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DEVICE FOR ANALYZING CHEMICAL OR
The invention relates to a method and to a device for analyzing chemical or biological samples. In particular, the 5 present invention relates to a novel support system for chemical or biological assays, in particular for use in DNA or RNA analysis, with it being possible for the biological probes or samples to be immobilized to be, for example, DNA, RNA, cDNA, oligonucleotides or PNA oligos. 10
Discovering new genetic information, or recognizing known genetic information, is an elementary molecular biological task, to achieve which a large number of different methods have already been proposed. High-throughput detection techniques are increasingly coming into use in 15 order to make it possible to handle the enormous amount of genetic information in biological systems. A popular representative of these techniques is the DNA CHIP, or the DNA array, which, by using a high density of applied probes (>1 000 per cm2), enables many thousand samples to be analyzed 20 simultaneously. In this connection, use is principally made of a conventional glass microscope slide whose planar surface is coated with DNA-binding reagents. In connection with genomic applications, the probes which have been loaded on and bound are termed target sequences or targets. The targets 25 have frequently already been identified genetically, which means that the gene sequence, and also in many cases the physiological function of the targets in the relevant organism, are known. This prior target information can be used to interrogate new systems, to establish relationships or differences, 30 to perform classifications or to investigate the biological purpose and function of the system. The biol. system to be investigated is applied, as the sample, to the slide and then hybridized. This involves the target DNA, which is fixed to the carrier surface, and the sample DNA, which is comple- 35 mentary to the target, locating each other and entering into a bond. If the sample DNA is, for example, labeled with a dye, the target can then subsequently be detected and the site or the position on the DNA array can be used to classify, and thus obtain preparatory information on, the target. Hybridization 40 to a DNA array can be used for qualitatively and/or quantitatively analyzing complex genetic information.
Northern and Southern blots, and also in-situ hybridization, are classical applications of this nature. For this, the samples are as a rule prepared appropriately and investigated 45 using defined DNA targets. Substances, i.e. what are termed labels, which can be identified using suitable detection methods are employed for labeling the samples. Radioactive labels, and also chemiluminescent or fluorescent labels, are particularly widely employed. In this connection, fluores- 50 cence methods, in particular, have a high standing in chemical and biological analysis and diagnosis. These methods are very powerful detection methods which can be performed without using any radioactivity and, if necessary, without using any toxic substances. There nowadays exist sensitive 55 detection systems which even make it possible to detect individual fluorescent molecules. In addition, a large number of very different fluorescent dyes are available, such that it is possible to have recourse to suitable fluorescent labels for most wavelength ranges in the visible spectrum and also in 60 the adjoining ultraviolet or infrared spectral range. It is frequently even possible to use several fluorescent dyes, having different excitation and/or emission wavelengths, in parallel when carrying out a measurement.
In the present instance, a solid support is understood as 65 being a material which has a rigid or semirigid surface. Supports of this nature can, for example, be particles, strands, in
particular fiber bundles, spherical bodies, such as spheres or spherules, precipitation products, gels, sheets, tubes, receptacles, capillaries, disks, films or plates. Flat supports are normally used.
As a result of progressive miniaturization, it has by now become possible to reduce the DNA spaces substantially such that it is nowadays possible to arrange a large number of spaces, which can be distinguished both from a point of view of process technology and measurement technology, on a single support. In imitation of semiconductor technology, reference is therefore made to chips, in particular biochips, gene chips, etc. The targets are bound to the support at as high a density as possible. In particular, the application of photolithographic manufacturing techniques derived from semiconductor technology has led to decisive advances in the production of these chips. The principle is based on lightdirected chemical solid phase synthesis in which photolithographic masks image the spaces (cf, for example, Fodoretal., "Light-directed, spatially addressable parallel chemical synthesis", Science, vol. 251, 767-773 (1991)). This method is particularly advantageous when the target DNA is to be synthesized from individual nucleotides in situ on the support. Thus, a particular building block can be selectively added on to the targets which are in the process of being synthesized on particular spaces while the probes on the remaining spaces remain unaffected. In this way, it is possible to produce, on a large scale, DNA oligochips which, when used combinatorially, enable new sequences to be discovered. In order to recognize sought-after sequences, the oligosynthesis chip requires elaborate pattern recognition. Methods for this purpose are described in detail in international patent applications WO 90/15070, WO 91/07087, WO 92/10092, WO 92/10587, WO 92/10588 and in U.S. Pat. No. 5,143,854.
In contrast to the synthesis arrays, spotting arrays, in which the previously produced DNA sequence is transferred in complete form to the support, are increasingly being used in practice. Different methods, such as inkjet spotting, solid pin spotting or microprinting, are used for applying the DNA solution. Arrays of this nature are suitable for a large number of applications, starting with the sequencing of DNA and proteins and proceeding all the way to DNA finger-printing and disease diagnosis. Commercial biochips, containing a large number of different cDNAs for hybridization, are by now being offered for sale. These cDNAs are nucleic acid sequences having lengths of from about 200 to 600 base pairs (bp. It is precisely in the area of gene expression profiling, that is identifying the state of activity of genes of interest, that the spotting chip comes into its own. In this connection, a control DNA pool is always compared with a stimulated DNA pool and changes in gene activity thus determined for the given problem or the relevant biological model. Chip technologies are increasingly being employed, in particular, for finding relevant biomolecules which, for example, have a key role in the organism.
While very many different substances or molecules can be immobilized on these planar chips, DNA arrays which are known today still suffer from problems in connection with handling and applicability. The essential reason for these problems is the lack of reproducibility of the overall process, which consists of many individual steps.
In addition, the lateral exchange of substances in the sample liquid which has been applied to the chip is controlled solely by diffusion, which means that there is no guarantee, at least within a practicable total period of measurement, that each species in the sample liquid is able to interact with each sample species which is immobilized on the chip surface.