EP1342071A1

EP1342071A1 - Method for characterising a surface, and device therefor

Info

Publication number: EP1342071A1
Application number: EP01270759A
Authority: EP
Inventors: Philippe Guedon; Yves Levy
Original assignee: Institut d'Optique Theorique et Appliquee
Current assignee: Genoptics SA
Priority date: 2000-12-13
Filing date: 2001-12-13
Publication date: 2003-09-10
Also published as: CA2431399C; CA2431399A1; AU2002219304A1; US7678584B2; US20070054392A1; WO2002048689A1

Abstract

The invention concerns a method for characterising the surface of a sensitive layer deposited on the base of an optical prism, said surface comprising active zones whereof the optical thickness is capable of varying with time, said method using a technique based on analysis of the reflective variation of the prism-sensitive layer interface. The method consists in causing: a collimated light beam with an angle of incidence enabling total reflection on the prism base to penetrate into the prism, said beam illuminating a fixed part of the interface corresponding to the part to be characterised; scanning at an angle so as to locate an incidence whereon the reflectivity of the active zones is minimal; determining an angle of incidence optimal for sensitivity of detection of the active zones; adjusting the incidence to the optimal value; and measuring the reflectivity with an imaging system. The invention also concerns a device for implementing said method.

Description

The present invention relates to a method for characterizing a surface, and to a device making it possible to implement this method. This process allows in particular a qualitative and / or quantitative measurement of interactions which can be physical, chemical, biochemical or biological.

Most of the existing methods in this field use fluorescent markers to reveal molecular interactions and require work under specific conditions. These specific conditions do not allow several types of interaction to be analyzed simultaneously. On the contrary, the method of the invention makes it possible in particular to follow in real time, and without markers, up to several hundred molecular interactions, both qualitatively and quantitatively.

The method of the invention allows the characterization of the surface of a sensitive layer, deposited on the basis of an optical prism, according to a technique based on the analysis of the variation of the reflectivity of the prism - sensitive layer interface. . This reflectivity is likely to vary with the angle of incidence of a light beam moving in the prism towards a part of the interface corresponding to a part to be characterized of said surface. This reflectivity also varies as a function of the optical thickness of the sensitive layer, so that the method of the invention makes it possible in particular to characterize the evolution of zones of the sensitive layer whose optical thickness is likely to vary over time . The part of the interface "corresponding" to the part to be characterized is of course the part which is opposite the part of the surface to be characterized.

The method of the invention can be used in particular either in techniques based on the resonance of surface plasmons, or in techniques based on interferometry and using the coupling between a prism and a waveguide.

The invention particularly relates to a method for characterizing the surface of a sensitive layer, said surface comprising active areas whose optical thickness is likely to vary over time, said sensitive layer being deposited on the basis of a optical prism, said method using a technique based on the analysis of the variation of the reflectivity of the prism-sensitive layer interface, in which; a) a light beam is brought into the prism through an entry face collimated with an angle of incidence allowing total reflection on the basis of the prism, said beam illuminating a fixed part of the interface corresponding to a part to be characterized, called useful part, of said surface, and the reflected beam emerging as an emerging beam by the exit surface of the prism, b) the useful part is scanned at an angle, by varying the angle of incidence of said beam, so as to identify an incidence for which the reflectivity of at least one part, or all of the active areas is minimum, c) an optimal angle of incidence is determined, for which the detection sensitivity of the active areas is maximum, d) the incidence is adjusted to the optimal value determined at previous step, and the reflectivity is measured using an imaging and detection system having a sensitive surface capable of receiving the entire emerging beam coming from the useful part. A collimated beam is a beam of which all the rays are parallel.

The prism is used, in the method and the device of the invention, as a coupling prism, which uses the phenomenon of total reflection on an interface.

The surface to be characterized of the sensitive layer is that of the external face of the sensitive layer, in contact with the external medium. In practice, the sensitive thin layer can be deposited either on the base of the prism, or on a flat transparent blade with parallel faces, having the same index or an index close to that of the prism, and fixed, or capable of being fixed, on the base of the prism, with the sensitive layer deposited on the external face, which is not in contact with the base of the prism. This external face can be considered as the true base of the prism in such a case, when it is fixed to the prism.

The sensitive layer can be a thin metallic layer, and in this case the method of the invention uses the phenomenon of surface plasmon resonance. Among the metals used, mention may in particular be made of gold and silver. An intermediate metallic thin layer, for example chromium, can be used to improve the adhesion of the gold layer to the glass of the prism. One can also deposit on the prism a layer of silver itself covered with a layer of gold, which protects the silver against oxidation.

The metallic sensitive layer can be deposited on the base of the prism, or on a thin layer, deposited on the base of the prism, with a dielectric of low index (index equal to or close to that of the prism).

The sensitive layer can also be a thin, transparent layer with a high index dielectric capable of serving as a waveguide. In this case, two thin dielectric layers are deposited on the base of the prism, first a thin layer (for example of silica) serving as a spacer medium, of intermediate index between that of the prism and that of the guiding layer, then the guiding layer, of high index. The guiding layer can be made for example with titanium or osmium oxides, stoichiometric or non-stoichiometric. The guide layer can itself be covered with a thin metallic layer.

When the sensitive layer is a dielectric layer serving as a waveguide, the method of the invention makes it possible in particular to use the waveguide as a resonant mirror system which requires a coherent light source. The principle and the applications of the resonant mirror are known; see for example R. Cush et al, Biosensors & Bioelectronics 8, 347-353 (1993); P. E. Buckle et al, Biosensors & Bioelectronics 8, 355-363 (1993); S. F Bier, Sensors & Actuators B, 29, 37-50 (1995); C. Stamm et al, Sensors & Actuators B, 31, 203-207 (1996); A. Bernard et al, Eur. J. Biochem., 230, 416-423 (1995). The content of these literature documents, as well as the documents mentioned below, is incorporated by reference into the present description.

The thin layers used in the implementation of the process of the invention can be deposited according to known methods, for example by vacuum evaporation, sputtering, CND, MOCVD, etc.

In what follows, reference will most often be made to the phenomenon of resonance of surface plasmons, to simplify the description, but specialists will readily understand that the method of the invention can be applied without difficulty to a device using a system with resonant mirror.

As indicated above, the method of the invention can be used in particular by exploiting the known phenomenon of surface plasmon resonance (RPS). This phenomenon is observed when a light beam undergoes a reflection at the glass-metal interface of a thin metallic layer, deposited on the base of the prism. When a ligand for an analyte of interest is attached to the external face of the metallic layer, and when the metallic layer is brought into contact with a liquid medium or gaseous, capable of containing the analyte, the latter, if it is present, will be fixed, by affinity, on the ligand and this results in a modification of the thickness of the molecular layers fixed on the metal surface, and therefore of the optical thickness, which is reflected, in particular due to the RPS phenomenon, by a variation in the reflectivity of the interface.

The optical phenomenon of surface plasmon resonance was discovered around thirty years ago (E. KRETSCHMA N & H. RAETHER; Z.Natiirforsch., 23a (1968), 615-617) and was used first place for the quantitative characterization of non-organic thin layers (WP CHEN & JM CHEN, J. Opt.Soc.Am, 71 (1981), 189-191). The optical principle is based on access to the variation of the optical thickness (product of the refractive index by the geometric thickness) via the measurement of the variation of reflectivity on a metal / dielectric interface generated by the fixing of material. on the external face of a metallic layer. Since the 1980s, the resonance phenomenon of surface plasmons has been used in biological applications, in particular in the quantitative characterization of biological interactions. Thus, it is possible, through knowledge of the variation in refractive index, to go back to the number of biological molecules that have interacted (JA DE FEIJTER et al, Biopolymers, 17 (1978), 1759-1772; A. BERNARD et al, Eur. J. Biochem., 230 (1995), 416-423; J. PIEHLER et al, J. of Immunol Methods, 201 (1997), 189-206; P. GUEDON et al, Anal. Chem ., (2000) 6003-6009), in their concentration as well as in real time kinetics characterizing the interaction of analytes with ligands previously immobilized on a thin metallic layer.

An advantage of the method and the device of the invention lies in the fact that it is possible to locally deposit different ligands, on a regular basis, on the sensitive layer, according to an ordered system (for example, matrix or hexagonal) in which each deposit confined to a small area, or spot, is spatially identifiable (so-called “addressed” ligands).

We know that the variation in reflectivity linked to the RPS phenomenon depends on the angle of incidence of the light beam undergoing total reflection. The phenomenon of total reflection on an interface takes place when a light wave passing through a medium of refractive index n _t meets a medium of refractive index nor encounters a medium of refractive index n ₂ (with n ₂ <ni ) with an incidence α such that α> α _c = Arcsin ^ / n. When the thickness of the sensitive layer increases (in particular when an analyte is fixed on ligands immobilized on the metal layer), the intensity of the reflected light decreases, then passes through a minimum, then increases again, this phenomenon being observed for a variation of the angle of incidence of several degrees. It is therefore desirable to identify, before contacting the analyte, the angle of incidence for which the intensity of the reflected light is minimum on the spots, which results in a notable contrast between the spots and the bare areas of the substrate. The precise knowledge of this angle for each spot allows the characterization of their surface condition. As a result, depending on the molar mass of the analyte or analytes, it is possible to determine an angle optimizing the detection sensitivity for each spot on which an analyte is fixed (or is fixed in greater quantity ).

When the optical thickness of the sensitive layer increases, for example when an analyte is fixed on the spots containing immobilized ligands, this causes a displacement of the resonance peak (minimum of reflectivity) towards the decreasing external angles of incidence ( here we call "external angle of incidence" the angle of incidence of the light beam in the external environment, before entering the prism). By choosing an external angle of incidence close to the initial angle of incidence of the peak, but slightly greater (corresponding to a reflectivity close to the minimum), this displacement of the peak will cause, for the angle of incidence thus chosen, an increase in reflectivity, which will result on the image by the appearance of a brighter spot (or by the variation of the gray level towards the lighter grays) while the spots whose optical thickness does not has not varied will remain dark. It is therefore easy to determine the optimum angle of incidence used in accordance with the method of the invention, for which the detection sensitivity is maximum for all of the spots whose optical thickness is likely to vary. Indeed, in the initial state, these spots most of the time have very similar resonance peaks. In addition, as the angular scanning made it possible to pinpoint the angle of incidence of the resonance peak for each spot, in the initial state, it is possible to determine, for each spot, by computer means, the value exact displacement of the peak for this spot, and therefore normalize the results collected by the detector of the imaging system which detects the photon flux coming from each of the spots.

In particular embodiments, the method of the invention can still have the characteristics, taken individually or, where appropriate, in combination, as defined in the claims.

In order to be able to characterize the evolution of the active zones of the useful part, the reflectivity as a function of time is measured, either continuously or at predetermined intervals.

The variation in reflectivity is proportional to the number of molecules which are fixed on the studs, this fixing corresponding to an increase in thickness on the stud considered.

The useful part of the sensitive layer can be fixed, for example: - antigens (for the search for specific antibodies),

- random peptides, for the search for specific mimotopes of a given antibody,

- anti-tumor antibodies, for the search for tumor antigens in cell shreds, - all specific ligands for particular cells, for cell sorting.

The method of the invention can also be used to study any interactions between the immobilized molecules and the analytes, including cells.

The process of the invention can also be used for the screening of small molecules which are immobilized on the active layer (for example oligosaccharides) which can inhibit the signaling or enzymatic role of a protein.

(the analyte is then the protein). Small molecules are selected by biological or biochemical affinity for proteins or cells.

The invention also relates to an imaging device making it possible to implement the method of the invention. This device can be used in particular for imaging the surface of a sensitive layer carrying multiple organic sensors, that is to say a surface on which immobilized one or more populations of ligands. These sensors can be deposited on the sensitive layer in the form of pads (or spots) forming a matrix of addressed ligands. These spots can be formed either by deposits on the surface of the sensitive layer, or by filling wells made on the surface of the sensitive layer (for example by laser abrasion).

The device of the invention comprises an imaging device, making it possible to implement the method of any one of the preceding claims, including:

- an optical prism having an entry face, an exit face and a base provided with a sensitive layer, said prism being characterized by its angle at the top and by the refractive index of the material which constitutes it, - a first afocal optical conjugation system, disposed on the side of the entry face of the prism, and whose optical axis, fixed relative to the prism, is oriented so that a collimated incident light beam having passed through it goes towards said entry face, refracts then strikes the base of the prism where it undergoes a total reflection and emerges after refraction on the exit face by forming an emerging beam, said first conjugation system having an orientation and an opening such that said incident beam refracted on the entrance face comes to illuminate the totality of at least a part called useful of said sensitive layer, and to undergo a total reflection therein when the direction of the incident beam varies, on both sides tre of said optical axis, over an angular range of at least 10 ° in total, said useful part containing active areas whose optical thickness is likely to vary over time,

a second afocal optical conjugation system arranged on the side of the exit face of the prism and whose optical axis, fixed relative to the prism, is oriented so that an incident beam parallel to the optical axis of the first conjugation gives an emerging beam parallel to the optical axis of the second conjugation system,

- a detection system having a plane sensitive surface which is capable of receiving and analyzing the light having passed through said second conjugation system, and which is perpendicular to the optical axis thereof, in which: (i) said second the conjugation system has an opening such that all of the light from the useful area passes through it when the direction of the incident beam varies over said angular range, and the second conjugation system is such that all of the light from of the useful area illuminates the sensitive surface of the detection system, and (ii) the apex angle and the prism index are such that when the direction of the incident beam is parallel to the optical axis of the first conjugation system, the intermediate image of the useful part, through the exit face of the prism, which constitutes the object for the second detection system, is perpendicular to the optical axis of it.

The angular scanning which must be carried out in order to be able to observe the resonance at various points in the active zone, and its displacement during the evolution of the system over time, varies in particular as a function of the metal, of the glass, of the length of wave, etc. It is generally at least around 10 °.

The study of the reflectivity, as a function of the external angle of incidence, of a metal-dielectric interface, requires an angular scanning generally comprised between 4 and 10 degrees, approximately, according to the wavelength used and the nature dielectric and metal. For example, with a glass-to-metal interface at the base of an angle prism at the top 30.6 degrees, index 1.776 and a thin layer of gold 45 nm thick, and a light source wavelength 660 nm, to fully describe the resonance of surface plasmons, sweep a range of about 8 degrees.

In addition, in the case where the metallic thin layer is provided with a plurality of studs consisting, for example, of polymers carrying immobilized biological species, the resonance for gold is broken, on all the parts of the sensitive layer bearing a stud, and the resonance then shifts towards the decreasing external angles of incidence. For example, for a 3 nm thick pad with an index of 1.7 to 660 nm, the resonance shifts by about 2 degrees. If the sensitive layer is then brought into contact with a fluid containing analytes capable of interacting with the biological species immobilized on the pads, a new shift in resonance can be observed which can go up to approximately 4 degrees.

The sum of the angular expanses to be traversed if one wishes to observe the resonances of gold, of the studs of polymers carrying biological species, and of some of these studs after they have interacted with the analytes, then requires an angular scan. total of 14 degrees. It is desired to be able to use an angular scan of 16 degrees, in order to be able to work with a certain comfort and to be able to modify at leisure the aforementioned materials, in particular in terms of index and thickness. In certain applications and with certain materials and certain wavelengths, the possibility of using an angular scan of 10 ° may suffice. But. the device of the invention makes it possible to use an angular scan which can go up to 16 °.

The light source is a coherent or non-coherent source. It is necessarily consistent in the case of a waveguide device. The collimation system can be any conventional optical system making it possible to transform a divergent light beam into a parallel beam.

The deflector optical system comprises for example a rotating plane mirror, which can be mounted on a stepping motor or a direct current motor. One can also use a galvanometric type mirror. It is known that when a rotating mirror rotates by a given angle, the reflected ray from a fixed incident ray is deviated by an angle twice from said given angle. The rotating plane mirror can in particular be able to pivot about an axis parallel to an edge of the prism. The line of intersection of the two planes containing the entry and exit faces of the prism respectively is called the edge of the prism.

The device of the invention may comprise a polarizing system, arranged for example between the collimation system and the rotating mirror, and making it possible to polarize the light parallel to the edge of the prism. The polarizing system is for example composed of a simple polarizer or a polarization splitter cube. In the case of the resonant mirror system, a second polarizer is of course necessary after the prism.

In particular embodiments, the device of the invention may also have the characteristics, taken in isolation or, where appropriate, in combination, as defined in the claims. In the present invention, the term “dioptric system” denotes a lens or a doublet.

The device of the invention makes it possible to perform the detection at the best angle of incidence of the variation in optical thickness (product of the geometric thickness by the refractive index of the material) on the metal-external environment interface, without having to modify the position of the elements of the device which are all fixed with respect to each other, with the exception of the rotating mirror. It is thus very easy to vary the angle of incidence during a preliminary study, by actuating the only rotating mirror, to determine for each angle of incidence the reflectivity of the sensitive area, and we can then carry out the measurements. proper by immobilizing the rotating mirror in the - position corresponding to this predetermined optimum angle of incidence.

In fact, according to a particular embodiment, the optical imaging device of the invention is such that the image of the useful area of the thin layer metal occupies a maximum surface of the CCD detection matrix, so that it is the entire surface of the useful area of the metal layer which can be analyzed simultaneously using the CCD matrix.

An important feature so that the detection device can receive all the light rays coming from the image of the useful surface of the metallic thin layer, although the imaging system and the detection system are fixed relative to the prism, is that the device of the invention comprises an optical conjugation system such as the beam which, after entering the prism, strikes the thin metallic layer, illuminates the entire useful surface of the CCD matrix, and this whatever the angle of incidence of said beam on said angular range up to at least about 16 °. Such an optical conjugation system can be developed, for example, in a known manner, using a device whose number of apertures, that is to say the ratio of the focal length to the useful diameter of the optic , is less than N = l / (2 * sin (16 ° / 2)) = 3.6 to accept all the beams reflected by the sensitive area, over the angular range of at least 16 °. The conjugation system can for example consist of a first focal lens 80 mm and diameter 31.5 mm and a second focal lens 65 mm and diameter 25 mm (the assembly having an opening of 2, 6), said lenses being arranged in such a way that the image of the useful part by the exit face of the prism is approximately in the focal plane object of the first lens, that the focal plane image of the first lens is coincident with the object focal plane of the second lens and finally that the CCD matrix is located in the image focal plane of the second lens; in this way, an afocal device has been created which combines. One can also for example use a third lens, commonly called "field lens" which makes it possible to bring the beams towards the optical axis, thus increasing the aperture of the system compared to an afocal system with two lenses (see M. BORN & E. WOLF, Principles of Optics, Chapter VI, Pergamon Press, 1959).

Another important characteristic of a particular embodiment of the device of the invention, which allows an analysis of each spot of the useful part with a satisfactory sensitivity, is that the angle at the top and the index of the prism are chosen to be so that the intermediate image of the useful area formed in the prism (i.e. the image of the useful surface through the plane diopter formed by the exit face of the prism, and which constitutes the object for the optical imaging system receiving the beam emergent) provides in the optical imaging system an image coinciding with the focal plane of the detection system (CCD matrix), or an image very close to this plane and parallel to it, or having an inclination relative to this plane low enough to avoid blurring the image over the entire surface. It is known that any optical imaging system has a certain depth of field, that is to say a longitudinal range, along the optical axis defined by the detector (this optical axis being perpendicular to the plane of the detector) for which the the image will appear sharp on the detector. The depth of field depends directly on the focal length of the optical system and its aperture. Thus, for a given focal length, the depth of field increases with the number of apertures (ratio of the focal length to the diameter of an optic). However, the number of openings must also be as small as possible to allow a maximum of rays to pass through the system, and thus allow significant angular acceptance. These two conditions being contradictory, it is necessary to find a compromise. To obtain a clear image on the detector, the inclination of the image of the useful area by the exit face of the prism relative to a plane perpendicular to the optical axis must be sufficiently small so that it is not not outside the depth of field defined by the detection system. However, the inclination of the incident beam in this system makes it necessary to straighten the image by an appropriate optical device, which can be achieved either by tilting the optics of the imaging system, or by tilting the detection system ( CCD camera) in order to have the image of the entire net surface on the CCD detector.

The problem of the inclination of the optic implies that said optic is very open to let all the rays pass through this optic, since the angles involved can be relatively large; for example, for an isosceles prism with an index 1.515 and an angle at the apex 60 °, this requires an opening number of the detection system of 0.55, which is prohibitive because such a system has many aberrations and very shallow depth of field. Similarly, the tilt of the camera would cause a loss of sharpness due to the fact that the system would no longer work under good imaging conditions, because we can no longer speak of planarism.

The originality of the prism and of the optics used in an embodiment of the device of the present application resides in the fact that, whatever the angle beam incidence at the prism / sensitive layer interface (within the variation limits indicated for the angle of incidence on the entry face of the prism), the inclination of the intermediate image by the face output from the prism varies little and remains almost perpendicular to the optical axis of the detection system. Thus, a completely reflected ray starting from the center of the useful area will present at the exit of the prism a small angle compared to the optical axis of the imaging system, while the corresponding image point on the detector will remain approximately in the limits of the depth of field defined by this system.

A major advantage of the device of the invention is that all of the optical elements are fixed (with the exception of the rotating mirror) and the optics are not very open.

(they therefore have fewer aberrations and allow a significant depth of field), and this without altering the performance of focusing and angular acceptance, which can range in particular up to 16 °, of the system.

Other objects and advantages of the invention will appear during the following description with reference to the accompanying drawings given by way of non-limiting example, in which:

FIG. 1 represents a diagram of the device according to the invention,

- Figure 2 shows a diagram of the reaction cell according to the invention. The device according to the invention comprises:

- a light source 1,

a first collimation system 2 of the incident light beam emitted by the light source,

- a reaction cell 3 comprising at least one prism 4, - an imaging and detection system 5 of the beam reflected by the base of the prism.

The non-coherent light source 1 is for example a light emitting diode of narrow spectral width. It may for example be a light-emitting diode with a wavelength of 660 nanometers and a spectral width of 30 nanometers.

The beam emitted by the light source 1 is collimated by a collimation system 2. This collimation system 2 is for example a system of two microscope objectives making it possible to make the light beam parallel. The light is then polarized by a polarizer 6 in order to be able to excite the surface plasmon.

This polarized light illuminates the entry face of a glass prism 4 of the reaction cell 3. In one embodiment, the polarized light is reflected by an oscillating galvanometric mirror 7 towards the entry face of the prism 4 such as shown in FIG. 1. The oscillating mirror 7 is conjugated on the base of the prism using two lenses 8 and 9. It is thus possible to vary the angle of incidence of the beam on the prism by the rotation of the mirror oscillating 7 around an axis 7a perpendicular to the plane of the drawing. The oscillating mirror 7 undergoes a partial rotation, controlled for example by a low frequency generator.

The rotational movement of the oscillating mirror rotation device 7 can be discretized using a continuous component in order to be able to fix the angle at which one wishes to carry out the experiments. The diameter of the mirror 7 is such that it allows the entire incident beam to be reflected on the prism 4.

In the embodiment considered, the diameter is at least 10 mm. The total travel of the mirror 7 is 16 ° for a frequency of 300 megahertz. During its rotation, the oscillating mirror 7 has a continuous stroke with a fixed equivalence. The lenses 8, 9 used to conjugate the mirror 7 with the prism 4 can for example be focal lenses F '= 75 mm with a diameter of 35 mm for an excursion of 19 °, or focal lenses F' = 50 mm with a diameter of 25 mm for an excursion of 17 °.

In Figure 1, there is shown in solid lines the limits of the light beam for the position of the mirror 7 shown, and in broken lines the extreme limits of this beam when the mirror 7 occupies the extreme positions of its angular travel.

The angle of incidence of the beam reflected by the mirror 7 on the prism 4 can be determined in two different ways. In a first embodiment, the part of the beam reflected by the entry face of the prism is collected by a strip of CCD charge coupled circuits 10 of sufficient length.

This CCD strip 10 makes it possible to detect the incidence of the beam. The beam thus reflected by the entry face of the prism is focused on the CCD strip 10 using a lens 11 whose focal length is chosen so as to use the entire surface of the strip 10.

For a CCD strip 10 of length L = 12.7 mm composed of 512 pixels, it is possible for example to use a lens 11 with focal length F '= 48 mm and diameter 30 mm.

In another embodiment, not shown, the rotation of the oscillating mirror 7 is ensured by a motor making it possible to determine the position of incidence for each position of the mirror. One can for example use a variable pitch motor. The reaction cell 3 comprises at least one glass prism 4 whose geometry and index are such that the intermediate image of the object plane in the prism 4 is almost parallel to the detection plane of the imaging and detection system 5 .

In one embodiment, the prism is made of glass of index 1.8 and has the characteristic of an apex angle of 40 °, a base of 10 mm in width and 25 mm in height. The distance between the two parallel faces of the prism is 8 mm.

A glass slide of 12 with the same index as the prism 4 is fixed to the base of the prism by means of an index adaptation oil.

On this glass slide 12, a thin layer of chromium 13, a thin layer of gold 14, and a layer to be characterized 15 are successively deposited. The layer of chromium 13 ensures the adhesion of the gold layer 14 on the glass slide 12 in the presence of an aqueous medium. The thickness of the thin layer of chromium 13 is for example between 1.5 and 2 nanometers.

The thickness of the thin layer of gold 14 is for example between 40 and 50 nanometers and preferably of the order of 45 nanometers. For an application to a qualitative and quantitative measurement of molecular interactions, the layer to be characterized is, for example, an organic layer 15.

The organic layer 15 can be deposited in the form of a continuous layer or in the form of spots or studs, as indicated above. In the case of a continuous layer, containing a single kind of ligand (for example an antibody), it solutions of analytes (for example peptides for the search for minotopes) should be deposited from time to time in the form of spots, then rinsed to remove the analytes which are not specifically bound.

In the case of an organic layer in the form of spots, the sensitive layer in contact with a fluid containing an analyte to be studied.

The ligands are immobilized on the gold layer in a known manner.

For example, for the study of interactions between oligonucleotides and single-stranded DNA fragments to be studied, the organic layer 15 used for the attachment of oligonucleotides to the gold layer 14 by means of a polymer such as for example polypyrrole, the oligonucleotides being grafted onto the polypyrrole. This polymer has the advantage of being very stable, which makes it possible to reuse the organic layer 15 comprising the probes (immobilized oligonucleotides) several times. The production and deposition of such a layer of polypyrrole grafted onto a layer of gold are for example described in patents EP 0 691 978 and FR 2 789 401.

Once the glass slide 12 is fixed on the base of the prism 4, it is hermetically covered with a tank 16.

This tank 16 is for example made of Teflon and makes it possible to pass solutions of analytes using one or more pipes 17. A peristaltic pump 18 with variable flow rate can then be used to circulate these solutions.

In order to optimize the introduction of solutions, the peristaltic pump 18 meets the following criteria:

- be remotely controllable;

- be able to generate flow rates between a few micro liters per minute and a few hundred micro liters per minute (of the order of 300 to 400 micro liters / min);

- allow input / output control of the rotation speed at the start and end of the injection.

The entire reaction cell 3 (prism 4 and tank 16) can be enclosed in an adiabatic enclosure 19 shown in FIG. 2, in order to control and fix the temperature of the system and of the products injected.

The temperature will for example be maintained at 37 ° in order to allow the detection of any type of biological molecules.

The adiabatic enclosure 19 preferably comprises an opening (not shown) allowing easy access to the detection cell 3.

It may in particular include a heating base 20, such as for example a copper resistor, to which the Teflon reaction vessel 16 will be fixed. This base 20 is covered with a cover 21 encompassing the entire detection cell 3.

Side portholes 22, 23 are provided in the cover 21 in order to allow the light beams to pass on either side of the prism. They are for example made of glass or any other material allowing light beams to pass through without disturbing them. A winding (not shown) wound around the pipe 17 of the tank 16 keeps the products injected at the temperature of the cell.

An imaging and detection system 5 is arranged on the side of the outlet face of the prism 4 in order to recover the beam reflected by the base of the latter.

This system includes an afocal conjugation and magnification system 24 and a CCD camera 25.

The afocal magnification system 24 makes it possible here to enlarge the image of the useful area of the gold blade 14 on the entire CCD camera 25.

The useful area of the gold strip 14 is for example of the order of approximately 25 mm 2. In the embodiment shown, an enlargement G = 1.6 is achieved and a digital opening system O.N. = 1.6 is used to combine the image on the plane of the CCD camera 25.

The afocal system 24 can for example comprise a lens 26 with a focal length F '= 50mm and a diameter of 35 mm, then another lens 27 with a focal length F' = 80 mm and a diameter of 40 mm.

The lens 26 is arranged so that the image of the useful part of the active layer after refraction on the exit face of the prism is located in the object focal plane of 26. As a result, the image is formed in the focal plane image of the lens 27.

The conjugation system can also consist, for example, of a first focal lens 80 mm and of diameter 31.5 mm, and of a second focal lens 65 mm and of diameter 25 mm (the assembly having an opening of 2.6). To design this optical system, commercial optical calculation software such as CODE V (Optical Research Associates) can be used.

The CCD camera 25 (or simply a CCD matrix) can for example have a sensitive surface of 6.4 mm x 5.8 mm composed of 768 x 576 pixels.

The CCD detector can also be, for example, a 1.5 inch format remote head camera, with a 6.4 x 4.8 mm ² sensitive surface, comprising 751 x 582 active pixels, with a signal sampled on 8 bits, or else a compact camera 2/3 inch format, sensitive surface 8.57 x 6.86 mm ² , comprising 1280 x 1024 active pixels with a 12-bit sampled signal. These are commercial cameras.

All the elements of the device according to the invention are controlled automatically by means of a computer chain not shown. The method of using said device comprises the following steps:

- Angle scanning of the surface of the glass slide 12 covered with the gold layer 14 and the layer to be characterized 15;

- Determination of the angle of incidence for which the sensitivity of the imaging and detection system 5 is maximum for the entire blade 12; - positioning of the oscillating mirror 7 to obtain this angle of incidence;

- measurement of the reflectivity by the imaging and detection system 5 as a function of time.

The device is then used to characterize the surface of the active areas to be characterized of layer 15. The device can be applied to the study of molecular interactions.

In order to measure the interactions between the organic layer 15 and the analytes, the step of measuring the reflectivity is carried out simultaneously with the introduction into the reaction cell 3 of (unlabeled) analytes, including the interactions with the ligands of the organic layer 15 are to be analyzed. The evolution of the interactions between the organic layer 15 and the analytes introduced is thus measured.

These measurements can be performed under non-selective conditions, so that all interactions can occur and be studied.

The imaging and detection system 5 makes it possible to measure the optical thickness, product of the geometric thickness and the refractive index of the medium, at all points of the useful part of the gold layer 14.

It is thus possible to visualize topologically the zones of equal optical thickness and to characterize the surface.

Thus, during the angled scanning of the surface of the glass plate 12 covered with the gold layer 14, the system 5 makes it possible to determine the geometric thickness of each point of the layer 14 and therefore to check the state of surface of it.

The contrast measured during scanning also serves as a reference level for real-time measurement.

During the measurement in real time, the imaging and detection system 5 measures the variation of the reflectivity relative to the reference level recorded during the angular scanning. This measurement of the variation of the reflectivity is carried out at every point of the useful part of the gold layer 14.

The reflectivity varying according to the molecular species present on the surface, this measurement allows a qualitative analysis by determining the different interactions between the organic layer 15, the gold layer 14 and the molecules introduced. Thus, when we position ourselves at the angle optimizing the sensitivity of the device, a positive variation in the reflectivity signifies the presence of analytes interacting with the ligands of the organic layer 15, a null variation signifying the absence of interacting analyte.

A negative variation then represents on the contrary a loss of material, that is to say a degradation of the layer 15.

Measuring the amplitude of the variation in reflectivity in real time allows access to the number of analyte molecules per unit area and therefore to the concentration at each point (on each spot) of the layer d or 14 of the analyte molecules introduced. The device according to the invention therefore also allows a quantitative analysis of the molecules having interacted with the ligands of layer 15.

Depending on the strength of the bond between the layer 15 and the gold layer 14 and the nature of the interactions studied, it will be possible at the end of the measurement to regenerate the organic layer 15 by eliminating all the analyte molecules which have reacted with it. It is then possible to reuse layer 15 to study interactions with new analyte molecules.

In order to optimize the sensitivity of the measurement, it is also possible to optimize the distribution of the spots fixed on the layer 15. It is indeed necessary that the number of probes (ligands) per spot is sufficiently high to be distinguished from the background noise of the measured signal, without the probes being too close to each other. Indeed, a large number of probes results in a large steric hindrance and hinders the interaction of molecules with neighboring probes. As part of the study of interactions between oligonucleotides and single-stranded DNA fragments to study the method according to the invention has many advantages. The interactions are studied under non-selective hybridization conditions, for example at a temperature of the order of 37 ° C., so that all of the interactions between the reagents can be observed simultaneously. The device makes it possible to follow several hundreds of molecular interactions at the same time and without markers. In particular, it is capable of discriminating the presence of a point mutation within a DNA fragment.

The discrimination technique is based on the fact that the molecular association can either be total if the DNA sequence is strictly complementary to that of the oligonucleotide, or partial if the DNA sequence has a mutated base.

The thermodynamics applied to DNA shows that depending on the type of mutation, for a fixed location in the sequence, the optical thickness varies. It follows that the device can precisely determine the type of mutation. Under non-selective conditions, if a normal sequence and the three sequences carrying the mutation of one of the bases of said sequence, that is to say the four sequences of which one of the bases has been mutated, are deposited in the form of four different pads on the gold layer 14, the device can measure a different signal for each of the immobilized sequences. Indeed, whatever the sequence of the fragment to be analyzed, the device measures a total interaction and three partial interactions.

Depending on the immobilized sequences, it suffices to refer to the existing tables giving the interaction strength between bases to go back to the exact nature of the DNA sequence. Consequently, if the detection system is calibrated, for example with respect to the value of a completely complementary signal, it is no longer necessary to immobilize the normal sequence and the three mutated sequences: the immobilization of a single sequence is sufficient.

The capacity to address DNA sequences on a solid substrate is therefore multiplied by four.

The device according to the invention following in real time and directly the molecular interactions, it is not necessary to carry out the measurement at a temperature promoting a single interaction as in the case of the methods using fluorescent markers.

Indeed, these methods only reveal a single interaction visible at a given temperature. If these methods were used at room temperature, all the markers would fluoresce at the same time and therefore could not be distinguished.

The following examples illustrate the invention. Examples of prism configuration

1) Data for a glass of index n = 1.776: - Material: SF11 (supplier: SCHOTT).

- Optimal sum angle: 30.6 °.

- Coefficient of anamorphosis: 0.479.

- Ratio between the size of the aberrations and the size of the image: 4.17%.

2) Data for a glass of index n = 2.20:

- Material: lithium niobate LiNbO (supplier: CRYSTAL TECHNOLGY, Inc., Palo Alto CA).

- Optimal apex angle: 77.4 °.

- Coefficient of anamorphosis: 0.7. - Ratio between the size of the aberrations and the size of the image: 1.57%.

3) Data for a glass of index n = 3.33:

- Material: GaAs (supplier: AXT, Inc., Palo Alto CA).

- Optimal apex angle: 123.7 °. - Coefficient of anamorphosis: 0.89.

- Ratio between the size of the aberrations and the size of the image: 0.5%.

The dimensioning of the prisms was carried out using the MATLAB software (MATH WORKS company), which makes it possible to establish appropriate algorithms, according to known methods. For a given index n, there is at least an angle at the top allowing the intermediate image of the base of the prism across the exit face to be perpendicular to the optical axis of the second afocal conjugation system. We then seek in the same way the couples (index - angle at the top) which give a minimum angular clearance for this intermediate image. The prisms can also be produced using single crystals such as lithium tantalate (LiTaO ₃ ), lithium niobate (LiNbO ₃ ).

These are birefringent materials, that is to say that they may have a different index depending on the state of light polymerization. This drawback can be overcome for example by orienting the axes of the crystal so that the effective refractive index, in transverse magnetic polarization, is the same regardless of the angle of incidence of the beam on the useful area.

Another solution is to use semiconductor materials such as gallium arsenide, germanium (index of the order of 4) or even silicon (index of approximately 3.9).

All of these materials have good infrared transmission, which is favorable given the interesting characteristics of surface plasmons at high wavelengths.

Examples of applications:

1) Discrimination of specific transfers

We have a sensitive zone provided with four DNA sequences 25 bases long, a normal or native sequence (WT) of the K-ras gene, a test sequence (CP) which will serve as a negative control and two mutated sequences, Ml and M5 which differ from the native sequence by only one base on the same codon.

We know that mutations in the K-ras gene almost always cause cancer, hence the need to identify individuals with a mutation. These four sequences were immobilized, in the form of four pads, covalently, by an electrochemical process described in patent applications WO 94/22889 and WO 00/47317.

The first step is to study the reflectivity of the sensitive area as a function of the external incidence, to determine the angle where to stand to observe the hybridizations with maximum sensitivity. Next, the complementary sequence of CP is injected into a hybridization buffer at 22 ° C (BPS, 137 mM NaCl), in order to demonstrate that only the CP pad reacts, which is the case. The experiment having guaranteed by the preceding stage that the system is selective, one injects then, after having regenerated the system with a 50 mM sodium hydroxide solution, the complementary sequence of WT, which reacts very well with the WT plot, little with the two plots corresponding to the mutated sequences and not at all with the CP plot. Then one regenerates and one injects the complementary sequence of Ml, which reacts very well with the plot Ml, little with WT and M5 and not at all with CP. The last injection gives the expected results: the complement of M5 reacts very well with M5, little with WT and Ml and not with CP.

We can measure the affinities for each of these hybridizations and affirm that according to the number of molecules having interacted on the native or mutated plots, and according to the value of the affinity, we can discriminate in this way a point mutation and even identify the type. of mutation.

After establishing the standard reflectivity curves, as a function of time, for the four types of hybridization, it is possible to detect, by comparison, a point mutation with only two probes, as indicated in the description.

2) Analysis of protein-protein interactions

There is a sensitive zone provided with four studs, three of which carry antibodies. Two antibodies are directed against hCG, the human pregnancy hormone which, when detected in humans, is very often indicative of testicular cancer. The third antibody is an anti-rabbit antibody, which does not cross with humans. It was simply absorbed on a polymer pad. The last pad contains only polymer, without antibodies.

First, bovine serum albumin is injected, which limits the passive adsorption of molecules not specifically recognized, until saturation. The hCG is then injected into a PBS buffer. The stud carrying only polymer has zero kinetics (no change in reflectivity as a function of time). The pad carrying the anti-rabbit antibody has a decreasing kinetics; this means that the antibodies, weakly bound to the polymers, are desorbed and evacuated from the reaction vessel. The two other studs each have an exponential kinetics, a sign of the recognition of hCG by the antibodies. However, the two anti-hCG antibodies being different, there is a difference in the slope at the origin of the interaction kinetics. Indeed, their affinity for hCG is not the same. For one of the antibodies, it has been deduced from the reflectivity results that the affinity is 8 × ^{10 −10} , ie the same value as that measured. independently by an ELISA technique.

The invention also relates to the application of the method described above to the qualitative and quantitative measurement of molecular interaction as described in the present application.

Claims

1. A method of characterizing the surface of a sensitive layer, said surface comprising active zones whose optical thickness is liable to vary over time, said sensitive layer being deposited on the basis of an optical prism, said method using a technique based on the analysis of the variation of the reflectivity of the prism-sensitive layer interface, in which: a) a collimated light beam is entered into the prism by an entry face with an angle of incidence allowing total reflection on the basis of the prism, said beam illuminating a fixed part of the interface corresponding to a part to be characterized, called useful part, of said surface, and the reflected beam emerging in a beam emerging from the exit surface of the prism, b) an angle scan of the useful part is carried out, by varying the angle of incidence of said beam, so as to identify an incidence for which the reflectivity of a u at least part, or all, of the active zones is minimum, c) an optimal angle of incidence is determined, for which the detection sensitivity of the active zones is maximum, d) the incidence is adjusted to the value optimal determined in the previous step, e) and the reflectivity is measured using an imaging and detection system having a sensitive surface capable of receiving the entire emerging beam from the useful part.

2. Method according to claim 1, in which, after step d), a fluid containing or capable of containing at least one analyte capable of binding with a population of ligands immobilized on the zones is brought into contact with said sensitive layer. active.

3. Method according to claim 1 or 2, wherein one measures, in step e), the variation of the reflectivity of the active areas as a function of time.

4. Method according to claim 3, in which the results of said measurements are used to quantitatively characterize the interactions of at least one corresponding ligand / analyte pair, the corresponding analyte being that to which the ligand is capable of binding.

5. Method according to any one of claims 2 to 4, wherein the ligands and / or analytes are chosen from wild type or mutated oligonucleotides, wild type or mutated proteins, protein fragments, oligopeptides of random sequence, antibodies or antibody fragments, non-protein antigens, haptens, agonists or antagonists of natural ligands, cells, biological receptors and oligosaccharides.

6. Method according to any one of the preceding claims, in which a wild type oligonucleotide and a mutated oligonucleotide having any point mutation in any given position are used as ligands, and a DNA fragment in which one is used as an analyte search for a mutation in said given position, we compare the reflectivity curves obtained with pre-established reflectivity curves for the wild type and the 3 possible mutations at said position, and we deduce the mutation present in the analyte, in the case where it has a mutation at said position.

7. An imaging device, making it possible to implement the method of any one of the preceding claims, comprising:

an optical prism having an entry face, an exit face and a base provided with a sensitive layer, said prism being characterized by its angle at the top and by the refractive index of the material which constitutes it,

- A first afocal optical conjugation system, disposed on the side of the entry face of the prism, and whose optical axis, fixed relative to the prism, is oriented so that a collimated incident light beam having passed through it directs towards said entry face, refracts then strikes the base of the prism where it undergoes total reflection and emerges after refraction on the exit face by forming an emerging beam, said first conjugation system having an orientation and an opening such that said incident beam refracted on the input face comes to illuminate the whole of at least one so-called useful part of said sensitive layer, and to undergo a total reflection therein when the direction of the incident beam varies, on either side of said optical axis , over an angular range of at least 10 ° in total, said useful part containing active zones whose optical thickness is likely to vary over time, - a second afocal conjugate system optical ison disposed on the side of the exit face of the prism and whose optical axis, fixed relative to the prism, is oriented so that an incident beam parallel to the optical axis of the first conjugation system gives an emerging parallel beam to the optical axis of the second system of conjugation,

a detection system having a plane sensitive surface which is capable of receiving and analyzing the light having passed through said second conjugation system, and which is perpendicular to the optical axis thereof, in which:

(iii) said second conjugation system has an opening such that all of the light from the useful area passes through it when the direction of the incident beam varies over said angular range, and the second conjugation system is such that the all of the light from the useful area illuminates the sensitive surface of the detection system,

(iv) the apex angle and the prism index are such that when the direction of the incident beam is parallel to the optical axis of the first conjugation system, the intermediate image of the useful part, across the face of output from the prism, which constitutes the object for the second detection system, is perpendicular to the optical axis thereof.

8. Device according to claim 7, in which the apex angle and the prism index are such that when the direction of the incident beam varies over said angular range, the angular movement of the intermediate image of the useful area formed at through the exit face does not exceed the limits of the depth of field of the second conjugation system.

9. Device according to claim 7 or 8, wherein said angular range is at least 12 °, or at least 14 °, or at least 16 °.

10. Device according to any one of claims 7 to 9, in which the magnification of the second conjugation system is such that all of the light coming from the useful zone illuminates the whole of the sensitive surface of the detection system.

11. Device according to any one of claims 7 to 10, in which said second afocal system comprises a first dioptric system, by which the beam emerges between, and a second dioptric system by which light exits to direct itself towards the sensitive surface. of the detection system, and in which said intermediate image through the exit face of the prism is in the object plane of said first dioptric system.

12. Device according to any one of claims 7 to 11, in which said deflector optical system comprises a rotating plane mirror able to pivot about an axis parallel to an edge of the prism.

13. Device according to any one of claims 7 to 12, in which the detection device is capable of analyzing light so as to measure and / or compare the reflectivity of any point region of the useful part.

14. Device according to any one of claims 7 to 13, in which the detection device comprises a CCD or C-MOS matrix.

15. Device according to any one of claims 7 to 14, wherein the metallic thin layer comprises, on the active areas, at least one population of immobilized ligands.