FR2993661A1 - METHOD AND DEVICE FOR MEASURING A MEDIUM OF INTEREST - Google Patents

Abstract

A method of measuring a medium of interest (20), comprising: - arranging (701) the medium of interest between a light source (11) and a substrate (12) comprising a layer (121) reflecting, - illuminating ( 702) the substrate (12) by incident electromagnetic radiation (302) from the light source (11), the reflective layer (121) producing reflected radiation (303) from the incident electromagnetic radiation (302), - measuring ( 703) by a sensor (13) an intensity of the reflected radiation, - detecting (704) from measurements made under constant illumination and measurement conditions, a change in the reflective properties of the assembly (40) comprising the medium of interest and the reflective layer (121), the emitted electromagnetic radiation (301) is in a spectral band included between 350 nm and 1000 nm, and the reflective layer (121) has a reflectivity between 0.2 and 0.7 and a sensiti more than 1 x 10 nm in the band.

Description

Field of the Invention The invention relates to a method and a system for measuring a medium of interest by electromagnetic radiation. State of the art Biosensors have many fields of application. There are methods and systems for measuring a medium of interest by biosensors. Biosensors are thus used for the quantification of biomolecules. Such biosensors rely on optical reading techniques.

Fluorescent biosensor techniques have high measurement sensitivity but require prior labeling of the molecules to be analyzed. This results in a significant additional cost and a considerable preparation time. There are also methods and systems for measuring a medium of interest by surface plasmon resonance. Surface plasmon (SP) is a mode of electron oscillation of conduction of a metal at the interface with a dielectric. It is manifested as a wave that propagates along the interface, the wave being associated with an evanescent electromagnetic field, that is to say it confined to the surface. In a measurement configuration of a so-called surface plasmon resonance (SPR) medium of interest, a first surface of a metal layer is illuminated by an incident light, for example a monochromatic plane wave. SP propagates at the interface between the metal layer and a tested ambient aqueous medium, at the level of the medium of interest, that is to say at a surface layer of a second surface of the layer. metal opposite the first surface. The second surface is functionalized; it can thus present fixed biomolecules forming the medium of interest, the biomolecules can selectively associate in the medium of interest with a molecule of interest whose presence is to be measured in the ambient environment tested. The coupling between the incident light and the SP is associated with an OSP resonance incidence angle. At this OSP resonance angle corresponds an abrupt drop and a minimum of the intensity of the beam reflected by the first surface resulting from the incident light. This OSP value strongly depends on the optical index of the medium of interest, nc, in the vicinity of the second surface, and therefore on the concentration of molecules at the level of the second surface, and therefore the association between the fixed biomolecules and molecules of interest. It is then possible to measure the presence of molecules of interest in the medium of interest, and therefore in the ambient environment tested, without performing preliminary marking. However, SPR imaging systems are relatively expensive and complex to implement. In addition, the paralleling of the measurements implies a sharp decrease in the sensitivity of the biosensor, whereas a high quality of measurement is necessary for this type of imaging. In addition, the measurement for these systems is long, which limits the possibilities of real-time monitoring. SUMMARY OF THE INVENTION An object of the invention is to provide a method and a device for measuring a medium of interest that does not have these disadvantages. For this purpose, there is provided a method for measuring a medium of interest, the method comprising the steps of: 2 - arranging the medium of interest between a light source adapted to emit electromagnetic radiation and a substrate comprising a layer having reflective properties, - illuminating the substrate by incident electromagnetic radiation from the light source, the reflective layer producing electromagnetic radiation reflected from the incident electromagnetic radiation, and - measuring by a sensor an intensity of the electromagnetic radiation reflected after the crossing of the medium of interest - detecting from measurements taken by the sensor under constant illumination and measurement conditions, a change in the reflective properties of the set comprising the medium of interest and the reflecting layer and determining a modification of an opt thickness ique (nc xd) of the medium of interest corresponding to a modification of a molecular quantity in the medium of interest, in which the emitted electromagnetic radiation is included in a spectral band included between 350 nm and 1000 nm, and the reflective layer has a reflectivity R between 0.2 and 0.7 and a sensitivity dR / d (nc xd) greater than 1 x 10-3 nm-1 in the spectral band. The invention is advantageously completed by the following characteristics, taken alone or in any of their technically possible combinations: the detection step comprises a determination by an abacus of the optical thickness of the medium of interest directly from the monitoring of the intensity of the electromagnetic radiation measured under the conditions of constant illumination and measurement, the constant illumination and measurement conditions for all the measurements comprise a fixed relative positioning of the light source, the reflective layer and the sensor, and the same electromagnetic radiation emitted by the light source for all the measurements, the medium of interest comprises a plurality of probe molecules immobilized on one surface of the reflective layer, the probe molecules being adapted to interact with target molecules, the interaction of probe molecules with molecules uthe targets changing the optical thickness of the medium of interest in the spectral band, - the medium of interest is subjected to a microfluidic flow, and the measuring step is repeated with a frequency higher than 1 Hz, - the layer the reflective layer comprises an opaque sub-layer having a smooth opaque surface facing the medium of interest; the opaque layer is a layer of gold, copper or stainless steel, the emitted electromagnetic radiation being included in a spectral band; embedded image between 350 nm and 500 nm, in which the reflective layer further comprises a transparent underlayer, the transparent underlayer being disposed between the medium of interest and the opaque sub-layer, the transparent layer has a transparent underlayer; thickness less than 1600 nm, the opaque sub-layer is a silicon layer and the transparent layer is a layer of silicon oxide or silicon nitride. The invention further relates to a device for measuring a medium of interest, the device comprising: a substrate comprising a layer having reflective properties; a light source, the medium of interest being arranged between the source; light source and the substrate, the light source being adapted to emit electromagnetic radiation and illuminate the substrate by incident electromagnetic radiation from a light source, the reflective layer producing an electromagnetic radiation reflected from the incident electromagnetic radiation, and a sensor adapted to measuring an intensity of the electromagnetic radiation reflected after passing through the medium of interest, - computing means adapted to detect from measurements made by the sensor under constant illumination and measurement conditions, a change in reflective properties of the set including the middle d interest and the reflective layer and determine a change in an optical thickness (nc xd) of the medium of interest corresponding to a modification of a molecular quantity in the medium of interest, in which the electromagnetic radiation emitted is included in a spectral band included between 350 nm and 1000 nm, and the reflective layer of the substrate has a reflectivity R between 0.2 and 0.7 and a sensitivity dR / d (nc xd) greater than 1 x 10-3 nm-1 in the spectral band.

Other features and advantages of the invention will appear in the following description of an embodiment. In the accompanying drawings: FIGS. 1a and 1b show details of devices according to two embodiments of the invention; FIG. 2 diagrammatically represents a method according to one embodiment of the invention; FIG. a graph representing the relative variations of reflectivity as a function of the thickness of the medium of interest on a substrate of a device according to the invention; - FIG. 4 is a graph representing the variations in detection sensitivity on different substrates and in different ambient media as a function of the wavelength of emitted electromagnetic radiation; FIG. 5a represents a schematic diagram of an interaction measured in an exemplary embodiment according to the invention; FIG. 5b is a graph representing a response of the measurement of an interaction in the exemplary embodiment according to the invention illustrated in FIG. 5a; FIG. 5c is a graph representing In the exemplary embodiment according to the invention illustrated in FIGS. 5a and 5b, FIG. 6b, respectively 6d, is a graph representing a response of the measurement of 6a and 6c respectively, in another embodiment of the invention, FIGS. 7a and 7b illustrate an electrografting on a gold microelectrode for producing a device according to one embodiment of the invention. FIGS. 8a to 8e illustrate an immunoassay in a microfluidic cell according to one embodiment of the invention. DESCRIPTION OF THE INVENTION Examples illustrating embodiments of the device With reference to FIGS. 1a and 1b, there is described a device 10 for measuring a medium of interest 20. Light source The device 10 comprises a light source 11. In an experimental configuration, the medium of interest 20 is disposed between the light source 11 and a substrate 12. The substrate 12 comprises a layer 121 having reflective properties. The light source 11 is adapted to emit electromagnetic radiation 301 and illuminate the substrate 12 by incident electromagnetic radiation 302 from the light source 11. The light source 11 may, for example, vertically illuminate a surface 124 of the reflecting layer 121 facing the medium of interest 20. The light source 11 can thus emit electromagnetic radiation in the form of a given numerical aperture light beam and at an average incidence of zero degrees with respect to the surface 124. The light source 11 can be polychromatic, or monochromatic. The light source 11 may comprise a spectral filter filtering a white light, for example a light produced by a halogen lamp. Alternatively, the light source 11 may comprise a light emitting diode.

The electromagnetic radiation emitted 301 by the light source is included in a spectral band included between 350 nm and 1000 nm. The light source 11 may comprise a microscope objective 111 or more generally an optical system comprising a combination of lenses. Substrate 7 2 99366 1 The reflective layer 121 of the substrate 12 has a reflectivity R of between 0.2 and 0.7. The reflective layer 121 further has a sensitivity dR / d (nc xd) greater than 1 x 10-3 nm-1 in the spectral band of the emitted electromagnetic radiation 301. The reflective layer 121 may comprise an opaque sub-layer 122 having a smooth opaque surface facing the medium of interest 20. This opaque sub-layer 122 may be metallic. The opaque sub-layer 122 is for example a layer of gold, copper, or stainless steel, the emitted electromagnetic radiation 301 then being included in a spectral band included between 350 nm and 500 nm. According to one embodiment, the metal sub-layer 122 constitutes the reflective layer 121, the smooth opaque surface being the surface 124 facing the medium of interest 20 and in contact with the medium of interest 20. According to another embodiment embodiment, the reflective layer 121 comprises, in addition to the opaque sub-layer 122, a transparent underlayer 123, the transparent underlayer 123 being disposed between the medium of interest 20 and the opaque sub-layer 122. The For example, transparent underlayer 123 has a thickness of less than 1600 nm. The opaque sub-layer 122 is a silicon layer and the transparent underlayer 123 is a silicon oxide or silicon nitride layer. By silicon oxide is meant an element of the SiOx form. Medium of interest The medium of interest 20 comprises, for example, a plurality of probe molecules 201 immobilized on the surface 124 of the reflective layer 121. The probe molecules are adapted to interact with target molecules 202, the interaction of the probe molecules 201 with the target molecules 202 changing the optical thickness of the medium of interest 20 in the spectral band. By interaction is meant a stabilizing interaction between a specific region (or atom) of a molecular entity and another molecular entity. It can be an interaction between a ligand and a receiver in the broad sense. Typical forms of interactions are for example obtained by hydrogen bonding, coordination, ion pair formation, or hydrophobic interaction.

Sensor The device 13 comprises a light sensor 13. The reflective layer 121 produces a reflected electromagnetic radiation 303 from the incident electromagnetic radiation 302. The sensor 13 is adapted to measure an intensity of the electromagnetic radiation reflected after passing through the medium of interest 30. The objective 111 of the microscope can for example collect a part situated within the limits of its numerical aperture of the luminous flux reflected and diffused by the reflecting layer 121 and the medium of interest 30.

The portion of the collected flow corresponds to an electromagnetic radiation 304 measured by the sensor 13. The sensor 13 may comprise an optical detector placed in the conjugate plane, with respect to the optical system of the objective 111, of a surface of the assembly comprising the reflective layer 121 and the medium of interest 20.

The sensor 13 may be a matrix detector comprising a plurality of pixels, such as a CCD or CMOS camera. The sensor 13 can then make it possible to map the electromagnetic radiation captured for each pixel. A clear image of the electromagnetic radiation 304 measured from the surface to be studied can thus be formed at the level of an associated reading system such as a user terminal. A single objective 111 can be used to transmit the electromagnetic radiation emitted by the light source 11 and the electromagnetic radiation after reflection by the reflective layer 121. The beam splitting corresponding to each electromagnetic radiation can be obtained via a 9 beam splitter. This separator is for example a blade or a cube. Means of calculation The device comprises calculation means 14. The calculation means 14 comprise for example a calculator. The calculation means 14 are adapted to detect a change in reflective properties of the assembly 40 comprising the medium of interest 20 and the reflective layer 121. This change is detected from measurements 703 made by the sensor 13 under conditions of constant illumination and measurement. This change in reflective properties characterizes a change in an optical thickness (nc xd) of the medium of interest corresponding to a modification of a molecular quantity in the medium of interest 20. The calculation means 14 can be directly connected to the sensor 13, as shown in Figures la and lb to detect the change of reflective properties in real time or directly after a data acquisition by the sensor 13. The calculation means 14 may alternatively be adapted to detect the change of reflective properties to from data from the sensor 13 and stored on storage means such as a memory. The calculating means 14 may comprise means for determining 705 by an abacus of the optical thickness of the medium of interest 20 directly from the monitoring of the intensity of the electromagnetic radiation measured under the conditions of constant illumination and measurement. Example illustrating an embodiment of the open system device Figure 1a illustrates an exemplary embodiment of the open system device. The objective 111 is immersed directly in the solution 10 constituting the ambient medium 50 to be tested, thus making it possible to carry out an immersion measurement. Example illustrating an embodiment of the closed system device FIG. 1b illustrates an exemplary embodiment of the closed system device. The ambient medium to be tested 50 and the substrate 12 are included in a microfluidic cell closed by a hood 501. The objective 111 may have an optical correction ring to correct the passage of the electromagnetic radiation from the reflective layer 121 through the hood 501. Examples illustrating embodiments of the method FIG. 2 describes a method of measuring the medium of interest 20. First step The method comprises a first step 701 consisting in arranging the medium of interest 20 between the light source 11 adapted to Emitting electromagnetic radiation 301 and the substrate 12 comprising the layer 121 having reflective properties, second step The method comprises a second step 702 of illuminating the substrate 12 by incident electromagnetic radiation 302 from the light source 11. The radiation electromagnetic incident is from the electromag radiation generated electromagnetic 301 after passing through the medium separating the light source 11 from the surface 124 of the reflective layer of the substrate 12. The emitted electromagnetic radiation 301 is included in a spectral band included between 350 nm and 1000 nm. The illuminated reflective layer 121 then produces a reflected electromagnetic radiation 303 from the incident electromagnetic radiation 302. The reflective layer 121 has a reflectivity R of between 0.2 and 0.7 and a sensitivity dR / d (nc xd) greater than 1 x 10-3 nm-1 in the spectral band. Third step The method comprises a third step 703 consisting in measuring by the sensor 13 an intensity of the electromagnetic radiation reflected after passing through the medium of interest 20. The measured electromagnetic radiation 304 is derived from the reflected electromagnetic radiation 303 after passing through the medium separating the surface 124 of the reflective layer and the sensor 13. The measurements are made by the sensor 13 under constant illumination and measurement conditions. By constant illumination and measurement conditions for the set of measurements 703 is meant a fixed relative positioning of the light source, the reflective layer and the sensor 13, and the same electromagnetic radiation emitted 701 by the light source 11 for all measures 703.

Fourth step The method comprises a fourth step 704 of detecting from measurements taken by the sensor 13, a change in the reflective properties of the assembly 40 comprising the medium of interest 20 and the reflective layer 121. The change of reflective property allows to determine a change in an optical thickness (nc xd) of the medium of interest corresponding to a modification of a molecular quantity in the medium of interest 20. The fourth detection step 704 may comprise a determination 705 of the optical thickness of the medium of interest 20. This determination is for example carried out by the computer 14 with the aid of an abacus stored in a memory, directly from the monitoring of 12 the intensity of the electromagnetic radiation measured under the conditions of constant illumination and measurement. Compared to the SPR, the described device is inexpensive and simple to perform. In particular, no prism is necessary. Moreover, such a device and such a method are easily adaptable to different substrates and to different reactions. Such a method allows in-situ and real-time growth monitoring of a medium of interest consisting of a biochemical layer. In particular, in an aqueous medium and under illumination at 490 nm, a thin layer can induce a 15% AR / R reflectivity change over gold, 12% over copper or stainless steel or 25% over silicon. In particular, the medium of interest can be subjected to a microfluidic flow. Such tracking is possible because unlike the SPR, the measurement step 704 can be repeated with a frequency greater than 1 Hz. Principle of the device and the method Detect a change of reflectivity by measuring the intensity of the radiation A reaction measured biochemical typically relates to an interaction between a target molecule 202 present in solution and a probe molecule 201 immobilized on the surface 124 of the reflective layer 121. The biochemical reaction consists, from the optical point of view, in the formation or evolution of a thin layer at the level of the medium of interest 20, of different refractive index (generally higher) than that of the ambient environment in which the light wave propagates. The target molecule 202 is for example an antibody and the probe molecule 201 an antigen. This therefore results in a change in the refractive index in the vicinity of the surface 124 of the reflective layer, at the level of the medium of interest 20. The crossing of the medium of interest 20 by the light beam 301 from the light source 11 modifies the characteristics of the reflected light beam 303, in particular its light intensity. By reflectance is meant the ratio of the intensity of the reflected beam 303, Ireflected, by that of the incident beam 302, incident. Reflectance is denoted R = Ireflectedilincident. For a given incidence and polarization of the emitted magnetic radiation 301, the reflectance can be calculated from a part of the optical characteristics, typically the optical indices of the ambient environment in which the wave propagates. For an ambient medium a, its real index nA is noted. The calculation of the reflectance also uses the characteristics of the reflective layer 121 at which the incident electromagnetic radiation 302 is reflected. For a reflective layer 121 corresponding to a medium s, a complex refractive index hs = ns + i ks is noted, of which ns is the real part of the complex refractive index and ks is the imaginary part of the refractive index. complex, is its extinction coefficient. The reflectance RAS is then defined as the square of the modulus of the reflection coefficient fAs of the electromagnetic wave. The reflection coefficient is a complex number which is expressed according to the Fresnel relations and is reduced, in normal incidence as proposed in the invention, to: 2 - they12 RAS = = nA + In the case of a biochemical interaction, the medium of interest constitutes a thin biochemical layer. A medium of interest 20 of a thickness d has, for example, a refractive index nc-1.47. During the crossing of the medium of interest 20, the global reflection coefficient r is given by a comparable expression accounting for the multiple reflections on each of the interfaces and the phase delay introduced by the crossing of the medium of interest 20 of thickness d. The coefficient of reflection 14 between the ambient medium 50 and the medium of interest 20 is denoted fAc. The coefficient of reflection between the medium of interest 20 and the reflecting layer 121 is denoted by fcs. The overall reflectance is then obtained as follows: 2 f'AC hise 21-2 "ncd R = I fl 2 = 211. - ncd 1 + ACrCSe When the flux of the incident radiation 302 is constant, the sensor 13 and the associated computer 14 make it possible to measure the evolution of the flow collected reflected radiation 304 at time t relative to a reference measurement, for example the intensity of the flux measured at the initial time: I reflected (I) / Ireflected (0) = 1 + AR / R. The device 10 and the method thus described thus measure the relative variation of the flux corresponding to the reflected reflected radiation 304 [I reflected-I reflected (0) 1 / I reflected (0) equal to the relative variation of the reflectivity AR / R. process is related to the optical properties of the reflective layer hoist 121, for example its reflectivity or its optical indices. DETERMINING A MODIFICATION OF A MOLECULAR AMOUNT IN THE MEDIUM OF INTEREST It has been observed that, in the described methods and devices, tracking of the measured reflected radiation intensity 304 makes it possible to follow the evolution of the optical thickness of the medium of interest and thus to measure an interaction between the probe molecules 201 of the medium of interest 20 and the target molecules 202. This effect is obtained in particular and optimally for an emitted radiation 301 included in a band spectral range between 350 nm and 1000 nm, and a reflective layer 121 having a reflectivity R between 0.2 and 0.7 and a sensitivity dR / d (nc xd) greater than 1 x 10-3 nm-1 in the Thus, the measurements by the sensor 13 of the intensity of the electromagnetic radiation reflected after passing through the medium of interest 20 under constant illumination and measurement conditions make it possible to detect er a change in the reflective properties of the assembly 40 comprising the medium of interest 20 and the reflective layer 121 and to determine a change in an optical thickness (nc xd) of the medium of interest 20 corresponding to a modification of a Molecular quantity in the medium of interest 30. Choice of a reference measurement As described above, the proposed method requires a reference measurement at which to compare the reflected intensity. The reference measurement can be, as described above, the measurement made in the initial state. Alternatively the measurement can be performed on a dedicated surface of the same nature as the surface 124 studied but which does not undergo transformation. Embodiments of the Reflective Layer Promising materials for the reflective layer 121 of the device are silicon and gold, but also copper or stainless steel. Reflective layer comprising silicon Silicon has a complex refractive index the real part of which is very high (n-4 and k <0.1 in the entire spectral band between 350 nm and 1000 nm) compared to the index of any biochemical layer or inorganic (its native oxide for example) which allows a follow-up with a very high sensitivity (<0.1nm) of the deposition of thin layers on its surface. FIGS. 3 and 4 show the theoretical evolution of the relative variation of reflectivity during the deposition of organic thin films of increasing thickness on Si. Curve 803 represents the evolution of the variation of reflectivity in the case of a layer reflector 121 of silicon irradiated with radiation having a wavelength of 490 nm, the ambient medium 50 consisting of water. The curve 804 represents the evolution of the reflectivity variation in the case of a reflective layer 121 of silicon irradiated with radiation having a wavelength of 634 nm, the ambient medium 50 being constituted by water. Note that the reflectivity change is periodic with the thickness d of the medium of interest 20. This is due to the complex exponential term in the expression of the global reflectance R.

FIG. 3 also makes it possible to assess the sensitivity dR / d (nc x d) of the detection device and of the method described. FIG. 4 shows the theoretical evolution of the sensitivity with respect to the thickness dR / d (d), which has a behavior similar to the sensitivity dR / d (nc xd) with respect to the optical thickness, as a function of the wavelength of the emitted radiation 301. In the case of silicon, the deposition of a transparent sublayer 123 makes it possible to modify R so as to control the sensitivity, dR / d (nc xd). The transparent layer 123 is for example made of silicon oxide and generated by heat treatment. The transparent layer 123 may also be made of silicon nitride. Transparent layer The curve 806 represents the evolution of the sensitivity as a function of the wavelength in the case of a reflective layer consisting solely of silicon. Curve 807 represents the evolution of the sensitivity in the case of a reflective layer consisting of an opaque sub-layer 122 of silicon and a transparent underlayer 123 of silicon oxide 40 nm thick. The curve 808 represents the evolution of the sensitivity in the case of a reflective layer consisting of an opaque sub-layer 122 of silicon and a transparent underlayer 123 of silicon oxide 120 nm thick. Thus the comparison of these curves shows that the presence of a thin transparent sub-layer 123 of silicon oxide, of at least 10 nm, on the opaque sub-layer 122 of silicon makes it possible to optimize the sensitivity of the detection. But thicker layers of oxides can also be used to form substrates with sensitivity variation in the spectral range. For a transparent underlayer 123 of silicon oxide 120 nm thick, it is noted that the sensitivity is maximum in the blue. Moreover, the light source 11 is characterized by a mean wavelength λ and a spectral width Δλ or Ao, with 6 = 1 / λ. From the expression of the reflectance as a function of the wavelength λ, we see that, for a monochromatic source, the conditions on R and dR / of, sensitivity with respect to the thickness e of the underlayer Transparency 123 and the medium of interest 20 may impose conditions on the working wavelength λ to be selected depending on the chosen substrate surface. In the case of a transparent layer of optical index of real part n and imaginary part k = 0, R and thus the sensitivity, dR / de, are periodic functions of the thickness e, of period λ / 2nc. For a source of Aλ spectral width, the sensitivity dR / de is conserved at low thicknesses, typically less than 100 nm, and damped for large thicknesses. The damping factor is greater than 0.5 when the optical thickness nc x e 1 / (4 x Ao). Sensitive detection therefore imposes an additional criterion on the thickness e of the transparent underlayer which can cover the substrate as a function of the spectral width of the source used. Typically, an interference filter placed in front of a halogen lamp makes it possible to produce a source of average wavelength of 500 nm with a spectral width of approximately 20 nm. Likewise, powerful LEDs typically have mid-height widths ranging from 20 to 50 nm over the 350-1000 nm spectral range. If Ao = 1000cm -1, that is, A = 25 nm at λ = 500 nm, or ΔA = 100 nm at λ = 1000 nm, the additional criterion on the thickness of the whole (d = d + e, with thickness of the underlayer and thickness of the medium of interest) results in: nc xd '1 / (4 x Aa) = 2.5 pm, or 1.6 5 pm for nc = 1.5. If Ao = 2000 cm-1, that is, A = 50 nm at λ = 500 nm, or λ = 200 nm at lambda = 1000 nm, the additional criterion for the thickness of the assembly is expressed by: nc xd '1 / (4 x Aa) = 1.25 pm, ie 0.8 pm for nc = 1.5. Thus, the transparent underlayer 123 preferably has a thickness of less than 1600 nm. Reflective layer with gold Gold also has remarkable optical properties. Unlike silicon, the deposition of a medium of interest comprising a biochemical layer on a reflective layer 121 of gold induces relative reflectivity variations strongly dependent on the wavelength of the emitted light radiation 301. FIG. shows the evolution of the relative variation of reflectivity of a reflective layer 121 of gold, in the case of a medium of interest 20 consisting of a homogeneous and isotropic thin film having a refractive index nc = 1, 47, the ambient medium being an aqueous medium of index nA = 1.33. Curve 802 represents such a change for emitted radiation 301 at a wavelength of 490 nm. Curve 801 represents such an evolution for emitted radiation 301 at a wavelength of 634 nm. Reflectivity variations are therefore fourteen times stronger in blue than in red. An optical detection based on the reflectometry in the blue thus makes it possible to characterize the growth of organic layers on a gold surface. Curve 805 of FIG. 4 represents the evolution of the sensitivity as a function of the wavelength in the case of a reflecting layer consisting solely of gold. Optimal sensitivity can thus be obtained in the case of a gold reflective layer 121 when the emitted radiation 301 is between 350 nm and 500 nm. The method and the device described make it possible, by image acquisition by a sensor 13, to quantify the evolution of the local distribution of a thin-film medium of interest on the surface 122 of a reflective layer 121. For example, the curve 802 of FIG. 3 shows that for a medium of interest 20 with a thickness of less than 30 nm and a reflective layer of gold illuminated by emitted radiation 301 of 490 nm, the reflectivity decreases with thickness of the medium of interest. Typically, in an aqueous medium a thin medium of interest of index nc = 1.47 induces a reflectivity variation of 0.38% per nm. For a medium of interest with a density of 1.35 g / cm 3, a typical value for organic thin films or biomolecules, a reflectivity variation of 0.38% corresponds to the deposition on the surface of 1.35 ng / mm 2. Detection of the immobilization of a protein 5 nm thick under the same conditions of the curve 802 results in a decrease in the reflected light intensity of -2%, that is to say by a surface 2% less luminous than before immobilization of the protein.

The analysis of a biochemical reaction on a substrate 12 requires the use of a stable light source 11. By stable is meant that has an intensity variation of less than 0.2% during the reaction. The sensor 13, such as an optical camera, must also under these conditions be able to detect a light intensity variation of less than 0.2%.

Examples of Applications Immunoassays The development of an immunoassay consists in immobilizing on a reflective layer 121 a molecule of biological interest, for example an antigen 201 on a reflective layer 121 of gold. The detection of the interaction between the immobilized antigen 201 and an associated antibody 202 is obtained by comparing the image obtained by the multi-pixel sensor 13, before and after potential interaction with the antibody 202.

The quantitative determination 704 of the relative reflectance variation is then obtained by comparison between an intensity measured on the first image, 10 (i, j) in each pixel of coordinates (i, j) with an intensity measured on the same pixel on the first pixel. image obtained at time t, It (i, j) and 1 + AR (t, i, j) / R = It (i, j) / Io (i, j). The real-time detection is simply carried out by monitoring in situ, in aqueous solution, for example a solution of a serum, a luminous intensity of the radiation reflected by the reflective layer 121. The quantitative determination of the quantity of material immobilized is directly obtained from the measurement of 1 + AR / R and its correlation presented by an abacus as shown in FIG. 3. This evolution is obtained with a sensor 13 such as a CCD camera, by successive recording of Reflected radiation images 301. A kinetic analysis can be obtained by tracking 1 + AR (t, i, j) / R in each pixel of the sensor 13. Example of an Ag-Ac antigen-antibody interaction Figure 5a represents an interaction between plactoglobulin 201 and its associated antibody 202 present in rabbit serum. FIG. 5b shows the evolution 809 of the reflectivity in a zone 5 × 5 μm 2 of a reflective layer 121 of gold. FIG. 5c represents the variation of reflectivity at t = 0 as a function of the dilution factor of the serum. Thus FIG. 5c represents the dynamic range of the immunoassay obtained by the slope (in s-1) at short times; the origin at t = 40s corresponds to the injection of the target. The sensitivity and the dynamic range of this immunoassay are obtained preferentially by the slope at the origin of the variation of the reflectivity with the reaction time. In the case of plactoglobulin and for a gold surface of 4 pm 2, the sensitivity corresponds to a dilution of 21/50000 of the patient's serum, a sensitivity comparable to that obtained in SPR, ie approximately 1/30000. The linear dynamic range of our detection ranges from serum dilutions ranging from 1/50000 to more than 1/1000.

The device and method described also allow a quantitative analysis of the probe-target biochemical reaction. It is thus possible to measure from the relative change in reflectivity the quantity of target molecules immobilized on the surface. For example in FIG. 5b, the reflectivity measured at long times over a region of 5 × 5 p.sup.2 corresponds to the adsorption of 4.5 ng / mm.sup.2 of antibody, ie a surface concentration of 3 × 10 -14 mol / mm.sup.2. The image of the entire surface makes it possible to map the homogeneity of coverage of the surface by the antibody throughout the imaged field, for example 400 × 400 μm 2 with a resolution of 0.4 μm.

It is also possible to determine from the in situ and real-time monitoring of the antigen-antibody interaction the kinetics and thermodynamics of this recognition process. The kinetics detected in FIG. 5b as a decay of reflectivity can be analyzed, as is done in many biosensors, for example in SPR, by a first order adsorption / desorption process. The variations of experimental reflectivity are compared with an exponential Langmuir-type evolution represented by curve 810 in FIG. 5b. The apparent rate of antibody-antigen coupling is extracted from the experiment / model comparison. Many models are known to those skilled in the art to account in particular for the transport of often limiting material. In the most simplified version of the kinetic model, the apparent constant is described by kapp = kaCo + kd where ka is the kinetic constant of the association phenomenon expressed in e1s-1, kd is the dissociation constant of the immune complex in terms of 1 and Co is the concentration of the target species present in solution, ie the antibody in Figure 5a. We obtain ka and kd and therefore the thermodynamic constant of association of the immune complex KA = ka / kd by observation of adsorption / desorption processes at several target concentrations in solution. In the example shown in FIGS. 5a to 5c, it is estimated from the kinetics of recognition for serum dilutions ranging from 1/1000 to 1/10000 kd = 8 × 10 -4 s-1, the slope of the variation. of kapp with the dilution factor is 8.2 / C * -m where C * is the concentration of the target antibody in the serum. The thermodynamic constant of the coupling reaction is obtained by KA = ka / kd, here KA = 104 / C * M-1. Antibody / anti-antibody interaction (IgG / Anti-IgG) - detection of complete immunological sandwiches Unlike SPR, the measurement of reflectivity 703 is not associated with the creation of an evanescent electromagnetic wave, called surface plasmon, propagating in the vicinity of the surface 124 of the reflective layer 121. While the SPR relies on detecting the disturbances of this evanescent field induced by the reflective layer 121, the detection by the measurement 703 of reflectivity is not limited to medium of interest 20 thin layers of thickness less than the penetration length of the evanescent wave, for example less than 150 nanometers with decreasing sensitivity when the thickness of the medium of interest 20 increases. Figure 3 shows that the reflectivity measurement does not exhibit this limitation. Oscillations are observed for such media of interest of increasing thickness. It is therefore possible to observe by the method and the device described for more complex biomolecular assemblies than with the SPR comprising for example several biochemical stacks or even to use the method and the device described for following more object couplings. such large assemblies or agglutination of nanoparticles or nano-objects on a reflective layer 121 of gold. The oscillating variation of the reflectivity signal 23 shows that a quantitative measurement of a thick medium of interest, for example with a thickness greater than 50 nm, requires the monitoring of the real-time evolution of the signal picked up by the sensor 13. demonstrates the presence of an antibody 201 immobilized on a surface 124 of a reflective layer by interaction with its associated antigen 202 present in solution (FIGS. 6a and 6b) but also by the subsequent interaction of the immobilized antibody 201 with its associated anti-IgG (Figures 6c and 6d). Such an example is illustrated in FIGS. 6a to 6d, which present the real-time and in situ monitoring of the Ag-Ac interaction of α-lactalbumin (Ag) in a solution with its antibody, Ac or anti-α- lactalbumin, immobilized on gold (FIG. 6b) or the follow-up of the interaction of Ac with anti-IgG (anti-anti-α-lactalbumin) (FIG. 6d). The measurements are made with 0.2 pmol / L of α-lactalbumin or a serum containing anti-IgG diluted 1/1000. The limit of detection of α-lactalbumin is estimated at 0.1 nmol / L and that of anti-IgG at a dilution of less than 1/30000. This example shows that the method and the device described allow, without loss of sensitivity, to detect complete immunological sandwiches (Ag / Ac / anti-Ac sequence), more difficult to detect by SPR. The method and the device described also make it possible to detect assemblies with larger objects using, for example, nanoparticles. Finally all these assemblies can be dissociated by contact with a solution of glycine or high ionic strength. This rapid dissociation can also be followed by in situ and real-time imaging of the reflectivity increase of the surface 124 of the reflective layer 121.

The examples presented here are illustrated on a planar gold surface 124 obtained by depositing a 100 nm gold reflective layer 121 on a silicon wafer. They can easily adapt to the monitoring of similar biochemical reactions on a silicon surface (a silicon wafer with an oxide or an associated nitride) or on surfaces of copper or stainless steel, for example covered with a thin layer 5 transparent protecting them from corrosion. Immunoassay formats in miniaturized format By microfabrication it is easy to deposit microstructures such as microdomains of gold, on any type of substrate (glass, polymer, etc.) and to address them electrically individually (gold microelectrode array). . The preparation of an immuno-chip high-throughput immunoassay involves immobilizing a large number of ligand proteins (different antigens, for example) on these microelectrodes. The selective immobilization of an antigen on a given microelectrode is made possible by the use of an electrochemical process: one can selectively and independently activate an electrode, by application of a potential or electric current, in order to to modify its surface chemistry. Several methods of electrografting or electrochemical surface functionalization have been described in the literature. There are various routes using the electrochemistry of diazoniums or thiols. The electrochemistry of the diazoniums makes it possible to graft onto an electrode: (1) a carboxyphenyl (C61-15C001-1) group from carboxyphenyldiazonium (S2C6H5COOH) or (2) an antigen from the corresponding diazonium of the antigen which can be prepared by peptide coupling between the antigen and the same carboxyphenyldiazonium (BP Corgier, CA Marquette, LJ Blum J Am Chem Soc., 2005, 127 (51), 18328-18332.). Lane (2) directly and selectively grafts an antigen onto any polarized electrode at a potential less than -0.4 V relative to the saturated calomel electrode. The route (1) likewise introduces the carboxyphenyl group selectively on any polarized electrode. The anchoring of the antigen is then done by peptide coupling, for example by using EDC / NHS type reagents between the surface COOH group and an antigen present in the solution covering the platform. In the case of the use of thiols, electrochemical pathways also allow the selective immobilization of antigen on an electrode. If the thiol adsorption on a gold surface is not selective, the thiol of gold electrodes can be selectively desorbed by applying a strongly reducing or oxidizing potential to the electrodes which it is desired to thus clean. or renew. Peptide coupling between an antigen and the thiol remaining on the electrochemically unsolicited electrodes allows selective immobilization of the antigen.

It is also possible to accelerate the thiol deposition on an electrode by electrochemical induction (by reduction and therefore application of a cathodic potential) and thus make the antigen deposition (after peptide coupling) selective. All these steps can be followed in real time and in situ by imaging the relative variations of the reflectivity of the activated surfaces. For example, FIGS. 7a and 7b show the selective electrografting of an organic diazonium layer on a gold microelectrode present in a microfluidic channel. An organic layer only grows on the electrochemically activated microelectrode, which is attested by the decrease in reflectivity of this single microelectrode during grafting. Figure 7a shows the real-time monitoring of the kinetics of the growth of a thin film of final thickness 10 nm obtained by electrografting of a diazonium salt. Figure 7b shows a reflectivity image showing the deposition of a thin film, 30 nm thick, selectively on the activated gold electrode. This type of device based on a measurement of the variation of reflectivity in real time makes it possible to detect in situ and in real time a biochemical reaction leading to an increase in the thin layer constituting the medium of interest and makes it possible to define an immunoassay. miniaturized microfluidic cell closed according to the steps shown schematically in Figure 8a to 8e. Reflectivity imaging makes it possible to simply report on the selectivity of each of the steps: COOH terminal function surface functionation shown in FIG. 8a by a solution of + N 2 -Ar-CO 2 H, selective anchoring of ligand represented in FIG. a solution of EDC / NHS and 8c by addition of ligand, selective detection of the target on the electrode functionalized with the corresponding antigen, but also and at the same time detection of the specificity of the reaction by measurement on the electrode functionalized with another antigen shown in Figures 8d and 8e.

This approach is developable in microfluidic system closed or open as indicated above. Nor is it limited to the simple platforms described here consisting of microelectrode networks implanted in the bottom of a mirofluidic channel. It is also adaptable to platforms consisting of microleviers ("microcantilevers" in English terminology) addressed individually. In this case, it is possible to use, in a formulation derived from that proposed in Optics Lett. 2011 (Am iot et al.), A 2-wavelength illumination to decouple the optical contributions related to the deformation of the lever and the biochemical reaction by identifying the dependent contributions and independent of the wavelength. Two-colored lighting is easily and cheaply obtained using LEDs. Selective activation of different levers is also made possible by electrochemical activation and by electrografting methods previously proposed.

The use of these electrochemically activatable platforms also makes it possible to multiply the number of detection (optical and electrochemical 27, even mechanical in the case of microleviers). This redundancy of interaction measurements makes it possible to increase the reliability of the proposed immunoassays. Large Format Immunoassays The proposed device 10 is not constrained by the size of the substrate 12 to be analyzed. Indeed the detection of the presence of a protein of interest is obtained by point-by-point comparison of an image of the substrate before and after, or during the immobilization of this protein on the substrate. The analysis of a large-sized substrate with an area greater than 5 × 5 cm 2, for example, is possible by reconstruction from images of different regions and constituting a succession of images or photographic and moving shots. of the surface in relation to the objective. This requires previously imaging all or part of the surface of the substrate before immobilization or before the immobilization step that one wishes to study. In this configuration, this prior acquisition is taken as a reference image. For example, it is possible to take as region of reference, in each elementary image, the parts of the image which have the largest intensity values. The images of the substrate obtained after immobilization will be compared to those of references and will make it possible to account for the immobilization step. Thus, each region corresponding to the presence of a protein or interaction on the surface is detected by a local decrease in the reflectivity of the surface. The analysis can be done ex situ to increase the detection sensitivity, but can also take place in situ. The observed field depends on the device used, with a device comprising an optical microscope, it depends on the lens used. For example, the field may be 0.5 x 0.5 mm 2 with an objective x 20, but may also be increased in a configuration using a system comprising lenses and a camera or a camera for example. This configuration is particularly suitable for detection in large protein chips. In these chips, different ligand proteins are immobilized individually at different positions of a substrate 12 in order, for example, to detect the interaction of each of them with one or more target (s) present in an ambient medium. liquid. This type of analysis exists in immunology, the immunological response of each of the ligand proteins to targets present for example in a serum being obtained by contacting a 2D support of ligands with a serum and then by revelation. secondary labeling of target antibodies that interacted with the ligands (themselves labeled with fluorescent or radioactive terminations). The method and the device proposed make it possible to transpose this strategy onto a gold surface 124 with detection in situ and without label. The method and the device described allow first of all to highlight the various steps of the immobilization of the ligands but also to reveal the presence and position of the ligands on the analysis substrate 12.

It is also possible to follow, by the device and the method described, in situ and in real time the target-ligand interaction. If the surface of the protein chip is reacted with a serum containing a single target, for example the 13-lactoglobulin antibodies, of only one of the ligands immobilized on the surface, for example 13-lactoglobulin, the imaging of the reflectivity of the gold surface after reaction with the serum must show a decrease in reflectivity only in the only region where the ligand has been previously immobilized. This measurement can be done in situ and in real time if one knows beforehand the location of the target on the surface. This is for example the case when one seeks to detect a known target in a complex mixture. It is thus possible to use this type of strategy to define a high-speed immunoassay on a reflective surface. This high-throughput immunoassay can also be miniaturized to operate in a microfluidic channel. On the gold surface where the ligands were immobilized is assembled a microfluidic channel into which a mixture of serum-forming target molecules is circulated. For example, the method used to assemble a flow cell proposed in WO2006 / 047591, which consists in attaching to the gold surface an adhesive Mylar layer in which a channel has been preformed and in overcoming it, can be used. a hood also in Mylar.

Claims (11)

REVENDICATIONS1. A method of measuring a medium of interest (20), the method comprising the steps of: - arranging (701) the medium of interest (20) between a light source (11) adapted to emit electromagnetic radiation (301) ) and a substrate (12) comprising a layer (121) having reflective properties, - illuminating (702) the substrate (12) with incident electromagnetic radiation (302) from the light source (11), the reflecting layer (121) ) producing reflected electromagnetic radiation (303) from the incident electromagnetic radiation (302), and - measuring (703) by a sensor (13) an intensity of the electromagnetic radiation reflected after passing through the medium of interest (20), - detecting (704) from measurements made by the sensor (13) under constant illumination and measurement conditions, a change in the reflective properties of the assembly (40) comprising the medium of interest (20) and the reflective layer (121) and determining a change in an optical thickness (nc xd) of the medium of interest (20) corresponding to a change in a molecular amount in the medium of interest (30), wherein the Electromagnetic radiation emitted (301) is in a spectral band included between 350 nm and 1000 nm, and the reflective layer (121) has a reflectivity R of between 0.2 and 0.7 and a sensitivity dR / d (nc xd). ) greater than 1 x 10-3 nm-1 in the spectral band. The method according to claim 1, wherein the detecting step (704) comprises a determination (705) by an abacus of the optical thickness of the medium of interest (20) directly from the monitoring of the intensity of the electromagnetic radiation measured under constant illumination and measurement conditions. The method according to any one of claims 1 and 2, wherein the constant illumination and measurement conditions for all the measurements (703) include a fixed relative positioning of the light source, the reflective layer and the sensor (13), and the same electromagnetic radiation emitted (701) from the light source (11) for all the measurements (703). The method of any one of claims 1 to 3, wherein the medium of interest (20) comprises a plurality of probe molecules (201) immobilized on a surface of the reflective layer (121), the probe molecules being adapted to interact with target molecules (202), the interaction of the probe molecules (201) with the target molecules (202) changing the optical thickness of the medium of interest (20) in the spectral band. 20 The method according to any one of claims 1 to 4, wherein the medium of interest (20) is subjected to a microfluidic flow, and the measuring step (704) is repeated with a frequency greater than 1 Hz. The method of any one of claims 1 to 5, wherein the reflective layer (121) comprises an opaque sub-layer (122) having a smooth opaque surface facing the medium of interest (20). The method of claim 6, wherein the opaque sub-layer (122) is a layer of gold, copper or stainless steel, the emitted electromagnetic radiation (301) being within a spectral band included between 350 nm and 500 nm. The method of any one of claims 6 and 7, wherein the reflective layer (121) further comprises a transparent underlayer (123), the transparent underlayer (123) being disposed between the medium of interest (20) and the opaque sub-layer (122). 9. The method of claim 8, wherein the transparent underlayer (123) has a thickness of less than 1600 nm. The method of any one of claims 8 or 9, wherein the opaque sub-layer (122) is a silicon layer and the transparent underlayer (123) is a silicon oxide or nitride oxide layer. silicon. A device for measuring a medium of interest (20), the device (10) comprising: - a substrate (12) comprising a layer (121) having reflective properties, - a light source (11), the medium of interest (20) being disposed between the light source (11) and the substrate (12), the light source (11) being adapted to emit electromagnetic radiation (301) and illuminate the substrate (12) by incident electromagnetic radiation (302) from the light source (11), the reflective layer (121) producing reflected electromagnetic radiation (303) from the incident electromagnetic radiation (302), and - a sensor (13) adapted to measure a radiation intensity electromagnetic signal reflected after passing through the medium of interest (30), 33- calculation means (14) adapted to detect from measurements (703) made by the sensor (13) under constant illumination and measurement conditions, u n change in reflective properties of the assembly (40) comprising the medium of interest (20) and the reflecting layer (121) characterizing a change in an optical thickness (nc xd) of the medium of interest (20) corresponding to a modification of a molecular amount in the medium of interest (20), wherein the emitted electromagnetic radiation (301) is in a spectral band included between 350 nm and 1000 nm, and the reflective layer (121) of the substrate (12) has a reflectivity R between 0.2 and 0.7 and a sensitivity dR / d (nc xd) greater than 1 x 10-3 nm-1 in the spectral band. 34