the wavelength of the light emitted. Under these conditions, the diffraction means operate according to the laws of geometric optics, and their characteristics (angles of incidence and emergence) are fixed by the laws of networks.
• a particular embodiment of the network consists in redirecting the light with a synthetic holographic network (that is to say resulting from the calculation of an optical transfer function according to the methods known to those skilled in the art).
• a synthetic holographic network that is to say resulting from the calculation of an optical transfer function according to the methods known to those skilled in the art.
• small bowls (or three-dimensional reliefs) 19 are formed in the upper part 10 of the support 1.
• Such reliefs can be produced according to different techniques for defining patterns (such as lithographs and serigraphs, exposure by electron beam (“e-beam”)), followed by techniques for forming reliefs o (such as chemical or dry etching, by plasma, by ion beam, by plasma and / or beam reactive ions), but also directly by punching ("embossing").
• each cuvette 19 After having deposited a reflective layer 20 (or a multilayer assembly having properties reflecting at wavelengths 5 of interest) on the bottom of each cuvette 19, it is filled with a filling material 18 of index n2, preferably high, then the chromophore 5 is placed on the upper surface of the filling material 18, substantially at the focal point (or center) of the bowl 19. Several (at least two) chromophores can possibly be placed above one or the same bowl .
• the reflecting nature of three-dimensional structures is obtained by metallic or dielectric deposits.
• the cuvettes are filled using any method leading to the desired material, preferably dielectric.
• a flat filling can be envisaged, as in the case of an epoxy resin solidifying well flat, either by planarization in the case of a convex deposit, or without intervention for example in the case of a concave dielectric.
• the bowls 19 are parabolic of revolution, the geometric focus being located at the 0 location provided for the chromophore (on the filling material 18), so as to allow an ideal collection of a maximum of emitted light.
• ⁇ c sin "1 (n1 / n2) and ⁇ c + 10 ° or 20 °.
• the bowl can also be elliptical revolution, so as to form upstream a real image of the chromophore located at the other focal point, in
• arrays of electronically addressed photodetector elements such as for example charge coupled devices (better known by the acronym CCD for “Coupled Charged Device”) or devices made up of detectors with MOS (Metal-Oxide Semiconductor) technology, among the least expensive of the matrix detectors currently available.
• CCD Charge Coupled Device
• MOS Metal-Oxide Semiconductor
• these detectors are used to directly form the support 1 or the substrate 2, which avoids any loss of light and makes it possible to keep the rays inclined beyond the critical angle of the support-air interface.
• a detector separated from the support by a medium with a low index, such as air these rays would be totally reflected and would not reach the detector.
• CCD arrays on a silicon substrate, for example, so that they operate in visible wavelengths, the near infrared, or even the near ultraviolet.
• These matrices can withstand certain thermal, fluidic or other treatments, necessary for the development of the device according to the invention.
• these photodetectors include photosensitive elements (or pixels) (as illustrated in the example of FIG. 7), the dimensions of which can be between 5 micrometers and 50 micrometers ( ⁇ m), and which, consequently, allow very high probe densities on the same support.
• a first solution is suitable for cases in which the luminescence decreases more slowly than the duration between two acquisitions. In these cases, it is possible to carry out a pulsed, rapid photo-excitation, in order to "empty" the detection pixels of the CCD matrix before acquisition.
• a second solution consists in adding mirrors, for example interference, or of another category (colored filters for example) or a combination of mirrors of different types, strongly rejecting the excitation radiation (typically less than 10 "4 , and if possible less than 10 " 5 ), while ensuring a transmission window for the radiation of chromophores 5.
• these two solutions can be combined together to further improve the collection results.
• S / N An improvement in the signal / noise ratio (S / N) can, moreover, be obtained by a differential method of the type described below.
• a first detection pixel is placed opposite each chromophore, and one or more other detection pixels are placed next to this first pixel to collect the rest of the emitted light, which has not been concentrated by the means of the invention, and stray lights of all kinds.
• a differential measurement of the signal received by the pixel associated with the chromophore and of the signal received by the neighboring pixel (s) gives a better S / N ratio and makes it possible to get rid of various sources of noise and parasites.
• FIGS. 3A to 3C describe another embodiment of a device according to the invention, operating according to the laws of wave optics.
• a double resonance is created so as to obtain a reinforcement of the collected light and excitement.
• the first resonance concerns the emission wavelength while the second resonance concerns the excitation wavelength.
• This double resonance is obtained by determining the coincidence of the bellies of the electric field for the two excitation and emission wavelengths.
• a support 1 is used, of the type described above with reference to FIG. 1, that is to say comprising a reflecting mirror 3 formed of dielectric or metallic layers.
• the mirror is said to be “close”. In other words, it is placed at a distance (d) from the chromophores which satisfies the criterion: d ⁇ n ⁇ / 2NA 2 .
• This close mirror condition is combined with a condition relating to the angle of incidence ⁇ of the excitation light with respect to the normal N at the upper surface of the support 1 (the collection of the concentrated light taking place substantially perpendicularly on the upper surface of the support 1).
• the spacing has bellies of the field at the wavelength ⁇ in a medium of index n illuminated by a wave whose incidence in the external medium, of index n ext , is ⁇ , is given by:
• FIG. 4A describes another embodiment of a device according to the invention, operating according to the laws of wave optics.
• a support 1 of the type described above with reference to FIG. 3A that is to say a substrate 2 comprising a near mirror, reflecting, formed of dielectric layers 3, for example, and a second semi-reflecting mirror 7 placed above the chromophores, at a chosen distance (d) from the upper surface 4 of the first mirror.
• the distance d between this upper surface 4 of the first mirror and the lower surface 8 of the second mirror 7, which faces it, is chosen so that the two mirrors define an asymmetrical resonant cavity, of the Fabry-Perot type.
• the second semi-reflecting mirror 7 allows the photons emitted by the chromophores 5 placed on the upper surface 4 of the first mirror 3, to pass through it, and therefore to be detected by a collection device (placed above the second mirror 7).
• a collection device placed above the second mirror 7.
• Such a second semi-reflecting mirror 7 can advantageously be formed by the underside of a collection objective. It may be directly this face or a coating deposited thereon. Adjusting the resonance wavelength with respect to 22
• an adjuster can be provided on one of the elements of the device (for example the support or a mirror outdoor), of the piezoelectric device type controlled by an in-situ optical measurement of the resonance. It can also be envisaged that this adjuster, coupled to the collection device, is mounted so as to allow scanning of the surface of the support 1.
• the second semi-reflecting mirror 7 can be formed on the underside of a strip, of the type used for microscopic observations. Due to the very small dimensions of the resonant cavities of the Fabry-Perot type, typically ⁇ / 2, the lamella can be placed above the chromophores 5 with the interposition of a fluid (constituted for example by the biological sample itself or by a wetting liquid), the control of the thickness of the cavity then being carried out by the control of the pressure applied to the strip, possibly coupled to an optical servo-control.
• a fluid constituted for example by the biological sample itself or by a wetting liquid
• a resonant cavity is produced in one piece, the upper layer and the upper mirror 7 being integral with the substrate 2, and the sites for positioning the chromophores 5 being located inside the structure in optically suitable positions, given above, relative to the resonant cavity.
• the resonant cavity is preferably of the Fabry-Perot type, asymmetrical, and more generally delimited by two mirrors 3, 7 spaced by a distance d chosen as a function of the working wavelength and of the materials used, in particular. These two mirrors are preferably of the type of those described above, that is to say in the form of a dielectric multilayer structure.
• the access of the chromophores 5 to the internal sites is carried out by any means making the upper layer 22 at least partially permeable, vertically and / or laterally.
• FIG. 5 describes another embodiment of a device according to the invention, in which the light concentration means operate according to the rules of wave optics.
• the support 1 is formed of a substrate 2, the upper surface 24 of which comprises a multiplicity of holes 11 each intended to receive a target.
• the holes allow the chromophores to be placed in locations where the field of the resonator is maximum. Their depth can vary between zero and the depth of the grooves of the resonator described below. These holes are preferably arranged in an orderly fashion.
• a planar resonator 12 is produced around each hole 11.
• Such a resonator may for example consist of a series of concentric grooves (or grooves) 13, opening at the level of the upper surface 24 and of widths and depths chosen so as to confer properties of confinement and extraction of light. emitted in their center. More precisely, this resonator is arranged so that the electric field has a belly at the level of the hole 11 containing the chromophore 5 and that it stores the electromagnetic energy, the light being recovered at the level of the grooves 13 (or grooves) and then transmitted in the part which is placed above the surface 4, following (approximately) the arrows, with a view to being collected there by an appropriate collection device.
• the depth of the grooves can be a few ⁇ m or equal to, or even greater than, the radius.
• the width of the grooves must be equal to approximately a fraction of the wavelength emitted, typically of the order of 0.1 ⁇ m. 24
• z mn is the nth zero of the Bessel function of order m, J m (z).
• neff is the underlying planar guide mode if necessary, otherwise it is the index of the solid in which the resonator is defined.
• planar resonator can be envisaged.
• lamellar networks with rectangular or parallel grooves having dimensions similar to those mentioned above.
• hollowed-out holes or columns placed in selected locations. It could be, for example, round holes forming a triangular network, with the omission of a certain number of them to form the resonant cavity.
• planar resonators if their dimensions are chosen for resonance with chromophore 5, present resonant modes which allow a large part of the emission of this chromophore. These resonant modes are shown very schematically by the dotted lines in FIG. 5. In addition, these planar resonators provide a coherent diffraction, similar to a Bragg type diffraction, which allows the energy of the mode to be redirected. resonating towards the collection device.
• a very small resonator typically 2 or 3 ⁇ / n.
• the resonator can be anti-resonant in the entire wavelength range of chromophore 5, which inhibits the emission in the plane and promotes the emission vertically, that is to say perpendicularly at the upper surface 24.
• a coupling to photodetector elements then makes it possible to collect the light.
• photonic crystal type structures we can use those known as "photonic band prohibited", which offer the property of prohibiting the propagation of light within them, thus repelling all the electromagnetic energy of the light emitted by chromophore 5 outside of themselves, even when chromophore 5 is located at a distance of a few wavelengths ( ⁇ ) inside the photonic crystal.
• Furrow or photonic crystal structures can, when they are no longer resonant, that is to say when they have sizes or radii greater than about 10 ⁇ , be used to diffract guided light from chromophore. These structures tend towards geometric optics and work well for a certain variety of incident waves and no longer for one or more privileged modes. This 26
• the target belly of the electric field must typically be about ⁇ / 2n in size, or less at around 200 nanometers.
• the extraction can be carried out through the substrate, on the back of which microlenses (operating according to the laws of geometric optics) can be placed so as to increase the amount of light collected.
• localized resonances of the type that occur in increased surface Raman diffusions are created.
• SERS Surface Enhanced Raman Scattering
• These resonances result from a local reinforcement of the electromagnetic field induced by the presence of nanometric structures, such as nanometric islands, or nanometric holes, produced in particular metals, such as in particular silver (Ag) or gold (Au ).
• nanometric structures such as nanometric islands, or nanometric holes, produced in particular metals, such as in particular silver (Ag) or gold (Au ).
• Local reinforcement concerns first of all the emission, and incidentally the excitement. More precisely, these resonances can be used either to reinforce the excitation light ( ⁇ exc), or to couple the light emitted by the chromophores ( ⁇ armes) to the collection means.
• FIG. 6 describes another embodiment of a device according to the invention, in which the light concentration means also operate according to the laws of wave optics.
• a planar waveguide is provided below the part of the support where the chromophores are fixed 5.
• the structure can remain guiding in the absence of the upper layer 16, as the knows the skilled person.
• More complex guiding structures can be envisaged, so as to position the guided modes of the light emitted by the chromophores in a particular way, for example so that the field is as strong as possible on the surface.
• a structure may include, for example, a substrate 2 of very low index (without upper layer 16) and a guiding layer of index and thickness chosen at the limit of the mode cut-off condition.
• this cut-off condition we can refer, in particular, to chapter 8 of the book "Light transmission optics (Van Nostrand, New York, 1972) by D. Marcuse".
• Such guides can be of the single mode or multimode type. In this last case (multimodes), if the number of modes is really important, and that consequently the thickness of the guide is very important, one is brought back to a structure functioning according to the laws of geometrical optics. Furthermore, this type of guiding structure can be very confining or not very confining depending on whether there is a strong or a small difference in index between the surrounding layers. On the other hand, these guiding structures can be optimized so as to capture a large fraction of the light, either in a single mode, or in a privileged part of these modes if it is multimode, so as to facilitate the extraction of the light. 28
• the guided modes are conveyed to the borders of the structure. It is then possible to collect the light by coupling the ends of the waveguide to an appropriate collection device, such as for example optical fibers, or microscope objectives.
• each chromophore structures In order to allow the collection of the guided modes on a single side of the support 1, it is possible to produce around each chromophore structures with grooves or grooves which open into the guiding structure, so as to produce a kind of blazed network which channels the light emitted towards a useful side of the guiding structure, where detection takes place.
• the guiding structure 14 it is also possible to add to the guiding structure 14 another structure forming a planar resonator, of the type described with reference to FIG. 5. It is possible, for example, to form grooves (or grooves) 15 concentric around the chromophore 5, so as to redirect the modes guided by the guiding structure 14 towards an area situated above said chromophores 5.
• the grooves or grooves 15 can be replaced by a two-dimensional or three-dimensional photonic crystal consisting of holes or columns, as described above.
• FIG. 7 describes another embodiment of a device according to the invention, also operating according to the laws of wave optics. 29
• the device according to the invention comprises a substrate 2, the upper part 10 of which is formed with relief structures 6, in hollow or in bumps.
• these structures 6 are grooves or ribs which are linear and parallel to each other, for example in the form of a U or a V (as in the example illustrated).
• the size of these structures is of the order of the working wavelength.
• the probes can be placed either at the top of the structures (as illustrated) or at the bottom of the structures, that is to say in the space which joins the lower parts of the structures 6. It is also possible to associate metals with dielectric supports, for example by depositing metallic patterns on all or part of the ribs or grooves.
• photodetector elements (or pixels) 17 below the relief structures 6, to which the chromophores 5 are fixed, it is possible to collect the photons emitted by the chromophores in the structures and channeled by them.
• the light emitted in the direction substantially opposite to the direction of the channeled light can be collected by other photodetectors placed above the chromophores 5.
• these photodetection pixels can be CCD elements forming, for example, a detection matrix.
• FIG. 8 The integration of a matrix of CCD photodetectors 17 into a substrate 2 carrying chromophores 5 is shown more completely in FIG. 8 where it can be seen that the substrate 2 comprises, under the chromophores 5, reflecting means 3 with a structure multilayer, and an absorbent layer 23 insensitive to the incidence and located between the reflective layers 3 and the photodetectors 17.
• the reflective layers 3 ensure a maximum field of excitation and emission at the level of the chromophores and prevent the light from excitation to propagate by simple transmission and by diffusion towards the matrix of photodetectors 17.
• the layer of absorbent material 23, such as that used for RGB filters in photography and in color CCD arrays, has an absorption band and a thickness chosen to reduce the crosstalk and / or increase the signal-to-noise ratio at the level photodetectors 17. A factor of 10 or 100 is thus gained on the extinction of the excitation light, without significantly degrading the collection of the fluorescence emitted by the chromophores 5.
• the chromophores 5 carried by the substrate 2 are excited by an evanescent wave.
• the substrate 2 comprises a guiding layer 25 similar to the layer 14 of FIG. 6, having a higher index than that of the surrounding layers and the upper face of which is very close to the chromophores 5 on the scale of the length d evanescent wave of the guided wave.
• the guide layer 25 is illuminated on its edge (after cleavage, or sawing and polishing), or by means of an array (for example, a thin layer 26 of photosensitive resin on the guide layer in a neutral area of the substrate 2, with a low thickness modulation, from 1 to 100 nm, making it possible to couple the light by illumination by means of a laser at the adequate coupling angle determined by means of the law of gratings.
• an array for example, a thin layer 26 of photosensitive resin on the guide layer in a neutral area of the substrate 2, with a low thickness modulation, from 1 to 100 nm, making it possible to couple the light by illumination by means of a laser at the adequate coupling angle determined by means of the law of gratings.
• An alternative embodiment relates to the detection of chromophores of different colors.
• the chromophores are illuminated by one or two excitation wavelengths ⁇ exd and possibly ⁇ exc2 and emit in response two wavelengths, ⁇ fluol, ⁇ fluo2 respectively. In that case, 31
• FIG. 10 Another variant embodiment shown diagrammatically in FIG. 10 allows the detection of chromophores of different colors at each point 5 on the substrate 2.
• the device repeats that of FIG. 9 and further comprises a second matrix 27 of CCD photodetectors 17 placed above above the chromophores, with the interposition of a lens 28 and a rejection filter 29 between the chromophores and the second matrix 27.
• the chromophores 5 are illuminated by two excitation wavelengths ⁇ exd and ⁇ exc2 and emit in response on two wavelengths ⁇ fluol and ⁇ fluo2.
• the absorbent layer 23 of the substrate 2 forms a spectral filter for rejection of the excitation wavelengths and of a wavelength ⁇ fluo2 emitted by the chromophores 5, letting through the other wavelength ⁇ fluol emitted by the chromophores 5.
• the filter 29 associated with the second matrix 27 stops the excitation wavelengths and the wavelength ⁇ fluol emitted by the chromophores 5, allowing the wavelength ⁇ fluo2 to pass. If necessary, the filter 29 can be associated with an absorbent layer 23 of the type already described.
• FIGS. 11 to 16 Various devices for collecting light are shown diagrammatically by way of example only in FIGS. 11 to 16.
• the collection device of FIG. 11 comprises a matrix 27 of photodetectors of the CCD or similar type receiving the luminescence emitted by the chromophores carried by the support 1 through a filter 29 for rejection of the excitation light and the emitted parasitic luminescence by the support 1.
• the excitation of the chromophores by a wavelength ⁇ exc takes place at a non-zero incidence compared to the normal to the support 1, by means of a coupler of the dark field type, commonly used in 32
• the objective 31 collects the luminescence emitted by the chromophores in the direction perpendicular to the support 1.
• the collection device of FIG. 13 corresponds to that mentioned above with reference to the device with resonant cavity of FIG. 4A: in this device, the second mirror 7 is formed on the underside of the entry lens 34 of a objective 35 of the collection system.
• the optical path between mirrors 3 and 7 is a multiple of ⁇ fl U0 / 2.
• the luminescence emitted by the chromophores 5 in an upper guiding layer 25 of the substrate 2 is recovered at one end by an objective shown diagrammatically at 36.
• the figure 15 shows the situation in which the points of the combined guide of the detector favor the selective detection of one of the chromophores, on which the focusing is the best, compared to the others, for which the emission is not in the plane and is not concentrated in the detector (or in the pixel of the detector array), which allows discrimination of the chromophores located at different distances from the edge of the guide.

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