Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-03-28
2003-03-25
Siew, Jeffrey (Department: 1037)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S006120, C435S007100, C435S091100, C435S091200, C435S287200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C356S394000
Reexamination Certificate
active
06537801
ABSTRACT:
DESCRIPTION
1. Technical Field
This invention relates to a biochip comprising several molecular recognition areas and a device for reading such a biochip. A biochip means any chip or support with one or several areas (called recognition areas) on its surface, equipped with molecules with recognition properties. Throughout the rest of this text, the term biochip is used improperly, independently of whether the chip is used for a chemical or a biological analysis.
For example, recognition molecules may be trace nucleotides, polynucleotides, proteins such as anti-bodies or peptides, lectines or any other ligand-receptor type system. In particular, recognition molecules may include fragments of DNA or RNA.
When the biochip is brought into contact with a sample to be analysed, recognition molecules can interact, for example by complexing or by hybridisation with “target molecules” of the sample. Thus, by equipping a biochip with several recognition areas with different recognition molecules selectively sensitive to different target molecules, it is possible to detect and possibly quantify a large variety of molecules contained in the sample. Each recognition area contains only one type of identical molecules.
Complexes formed on the biochip may be identified by means of fluorescent marking applied to target molecules of the sample.
The read device according to the invention is intended to facilitate the operation to read marked or unmarked molecules that may be present in chip recognition areas.
Recognition areas may be read without the presence of any marker, and this type of technology is already known in the state of the art. In particular, some direct methods of detecting hybridisation include detection of a variation of the mass, a variation of the thickness, and a variation of the index. Photothermal methods are also known, and are described in document
1
that is mentioned in the references at the end of this description. Finally, Boccara et al. have described a photothermal deflection technique in documents
2
and
3
. Improvements to this technique were then described in document
4
. The references of these documents are given at the end of the description.
Thus, the invention is used for applications in the biological and chemical analysis fields.
Particular applications in the biological analysis field may include the search for polymorphisms and mutations, sequencing by hybridisation and monitoring the expression of genes.
2. State of Prior Art
The number of recognition areas in a chip varies depending on the type of analysis to be carried out. Thus, a distinction is made between “low-density” chips that comprise a few tens to a few hundreds of recognition areas, and “high-density” chips that may comprise several thousand or several hundred thousand areas of this type.
The size of recognition areas on high-density chips is small. The dimensions of these areas are less than 100 &mgr;m, or possibly even less than 10 &mgr;m.
As mentioned above, complexes formed on biochips are marked using fluorescent markers. For example, markers such as fluoresceine or phycoerythrine may be coupled directly on target molecules of the sample to be analysed. Target molecules may also be marked by means of indirect recognition groups such as biotin or digoxigenin.
Thus, when recognition molecules for a given recognition area have interacted or hybridised with marked target molecules, the fluorescent marker is fixed on these areas.
Reading a biochip includes excitation of fluorescent markers under the effect of light called the excitation light directed at the chip, and then recording of the fluorescence caused by the excitation light for each recognition area.
Detection of fluorescence in a recognition area enables a decision about the presence of target molecules (marked molecules) that could interact with the known recognition molecule, in the sample to be analysed, knowing the type of recognition molecule present in this area. The intensity of the fluorescence may possibly be measured to deduce the concentration of the target molecules concerned in the sample.
The reader can refer to documents such as 6, 7 and 8 for examples of these techniques, particularly for genetic biology applications. The references of these documents are given at the end of this description.
For low-density biochips, recognition areas may be read with imagery stations equipped with charge coupled devices (CCD). These stations are not very well adapted to high-density chips. CCD cameras should have a larger number of detection pixels, and since fluorescence light fluxes are particularly low for high-density chips, the cameras also need to be cooled to improve their signal to noise ratio.
Thus, for high-density chips, fluorescence scanners are used to scan the chip so that the recognition areas can be analysed in sequence.
These scanners are provided with a confocal optical system associated with a photo-electric sensor, to record the fluorescence in each area. The scanner can be used to observe objects with a very good spatial resolution (from 1 to 10 &mgr;m) and the confocal optical system is a means of overcoming parasite light emission effects (auto-fluorescence, specular reflection, etc.).
As an illustration of a scanner for high-density chips, the reader could refer to document 4, the reference of which is also given at the end of the description.
Fluorescence scanners output electrical signals that are acquired to form a two-dimensional image of the biochip. The signals are also used to recognise the spatial structure of the surface of the biochip and to identify and delimit the recognition areas located on it.
Finally, the intensity of the signal for each recognition area is recorded as the result of the analysis.
These analysis results can then be subjected to an appropriate computer processing to obtain biologically or chemically relevant information.
It is found that the signal processing mentioned above has the disadvantage that a larger number of measurement points, or pixels, are required for each recognition area.
A precise delimitation of each recognition area requires a sufficiently high pixel-image density for each recognition area.
In practice, it is found that the number of pixels needs to be of the order of 36 to 64 for each recognition area, depending on its size, to be able to perform signal processing under acceptable conditions.
Thus, signal processing for high-density biochips requires major computer data processing and storage means. Therefore, processing is expensive.
Furthermore, segmentation of the image based on recognition areas is not perfectly reliable.
Documents 9 and 10 mentioned in the references at the end of this description describe other possible means of reading a biochip that avoid some of the difficulties mentioned above.
Document 9 describes how to place recognition and marking elements on the chip to make reading easier, making it possible to envisage reading biochips using reading devices such as compact optical disk players (CD-ROM).
In document 9, the authors describe a chemical treatment for biochips to make them readable on a reading device distributed to the general public (and particularly CD-ROM compact optical disk players). Before analysing the sample containing target molecules to be analysed, molecular recognition areas on the biochip are covered with a reflecting film formed by metallic balls anchored to its surface by “bridge” molecules. These reflecting balls are functionalised to make them bond to target molecules in the sample. Therefore during hybridisation, the same target molecule needs to be bonded at two points; firstly to the surface of the biochip on molecular recognition areas, and secondly to the surface of one of the metallic balls located above this recognition area. After hybridisation, an appropriate chemical treatment breaks the bridge molecules anchoring the metallic balls to the surface, and the surface of the biochip is rinsed. Only the metallic balls retained at the surface by a minimum number of target molecules are present at thi
Fargeix Alain
Ida Michel
Lagrange Alexandre
Perraut François
Commissariat a l'Energie Atomique
Siew Jeffrey
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