Contrast-amplifying carriers using a two-dimensional material

Abstract

A contrast-amplifying carrier for observing a sample, includes a transparent substrate bearing at least one absorbent coating suitable for behaving as an antireflection coating when it is illuminated at normal incidence at an illumination wavelength λ through the substrate and when the face of the coating opposite the substrate is in contact with a medium referred to as a transparent ambient medium, the refractive index n.sub.3 of which is lower than that of the refractive index n.sub.0 of the substrate. The absorbent coating comprises: an absorbent sublayer referred to as the contrast sublayer, deposited on the surface of the transparent substrate; and an absorbent layer referred to as the sensitive layer, distinct from the contrast sublayer and comprising between 1 and 5 sheets of a graphene-type material. Methods for producing and for using such a contrast-amplifying carrier are also provided.

Claims

1. A contrast-amplifying carrier for observing a sample, comprising a transparent substrate bearing at least one absorbent coating suitable for behaving as an antireflection coating suppressing reflection through destructive interference when it is illuminated at normal incidence at an illumination wavelength λ through said substrate and when the face of said coating opposite said substrate is in contact with a transparent ambient medium, a refractive index n.sub.3 of which is lower than that of a refractive index n.sub.0 of said substrate, wherein said absorbent coating comprises: a contrast sublayer that is absorbent at said illumination wavelength λ and which exhibits an antireflection behavior at said wavelength, deposited on a surface of said transparent substrate; and an absorbent layer, being a sensitive layer, distinct from said contrast sublayer and comprising between 1 and 5 sheets of at least one two-dimensional material, each sheet having a monoatomic or mono-molecular thickness, wherein, a thickness e.sub.1 of the contrast sublayer is expressed as a dimensionless parameter δ.sub.1 which equals 2πn.sub.0*e.sub.1/λ, for said illumination wavelength λ, and for said refractive index n.sub.0 of the substrate, said contrast sublayer having a complex index of refraction N.sub.1=n.sub.1−jk.sub.1, having a real part n.sub.1 and an imaginary part k.sub.1, the thickness e.sub.1 of the contrast sublayer is a first thickness wherein dimensionless parameter δ.sub.1 meets the following conditions: ${\delta }_1\cong \frac{\left(\frac{n_0}{n_3}-1\right)}{2v_1{\kappa }_1}\left[1-e^{-\frac{{\kappa }_1}{K}}\right]$ where: $v_1=\frac{n_1}{\sqrt{n_0{n}_3}};{\kappa }_1=\frac{k_1}{\sqrt{n_0{n}_3}};\mathrm{and}K={\left\{\left[\pi /\left(n_0/n_3-1\right)\right]\sqrt{n_0/n_3}\right\}}^{-1},$ or the thickness e.sub.1 of the contrast sublayer is less than said first thickness, where:
ν.sub.1.sup.2≠1+κ.sub.1.sup.2.

2. The contrast-amplifying carrier as claimed in claim 1, wherein said contrast sublayer is chosen from: a layer of impurities implanted into said substrate; a metal layer or made of gold; a semiconductor layer; a metal/semiconductor composite alloy; a magnetic absorbent layer; a layer of metal nanoparticles; a nonmetal conductive layer; a scattering layer; a polymer or photoresist layer containing pigments or dyes; an inorganic dielectric layer containing color centers; a composite hybrid layer comprising a continuous phase throughout which nanoparticles are dispersed; and a multilayer structure.

3. The contrast-amplifying carrier as claimed in claim 1, wherein said at least one two-dimensional material is a graphene-type material.

4. The contrast-amplifying carrier as claimed in claim 3, wherein said sensitive layer is chosen from: a single sheet of a graphene-type material; a stack of 2 to 5 sheets of at least one graphene-type material; a single sheet or a stack of 2 to 5 sheets of at least one surface-functionalized graphene-type of material; and a stack of 2 to 5 sheets of at least one graphene-type material, at least one of which is functionalized on both faces.

5. The contrast-amplifying carrier as claimed in claim 3, wherein the graphene-type material of said sensitive layer is chosen from: raw graphene; graphene oxide; reduced graphene oxide; and doped graphene.

6. The contrast-amplifying carrier as claimed in claim 1, wherein said absorbent coating is discontinuous, forming a plurality of pads on the surface of said substrate.

7. The contrast-amplifying carrier as claimed in claim 1, wherein said contrast sublayer is continuous and said sensitive layer is discontinuous, forming a plurality of pads on the surface of said substrate.

8. The contrast-amplifying carrier as claimed in claim 1, wherein said sublayer is conductive and is linked by a conductive line to a contact pad allowing the application of an electrical potential.

9. The contrast-amplifying carrier as claimed in claim 1, wherein said antireflection absorbent coating exhibits, at normal incidence, a transmittance higher than or equal to 80%, or higher than or equal to 85% or higher than or equal to 90% at said wavelength λ.

10. The contrast-amplifying carrier as claimed in claim 1, wherein said antireflection absorbent coating exhibits, at normal incidence, at said wavelength λ, a reflectivity lower than or equal to 1%, or lower than or equal to 0.5% or lower than or equal to 0.1% or lower than or equal to 0.05%.

11. The contrast-amplifying carrier as claimed in claim 1, wherein said sensitive layer is functionalized by the addition or the natural presence of ligands that are capable of binding at least one chemical or biological species.

12. The contrast-amplifying carrier as claimed in claim 11, forming the bottom of a Petri dish or of a fluidic cell.

13. The contrast-amplifying carrier as claimed in claim 1, wherein k.sub.1≥0.01.

14. The contrast-amplifying carrier as claimed in claim 1, wherein k.sub.1≥0.15.

15. A method for observing a sample including the following steps: A. placing said sample on the sensitive layer of a contrast-amplifying carrier as claimed in claim 1 and bringing it into contact with a transparent ambient medium, the refractive index of which is lower than that of the substrate of said contrast-amplifying carrier; B. illuminating said sample at normal incidence through said ambient medium, with an illumination cone including the normal incidence, with light radiation including at least one wavelength λ such that the absorbent coating of said carrier behaves as an antireflection coating; and C. observing the sample thus illuminated, also through said ambient medium.

16. The method as claimed in claim 15, wherein said step C. is carried out by detecting fluorescence radiation or Raman scattering.

17. A method for detecting or assaying at least one chemical or biological species or nanoparticles including the following steps: I. providing a contrast-amplifying carrier as claimed in claim 14, comprising a functionalized sensitive layer that is capable of binding at least one chemical or biological species or nanoparticles; II. bringing said functionalized surface or layer into contact with at least one solution containing a chemical or biological species that is capable of binding to said functionalized surface or layer, said solution being substantially transparent and exhibiting a refractive index that is lower than that of the substrate of said contrast-amplifying carrier; III. illuminating said sample at normal incidence through said ambient medium at an illumination wavelength λ such that the absorbent coating of said carrier behaves as an antireflection coating; and IV. observing said contrast-amplifying carrier thus illuminated, also through said substrate.

18. The method as claimed in claim 17, wherein said step IV. is carried out by detecting fluorescence radiation or Raman scattering.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Other features, details and advantages of the invention will become apparent upon reading the description provided with reference to the appended drawings, which are given by way of example and in which, respectively:

(2) FIG. 1 illustrates the operating principle of an absorbent antireflection layer according to the prior art;

(3) FIGS. 2A, 2B and 2C show experimental results demonstrating the feasibility of the invention and the real nature of its technical effect;

(4) FIG. 3 shows a contrast-amplifying carrier using an absorbent antireflection layer according to one embodiment of the invention; and

(5) FIGS. 4A to 4E show various embodiments of the invention.

DETAILED DESCRIPTION

(6) The figures are not to scale. In particular the thickness of certain layers (and in particular the sensitive layer made of a graphite-type material) has been greatly enlarged for the sake of clarity.

(7) Hereinafter:

(8) the term “two-dimensional material” will refer to a material taking the form of a single sheet of monatomic or monomolecular thickness, or of a stack of a small number (at most 10) of such sheets, potentially of different natures; a two-dimensional material may be crystalline or amorphous. Examples of two-dimensional materials are graphene and graphene-type materials (see below) as well as other materials of different chemical nature such as WS.sub.2, MoS.sub.2, WSe.sub.2, conductive two-dimensional polymers, etc. See in particular the article by Hua Zhang “Ultrathin Two-Dimensional Nanomaterials”, ACS Nano, Vol. 9, No. 10, 9451-9469 (2015) and, more specifically for conductive two-dimensional polymers, the article by J-J. Adjizian et al. “Dirac Cones in two-dimensional conjugated polymer networks” Nat. Commun. 5:5842 Dec. 18, 2014.

(9) “graphene”, unless further specified, will refer to a single sheet (i.e. a layer of monatomic thickness) of pure carbon, forming a hexagonal lattice;

(10) a “graphene-type material” (or “graphene-related material”, GRM) will refer to pure graphene, to a single sheet of a material derived from graphene such as graphene oxide, reduced graphene oxide, graphene doped by substituting certain carbon atoms with other atoms, graphane, graphyne, etc.; or to a stack of a small number (at most 10) of such sheets, potentially of different natures. Graphene oxide is particularly advantageous because it can be handled easily in solution, because it is “naturally” functionalized with a high density of epoxy, carboxyl and hydroxyl groups and because it lends itself particularly well to all sorts of surface functionalizations, for example by attaching amine groups to the carboxylic groups. See in this regard the article by Nan-Fu Chiu et al. “Graphene Oxide Based Surface Plasmon Resonance Biosensors”, Chapter 8 of “Advances in Graphene Science”, InTech, 2013;

(11) a material will be considered absorbent at a wavelength λ when the imaginary part of its refractive index at this wavelength is greater than or equal to 0.0001, preferably than 0.001, and more preferably still than 0.01. In the opposite case, it will be considered transparent. Using this definition, for example, all of the TCOs (transparent conducting oxides) are transparent.

(12) FIG. 1 presents an opportunity to recall the theory of absorbent antireflection layers, as already explained in the aforementioned international application WO2015/055809. It illustrates a beam of parallel light FL (which may be locally likened to a plane wave) that is monochromatic at a wavelength (in vacuum) λ, at normal incidence on a structure consisting of: a semi-infinite medium referred to as the incident medium MI, from which the light beam originates, which is transparent and characterized by a real refractive index n.sub.0; an absorbent layer CA of thickness e.sub.1, characterized by a complex refractive index N.sub.1=n.sub.1−jk.sub.1 (“j” being the imaginary unit); and a semi-infinite medium referred to as the emergent medium, or ambient medium, ME, located of the side of the layer opposite that from which the light originates, which is transparent and characterized by a real refractive index n.sub.3<n.sub.0. The incident medium may in particular be a substrate, for example made of glass, on which the layer CA is deposited. A sample (not shown) having a real refractive index n.sub.2, or having a complex refractive index N.sub.2=n.sub.2−jk.sub.2, may be deposited on the layer CA, on the emergent medium side. As explained above, to maximize the contrast with which the sample is observed, it is necessary to eliminate the reflectance of the incident medium MI/layer CA/emergent medium ME assembly in the absence of a sample.

(13) The complex reflection coefficient of a structure of the type illustrated in FIG. 1 (layer of thickness e.sub.1 comprised between two semi-infinite media) is given by the Airy formula:

(14) $\begin{array}{cc}{r}_{013}=\frac{r_{01}+r_{13}{e}^{-2j{\beta }_1}}{1+r_{01}{r}_{13}{e}^{-2j{\beta }_1}}& \left(2\right)\end{array}$

(15) where r.sub.ij is the Fresnel coefficient at the i-j interface (i,j=0, 1 or 3, “0” corresponding to the incident medium, “1” to the layer CA and “3” to the emergent medium) and β.sub.1=2πn.sub.1e.sub.1 cos θ.sub.1/λ is the phase factor associated with said layer, θ.sub.1 being the angle of refraction in the layer. In a first instance, a transparent layer having a real index n.sub.1 is considered, the generalization to the case of an absorbent layer being dealt with below. Still in a first instance, an incidence which may not be normal is considered.

(16) The Fresnel coefficients for the “p” (TM) and “s” (TE) polarizations are:

(17) $r_{\mathrm{ij}}^{\left(p\right)}=\frac{\left(n_j\cos {\theta }_i-n_i\cos {\theta }_j\right)}{\left(n_j\cos {\theta }_i+n_i\cos {\theta }_j\right)}\mathrm{and}{r}_{\mathrm{ij}}^{\left(s\right)}=\frac{\left(n_i\cos {\theta }_i-n_j\cos {\theta }_j\right)}{\left(n_i\cos {\theta }_i+n_j\cos {\theta }_j\right)}$

(18) The antireflection condition corresponds to r.sub.013=0 which, in the case of transparent media (real indices) gives two families of solutions:

(19) the layers referred to as “λ/2” layers, for which

(20) $e_1=\frac{m\lambda }{\left(2n_1\cos {\theta }_1\right)}$
where m is an integer, which exist only if n.sub.0=n.sub.3; and

(21) the layers referred to as “λ/4”, for which

(22) $n_1{e}_1=\left(2p+1\right)\frac{\lambda }{4}$
(p being an integer).

(23) In the case in which the medium 1 (layer CA) is absorbent, its refractive index N.sub.1=n.sub.1−jk.sub.1 is complex; the angle of refraction—which is then denoted by Θ.sub.1—and the phase coefficient—B.sub.1—are also complex. In this case, r.sub.013=0 dictates: r.sub.01,s.Math.r.sub.13,p=r.sub.01,p.Math.r.sub.13,s; this equality can only hold if one of the three following conditions: Θ.sub.1=0 (normal incidence), N.sub.1.sup.2=n.sub.0.sup.2 (no layer) or n.sub.0.sup.2=n.sub.3.sup.2 (identical incident and emergent media) is met. Consequently, in the case of any extreme media, the antireflection condition can be met only at normal incidence. Knowing that r.sub.011,p=−r.sub.01,s and r.sub.13,p=−r.sub.13,s, equation (2) becomes:

(24) $\begin{array}{cc}{N}_1^2-j\frac{\left(n_3-n_0\right)}{\tan B_1}{N}_1-n_0{n}_3=0& \left(3\right)\end{array}$

(25) Equation (3) is transcendental and it has no closed-form solution. However, solutions corresponding to the extreme cases may be found: namely that of a strongly absorbent layer and that of a weakly absorbent layer.

(26) In the strongly absorbent case, it can be assumed that e.sub.1<<λ since light would not propagate through a very absorbent and thick layer; consequently, |B.sub.1|<<1 and it is then possible to write, to the second order in B.sub.1: tan B.sub.1≈B.sub.1=√{square root over (n.sub.3/n.sub.0)}(N.sub.1/√{square root over (n.sub.0n.sub.3)})δ.sub.1, where δ.sub.1=(2πn.sub.0/λ)e.sub.1. It is useful to separate the real and imaginary parts of the equation, and to use the “reduced” variables ν.sub.1=n.sub.1/√{square root over (n.sub.0n.sub.3)} and κ.sub.1=k.sub.1/√{square root over (τn.sub.0n.sub.3)}. Equation (3) can then be written in the form of the following system:

(27) $\begin{array}{cc}{v}_1^2-1+{\kappa }_1^2& \left(4a\right)\end{array}$$\begin{array}{cc}{\delta }_1=\frac{\left(\frac{n_0}{n_3}-1\right)}{2v_1{\kappa }_1}& \left(4b\right)\end{array}$

(28) Given that δ.sub.1 must be real and positive, there is the condition n.sub.0>n.sub.3 (“reversed geometry”). By taking n.sub.0=1.52 and n.sub.3=1.34—this corresponding to the glass/water case customarily used in biophotonics—a thickness e.sub.1=(λ/2π)(n.sub.0−n.sub.3)/2n.sub.1k.sub.1 of the order of a nanometer is found, thus confirming the initial assumption. It is interesting—and unexpected—that equation (4a) should tend toward the conventional index condition as κ.sub.1—and therefore k.sub.1—tends toward zero. A comparison with numerical results makes it possible to verify that equation (4a), although derived under the assumption of a strongly absorbent layer, is approximately valid for any value of k.sub.1. However, the value of e.sub.1 obtained from equation (4b) does not tend toward λ/4n.sub.1, consequently, equation (4b) does not have general validity.

(29) In the weakly absorbent case, let B.sub.1=π/2−ε.sub.1 (where ε.sub.1 is a complex variable), thus implying:

(30) ${\epsilon }_1=\pi /2-\sqrt{\frac{n_3}{n_0}}\left(v_1-j{\kappa }_1\right){\delta }_1.$
It is then possible to write, to the second order in κ.sub.1:

(31) $\begin{array}{cc}{v}_1^2=1+\frac{\pi }{2}\sqrt{\frac{n_3}{n_0}}\left(\frac{n_0}{n_3}-1\right){\kappa }_1-3{\kappa }_1^2+o\left({\kappa }_1^3\right)& \left(5a\right)\end{array}$$\begin{array}{cc}{\delta }_1\simeq \frac{\pi }{2}\sqrt{\frac{n_0}{n_3}}\frac{1}{v_1}\left\{1-\frac{4}{\pi }\frac{\sqrt{n_0/n_3}}{\left(n_0/n_3-1\right)}{\kappa }_1+{\kappa }_1^2+o\left({\kappa }_1^3\right)\right\}& \left(5b\right)\end{array}$

(32) In practice, equation (5a)—the domain of validity of which has proven to be very limited—is of little interest since, as mentioned above, equation (4a) constitutes a satisfactory approximation for any value of κ.sub.1. By calculating the numerical solution to equation 3 it may be seen that the solution obtained for high κ.sub.1 does not constitute an acceptable approximation κ1 for low κ.sub.1. However, there exists a semi-empirical equation which has proven to be satisfactory in all cases and which is given by equation 6b below. Equation 6a is simply equation 4a which, as was shown above, can be considered general and used as a replacement for 5b even for low κ.sub.1:

(33) $\begin{array}{cc}{v}_1^2=1+{\kappa }_1^2& \left(6a\right)\end{array}$$\begin{array}{cc}{\delta }_1\cong \frac{\left(n_0/n_3-1\right)}{2v_1{\kappa }_1}\left[1-e^{-\frac{{\kappa }_1}{K}}\right]& \left(6b\right)\end{array}$

(34) where K={[π/(n.sub.0/n.sub.3−1)]√{square root over (n.sub.0/n.sub.3)}}.sup.−1

(35) In practice, while being less restrictive than condition 1a that applies to transparent antireflection layers, condition 6a is still very restrictive. In practice, it is often possible to settle for meeting condition 6b in an approximate manner. In the case in which the contrast sublayer is made of a relatively unabsorbent material (k.sub.1<0.15), the tolerance for e.sub.1 could be of the order of λ/100, λ/50, λ/20 or λ/10, or even λ/5; in terms of e.sub.1 this means a tolerance of the order of 0.01, 0.02, 0.05 or 0.1 or even 0.2. The tolerance for e.sub.1 could be much higher in the case in which the contrast sublayer is made of a material that may be considered highly absorbent (k.sub.1>0.15). In this case, it could reach 0.3 or even 0.6, although it will preferably be of the order of 0.1 or even 0.01.

(36) It is therefore particularly useful to introduce a hierarchy between conditions 6a and 6b, which is explained by the following analysis:

(37) The destructive interference between the beams reflected by the two surfaces f′ of an absorbent antireflection layer results from two contributions:

(38) i) the amplitude of the reflection coefficients of these two surfaces;

(39) ii) the phase shift between the beams reflected by the two surfaces, to which the difference in phase shift on reflection by each of the two surfaces and the optical path separating them contributes.

(40) Equation 6a reflects the fact that the amplitude of the reflection coefficients is identical. Equation 6b reflects the fact that the emergent beams are in phase opposition.

(41) If both equations are satisfied at the same time, extinction is total.

(42) If only equation 6a is satisfied, the fluctuations in the intensity reflected by the layer with its thickness are maximum, which guarantees the possibility of perfect extinction, but which does not guarantee extinction, not even satisfactory extinction.

(43) If only equation 6b is satisfied, extinction is not necessarily perfect, since the amplitudes reflected by the two surfaces may be different, but the thickness of the layer is such that extinction is satisfactory due to the optical path being adjusted so that these two reflections are in phase opposition, or “nearly” in phase opposition. It could be referred to as a antiresonant layer in this case. The term “nearly” reflects the fact that the differences in condition 6a affect the phases of the amplitudes reflected by the two surfaces, but that they affect them only slightly. It is for this reason that it is more important to approach condition 6b than to approach condition 6a. In practice, the index N.sub.1=n.sub.1−jk.sub.1 varies substantially with wavelength and the product ν.sub.1κ.sub.1 is subject to a period [p1, p2]. It is therefore necessary to choose the target wavelength for which the thickness of the layer is optimized. The variations in the product ν.sub.1κ.sub.1 with wavelength are enough, through the choice of working wavelength, to make up for the error in the thickness by using equation 6b with the target wavelength when equation 6a is not satisfied.

(44) It is therefore particularly judicious:

(45) i) to approach condition 6a as far as possible (for example with a tolerance lower than or equal to 20%, preferably lower than or equal to 5%, and more preferably still lower than or equal to 1%, for n.sub.1 and k.sub.1, but it will sometimes be necessary to accept higher tolerances) taking into account the constraints imposed on the materials;

(46) ii) to select a target wavelength such that the corresponding product ν.sub.1κ.sub.1 is located toward the middle of the period [p1, p2];

(47) iii) to determine δ.sub.1 by means of equation 6b applied to this target wavelength;

(48) iv) to select the working wavelength that provides the best extinction.

(49) In the case of a contrast sublayer, and with a view to obtaining even higher sensitivity, it might be advantageous to select a sublayer thickness that is less than the ideal thickness given by equation 6b, for example half of this thickness. It could be referred to as a sub-antiresonant sublayer in this case.

(50) Lastly, it is worth specifying that the antireflection conditions described by equations 6a and 6b, which relate to normal incidence, also make it possible to obtain very low reflectivity for large illumination angles (for example up to a half-angle of 60°) and above all for observation, so much so that they allow highly sensitive imaging at high optical resolution.

(51) The inventors became aware that the performance of a nonideal absorbent antireflection layer, i.e. one completely or nearly meeting condition 6b (an “antiresonant” layer) but not condition 6a, for example made of metal, could be enhanced by depositing a small number (between 1 and 5) sheets of a graphene-type material on top thereof. This is illustrated by FIGS. 2A to 2C, which show sheets of graphene oxide (GO) and reduced graphene oxide (rGO) on an “optimal” absorbent antireflection layer made of Cr/Au on a glass substrate. The observation is made under the conditions of FIG. 1; it can be seen that the GO- or rGO-covered regions appear darker than those that are uncovered (negative contrast); this indicates that the presence of a graphene-type material makes it possible to get even closer to a condition for reflection extinction than what is possible using only a metal layer.

(52) The invention makes use of this discovery, by proposing an absorbent antireflection coating comprising an “antiresonant” or “sub-antiresonant” absorbent sublayer made of a material other than a two-dimensional material, supporting a surface layer made of a two-dimensional material, and in particular a graphene-type material. As mentioned above, this makes it possible both to obtain better reflection extinction and to benefit from the excellent physical and chemical properties of graphene-type materials, or of other two-dimensional materials, the exclusive use of which for producing absorbent antireflection layers has proven to be troublesome.

(53) Advantageously, an absorbent sublayer according to the invention exhibits an antireflection behavior that is close to optimal, with a reflectivity at normal incidence that is lower than or equal to 1%, preferably lower than or equal to 0.5% and more preferably still lower than or equal to 0.1% or even 0.05% at the target wavelength (or more generally one wavelength of the spectral band of the illumination) λ. Furthermore, its transmission coefficient at normal incidence will preferably be higher than or equal to 80%, preferably 85%, and more preferably still 90%, which may be obtained, in particular, by virtue of a low thickness, advantageously lower than or equal to 10 nm.

(54) FIG. 3 shows a contrast-amplify carrier SAC comprising a transparent substrate SS, for example made of glass or of transparent plastic such as a polycarbonate or a polystyrene, serving as the incident medium, an antireflection absorbent sublayer SCC, referred to as the contrast sublayer (the structure of which may in fact be composite, in particular consist of a multilayer stack), deposited on said substrate, an absorbent layer CS made of a graphene-type material, referred to as the sensitive layer, deposited on the contrast sublayer and in contact with an emergent medium, or ambient medium, ME, for example an aqueous solution or air. A sample ECH is placed on a portion of the sensitive layer CS, on the emergent medium side. The substrate is illuminated at normal incidence by a light beam FL which is, in the example under consideration here, a Gaussian laser beam, focused by a lens LE onto the antireflection layer. It is in effect known that, in its focal region (beam waist), a Gaussian beam exhibits a planar phase front, and therefore may be locally likened to a plane wave (which case is considered in the theoretical discussions above). A semitransparent mirror MST diverts a portion of the light reflected by the substrate SS/contrast sublayer SCC/sensitive layer CS/Sample ECH/emergent medium ME assembly, to direct it toward an objective LO, allowing said sample to be observed. In this configuration, the use of a diffusing disk rotating on the incident laser beam allows speckle-type parasitic interference to be removed. Observation may be performed by scanning, in particular using a confocal or “full-field” microscopy device. As a variant, it is possible to use a wide beam of parallel light for the illumination and to observe the intensity or color modulations over the cross section of the reflected beam or through imagery by means of a telecentric vision system. In the majority of cases an illumination cone having a half-angle potentially reaching 60°, but typically of the order of 20° to 30° or less, centered around the direction of normal incidence or in any case including this direction, will be used.

(55) It should be noted that the spatial coherence of the incident light and its polarization state are of no importance; the illumination will therefore typically not be polarized. However, if it is desired to observe and/or to measure an intensity contrast, narrowband illumination should be used, or illumination consisting of multiple disjunct narrow spectral bands; polychromatic illumination resulting in a contrast which is a color rather than intensity contrast (the sample being observed with a different color than that of the background and different colors according to the thickness or the index of the sample).

(56) In the setup of FIG. 3, the lenses LO and LE are interchangeable. Moreover, the parasitic reflection on the front face of the substrate can be usefully attenuated by techniques such as: immersion in an oil, the presence of a bevel between the front face and the rear face, spatial filtering, and conventional antireflection treatment.

(57) The contrast-amplifying carrier according to the invention may also be used in fluorescence or Raman scattering microscopy. In this case, its antireflection properties are used to attenuate to a high degree reflection at the illumination wavelength so as to facilitate the detection of the less intense fluorescence or Raman scattering radiation.

(58) To design a contrast-amplifying carrier of the type illustrated in FIG. 3, the following operations are performed:

(59) First, a first material intended to form the substrate and a material intended to form the “ambient” or “emergent” medium are chosen. Typically, the choice of ambient medium is determined by the application under consideration (generally an aqueous solution for biological applications); the choice of material forming the substrate is dictated by technological considerations and by the constraint n.sub.3<n.sub.0 at the wavelength λ used for illumination and/or observation. Typically, a transparent plastic or glass substrate will be chosen, along with an ambient medium consisting of air (n.sub.3/n.sub.0 ratio comprised between 1.45 and 1.7) or water (n.sub.3/n.sub.0 ratio comprised between 1.1 and 1.3).

(60) Second, the illumination wavelength (or the shortest illumination wavelength, if the illumination is polychromatic) λ is determined according to the application under consideration or various technology constraints.

(61) Third, equation 6a is used to determine the relationship linking the real part and the imaginary part of the refractive index of the constituent material of the contrast sublayer. A material approximately satisfying this relationship is then chosen or designed. For example, a transparent starting material may be chosen according to various technological considerations: for example a polymer, taking the real part of its refractive index as an imposed value, and modifying the imaginary part of said refractive index by adding impurities (dyes, nanoparticles, etc.) so as to get as close as possible to equation 6a.

(62) Next, the thickness of said sublayer is determined by applying equation 6b (or either of equations 4b and 5b, which constitute particular cases thereof).

(63) A two-dimensional material is then chosen, for example a graphene-type material, which is intended to form the sensitive layer. The choice may be dictated by various considerations, namely optical (maximizing reflection extinction, potentially with the aid of numerical simulations) and/or physicochemical (obtaining a smooth surface on the atomic scale and/or a particular interaction with the sample) considerations. The sensitive layer may in particular be functionalized, i.e. it may bear molecules (ligands) that are capable of binding certain chemical or biological species.

(64) Next, the carrier is produced using conventional techniques, such as spin coating, or coating by immersion, rolling, sedimentation, or evaporation; physical or chemical vapor deposition, ion implantation, electrodeposition, Langmuir-Blodgett transfer, or bubbling method (J. Azevedo et al. “Versatile Wafer-Scale Technique for the Formation of Ultrasmooth and Thickness-Controlled Graphene Oxide Films Based on Very Large Flakes”, ACS Appl Mater Interfaces. 2015 Sep. 30; 7(38):21270-7), etc.

(65) The contrast sublayer may be made of metal (and in particular of gold), of a semiconductor, of a nonmetal conductor, of a polymer containing pigments or dyes, of an inorganic (mineral) material containing color centers, etc. Among the semiconductor materials suitable for producing absorbent antireflection layers are: germanium (for near-ultraviolet (UV) applications, for example at 354 nm), TiO.sub.2 (also in the near-UV), molybdenum silicide, nickel silicide or titanium silicide (in the visible spectrum), tungsten silicide (in the near-infrared or in the near-UV), zirconium silicide (in the visible spectrum or in the near-UV) tantalum or vanadium (in the visible spectrum), etc. It may also contain metal nanoparticles. It may be magnetic, which is advantageous for studying samples which are themselves magnetic. The use of conductive layers, whether metal or not, makes it possible to apply a controlled potential difference to the sample and/or to carry out “electrochemical imaging” allowing electrodeposition, corrosion, catalysis, etc. phenomena to be studied and/or the ligands of the functionalized sensitive layer to be activated/deactivated. One particularly advantageous variant consists in producing a monolithic carrier, wherein the sublayer is a layer of implanted impurities, for example implanted by low-energy ion implantation, in the surface of the substrate. Although qualified as “absorbent”, the contrast sublayer does not necessarily have to be absorbent in the strict sense: as a variant, it may be a scattering layer, the scattering “imitating” absorption and potentially also being modelled by a complex refractive index. Additionally, the contrast sublayer may be formed of a stack of elementary layers. Lastly, it may be advantageous for the contrast sublayer itself to be functionalized, for example by a layer of mercaptooctanoic acid (O.sub.8H.sub.16O.sub.2S) in the case in which it is a layer of gold (see in this regard the aforementioned article by Hua Zhang).

(66) Numerous techniques make it possible to fabricate the sensitive layer of graphene-type material, such as chemical vapor deposition (CVD), molecular beam epitaxy (MBE) and liquid phase techniques. See for example F. Bonaccorso et al. “Production and processing of graphene and 2d crystals” Materials Today, Vol. 15, n.sup.o 12 (December 2012), pages 564-589.

(67) The sensitive layer may be formed of a stack of sheets of the same or different type.

(68) A contrast-amplifying carrier such as described above, having a functionalized sensitive layer, makes it possible to produce biochips for detecting and/or assaying chemical or biological species or nanoparticles, or even nanoparticles which are themselves carrying chemical or biological species to be detected that were captured by the nanoparticles in a prior step. In this application, the use of infrared or ultraviolet illumination is particularly advantageous. Specifically, the majority of captured species have absorption bands in the infrared or in the ultraviolet which allow them to be detected specifically, i.e. to be recognized. This specific detection cooperates with the specific capture performed by the ligands, and reinforces it by superposing one specificity on top of the other.

(69) Chemical or biological species or nanoparticles may be detected/assayed directly by capturing the species to be detected/assayed, or indirectly by replacing or removing a species captured previously by the ligands.

(70) To facilitate applications to the detection of chemical or biological species, including nanoparticles, the carrier may advantageously constitute the bottom of a Petri dish or else of a fluidic cell comprising one or more channels having a minimum diameter of 1 micron, allowing the analyzed gases or liquids to be handled economically and in a perfectly controlled manner.

(71) FIG. 4A illustrates one embodiment wherein both the contrast sublayer and the sensitive layer are discontinuous and form pads P. A functionalized layer CF is deposited on the sensitive layer of each pad; typically, different pads receive different functionalizations allowing the selective binding of different chemical or biological species. The functionalized layers are brought into contact with a solution S, for example an aqueous solution, or else with a gas, containing the one or more chemical or biological species to be detected ECD. These species are bound by the functionalized layers of the respective pads and form additional thin films CE, constituting the sample to be observed (the case of a single pad is shown). By observing the biochip under a microscope, under the conditions described above, it is easily possible to identify the species that are actually present in the solution.

(72) FIG. 4B illustrates one variant wherein the contrast sublayer is continuous, and the pads P′ are formed by a discontinuous sensitive layer CS.

(73) FIG. 4C illustrates another variant wherein both the contrast sublayer and the sensitive layer are continuous, and the pads P″ are defined solely by localized functionalization layers CF. In this case it may be advantageous to provide, outside the pads, a passivation layer preventing the binding of any chemical or biological species contained in said solution (“chemical passivation”). It is possible to use for example a polyethylene glycol, a fluoropolymer, or a fluorinated alkyl.

(74) FIG. 4D schematically illustrates the case in which the contrast sublayer is made of metal (or more generally is conductive) and is connected via a conductive line LC to a contact pad PC allowing a voltage to be applied, for example to activate/deactivate in a selective manner the ligands of a functionalization layer or to promote/discourage the capture of a charged target by electrostatic or electrokinetic action (such as electrophoresis). The electroactivation of ligands is described, for example, in the article by Lucian-Gabriel Zamfir et al. “Synthesis and electroactivated addressing of ferrocenyl and azido-modified stem-loop oligonucleotides on an integrated electrochemical device” Electrochimica Acta 164 (2015) 62-70.

(75) FIG. 4B schematically illustrates a sensitive layer CS formed of three sheets of a graphene-type material, two of which exhibit a functionalization on both of their faces. To achieve this, the graphene oxide may be functionalized in solution, then deposited by applying the bubbling method a number times. Under these conditions, an intercalation phenomenon occurs which augments the effectiveness of capture of the species to be detected by the ligands. Detection sensitivity is greatly increased thereby.