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
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Definitions

• the invention relates to a method for amplifying a fluorescence signal emitted by a surface sample supported by a support in response to an excitation signal. It also relates to a device amplifying the fluorescence emitted by such a sample.
• Fluorescence measurement is a measurement method used in many technical fields. It is used in particular to exploit the chemical and / or biological analysis devices known under the name of biochip.
• the measurement of the biological activity with such a chip is done by a measurement of the fluorescence emission of a molecule attached to the biological sample, which is in the form of a surface coating, using for example an epifluorescence microscope, a scanner, a fluorimeter.
• the chip can be placed in a special chamber or cartridge. It can also be placed directly on the bottom of a Petri dish. It can still be placed in the air thanks to a mechanical support.
• the surface sample is read in so-called rear face mode, that is to say that the reading is done by crossing a support supporting on one of its faces the surface sample, the support necessarily being transparent to the fluorescence signal.
• the fluorescence excitation signal is generally directed towards the sample through its support which advantageously has the shape of a slide.
• Another way to excite the fluorescence is to generate evanescent waves by injecting the excitation light through the edge of the slide or by coupling with a prism.
• the surface sample is read in so-called front face mode, the reading taking place directly on the surface sample disposed on the support.
• the surface sample can be formed from a biospecific surface formed on one face of the support and serving as a capture phase for a body carrying a fluorescent marker. It is thus possible to form a complex, for example a duplex of nucleic acids.
• the fabrication of the biospecific surface can be carried out by a combinatorial synthesis of the probes, by deposition of the probes by a projection technique or in another way.
• This complex can also be an antibody-antigen association, the antibodies deposited on the face of the support forming the biospecific surface.
• the thickness of the surface forming the complex is between a few nanometers and a few hundred nanometers.
• the complex can also be brought to the surface after its formation, for example by drying or adsorption of the complex on the face of the support.
• the gain in fluorescence is then a function of the position of the molecules relative to the silver islands. If there is contact, the molecules are adsorbed on the surface and the fluorescence is inhibited as indicated in the article by K. SOKOLOV et al. entitled “Enhancement of molecular fluorescence near the surface of colloidal metal film", Analytical Chemistry, Vol. 70, n ° 18, pages 3898 to 3905, September 15, 1998.
• International application WO-A-99 / 23,492 discloses a fluorescence amplification technique which uses a surface capable of enhancing fluorescence, this surface being interposed between a support and the biological complex deposited on the support, the measurement being carried out through the support. This intermediate surface must however be textured and, for this, the material forming this surface is chosen from nylon membranes, material and texture causing the diffusion of the fluorescence signal, phenomenon which the invention avoids.
• Document US-A-5 822 472 discloses a method for detecting evanescently excited luminescence.
• the method uses a transparent substrate supporting a transparent guide layer wave.
• the material of the transparent layer has a refractive index greater than the refractive index of the material of the transparent substrate.
• the device also has two coupling networks. One of these networks makes it possible to introduce an excitation light beam, passing through the transparent substrate, into the transparent layer.
• the excitation beam is carried by the transparent layer forming the waveguide. Substances in contact with the transparent layer and having luminescent properties are then excited in the evanescent field of the waveguide layer.
• the coupling network ensures the exit of the excitation beam from the transparent layer, the outgoing beam then passing through the transparent substrate.
• the transparent waveguide layer has a thickness less than the wavelength of the excitation light. Its refractive index is ⁇ 1.8.
• the excitation beam is therefore transported by the transparent layer between a beam introduction zone and a beam exit zone (the coupling networks).
• the guidance of the beam by the transparent layer necessarily implies a loss of part of the fluorescence signal.
• the invention proposes another way of obtaining the amplification of a fluorescence signal while avoiding the drawbacks of the methods or devices of the known art.
• the invention makes use of the fact that molecules placed on a surface constitute a discontinuity in the refractive index and that they emit most of their fluorescence in the medium which has the highest index.
• the angular distribution of fluorescence of molecules on the surface is very different from that of molecules in volume.
• the fluorescent emission is isotropic because the fluorescent molecules are not oriented relative to each other (random distribution of the orientation of the dipole moments).
• the phenomenon is different when the fluorescent molecules constitute a thin layer of ten nanometers to a few hundred nanometers.
• the signal emitted in fluorescence is a function of the orientation of the electric dipoles formed by the fluorescent molecules. It is therefore favorable, for surface fluorescence, to place the fluorescent molecules at the interface of two very different refractive index media.
• the excitation and fluorescence signals pass through the thin layer at near normal incidence, which is therefore not used as a waveguide. There can therefore be no loss of part of the fluorescence signal.
• this thin layer composed of a stack of sublayers of different materials, this thin layer having the same optical properties as the previous thin layer, in particular as regards the refractive index and the transparency to the signal. fluorescence.
• a first object of the invention is a method for amplifying a fluorescence signal emitted by a surface sample supported by a support in response to an excitation signal, the support transmitting all or part of the fluorescence signal, consisting of interposing a thin layer between the support and the surface sample, the thin layer having a refractive index greater than the index of refraction of the support and the refractive index of the medium surrounding the surface sample, the thickness of the thin layer being chosen so that the thin layer transmits all or part of the fluorescence signal which is measured after having passed through the transparent support.
• the surface sample can be supported by a support made of a material chosen from glass, quartz, silica, plastics such as polystyrene, polypropylene, polycarbonates, polymethylmetacrylates.
• the method may consist in interposing, between the support and the surface sample, a thin layer of a material chosen from silicon nitride, silicon carbide, titanium oxides, aluminum oxide, Zr0 2 , Zr0 4 Ti, Hf0 2 , Y 2 0 3 , diamond, MgO, oxynitures (Si x 0 y N z ), fluorinated materials like YF 3 or MgF 2 -
• This thin layer can also be obtained by stacking several sub -layers whose optical properties and thickness give the assembly represented by the latter the necessary characteristics (see A. HERPIN, CR Acad. Sciences, Paris, 225, 182, 1947).
• the thin layer can be a layer obtained on the support by one of the following methods: vacuum evaporation, replication, transfer, film deposition, by CVD type processes (LPCVD, PECVD ...) or PVD type, by film transfer, by sol-gel process. It can be a layer carried over the support by one of the following methods: bonding and molecular adhesion.
• said thin layer obtained is annealed on the support.
• the surface sample can be formed by a complex associating a biospecific surface with sample molecules carrying a fluorescent marker.
• the medium surrounding the sample can be a liquid, a gel or a gas.
• a second object of the invention is a device amplifying the fluorescence emitted by a surface sample by one of the above methods, the device comprising a support transmitting all or part of the fluorescence signal and intended to support the surface sample, a thin layer of a material being interposed between the support and the surface sample, the material of the thin layer having a refractive index higher than the refractive index of the support and the refractive index of the medium surrounding it surface sample during a fluorescence measurement, the thickness of the thin layer being chosen so that the thin layer transmits all or part of the fluorescence signal which is measured after having passed through the support.
• a third object of the invention consists of a biochip, characterized in that it comprises the above device, the device supporting a plurality of surface samples constituting as many recognition zones.
• FIG. 1 represents a device according to the present invention in a first configuration for reading a fluorescence signal
• FIG. 2 shows a device according to the present invention in a second configuration for reading a fluorescence signal
• FIG. 3 shows a device according to the present invention in a third configuration for reading a fluorescence signal.
• the invention consists in interposing a thin layer between a support transparent to the fluorescence signal and a surface sample immersed in a medium and in measuring the fluorescence through the support.
• the material constituting the thin layer is chosen so that its refractive index is greater than the refractive index of the material constituting the support and greater than the refractive index of the medium surrounding the surface sample.
• the medium surrounding the surface sample is for example a liquid buffer if one wishes to perfectly control the environment for fluorescence (pH, salinity).
• the medium can be a gel if it is desired to reduce the photodestruction or "bleaching" of the fluorescent molecules.
• This medium can also be a gas (air, neutral gas) if the fluorescent complex requires such reading conditions.
• the support is a silica blade (Si0 2 ) 700 ⁇ m thick and with a refractive index 1.485 at 650 nm.
• the thin layer may be a layer of silicon nitride (Si 3 N 4 ) 150 nm thick and with a refractive index 1.997 for the same wavelength.
• the surface sample can be a DNA-based complex labeled with a fluorescent cyanine (Cy5-Amersham, registered trademark).
• the medium bathing the sample may consist of a liquid buffer for washing (SSPE 6X / Triton x 100 at 0.005%), of index 1.34.
• an amplification of the fluorescence of 60% is measured compared to the measurement carried out under the same conditions for the complex deposited directly on the support, without the presence of the thin layer.
• the measurement is made with an epifluorescence microscope and a CCD camera.
• the fluorescence excitation is centered on 635 nm and the measurement of the emission is centered on 670 nm.
• the value of the gain depends on the measurement system (wavelength, digital aperture of the optics), on the marker used (orientation of the dipole moment) and on the characteristics of the thin layer (refractive index, thickness).
• FIG. 1 represents a device according to the invention arranged in a housing in order to carry out the fluorescence measurement.
• the device 10 consists of a transparent support 11, in the form of a blade, covered on one of its faces with a thin layer 12. A surface sample 13 is deposited on the free face of the thin layer 12.
• the device 10 is for example made up of the elements described above.
• the device 10 is placed, for the measurement of fluorescence, in a housing provided in the upper wall of a housing 20. It is placed so that the surface sample 13 is directed towards the interior of the housing 20.
• the housing has an inlet orifice 21 and an outlet orifice 22 in order to bring the surface sample into contact with a liquid 30 constituting the medium surrounding the surface sample.
• the fluorescence signal is collected by a measuring instrument 40. As clearly shown in FIG. 1, the fluorescence signal reaches the measuring instrument 40 by passing through the thin layer 12 and the transparent support 11. This is the reading mode says on the rear panel.
• FIG. 2 shows the same device 10 placed on the bottom of a Petri dish 50 containing the medium 30.
• the fluorescence reading by means of the measuring instrument 40 is also done on the rear face.
• FIG. 3 represents the same device 10 placed in the air to carry out the fluorescence measurement.
• the device 10 is held by its periphery by means of a mechanical support comprising a plate 60 pierced with an opening 61 allowing the surface sample to be in contact with the air 62.
• the support can be a glass, silica or polystyrene plate. It is transparent for the spectral range of the fluorescence measurement.
• the thin layer deposited on one face of the support can be manufactured by evaporation under vacuum - or by replication (optical thin layer techniques) for materials such as silicon nitride, titanium oxide, aluminum oxide. It can be deposited in the form of a film or transferred to the support (by bonding, by molecular adhesion) in the case of a thin layer of thickness greater than a few ⁇ m.
• Various tests have been carried out to demonstrate the effectiveness of the invention. The tests covered several devices:
• the thin layer of which is made of silicon nitride
• the thin layer of which is made of annealed silicon nitride (the annealing carried out under nitrogen makes it possible to densify the nitride layer).
• the fluorescence reading was carried out by means of an epifluorescence microscope equipped for the Cy5 marker and by means of a cooled digital CCD camera. Two measurements were made per device: a measurement on the front panel and a measurement on the rear panel.
• the front panel measurement is carried out by placing the surface sample in front of the measuring instrument, the fluorescence signal therefore not passing through the support or the device.
• the measurements performed show a 1.6-fold increase in fluorescence for a thin-film Si 3 N 4 device compared to a simple glass support, and a 2.4-fold increase in fluorescence for a coated device. thin in Si 3 N having undergone annealing compared to the simple glass support.
• the device of the invention can be used in a biochip comprising a plurality of molecular recognition zones.
• biochip is meant a chip or a support having on its surface one or more zones, called recognition zones, equipped with molecules having recognition properties.
• Recognition molecules can be, for example, oligonucleotides, polynucleotides, proteins such as antibodies or peptides, lectins or any other ligand-receptor type system.
• the recognition molecules can contain fragments of DNA or RNA.
• the recognition molecules are capable of interacting, for example by complexation or by hybridization with molecules called "target molecules" of the sample.
• target molecules molecules of the sample.
• the complexes formed on the biochip can be identified by means of fluorescent labeling applied to the target molecules of the sample.
• the support of the biochip is the support of the device of the invention coated with the thin layer and the recognition zones of the biochip are the surface samples.

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• Health & Medical Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Physics & Mathematics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

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• 1999
  • 1999-12-02 FR FR9915193A patent/FR2801977B1/fr not_active Expired - Fee Related
• 2000
  • 2000-12-01 EP EP00985381A patent/EP1234170A1/fr not_active Withdrawn
  • 2000-12-01 US US10/129,948 patent/US6893876B2/en not_active Expired - Fee Related
  • 2000-12-01 AU AU21817/01A patent/AU2181701A/en not_active Abandoned
  • 2000-12-01 WO PCT/FR2000/003359 patent/WO2001040778A1/fr not_active Ceased
  • 2000-12-01 JP JP2001542193A patent/JP5281222B2/ja not_active Expired - Fee Related

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