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
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
  • G01N21/274—Calibration, base line adjustment, drift correction
  • G01N21/278—Constitution of standards
• • 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

Definitions

• Standard micro-component for calibrating or calibrating fluorescence measurement equipment and biochip comprising it.
• the invention relates to a standard micro-component for calibrating and calibrating fluorescence measurement equipment comprising a substrate on which is arranged at least one thin layer comprising fluorescent components, said micro-component comprising at least first and second fluorescence levels.
• the invention also relates to a biochip comprising said micro-component.
• the invention also relates to a method of manufacturing said microcomponent comprising the deposition on a substrate of at least one thin layer comprising fluorescent components.
• a known standard micro-component (FIG. 1) comprises a non-fluorescent glass substrate 1, on which is deposited a layer 2 of fluorescent organic material, 3 microns thick. It also has openings 3 formed in layer 2 by etching. This type of micro-component makes it possible to obtain a level of fluorescence corresponding to that of layer 2. However, the openings 3 are approximately 4 microns wide and are spaced from each other by 8 microns, which is not satisfactory for the calibration of the instruments generally used.
• the document WO-A-0159503 describes a standard micro-component comprising a fluorescent layer deposited on a substrate. It is generally used to establish a reference base between different microscopes and to characterize an image quality, for example in terms of resolution, contrast, depth of field and distortion.
• the layer is covered by a thin mask, having openings and not fluorescent.
• the mask and the fluorescent layer are plated one on top of the other, which requires three manufacturing operations: the production of the layer, the production of the mask and their assembly.
• the mask and the layer being made of two different materials, they cannot be placed on the same optical plane, at the risk of distorting the optical image of the observed area.
• Document DE-A-10200865 describes a standard for fluorescence detector, the standard comprising several levels of fluorescence respectively defined by zones of different thicknesses. Each zone of a predetermined thickness corresponds to the superposition of a predetermined number of thin polymer layers.
• the fluorescence characteristic of a thin layer depends on the rate of crosslinking of the thin layer of polymer, the rate of crosslinking being obtained by exposure of the thin layer during a photolithography step. It is also indicated that the phenomenon of oxidation of the fluorescent components due to insolation (so-called "bleaching" phenomenon) is a harmful phenomenon which is avoided in the standard described in document DE-A-10200865. Such a standard is however impractical to implement, its manufacture requiring a succession of long and tedious production steps and the standard thus produced can prove to be bulky. Subject of the invention
• the object of the invention is to provide a standard micro-component which does not have the drawbacks of standard micro-components of the prior art and which is easy to produce.
• first and second levels of fluorescence are respectively defined by a non-exposed part and by at least one exposed area of said thin layer, the second level of fluorescence being lower than the first level of fluorescence .
• the thin layer comprises at least one opening defining a third level of fluorescence lower than the first and second levels of fluorescence.
• the thin layer comprises a plurality of exposed areas so as to define a plurality of different fluorescence levels.
• the standard micro-component comprises a plurality of superimposed thin layers, so as to define a plurality of fluorescence levels.
• the invention also relates to a biochip comprising, on the same substrate, at least one biological probe and at least one standard micro-component as described above.
• the object of the invention is also a method of manufacturing such a standard microcomponent.
• this object is achieved by the fact that the method consists in exposing at least one area of the thin layer so that first and second levels of fluorescence are defined respectively by the non-exposed portion and by the area insolated from the thin layer.
• Figure 1 is a schematic representation of a standard micro-component according to the prior art.
• FIG. 2 schematically represents a first embodiment of a standard micro-component according to the invention.
• Figures 3 and 4 show a second embodiment of a standard microcomponent according to the invention, respectively before and after etching of a second thin layer.
• Figure 5 is a schematic representation of a biochip comprising a standard micro-component according to the invention.
• Figures 6 and 7 show third and fourth embodiments of a standard micro-component comprising a thin protective layer according to the invention. Description of particular embodiments.
• a standard micro-component 4 intended for calibrating or calibrating fluorescence measurement equipment such as confocal or non-confocal fluorescence microscopes, comprises a substrate 1, non-fluorescent, on which is placed at least one thin layer 2.
• the substrate 1 is preferably made of a material chosen from silicon, silica, quartz, plastics and glasses.
• the thin layer 2 comprises fluorescent components defining a first level of fluorescence. It can be made of fluorescent material or include particles or fluorescent molecules. Thus, it can be constituted by a photosensitive resin, fluorescent or comprising fluorescent particles, such as Duramide® 7505 sold by the company OLIN Microelectronic Material.
• the thin layer 2 is deposited on the substrate 1, by any type of known method.
• it can be deposited by a chemical vapor deposition process, under low pressure called the LPCVD process (“Low Pressure Chemical Vapor Deposition”), or under plasma also called process
• the thin layer 2 can also be produced by a deposition of tetraethoxysilane (Si (OC 2 H 5 ) 4 or TEOS), by a process of deposition by centrifugation of a layer of photoresist known under the Anglo-Saxon terms of “spin- coating ”, by localized deposition of resin (“ lift-off ”process), by evaporation, by spraying or by dip-stretching.
• the thin layer 2 preferably comprises at least one opening 3 freeing the surface of the substrate 1. In FIG. 2, seven openings 3 are formed in the thin layer 2 and they define a second level of fluorescence corresponding to the level of fluorescence of the substrate 1.
• the level of fluorescence of the substrate is at least 10 times lower than the first level of fluorescence of the thin layer 2, and preferably 100 lower than the first level of fluorescence.
• All of the openings 3 form patterns and they are produced by any type of known means. They are, for example, formed by etching, by photolithography, by photolithography followed by etching ("lift-off" process). Thus, for a thin layer 2 of photosensitive resin, the openings 3 are preferably produced by a conventional photolithography step (exposure then chemical development).
• the thin layer 2 comprises at least one zone 2a exposed by a light source 5, which is, for example, a mercury vapor lamp. Two zones
• the zones 2a then have an intermediate level of fluorescence, lower than the first level of fluorescence defined by the non-exposed part of the layer thin 2 not exposed and, in the example described, greater than the second level of fluorescence of the openings 3.
• the choice of parameters such as the wavelength, the power and the time period of the light radiation emitted by the light source 5 determine the intermediate level of fluorescence, so that it is lower than the first level of fluorescence of the non-exposed thin layer and higher than the second level of fluorescence, that is to say, most often, not zero. These parameters are adjusted according to the type of material constituting the thin layer as well as the thickness thereof.
• the fluorescence level of a thin layer of Duramide® 7505 resin of approximately 10 microns thick can be halved by insulating the thin layer with a mercury vapor lamp, with a power of 14500W / m 2 and an exposure time of 240 minutes.
• the micro-component 4 has the advantage of being easy to produce. Indeed, the implementation techniques are usual techniques in microelectronics which make it possible to achieve pattern dimensions of the order of 0.3 ⁇ m. They make it possible to collectively manufacture a large number of standard microcomponents on the same substrate and the number of production steps is limited.
• a method of manufacturing a micro-component consists in depositing on a substrate at least one thin layer comprising fluorescent components and in exposing at least one area of the thin layer so that first and second fluorescence levels are respectively defined by the non-exposed part and by the exposed part of the thin layer.
• the thin layer 2 may include a plurality of exposed areas, so as to define a plurality of different intermediate levels of fluorescence. Fluorescence levels intermediates are determined according to the global local characteristics of sunshine (power and duration of sunshine). These global characteristics are obtained during one or more successive, independent or complementary sunstrokes
• the standard micro-component can additionally comprise a plurality of superimposed thin layers, which can be totally, partially or not exposed, so as to define a plurality of fluorescence levels.
• Each thin layer has at least one opening 3 and the openings 3 of at least two layers can be superimposed.
• the method of manufacturing such a micro-component then comprises depositing, on the substrate, a plurality of superimposed thin layers.
• This has the advantage of producing a standard micro-component having dimensions equivalent to those of the objects that the reader, whose calibration or calibration is desired.
• the thickness of the fluorescent material constituting the patterns is close to that of the areas to be measured on biochips, for example. This makes it possible to calibrate the reader under optical conditions equivalent to those of usual readings.
• the thickness of the standard micro-component is preferably less than 50 microns, see 10 microns.
• a second thin layer 6 is deposited by any type of suitable means on the standard micro-component 4 comprising a first layer 2 such as that described in FIG. 2.
• the second layer 6 then covers the openings 3 , the first thin layer 2 and the exposed areas 2a.
• the first and second layers 2 and 6 have distinct fluorescence characteristics, either by the nature of the respective fluorescent components that they contain, either by their respective concentrations of fluorescent components.
• a part of the second layer 6 is then removed (FIG. 4) by any type of suitable means, so as to form zones 6a, 6b and 6c, respectively covering part of the zones 2a of the first thin layer 2, part of the openings 3 and part of the thin layer 2.
• the zones 6b define a third level of fluorescence corresponding to the fluorescence characteristic of the second thin layer 6.
• the accumulation of several thin fluorescent layers on each other increasing d 'As much the level of fluorescence, the zones 6a and 6c, respectively superimposed on the zones 2a and on the thin layer 2, define a fourth and a fifth level of fluorescence.
• the fourth and fifth levels of fluorescence are greater than the highest level of fluorescence of the first and second layers 2 and 6 which are not exposed.
• the standard micro-component 4 according to FIG. 4 then has 5 different fluorescence levels.
• a biochip 7 comprises a substrate 1, on which biological probes 8 and the standard micro-component are deposited 4. It is then possible to produce biochips comprising, on the same substrate, at least one micro - standard component and at least one biological probe.
• the fluorescence levels of the standard micro-component can also be stabilized over time, by depositing, after exposure, at least one thin protective layer, on at least part of the thin layers. of the standard micro-component.
• the thin protective layer makes it possible to isolate, from the external environment, at least part of the thin layers.
• a micro-component 4 of the type represented in FIG. 2 comprises a non-fluorescent substrate 1, on which is arranged at least a first structured thin layer 2.
• the thin layer 2 can also consist of biological molecules marked by particles or fluorescent molecules.
• this layer is produced and defined by any type of process known in the field of biochips (functionalization, hybridization, adsorption, etc.).
• the micro-component comprising this type of thin layer can then serve as a reference biochip.
• the first thin layer 2 is covered with a thin protective layer 9, intended to isolate the first thin layer 2 from the external medium in which the micro-component 4 is located.
• the external medium is generally air.
• the protective layer 9 prevents oxidation of the fluorescent components contained in the thin layer 2, which makes the fluorescent components stable over time,
• the protective layer can be opaque or semi-transparent, when the micro-component is read through the substrate.
• the substrate is then transparent to the optical reading signals and which can be, for example, glass, silica or plastic.
• the protective layer 9 must be transparent to the optical reading signals received and returned by the first thin layer 2. This makes it possible to excite and to observe the phenomenon of fluorescence, without disturbing it.
• the thin protective layer 9 is produced by any type of process suitable for the requirements of the protective layer 9. For example, it can be produced by an LPCVD, PECVD process, by evaporation, by spraying or by "spin- coating ".
• the protective layer 9 can be structured by any type of means known in microelectronics, so as to cover, for example, at least part of the fluorescent zones.
• the thin layer 2 can be covered by a plurality of superimposed thin protective layers.
• the thin protective layer or layers can be used to reinforce the fluorescence characteristics of the thin layer 2.
• the thin protective layers can be of the type of thin layers described in document WO-A-0248691 .
• the material forming the thin protective layer can be chosen from the following materials: Ti0 2 , Ta 2 0 5 . Hf0 2 , Zr0 2 , MgO, Si0 2 , Si 3 N 4 , MgF 2 , and YF 3 ⁇ Al 2 0 3> Zr0 4 Ti, Y 2 0 3 , diamond and oxynitrides.
• the optical thickness corresponds to the product of the refractive index n with the thickness of the thin layer considered, for the wavelength considered.
• the standard micro-component can, as in FIG. 4, comprise a plurality of thin layers 2 and 6 superimposed, so as to define a plurality of levels of fluorescence.
• the protective thin layer 9 is deposited on the micro- component 4, so as to completely cover, for example, layers 2 and 6 and the exposed parts of the substrate 1.
• microcomponents such as standard chips or standard micro-components, having fluorescence characteristics stable over time, which makes it possible to make comparisons between several measurements spread over the time or between different measuring devices, with respect to a time invariant reference.
• the invention is not limited to the embodiments described above.
• at least part of the second thin layer 6 can also be exposed, at the same time as the zones 2 a, before or after, with exposure parameters, such as the wavelength, the duration or the power d sunstroke, different or identical.

Landscapes

• Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• Pathology (AREA)
• General Physics & Mathematics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Theoretical Computer Science (AREA)
• Mathematical Physics (AREA)
• Engineering & Computer Science (AREA)
• Spectroscopy & Molecular Physics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
• Sampling And Sample Adjustment (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=32338898

Family Applications (1)

Country Status (6)

Families Citing this family (6)

Family Cites Families (3)

• 2002
  • 2002-12-17 FR FR0216012A patent/FR2848670B1/fr not_active Expired - Fee Related
• 2003
  • 2003-12-10 AU AU2003296803A patent/AU2003296803A1/en not_active Abandoned
  • 2003-12-10 EP EP03815387A patent/EP1573304A1/de not_active Ceased
  • 2003-12-10 US US10/538,866 patent/US20060060931A1/en not_active Abandoned
  • 2003-12-10 WO PCT/FR2003/003656 patent/WO2004065946A1/fr not_active Ceased
  • 2003-12-10 JP JP2004566985A patent/JP2006510916A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events