FR2843634A1 - Support device for chromophore elements with a flat mirror coated with a multi-thickness banded layer of material transparent to a wavelength to be detected, making it possible to vary fluorescence intensity - Google Patents

Abstract

Support device for chromophore elements comprises a flat mirror coated with a material layer (12) transparent to the wavelengths to be detected. The layer incorporates a number of bands (14) provided with chromophore elements and subdivided into a plurality of areas (20) having different thickness, making it possible to vary the fluorescence intensity emitted by the chromophore elements by means of destructive or constructive interference respectively.

Description

DEVICE FOR SUPPORTING CHROMOPHORE ELEMENTS

  The invention relates to a device for supporting chromophoric elements. In devices of this type, which are commonly called "biochips", the chromophoric elements are chemical or biological molecules which are generally fixed on a substrate after a hybridization or affinity reaction in a liquid, or else 10 coloring elements added or grafted to these molecules or certain types of semiconductor nanostructures such as wires or quantum dots, each chromophore element being capable of emitting, in response to a light excitation, a fluorescence over a length

  wave which depends on the nature of this chromophore element.

  Support devices for chromophoric elements are described in particular in application WO-A-02/16912 in the names of Claude WEISBUCH and Henri BENISTY and include means making it possible to reinforce the light intensity of excitation of the chromophoric elements and to increase the light intensity emitted by these elements, by interference effects produced by stacks of layers of judiciously chosen materials and by effects of light extraction guided by lateral structures having dimensions of the same order of magnitude as the wavelength of guided light, such as

than photonic crystals.

  In these biochips, the chromophoric elements fixed on the

  substrate are divided into areas (called "spots" in English terminology) separated from each other and arranged regularly, in particular in rows and columns. These ranges have dimensions, for example of a few tens or hundreds of μm, which are clearly greater than the wavelengths considered.

  They are delimited for example by a deposition technique of the "spotting" type which comprises a physico-chemical treatment of the surface of the overall substrate, the "spot" then being determined by the area wetted by the fluid deposition, or by a treatment selective spatial, for example by selective silanization, the "spot" being determined by this treatment. The ranges generally include chromophoric elements of different types, which emit on different wavelengths. The signals emitted are picked up by suitable photodetectors, in particular by arrays or arrays of CCD photodetectors which also pick up an overall background noise formed by an excitation light incompletely.

  filtered, by fluorescence coming from chromophoric elements of neighboring ranges according to grazing or guided rays, etc., this background noise being difficult to eliminate with precision for each wavelength considered and being able to represent a significant part of the intensity signals received.

  The present invention aims in particular to provide a simple and effective solution to the problem of determining and

the elimination of this background noise.

  It relates to a device for supporting chromophoric elements, which allows, at any point on its surface, a determination

  reliable, precise and automatic of the above-mentioned background noise.

  To this end, it proposes a device for supporting chromophoric elements capable of emitting fluorescence in response to a light excitation, the wavelength emitted by each chromophore element depending on the nature of this element, the device comprising a plane mirror covered with a layer of transparent material with wavelengths emitted and on which the chromophoric elements are distributed in ranges separated from each other and having lateral dimensions greater than the wavelengths of the fluorescence emitted, characterized in that said layer of transparent material has a thickness of the same order of magnitude as the wavelengths of the fluorescence emitted and comprises, for each range of chromophoric elements, at least two zones having different thicknesses, the thickness of a first of these zones being determined to generate a minimum intensity by a destructive interference phenomenon of the fluorescence emitted on a first wavelength by elements

chromophores of said area.

  In this device, if the thickness of the first zone is correctly adjusted, the fluorescence emitted on the surface of this zone is at a minimum value which is zero or substantially zero. Consequently, the light signal picked up on the surface of this zone represents the noise

  overall at the wavelength considered.

  The cancellation of the fluorescence in the aforementioned area is due to: - either a round-trip optical path to the mirror, which is equal to an odd multiple of the half-wavelength of the fluorescence emitted, taking account of the phase shift at reflection on the mirror and / or of the penetration of the wave in the mirror, - either to an optical path on a round trip to the mirror, which is equal to an odd multiple of half excitation wavelength, taking into account the phase shift at reflection on the mirror and / or the penetration of the wave in the mirror, - either a combination of the two cases mentioned above, - either, when the two above conditions are close and correspond to thicknesses which typically differ by less than 30 nm, at any intermediate condition between the two above conditions. According to another characteristic of the invention, the layer of transparent material comprises, for each range, at least one other zone having a thickness different from that of the first zone, such as the difference in the optical paths in these two zones for a wavelength considered is equal to an odd multiple of a quarter of this wavelength. The wavelength considered for determining the difference in thickness between the two zones can be, as indicated above, either the wavelength of the fluorescence emitted, the excitation wavelength, or at both the wavelength of the fluorescence emitted and the excitation wavelength (to obtain on said other zone a maximum of excitation intensity and a maximum of intensity of fluorescence emitted), that is to say another condition intermediate when the 10 thicknesses of the two zones calculated for the two wavelengths are

very close to each other.

  The fluorescence emitted by said other zone then having a maximum intensity or close to a maximum value, the light signal picked up on the surface of this other zone corresponds to the sum of the maximum intensity of the fluorescence emitted at the first length d on the

  surface of this other area and overall background noise. By subtracting from this signal the background noise obtained by picking up the signal on the surface of the first zone, an intensity value is obtained corresponding approximately to the maximum intensity of the fluorescence emitted, on the first wavelength, at the surface of said other zone.

  According to other characteristics of the invention, which aim to generalize the aforementioned characteristics: - the layer of transparent material comprises, for each range of chromophoric elements, a plurality of the aforementioned zones 25 of different thicknesses, making it possible to sample between a minimum value and a maximum value the intensity of the fluorescence emitted on said first wavelength by chromophoric elements of said range, - the layer of transparent material comprises, for each range of chromophoric elements, a plurality of zones above of different thicknesses, making it possible to vary the intensity of the fluorescence emitted on different wavelengths by elements

  chromophores of different types from said range.

  Thus, with a series of zones of different thicknesses known in each range of chromophoric elements, it is possible to have a linear combination of the significant signals emitted by the different

  chromophoric elements present and background noise.

  According to yet other characteristics of the invention, said zones are arranged in lines or in strips parallel to the surface of

  said layer of transparent material.

  As a variant, these zones may have a matrix arrangement in rows and columns on the surface of the layer of transparent material.

  This then comprises, over its entire surface, a plurality of zones of different thicknesses with preferably regular distribution which form a structure of the paving type or the like.

  As a variant, these zones of different height can be formed on the aforementioned reflecting layer or on an intermediate layer of different index interposed between the transparent layer and the

reflective layer.

  The means for capturing the fluorescence emitted by the chromophoric elements may be above the support device for these chromophoric elements, or even below, as already

  described in the aforementioned international application WO-A-02/16912.

  In this case, these capture means comprise a matrix of photodetectors of the CCD or CMOS type which is fixed under the device, the latter comprising a first layer of material highly reflecting at the excitation wavelength and a second layer of material selectively absorbing the excitation radiation, the first layer being deposited on the second, so that the emitted fluorescence easily reaches the detectors, but not the excitation radiation. The reflection of the excitation radiation on the first layer can then possibly provide the aforementioned effect of strengthening the

fluorescence emitted.

  A weakly resonant cavity is formed between the diopter on the upper surface of the layer carrying the chromophoric elements and said first reflective layer (at the excitation wavelength) when the latter also has a non-negligible reflectivity at wavelength of the fluorescence emitted. This effect can be used to increase

  the intensity of the fluorescence channeled towards the photodetectors.

  Preferably, the upper layer of this device is made of a material with a high refractive index. This promotes the formation of said weakly resonant cavity and therefore good detection of fluorescence.

  by the photodetectors fixed under the device.

  It is moreover known that the radiative emission towards the medium on which a chromophore element is placed is favored all the more as the index of this medium is high.

  The invention will be better understood and other characteristics, details and advantages thereof will appear more clearly on reading the description which follows, given by way of example with reference to the drawings

  appended in which: - Figure 1 is a schematic representation from above of part of a device according to the invention; - Figure 2 is a partial view on a larger scale of Figure 1; - Figure 3 is a view corresponding to Figure 2, for an alternative embodiment of the invention; - Figure 4 is a partial schematic perspective view of yet another alternative embodiment; - Figure 5 is a partial schematic sectional view of a device according to the invention; - Figure 6 is an enlarged view of part of the section shown in Figure 5; - Figure 7 is a graph schematically representing the intensities of the fluorescence emitted on the surface of three different zones of the device of Figure 6; - Figures 8 to 13 are schematic sectional views of other alternative embodiments of the device according to the invention.

  The device shown schematically in FIG. 1 comprises a substrate 10 of generally rectangular shape, the upper face 12 of which comprises a plurality of areas 14 occupied by chromophoric elements, these areas 14 forming a set o they are 10 distributed regularly in lines and in columns for example.

  Typically, the dimensions of the pads 14 (their diameter d) are of the order of 30 to 400 μm, and the spacing D between adjacent pads is of the order of 40 to 500 μm. As indicated above, the dimensions of the areas are determined by fluid deposition or by selective spatial treatment.

  The upper part of the substrate 10 comprises a layer 12 of a material which is transparent to the wavelengths of the fluorescence emitted by the chromophoric elements of the pads 14 in response to a light excitation, this material preferably having a refractive index. relatively high and being for example formed of TiO2 having a refractive index of between 2.2 and 2.5 depending on the shape

crystalline used.

  This layer 12 of material has a thickness which is of the same order of magnitude as the wavelengths of the fluorescence emitted by the chromophoric elements and covers a plane mirror which can be reflective for the excitation wavelength, this mirror plan being

  especially reflecting for the wavelengths of the fluorescence emitted.

  The free surface or upper surface of the layer 12 is structured for example as shown diagrammatically in FIGS. 2, 30 3 or 4.

  In FIG. 2, this layer 12 comprises a plurality of parallel bands 16 of different thicknesses, these bands having a width in the plane of the layer 12 which is greater than the wavelengths of the fluorescence emitted by the chromophoric elements. The thicknesses of the 5 different bands 16 are determined so that one of these thicknesses produces, in each range 14, a destructive interference effect on the surface of the layer 12 for an excitation wavelength and / or for a given wavelength of the fluorescence emitted by the chromophoric elements. The other bands 16 have different thicknesses, 10 of which corresponds to a constructive interference effect on the upper surface of the layer 12, for the excitation wavelength and / or for the wavelength of the fluorescence emitted. The bands 16 of different thicknesses are formed alternately in the layer 12, their thicknesses being determined to produce the aforementioned effects of destructive interference and constructive interference for one or preferably for several wavelengths emitted by the different chromophoric elements present in ranges 14 and / or for wavelengths

corresponding excitement.

  It is therefore possible, considering a given wavelength 20 emitted by chromophoric elements of a range 14, to determine two thicknesses corresponding to the two aforementioned interference effects and one or more intermediate thicknesses, which makes it possible to sample the intensity light emitted at this wavelength between a value

minimum and maximum value.

  It is also possible to determine other thicknesses of bands 16 which correspond to destructive and constructive interference effects for another or for several other wavelengths emitted by the chromophoric elements and / or for the wavelengths

corresponding excitement.

  It is also possible, as shown diagrammatically in FIG. 3, to form in layer 12 strips 16, 18 of different thicknesses, which extend in two perpendicular directions. The strips 16 parallel to each other and of different thicknesses are transverse strips, and are cut at right angles by longitudinal strips 18, which are parallel to each other and have different thicknesses. A structure such as that shown schematically in perspective in FIG. 4 is then obtained at each area 14, where each area 14 comprises a plurality of adjacent plates 20 of square or rectangular shape which have different heights. The dimensions of these plates 20 at the upper surface of

  the layer 12 can be identical from one tray to another or different.

  As seen in FIGS. 5 and 6, the layer 12 of transparent material carrying the chromophoric elements C is formed on a plane mirror 22 which is highly reflective at least for the fluorescence 15 emitted by the chromophoric elements. The plane mirror is formed by one or

  several layers of a reflective metal or a dielectric material such as for example a semiconductor material, an oxide or a glass, a nitride, an organic polymer or a compound obtained by sol-gel route from organometallic compounds . In a particular embodiment, the plane mirror 22 is made of silicon.

  In an alternative embodiment, the plane mirror 22 comprises at least one metal layer deposited on silicon.

  In yet another alternative embodiment, this plane mirror comprises at least two layers of oxides such as, for example, SiO2 and TiO2.

  In practice, the determination of the thicknesses of the bands 16 leading to constructive and destructive interference respectively must take into account the penetration length of the excitation or fluorescence at the wavelength considered in the mirror 22, as well as 30 the reflectivity of this mirror and the index of the transparent layer 12.

  The signals which can be picked up above the zones of different thicknesses of a range 14 have been shown diagrammatically in FIG. 7, the intensity I being represented on the ordinate and a dimension in the plane in the

  layer 12 being shown on the abscissa.

  The curve of FIG. 7 comprises a first part 24 of minimum intensity corresponding to a destructive interference effect, a part 26 of maximum intensity corresponding to a constructive interference effect, and a part 28 of medium intensity which corresponds for example the signal that would be obtained in the absence of a plane mirror 22, that is to say in the absence of any interference phenomenon. It is clear, for the person skilled in the art, that it is possible, for each wavelength considered of the fluorescence emitted by chromophoric elements, to take into account the minimum 24 and maximum 26 values of sensed intensity and make their difference in eliminating the overall background noise, which includes both local noise and background noise which does not originate from the area considered. Knowledge of the structure of the upper layer 12, that is to say of the locations of the bands 16 which correspond to destructive interference effects for one or more wavelengths and 20 of those of the bands 16 which correspond to a constructive interference effect for this or these wavelengths, makes it possible to directly take into account the signals of minimum intensity and the signals of maximum intensity and greatly simplifies the analysis. If a matrix of CCD photodetectors is used to capture the fluorescence emitted, it is no longer necessary to know which photodetector of the matrix is associated with

  this or that zone of a range 14, because the analysis of the image itself, associated with the knowledge of the structure of the surface of the layer 12, makes it possible to locate and identify the zones of minimum intensity and maximum.

  A variant of the invention consists in structuring the mirror 22 in zones by removing the reflective layer in certain zones.

  In this case, pairs of signals 26 and 28, or else 24 and 28, will be used as modulation of the signal to be detected, depending on the thickness chosen for the

  layer 12, with flat upper face.

  FIG. 8 shows an alternative embodiment of the invention, in which a set of photodetectors 30 of the CCD 5 type or the like is located under the substrate 10, on the face opposite to that which carries the chromophoric elements C. This embodiment has the advantage of not requiring an imaging objective on the

photodetectors 30.

  In this embodiment, the downward fluorescence emission 10 towards the photodetectors 30 is modulated by the same interference effects as those described above, but the amplitude of these effects is determined by a different physical mechanism and is generally weaker: it is multi-wave interference linked to the fact that the layer 12 forms a weakly resonant cavity, one of the mirrors of which is formed by the diopter with the air or the surrounding medium at the upper surface of the layer 12 and the other mirror of which is formed by a layer 32 which is semi-reflective at the fluorescence wavelength which it is desired to detect and which is very reflective for the wavelength of excitation of chromophoric elements. The strength of the resonance of such a cavity and the amplitude of the modulation of the collected fluorescent signals are all the greater the higher the product of the amplitude reflectivities of the two mirrors. It is therefore advantageous to use an upper layer 12 of high index, for example of TiO2 having an index of between 2.2 and 2.5 depending on its crystalline form. The amplitude reflectivity of the TiO2 / air diopter is approximately 0.4. In addition, it is known that the radiative emission towards the medium on which the chromophoric elements are found is all the more favored the higher the index of this medium. These two effects therefore combine and promote good signal detection by the

photodetectors 30.

  In the example shown, a photodetector 30 is located under each zone or strip 16 of height different from a range 14. It is therefore known directly which photodetector 30 is located opposite a particular zone or strip 16 corresponding to a maximum or minimum emission. Of course, it is possible to provide several photodetectors 30

  under each zone or strip 16 of different height.

  In the alternative embodiment of FIG. 9, the layer

  upper 12 of transparent material has a flat upper surface and the mirror 22 is formed on a structured upper face 34 of the substrate 10.

  It is this surface 34 which carries the zones 16, 18, 20 previously

  described in layer 12 of the previous embodiments.

  When, as shown in the drawing, the thickness of the reflective layer 22 is constant in each zone, the abrupt discontinuities between the zones can form parasitic channels for the excitation wavelengths and possibly for the fluorescence emitted. A more continuous variation (with a triangular or wavy profile for example) in the thickness of the substrate overcomes this drawback. In this case, the zones are defined by the fact that the

  desired interference condition is substantially achieved there.

  In another variant shown in FIG. 10, the substrate 10 of the device comprises a plane mirror 22 and a planar upper layer 20 12, with an intermediate layer 35 of different index, which is structured with zones of different thickness, corresponding to the zones 16 above and giving rise to variations in intensity of fluorescence by phase shift. In another variant shown in FIG. 11, the device 25 comprises one or more upper layers deposited on a transparent intermediate layer 37 to form a waveguide 36 for the excitation radiation, for example with propagation along the above-mentioned bands formed by the mirror 22, so as not to cause the excitation radiation to escape through the discontinuities between bands and to diffuse it undesirably towards the photodetectors. It is also possible, to avoid this drawback, to give a sufficiently large thickness to the layer of material which covers the structured mirror 22, which makes it possible to move the profile of the guided mode and its evanescent part away from the structured surface of the

mirror 22.

  In the variant embodiments of FIGS. 12 and 13, the device comprises an opaque layer 38, 40 respectively, for example metallic, which limits the useful surfaces (corresponding to the areas 14) the illumination by the excitation radiation or the transmission. of the

  fluorescence towards photodetectors.

  In FIG. 12, this opaque layer 38 covers the layer 12 10 of transparent material and has orifices corresponding to the pads 14.

  As a variant, this opaque layer 38 is inside the layer 12, between the upper face of the latter and the reflective layer 22 and its orifices are aligned with the areas 14 or with the aforementioned areas 16 15.

  In FIG. 13, the opaque layer 40 is located inside the substrate 10 and has orifices facing the fixed photodetectors 30

under the substrate.

Claims (18)

  1 / Device for supporting chromophoric elements capable of emitting fluorescence in response to a light excitation, the wavelength emitted by each chromophore element depending on the nature of this element, the device comprising a reflective layer (22) covered a layer (12) of material transparent to the wavelengths emitted by the chromophoric elements and on which these elements are distributed in areas (14) separated from each other and 10 having dimensions greater than the wavelengths emitted, characterized in that said layer (12) of transparent material has a thickness of the same order of magnitude as the wavelengths emitted and comprises, for each range (14) of chromophoric elements, at least two zones (16) having different thicknesses, the thickness of a first of these zones (16) being determined to generate, by a destructive interference effect, a minimum of fluorine intensity scence emitted on a first wavelength by chromophoric elements   located on said first zone (16).   2 / Device according to claim 1, characterized in that said layer (12) of transparent material comprises at least one other zone (16) having a thickness different from that of the first zone and such that the difference of the optical paths in these two zones for said first wavelength is equal to an odd multiple of a quarter of this wavelength and / or a corresponding excitation wavelength. 3 / Device according to claim 1 or 2, characterized in that said zones (16) have dimensions, in the plane of the layer (12) of transparent material, which are greater than the wavelengths   emitted by the chromophoric elements.   4 / Device according to one of claims 1 to 3, characterized in that the layer (12) of transparent material comprises, for each range (14) of chromophoric elements, a plurality of the aforementioned zones (16, 5 20) d '' different thicknesses, allowing to vary between two values   substantially different the intensity of the fluorescence emitted at said first wavelength by the chromophoric elements of said range (14).   5 / Device according to one of claims 1 to 3, characterized   in that the layer (12) of transparent material comprises, for each range (14) of chromophoric elements, a plurality of the aforementioned zones (16, 20) of different thicknesses, making it possible to vary between a minimum value and a value maximum the intensity of the fluorescence emitted at said first wavelength by the chromophoric elements of said range (14).   6 / Device according to one of the preceding claims, characterized in that the layer (12) of transparent material comprises, 20 for each range (14) of chromophoric elements, a plurality of zones   mentioned above (16, 20) of different thicknesses, making it possible to vary the intensity of the fluorescence emitted on different wavelengths by chromophoric elements of different types located on said range (14).   7 / Device according to one of the preceding claims, characterized in that the areas (14) are arranged in lines and / or   columns on said layer (12) of transparent material. o ^ A, 2843634   8 / Device according to one of claims 1 to 7, c aractérisé in that said areas are arranged in rows (16) and / or columns   (18) on the surface of said layer (12) of transparent material.   9 / Device according to one of the preceding claims,   characterized in that the zones (20) of different thicknesses form a   regular structure such as for example a paving.   / Device according to one of the preceding claims, 10 characterized in that the reflective layer (22) comprises at least   a layer of highly reflective material at the wavelengths emitted by the chromophoric elements, for example of reflective metal, or one or more layers of dielectric material such as for example a semiconductor material, an oxide, a glass, or a nitride , An organic polymer or an organometallic compound obtained by the sol-gel route.   11 / Device according to claim 10, characterized in that the reflective layer (22) is made of silicon or comprises at least one metallic layer deposited on silicon.   12 / Device according to claim 10, characterized in that the reflective layer (22) comprises at least two layers of oxides such as SiO2 and TiO2.   13 / Device according to one of the preceding claims, characterized in that it is associated with means for capturing the light emitted by the chromophoric elements and with means for eliminating background noise, these means elimination comprising means of 30 capture, for a given wavelength, of a corresponding signal wavering at   a minimum light intensity emitted by a destructive interference zone at this wavelength, and means for subtracting this   signal of the signal received on another zone at this wavelength.   14 / Device according to claim 13, characterized in that 5 the capture means are opposite the layer (12) of transparent material. / Device according to claim 13, characterized in that the capture means are on the side opposite to the layer (12) of transparent material and comprise a matrix of photodetectors (30) of CCD or CMOS type fixed under the device, this one ci comprising a layer (32) of material reflecting at the excitation wavelength and a   layer of absorbent material at this excitation wavelength.   16 / Device according to claim 15, characterized in that a weakly resonant cavity is formed between said reflective layer (32) and the diopter on the upper surface of the layer (12)   carrying the chromophoric elements.   17 / Device according to claim 15 or 16, characterized in that said reflective layer (32) comprises a plurality of layers deposited on a plurality of layers of selectively absorbent material the excitation wavelength.   18 / Device according to one of the preceding claims,   characterized in that the upper layer (12) of the substrate is in one   material with a high refractive index, such as, for example, TiO2.   19 / Device according to one of the preceding claims, characterized in that said zones (16) are formed by variations   in height from the surface of the transparent layer (12) carrying the chromophoric elements or of the aforementioned reflecting layer (22) or of an intermediate layer (35) of different index between the layer   transparent and reflective layer.   20 / Device according to claim 19, characterized in that it comprises one or more upper layers forming a waveguide   (36) for the radiation of excitation.   21 / Device according to one of claims 15 to 20, 10 characterized in that it comprises an opaque layer, for example   metallic, having openings corresponding to the aforementioned areas or to the photodetectors (30) fixed under the substrate (10) or to the aforementioned areas (16).