Biochip device

Abstract

A biochip device comprising a substrate constituted by at least one plate of material forming a multimode planar waveguide and carrying chromophore elements suitable for emitting fluorescence in response to excitation by guided waves having an evanescent portion, the device being characterized in that it includes coupling means for coupling excitation light with the waveguide in the form of guided waves, the coupling means being substantially non-directional.

Claims

1. A biochip device for excitation by an excitation light having a wavelength, the biochip device comprising: a multimode waveguide that is formed as a plate, with a thickness in a range supporting multiple light-propagating modes at the excitation wavelength, the multimode waveguide having a top face, and a side wall connecting the top face and the bottom face, the top face including a first portion and a second portion, the second portion carrying chromophore elements emitting fluorescence in response to the excitation light by guided waves having an evanescent portion, the chromophore elements being used to detect molecular attachments; a light-diffusing element configured to diffuse the excitation light incoming from a direction out of the plane of the waveguide into the waveguide in a plurality of modes propagating inside the waveguide in the plane of the waveguide as guided waves and having a disordered spatial distribution of refractive index, the light-diffusing element diffusing the excitation light into the waveguide at one of: (i) the first portion of the and (ii) a portion of the bottom face opposite the first portion of the top face through the waveguide to the second portion carrying the chromophore elements, the light-diffusing element being a frosted portion formed as part of the one of: (i) the first portion of the top face, and (ii) the portion of the bottom face opposite the first portion of the top face at which the excitation light is diffused into the waveguide; and a layer disposed on the top face or the bottom face of the waveguide, the layer having a refractive index smaller than the refractive index of the waveguide material such that some of the light-propagating modes are extracted from the waveguide and refracted inside the layer, the layer being separated from the light-diffusing element.

2. The device according to claim 1, further comprising means for absorbing light, the layer interposed between the top face or the bottom face of the waveguide and the means for absorbing light, the layer and the means for absorbing light together eliminating the some of the light-propagating modes from the waveguide.

3. The device according to claim 2, wherein the layer is located between the light-diffusing structure and the second portion carrying the chromophore elements.

4. The device according to claim 3, wherein the means for absorbing is disposed on the index-matching layer.

5. The device according to claim 1, further comprising a means for deflecting light placed on the layer.

6. The device according to claim 5, wherein the means for deflecting comprises a prism.

7. The device according to claim 2, wherein said waveguide has a thickness e and a guided mode having the greatest effective index reflecting inside the waveguide has a reflection angle θ, and the layer and the means for absorbing extend over a distance that is longer than 2×e×tan θ.

8. The device according to claim 2, wherein the layer extends upstream from the second portion carrying the chromophore elements and in part within the second portion.

9. The device according to claim 1, further comprising a hybridizing chamber disposed on the top face of the waveguide and surrounding the second portion carrying the chromophore elements and a hybridizing fluid contained in the chamber, wherein the refractive index of the layer is greater than or equal to the greatest refractive index of the hybridizing chamber and the hybridizing fluid.

10. The device according to claim 9, wherein the value of the refractive index of the layer lies in the range n=1.30 to n=1.45.

11. The device according to claim 1, wherein the layer is configured to extract guided modes for which the effective index is less than or equal to a predetermined threshold value from the plurality of modes from the waveguide.

12. The device according to claim 1, further comprising a plurality of pads disposed at the second portion, the plurality of pads being configured to receive probe molecules deposited thereon, the chromophore elements being configured to hybridize with the probe molecules deposited on the pads.

13. The device according to claim 1, wherein the frosted portion has a grain size in the range of 0.1 μm-50 μm in the plane of the waveguide and perpendicularly thereto.

14. The device according to claim 1, wherein the value of the refractive index of the layer lies in the range n=1.30 to n=1.45.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Other advantages and characteristics of the invention appear on reading the following description made by way of non-limiting example and with reference to the accompanying drawings, in which:

(2) FIG. 1 is a diagrammatic section view of a prior biochip art device;

(3) FIGS. 2 and 3 are diagrammatic section views of a biochip device of the invention including filter means integrated in the waveguide;

(4) FIG. 4 is a diagrammatic section view of a biochip device of the invention in which the waveguide carries the filter means;

(5) FIGS. 5 and 6 and diagrammatic section views of two variants of the FIG. 4 device;

(6) FIGS. 7A, 7A′, 7B, and 7C are diagrams of the portion of the waveguide where guided waves are formed having an evanescent portion; and FIGS. 8 to 13 show various setups enabling guided waves to be generated in the waveguide with the device of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(7) Reference is made initially to FIG. 1, which shows a prior art biochip device 10 comprising a substrate 12 including a top layer 14 forming a waveguide. An excitation light 16 is directed to coupling means 18, e.g. such as a grating formed on the surface of the waveguide so as to cause a guided wave to propagate inside the waveguide 14. At a distance from the grating 18, the waveguide 14 caries a hybridizing chamber 20 containing a solution 22 including target molecules marked by chromophore elements and suitable for hybridizing with probe molecules deposited on pads 24 on the surface of the waveguide 14.

(8) Detector means are provided, e.g. on the face of the substrate 12 that is opposite from its face carrying the hybridizing chamber 20, and they comprise a camera 26 such as a charge coupled device (CCD) or a complementary metal oxide on silicon (CMOS) camera and a filter 28 for rejecting the light for exciting the chromophore.

(9) In such a device, the evanescent portion of the guided wave excites the chromophores carried by the waveguide 14. Nevertheless, and as mentioned above, that type of device can be difficult to implement because of the difficulty of achieving appropriate optical coupling between the incident light and the waveguide, since the coupling requires great precision on the collimation angle of incidence of the excitation light if coupling is performed by a conventional resonant grating in guided optics, or else it requires submicron mechanical precision if the coupling is directly via the edge face. Furthermore, and above all, when the waveguide is suitable for having a plurality of guided modes propagate therein, i.e. a waveguide typically having a thickness greater than the wavelength of the guided waves for index steps of about 1, then guided waves having an effective index of less than the index of the elements surrounding the chromophores, such as the material of the hybridizing chamber 20 or the hybridizing solution 22, are extracted from the waveguide 14 and excite target molecules that are present in the hybridizing solution 22 but that are not attached to probe molecules. This results in a decrease in the signal-to-noise ratio when measurement of the luminescence emitted by the chromophores is performed in real time.

(10) The device of the invention proposes using coupling means 19 that are substantially non-directional and of dimensions that are not very critical (e.g. 0.1 millimeter (mm) to 10 mm) providing optical coupling between the light source and the waveguide so as to generate waves that are guided in a plurality of directions inside the waveguide, e.g. from an excitation light beam that is not collimated. By way of example, such coupling means may be of diffusing structure and they are described in greater detail below in the description.

(11) Advantageously, the coupling means are combined with mode filter means so as to extract from the multimode planar waveguide 14 those guided modes for which the effective index is less than or equal to a predetermined threshold value.

(12) These filter means may either be formed by the waveguide itself (FIGS. 2 and 3) or else they may be carried thereby (FIGS. 4 to 6). Reference is now made to FIG. 2, which shows a stratified structure comprising three superposed layers, with the top layer 30 serving as a planar waveguide without absorption and having an index n.sub.1. The second layer 32 is interposed between the top layer 30 and an absorption bottom layer 34. The index-matching intermediate layer 32 has an index n.sub.2 that is substantially equal to the predetermined threshold index value.

(13) In this embodiment, the top layer 30 has the coupling means 19 at one end and has an optofluidic portion 36 at another end. The intermediate and absorption layers 32 and 34 extend over the entire length of the guiding top layer 30 so as to perform filtering over the entire length L between the substantially non directional coupling means 19 and the optofluidic portion 36. The index n.sub.3 of the absorbent layer 34 is selected to be greater than n.sub.2, while the thickness of the layer is appropriate in application of the rules of guided optics and n.sub.1 is sufficiently large given the contrast firstly with n.sub.2 and secondly with the index of the hybridizing solution 22 to be capable of accepting at least one mode of index higher than the desired threshold.

(14) The thickness z.sub.2 of the intermediate layer of index n.sub.2 is sufficient to ensure that this mode does not escape over the distance L: the exponential decay factor in z.sub.2,
f=exp[2xn:x(z/A)xn;ff−n.sup.2>1
must be at least three times greater than the ratio L/z.sub.1 of L to the thickness z.sub.1 of the layer 30 of the index n.sub.1 for the intermediate layer to perform its role over the length L. The modes of index neff<n.sub.2 are obliged to propagate in the absorbent third layer of thickness z.sub.3 and to travel a distance of about L therein. It then suffices to provide this third layer 34 with an absorption coefficient 0:3 that is greater than 2/L in order to attenuate the undesirable modes of index neff<n.sub.2

(15) By way of example, these three stratified layers may be made of polymer. A typical index sequence may be n.sub.1=1.55, n.sub.2=1.42, and n.sub.3=1.55. The thicknesses may be z.sub.1=5 μm to 50 μm for the layer 30, z.sub.2>3 μm for the layer 32, and z.sub.3 greater than or equal to z.sub.1 for the layer 34, e.g. being about 500 μm for a length L of 1 centimeter (cm). The absorption may be obtained by means of an organic or inorganic dye dispersed in the third layer 34. In practice, in order to absorb the light at a wavelength of 532 nanometers (nm) that is used for exciting 6hromophores such as Cyanine 3, it is possible to perform doping with absorbents that are stable, such as Fe.sup.3+ salts of iron.

(16) FIG. 3 shows a second embodiment of coupling means formed by the waveguide. In this embodiment, the top and bottom faces 38 and 40 of the waveguide 42 are plane and they diverge from each other going from the coupling means 19 towards the optofluidic portion 36, e.g. in the form of a wedge.

(17) The top and bottom faces 38 and 40 of the waveguide 42 form an angle o: at the edge 44 where they intersect, situated outside which edge in the present example is the device. Such a waveguide 42 is referred to as a “wedge” waveguide. The light rays coming from the coupling means 19 that are reflected on the bottom face 40 become increasingly inclined relative to the top face 38, by an angle 2a for each pair of reflections. This filtering is “dynamic” filtering in which the energy of the modes is shifted to ever increasing effective indices as the light rays propagate. In order to understand the operation of such filtering, it is possible to make use of the laws of geometrical optics. It is thus possible to predict that the successive images 46 of the coupling means 19 are turned through successive angles of 2a on each pair of reflections and that they move away towards the edge 44 by following a semicircle centered on the edge 44. This implies a limit on the angle of incidence that can be achieved at the optofluidic portion, as a function of the two pertinent distances in the geometrical optics problem, namely L′ corresponding to the distance of the edge 44 from the coupling means 19, and the distance L from the coupling means 19 to the optofluidic portion 36.

(18) Constructing a right-angled triangle ABC with A at the edge 44 and of radius L′ shows that the maximum angle of incidence e of a light ray on the optofluidic portion is given by:
sin(90°−8)=cos 8=L′/(L+L′}
since the right-angled triangle ABC has the angle 90°−0 at the vertex C. Determining this angle thus requires a minimum value for the effective index that is given by neff=nxsin(8). In practice, it is desirable to aim for angles 8 of about 70° (neff=1.41 for a waveguide material having an index of about n=1.5). This imposes the following ratio:
L/L′=1−1/sin(90°−arcsin(neff/n)}=2.
It should be observed that this result is independent of the angle a of the wedge. This angle may be selected in practice to lie in the range 2° to 10°. The overall extent of the coupling means 19, e.g. diffusers, needs to be taken into account, seeking merely to ensure that the desired condition applies for the most unfavorable of the light rays coming from said means, i.e. those from the source 19 that are the closest to the hybridizing chamber 20.

(19) In other variants (not shown), the top and bottom faces may diverge from each other, but without being plane, as would be the case for example with curved faces that are concave. The top face may also be plane while the bottom face may be curved, for example it may present a concave curve.

(20) In the embodiments shown in FIGS. 4 to 6, the filter means are carried by the waveguide and they are interposed between the coupling means 19 and the zone 36 of the waveguide 14 carrying the hybridizing chamber 20. Advantageously, these means comprise a layer 48 in contact with the top surface 50 that is a top face of the waveguide 52. The waveguide 52 has a bottom face 94 and a side wall 92 connecting the top surface 50 and the bottom face 94, illustrated in FIG. 5. The layer 48 is selected so that its refractive index is equal to the predetermined threshold value (FIG. 4). Means 53 for absorbing or deflecting the guided modes that are extracted from the waveguide 52 are advantageously placed on the layer 48.

(21) In a practical embodiment of the invention, the absorption means may, for example, comprise a filter 54 having a wide absorption spectrum that performs volume filtering on the guided wave. This type of filter is very suitable since it reflects only very little of the guided light waves at its interface with the layer 48 (FIG. 5). The deflection means may, for example, comprise a prism 56 of index selected to deflect the extracted guided light waves as shown at 58 in FIG. 6.

(22) Because of the presence of an layer 48 between the coupling means and the zone 36 of the waveguide carrying the hybridizing chamber 20, the guided modes that are of effective index that is less than the index of the layer 48 are extracted from the waveguide and are refracted inside said layer.

(23) The presence of absorption means 54 or deflection means 56 on the layer 48 prevents any reflection of the guided modes that have been extracted via the top interface of the layer 48, which would lead to the extracted guided modes being re-introduced into the inside of the planar waveguide 52.

(24) In guided order modes to guarantee optimum of effective index filtering of less than the predetermined threshold value, the layer 48 and the absorption means or the deflection means must extend, between the zone 19 where the guided waves are generated and the zone 36 carrying the hybridizing chamber 20, over a distance that is greater than or equal to 2×e×tan 8, where e is the thickness of the waveguide and 8 is the reflection angle inside the waveguide relative to the normal to the waveguide 52 for the guided mode for filtering that has the greatest effective index.

(25) In this way, it is possible to guarantee that all of the guided modes of effective index less than the predetermined threshold value are subjected to at least one reflection at the interface between the waveguide 52 and the layer 48.

(26) In a variant, the filter means may also extend at least in part under the pads 24 in the hybridizing chamber so as to filter out the photons that the edges of the chamber might diffuse towards uncontrolled index odes of the waveguide that could then escape and excite the solution.

(27) In an embodiment of the invention, the hybridizing chamber is made of polydimethylsiloxane (PDMS) having a refractive index n=1.42, and the hybridizing solution 22 is water-based having an index of about n=1.33. Under such circumstances, the index-matching medium 36 is selected to have an index that is not less than the highest index in the environment of the chromophores, i.e. n=1.42.

(28) In practice, the threshold index value is selected to lie in the range n=1.30 to n=1.45, which corresponds to the index values commonly encountered for the materials of the hybridizing chamber 20 and also for the hybridizing solution 22.

(29) There follows a more detailed description of the means used for generating a plurality of guided inside the waves planar waveguide and having an portion for evanescent exciting the chromophore elements fixed to the pads 24.

(30) In a first embodiment, the device has a diffusing structure formed in the waveguide or on the waveguide and that is to be illuminated by the excitation light.

(31) This diffusing structure (FIGS. 7 and 8) presents a disordered spatial distribution of index so as to diffuse the excitation light into the waveguide in a plurality of directions. Such a structure makes it easier to convert the excitation light into guided light and to do so with efficiency that depends little on the excitation conditions, and in particular on the angle of incidence. Thus, with such a diffusing structure, the angle of tolerance is quite large, being about 10 degrees, which does not require a high precision mechanical and optical setup, as compared with tolerance of about 0.1 degrees when using a grating 18.

(32) This diffusing structure may be on one or other of the faces of the waveguide 14 that is transparent to the excitation light 16.

(33) In a first embodiment of the diffusing structure 60, it is constituted by a layer deposited on the waveguide and presenting an internal structure that is disordered.

(34) The layer may consist in a deposit of metallic or colloidal particles 60 (FIG. 7A), or indeed a deposit of “Teflon” (polytetrafluoroethylene).

(35) It is also possible to deposit a layer 62 made up of a matrix containing diffusing particles 64 (FIG. 7B). The matrix may consist in a resin of the same type as that used for paints or varnishes, such as for example acrylic or glycerophthalic resins or indeed resins fluoropolymers.

(36) In order to guarantee good conversion by diffusion of the excitation light 16 into guided waves with a thin diffusion layer, i.e. a diffusion layer with a thickness of about 15% to 60%, it is desirable for the refractive index of the matrix to be less that the index of the diffusing particles 64 by at least ˜n=0.5.

(37) A diffusing structure 66 may also be obtained by making microcavities, e.g. spheroidal microcavities, inside the waveguide 14 or by locally modifying the material of the waveguide 14 by changing its degree of oxidation or by changing its phase, from amorphous to crystalline or from crystalline to amorphous, e.g. by means of laser pulses having a duration lying in the range 0.1 picoseconds (μs) to 1 microsecond (μs) with typical energy lying in the range 1 nanojoule (nJ} to 100 to microjoules (μJ). This type of structure thus presents index discontinuities suitable for diffusing the excitation light in a plurality of directions. There also exist methods of nucleating pores in a sol-gel phase, and these methods are often used for making layers of low dielectric constant in microelectronics.

(38) In another embodiment of a diffusing structure, it consists in a layer 68 of fluorescent and diffusing material such as the phosphors of white light-emitting diodes (LEDs) that respond to light excitation by generating guided waves having an evanescent portion and

(39) propagating in a plurality of directions.

(40) The fluorescent material may consist in an ordered or disordered layer of fluorophores, in particular such as those based on quantum dots, organic fluorophores, or fluorophores based on rare earth.

(41) The layer of fluorescent material may also consist in a layer comprising a binder such as an organic or inorganic powder with grain size lying in the range 0.1 μm to 50 μm or a polymer matrix belonging to the families used for makeup and for paint and varnishes, such as acrylic resins, glycerophthalic resins, etc., and fluorophore elements such as those described in the paragraph above.

(42) The layer of fluorescent material may also include diffusing elements of the high-index particle type (e.g. oxides of titanium or carbonates of calcium or barium sulfate) and fluorophores such as those mentioned above with the diffusing structure made in these ways, the process of creating guided light waves possessing an evanescent portion may be thought of as emitting a set of optical dipoles at the surface of the waveguide or inside it. With a diffusing structure as shown in FIGS. 7A, 7B, and 7C, these dipoles oscillate at the same frequency as the excitation light 16, and with a structure that diffuses by fluorescence, the dipoles oscillate after frequency conversion at the frequency at which the fluorophores fluoresce. Under such circumstances, the

(43) fluorescence frequency is selected so as to be suitable for exciting the chromophores carried by the pads 24.

(44) The diffusing structure may be excited by the light 16 either directly or indirectly, as shown in FIGS. 8 to 13.

(45) FIG. 8 shows excitation of the diffusing structure by transmission of the excitation light 16 through the diffusing structure 68. This is possible only when the diffusing structure 68 presents little opaqueness in order to avoid the excitation light 16 being absorbed by the diffusing structure 68.

(46) FIG. 9 shows the diffusing structure being excited by the excitation light 16 being transited through the thickness of the waveguide.

(47) In another configuration, shown in FIG. 10, a prism 72 may be optically coupled to the waveguide 52 via an index-matching layer 74. It is also possible for the prism 72 to be integrated with the waveguide 52 by molding. The excitation light 16 is thus deflected by the prism into the inside of the waveguide and it excites the diffusing structure 70

(48) In a similar embodiment, shown in FIG. 11, a chamfer 76 is made at the end of the waveguide that carries the diffusing structure 70. For the two Embodiments shown in FIGS. 10 and 11, the excitation light 16 is oriented parallel to the plane of the waveguide towards the sloping surface of the prism 72 or the chamfer 76. The light 16 is then deflected towards the diffusing structure 70 in order to generate guided light waves having an evanescent portion.

(49) In the embodiment of FIG. 12, the diffusing structure 70 is carried by the chamfered surface 62 of 15 the waveguide and the excitation light may be at any orientation relative to the plane of the waveguide carrying the pads 24. The excitation light 78 may thus be oriented perpendicularly to the plate or it may be inclined relative to the vertical to the plate (as shown 20 at 80). It passes through a portion of the waveguide in order to illuminate the diffusing structure. The excitation light 82 may also excite the waveguide by diffusion/transmission through the diffuser 70.

(50) In a last embodiment, shown in FIG. 13, the 25 excitation light 16 is transmitted to the diffusing structure 70 through the polished or unpolished edge face of the waveguide 14.