Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
  • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/55—Specular reflectivity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

• the invention relates to a method and a system for measuring a medium of interest by electromagnetic radiation.
• 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.
• SP Surface plasmon
• a first surface of a metal layer is illuminated by an incident light, for example a monochromatic plane wave.
• SP spreads at the interface between the metal layer and an ambient aqueous medium tested, at the level of the medium of interest, that is to say at a surface layer of a second surface of the metal layer opposite to 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 a resonance incidence angle of 9SP.
• ⁇ SP corresponds an abrupt drop and a minimum of the intensity of the beam reflected by the first surface resulting from the incident light.
• This value 9SP strongly depends on the optical index of the medium of interest, n c , 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.
• 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.
• a method for measuring a medium of interest comprising the steps of: disposing the medium of interest between a light source adapted to emit electromagnetic radiation and a substrate comprising a layer having reflective properties,
• the reflective layer illuminating the substrate by incident electromagnetic radiation from the light source, the reflective layer producing an electromagnetic radiation reflected from the incident electromagnetic radiation, and
• the emitted electromagnetic radiation is in a spectral band included between 350 nm and 1000 nm
• the reflective layer has a reflectivity R between 0.2 and 0.7 and a sensitivity c / R / c / (n c xd ) greater than 1 x 10 "3 nm " 1 in the spectral band.
• 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;
• Constant illumination and measurement for all measurements include relative positioning fixed light source, reflective layer and sensor, and the same electromagnetic radiation emitted by the light source for all 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 the probe molecules with the target molecules 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 greater than 1 Hz,
• the reflecting 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 included between 350 nm and 500 nm,
• 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 thickness of less than 1600 nm
• the opaque sublayer 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,
• the medium of interest being disposed between the 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 radiation; electromagnetic radiation reflected from incident electromagnetic radiation, and
• a sensor adapted to measure an intensity of the electromagnetic radiation reflected after passing through the medium of interest
• calculating means adapted to detect, from measurements taken by the sensor under constant illumination and measurement conditions, a change in the reflective properties of the assembly comprising the medium of interest and the reflecting layer and to determine a modification an optical thickness (n c xd) of the medium of interest corresponding to a modification of a molecular quantity in the medium of interest,
• the electromagnetic radiation emitted is in a spectral band included between 350 nm and 1000 nm
• the reflective layer of the substrate has a reflectivity R of between 0.2 and 0.7 and a sensitivity c / R / c / (n c xd) greater than 1 x 10 "3 nm " 1 in the spectral band.
• FIGS. 1a and 1b show details of devices according to two embodiments of the invention
• FIG. 2 schematically represents a method according to one embodiment of the invention
• FIG. 3 is 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 showing 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 a response operation of the measurement of an interaction in the exemplary embodiment according to the invention illustrated in FIGS. 5a and 5b, FIGS. 6b and 6d, respectively, is a graph representing an answer.
• the measurement of an interaction shown diagrammatically in FIG. 6a, respectively 6c, in another exemplary embodiment according to 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
• FIGS. 1a and 1b 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.
• the device 10 comprises a light source 1 1.
• 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 1 1 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 can 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 digital aperture light beam. given at an average incidence of zero degrees with respect to the surface 124.
• the light source 11 may 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.
• 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 11 or more generally an optical system comprising a combination of lenses.
• 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 c / R / d (n c xd) greater than 1 x 10 -3 nm- 1 in the spectral band of 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.
• 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.
• 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 underlayer opaque 122.
• the transparent underlayer 123 has for example a thickness of less than 1600 nm.
• the opaque sub-layer 122 is a silicon layer and the transparent underlayer 123 is a layer of silicon oxide or silicon nitride. By silicon oxide is meant an element of the form SiO x .
• 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.
• interaction is meant a stabilizing interaction between a specific region (or atom) of a molecular entity and another entity molecular. 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.
• 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 1 1 1 of the microscope can for example collect a portion located within the limits of its numerical aperture of the light flux reflected and diffused by the reflective layer 121 and the medium of interest 30.
• the portion of the flux collected corresponds to a radiation electromagnetic sensor 304 measured by the sensor 13.
• the sensor 13 may comprise an optical detector placed in the plane conjugate, with respect to the optical system of the lens 1 1 1, a surface of the assembly comprising the reflective layer 121 and the environment 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 lens 1 1 1 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 separation of the beams corresponding to each electromagnetic radiation can be obtained by means of a beam splitter.
• This separator is for example a blade or a cube.
• 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 constant illumination and measurement conditions.
• This change in reflective properties characterizes a change in an optical thickness (n c xd) of the medium of interest corresponding to a modification of a molecular quantity in the medium of interest 20.
• this change in reflective properties can be a variation of reflective properties, and can characterize a variation of an optical thickness of the medium of interest which is representative of a modification, for example of a variation of a molecular quantity in the medium of interest 20.
• the calculation means 14 may be directly connected to the sensor 13, as shown in FIGS. 1a and 1b, in order to detect the change of reflective properties in real time or directly after a data acquisition by the sensor 13.
• the calculation means 14 can alternatively be adapted to detect the change of reflective properties 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.
• Figure 1a illustrates an exemplary embodiment of the device in open system.
• the objective 1 1 1 is immersed directly in the solution constituting the ambient medium 50 to be tested, thus making it possible to carry out an immersion measurement.
• FIG. 1b illustrates an exemplary embodiment of the device in a closed system.
• the ambient medium to be tested 50 and the substrate 12 are included in a microfluidic cell closed by a cover 501.
• the lens 1 1 1 may have an optical correction ring to correct the passage of the electromagnetic radiation from the reflective layer 121 through the cover 501.
• FIG. 2 describes a method for measuring the medium of interest 20.
• the method comprises a first step 701 of arranging the medium of interest 20 between the light source 1 1 adapted to emit electromagnetic radiation 301 and the substrate 12 comprising the layer 121 having reflective properties, Step 2
• the method comprises a second step 702 of illuminating the substrate 12 by incident electromagnetic radiation 302 from the light source 11.
• the incident electromagnetic radiation is derived from the electromagnetic radiation emitted 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 between 0.2 and 0.7 and a sensitivity c / R / c / (n c xd ) greater than 1 x 10 "3 nm " 1 in the spectral band.
• 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 electromagnetic radiation measured 304 is derived from the reflected electromagnetic radiation 303 after passing through the medium separating the surface 124 from the reflective layer and the sensor 13.
• the measurements are made by the sensor 13 under constant illumination and measurement conditions.
• 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 emitted electromagnetic radiation 701 by the light source 11 for all measures 703.
• 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 in reflective property makes it possible to determine a modification of an optical thickness (n c 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 using an abacus stored in a memory, directly from the monitoring the intensity of electromagnetic radiation measured under constant illumination and measurement conditions.
• 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.
• a thin layer can induce a 15% AR / R reflectivity change over gold, 12% over copper or stainless steel or 25% over silicon.
• the medium of interest can be subjected to a microfluidic flow.
• the measurement step 704 can be repeated with a frequency higher than 1 Hz.
• a measured biochemical reaction 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, with a refractive index different (generally higher) than that of the ambient medium in which propagates the light wave.
• the target molecule 202 is for example an antibody and the probe molecule 201 an antigen.
• 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. It is noted for an ambient environment a, its real index n A.
• the calculation of the reflectance also uses the characteristics of the reflective layer 121 at which the incident electromagnetic radiation 302 is reflected.
• the reflectance RAS is then defined as the square of the modulus of the reflection coefficient 3 ⁇ 4 of the electromagnetic wave.
• the reflection coefficient is a complex number which is expressed according to Fresnel relations and is reduced, in normal incidence as proposed in the invention, to:
• the medium of interest constitutes a thin biochemical layer.
• a medium of interest 20 of a thickness d has, for example, a refractive index n c ⁇ i, 47. then crossing the medium of interest 20, the overall reflection coefficient 'f is given by an expression comparable realizing multiple reflections on each of the interfaces and the phase delay introduced by the passage through the medium of interest 20 thickness d.
• the coefficient of reflection between the ambient medium 50 and the medium of interest 20 is noted TAC.
• the reflection coefficient between the medium of interest 20 and the reflecting layer 121 is noted.
• the device 10 and the method described thus measure the relative variation of the flux corresponding to the collected reflected radiation 304 [Irefiected (t) - Irefiected (O)] / 1 re fiected (0) equal to the relative variation of the reflectivity AR / R.
• the sensitivity of the method is related to the optical properties of the reflective layer 121, for example its reflectivity or its optical indices.
• the monitoring of the intensity of the measured reflected radiation 304 made 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 spectral band included between 350 nm and 1000 nm, and a reflective layer 121 having a reflectivity R of between 0.2 and 0.7 and a sensitivity c / R / c / (n xd c) greater than 1 x 10 "3 nm" 1 in the spectral band.
• 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 a change in the reflective properties of the assembly 40 comprising the medium of interest 20 and the reflecting layer 121 and determining a change in an optical thickness (n c xd) of the medium of interest 20 corresponding to a modification of a molecular quantity in the medium of interest 30.
• 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.
• the measurement can be performed on a dedicated surface of the same nature as the surface 124 studied but which does not undergo transformation.
• 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 or inorganic layer (its oxide native for example) which allows a monitoring with very high sensitivity ( ⁇ 0.1 nm) 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.
• the curve 803 represents the evolution of the variation of reflectivity in the case of a reflective layer.
• 121 of radiation-irradiated silicon having a wavelength of 490 nm, the ambient medium 50 being 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.
• Figure 3 also allows to assess the sensitivity c / R / a / (n c xd) of the detection device and method described.
• FIG. 4 shows the theoretical evolution of the sensitivity with respect to the thickness c / R / c / (d), which has a behavior similar to the sensitivity c / R / c / (n c xd) with respect to optical thickness, as a function of the wavelength of the emitted radiation 301.
• 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.
• Curve 806 represents the evolution of the sensitivity as a function of the wavelength in the case of a reflective layer composed 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.
• Curve 808 represents the evolution of 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.
• the sensitivity c / R / c / e is retained at low thicknesses, typically less than 100 nm, and damped for large thicknesses.
• the damping ratio is greater than 0.5 when the optical thickness n c xe ⁇ 1 / (4 x ⁇ ).
• 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.
• 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.
• the LEDs typically have half-widths ranging from 20 to 50 nm over the 350-1000 nm spectral range.
• the transparent underlayer 123 preferably has a thickness of less than 1600 nm.
• Reflective layer comprising gold
• Gold also has remarkable optical properties.
• a medium of interest 20 comprising a biochemical layer on a reflective layer 121 of gold induces relative reflectivity variations strongly depending on the wavelength of the emitted light radiation 301.
• Curve 802 represents such a change for emitted radiation 301 at a wavelength of 490 nm.
• Curve 801 represents such a change 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.
• 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.
• 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.
• 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.
• 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
• 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 sensor 13 comprising several pixels, before and after potential interaction with the antibody 202.
• 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 amount of immobilized material is directly obtained from the measurement of 1 + AR / R and its correlation presented by an abacus as represented in FIG. 3. This evolution is obtained with a sensor 13 such that a CCD camera, by successive recording of images of the reflected radiation 301.
• a kinetic analysis can be obtained by tracking 1 + AR (t, i, j) / R in each pixel of the sensor 13.
• FIG. 5a shows an interaction between ⁇ -lactoglobulin 201 and its associated antibody 202 present in rabbit serum.
• FIG. 5b represents the evolution 809 of the reflectivity in a zone of 5 ⁇ 5 ⁇ 2 of a reflective layer 121 of gold.
• 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.
• the sensitivity corresponds to a dilution of 1/50000 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.
• the reflectivity measured at long times over an area of 5 x 5 ⁇ 2 corresponds to the adsorption of 4.5 ng / mm 2 antibody is a surface concentration of 3 ⁇ 10 "14 mol / mm 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 x 400 ⁇ 2 with a resolution of 0.4 ⁇ .
• the measurement 703 of reflectivity 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 thin media of interest of thicknesses less than the length of the reflective layer 121. penetration of the evanescent wave, for example less than 150 nanometers with a decreasing sensitivity when the thickness of the medium of interest 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 process 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 couplings of larger objects such as assemblages or agglutination of nanoparticles or nano objects on a reflective layer 121 of gold.
• the oscillating variation of the reflectivity signal 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.
• FIGS. 6a to 6d show the real-time and in situ monitoring of the Ag-Ac interaction of ⁇ -lactalbumin (Ag) in a solution with its antibody, Ac or anti- ⁇ -lactalbumin, immobilized on gold (FIG. 6b) or the follow-up of the interaction of Ac with anti-IgG (anti-anti- ⁇ -lactalbumin) (FIG. 6d).
• the measurements are carried out with 0.2 ⁇ / L of ⁇ -lactalbumin or a serum containing anti-IgG diluted 1/1000.
• the detection limit of ⁇ -lactalbumin is estimated at 0.1 nmol / L and that of anti-IgG at a dilution of less than 1/30000.
• microfabrication it is easy to deposit microstructures such as gold microdomains, 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 consists in 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 an electric current, in order to to modify its surface chemistry.
• electrografting or electrochemical surface functionalization have been described in the literature. There are various routes using the electrochemistry of diazoniums or thiols.
• a carboxyphenyl group ( " C6H 5 COOH) from carboxyphenyldiazonium ( + N 2 C 6 H 5 COOH) or (2) an antigen from the corresponding diazonium of the antigen which can be prepared by peptide coupling between the antigen and the same carboxyphenyl diazonium (BP Corgier, CA Marquette, LJ Blum J. Am Chem Soc, 2005, 127 (51), 18328-18332.).
• the route (2) makes it possible to directly and selectively graft an antigen onto any polarized electrode at a potential of 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.
• 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.
• 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 reduction of the 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 20 and makes it possible to define a miniaturized immunoassay in closed microfluidic cell according to the steps shown diagrammatically in FIGS. 8a to 8e.
• the 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 ligand anchoring represented in FIGS.
• 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 photographic images. displacements of the surface with respect 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• a microfluidic channel into which a mixture of serum-forming target molecules is circulated.
• 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.
• the method may further comprise a step of measuring by a second sensor an electrical quantity whose modification, in particular a variation, is representative of a modification of a molecular quantity in the medium of interest, the electrical quantity being representative of electric charge transport properties between the surface 121 and the medium of interest, a physical phenomenon distinct from the measurement of the reflected electromagnetic radiation, the measurement step being performed simultaneously with the measurement step 703 of the intensity of the reflected electromagnetic radiation. It is thus possible to carry out, in parallel and / or with the aid of a common device, electrical measurements making it possible to account for or detect magnetic, electrical or electrochemical phenomena. Electrical measurements corresponding to such phenomena can for example include conductimetry, impedance, potentiometric and / or amperometry measurements, of the same molecular or biomolecular phenomenon observed.
• the configuration and geometry of a device for measuring a medium of interest as described above is particularly suitable for coupling with other types of measurements.
• the measurement device described allows a measurement of an electromagnetic field, typically an optical measurement, non-destructive vis-à-vis the medium of interest.
• the method and the device described thus allow easy implementation of other analysis techniques.
• the device for measuring a medium of interest proposed above can thus also comprise at least a second sensor adapted to measure a physical quantity representative of electric charge transport properties between the surface 121 and the medium of interest, typically for perform a different type of measurement as described above, for example an electrical measurement.
• Such electrical measurements can thus be performed by second dedicated sensors.
• the second sensors can be connected to an electrical measuring device.
• the electrical measurement device may be an external electrical measurement device, or be included in the device for measuring a medium of interest as described above.
• Such electrical measurements can be made by means of at least 2 electrodes.
• a dedicated sensor can form an electrode.
• One of the electrodes may be formed by one of the aforementioned surfaces, for example the surface 124 of the reflective layer 121.
• a second electrode can be introduced into the solution or the environment.
• such a combination of measurements of intensity of electromagnetic radiation on the one hand and electric on the other hand can be implemented by a sensor or a plurality of sensors having an electronic conductivity corresponding to that a metal, for example gold as described above, an alloy of metals, or silicon, in particular doped silicon.
• a second sensor forming an electrode may be placed in direct contact with the medium of interest or the solution.
• the measured electrical response corresponds to or makes it possible to detect the presence or absence of a given layer, for example a layer of biological material corresponding to or responsible for a reaction involving a change in the optical thickness of a layer, such as immunoassay or immunoassay.
• the second electrical sensor forming the electrode 121 may also be buried under a thin dielectric layer, for example an insulating layer, for example a transparent layer such as the transparent underlayer 123, for example a silicon oxide, for example SiO 2, the second electric sensor can thus be arranged without direct contact with the solution and / or the medium of interest.
• the device can be made in such a way that the optical properties of a measurement zone of the radiation intensity sensor 13, for example a specific surface of the sensor, are compatible with the presence of the dielectric layer to give a sensitivity response.
• the device for measuring a medium of interest and / or the measurement method described above is made so that the electrical measurement, by the second electrical sensor, electrical charge transport properties between the layer 121, the dielectric layer 123 and the medium of interest have an equally sufficient electrical sensitivity, that is, the sensitivity for detecting the same reaction as measured by the radiation intensity sensor 13.
• This can be obtained for example by a dielectric layer sufficiently thin to allow an electrical measurement, for example an impedance measurement, that is to say a measurement of high frequency capacitance, sensitive to the layer possibly formed and resulting of the reaction that is to be detected, for example a deposit induced by the immunoassay which alters the capacity of the second sensor.
• a so-called "contactless" measurement of capacitance can be performed, for example in TouchChip TM fingerprint sensors (TCS).
• the detection step 704 can then be performed from the electrical measurements made by the second sensor.
• the calculation means 14 can then be adapted to perform the detection as a function of the measurements described above and the measurements from the second sensor.
• the optical thickness modification whose method and device aim at determining the presence, results in a variation of a signal: an electromagnetic radiation, in particular an optical signal, with respect to the measurement by the radiation sensor described above, an electrical signal in the context of an electrical measurement, for example resistance, impedance, that is to say resistance and / or capacity, potential or current.
• the method may thus comprise a step of comparing the various measurements, for example the measurement of electromagnetic radiation and at least one electrical measurement.
• a comparison step may be obtained by standard logic electronic tests, for example implemented by the calculation means 14.
• Such a step may comprise a measurement correlation step.
• a such a comparison step may make it possible to increase the robustness of the detection.
• calculation means may be adapted to filter the data from the detection step from the data from the second sensor, for example from detection data calculated by the calculation means 14 from data from the second sensor.
• Such a coupling between the measurement of an electromagnetic radiation described above and the measurement of another phenomenon, for example an electrical measurement, or of the detection data resulting from these measurements, can be implemented by a device such as the device provided, further comprising a second electrical sensor or a plurality of second electrical sensors.
• the plurality of different types of measurements of the same phenomenon on the same surface makes it possible to significantly increase the reliability of the results of a test thus implemented by the proposed method or device.
• Such a coupling may for example be implemented as part of a false positive detection step using the results of the various measurements.
• Such a step may be implemented by the calculation means described, the calculation means outputting a corrected value, for example by removing the false positives detected by the different types of measurements.
• the modification detected during the detection step can be corrected during a correction step from the data from sensors and / or from different measurements and / or of different type.
• the microelectrode used to carry out electrografting may allow the realization of an electrochemical measurement in parallel with the steps previously described for the measurement of the medium of interest by electromagnetic radiation.
• the example of electrografting shown in FIG. 7b is produced by selective connection of the activated electrode. We can then follow the current that passes through each of these electrodes during this electrografting.
• the inactivated electrode has a current ⁇ 1 pA while the activated electrode has a current> 100 pA.
• the electrografting results in a measurable reflectivity decrease (-4% in Figure 7a) on the active electrode and in a reflectivity variation lower than the optical sensitivity on the active electrode.
• the comparison of the two measurements can thus allow a more robust detection of the presence or absence of a deposit on the two electrodes, by means of the method described having the two measuring steps and / or the device described presenting the two sensors.
• the divergent results between the two types of measurement can for example be considered as false positives or false negatives.

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