Miniaturized ligand-binding assays: theoretical considerations

Abstract

Markus F. Templin Dieter Stoll Monika Schrenk Petra C. Traub Christian F. Vöhringer and Thomas O. Joos* NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, 72770 Reutlingen, Germany. *e-mail: Joos@nmi.de The fundamental principles of miniaturized and parallelized microspot ligand-binding assays were described more than a decade ago. In the ‘ambient analyte theory’, Roger Ekins and coworkers [1–4] explained why microspot assays are more sensitive than any other ligand-binding assay. At that time, the high sensitivity and enormous potential of microspot technology had already been demonstrated using miniaturized immunological assay systems. Nevertheless, the enormous interest that microarray-based assays evoked came from work using DNA chips. The possibility of determining thousands of different binding events in one reaction in a massively parallel fashion perfectly suited the needs of genomic approaches in biology. The rapid progress in whole-genome sequencing (e.g. [5,6]) and the increasing importance of expression studies [expressed sequence tag (EST) sequencing] was matched with efficient in vitro techniques for synthesizing specific capture molecules for ligand-binding assays. Oligonucleotide synthesis and PCR amplification allow thousands of highly specific capture molecules to be generated efficiently. New trends in technology, mainly in microtechnology and microfluidics, newly established detection systems and improvements in computer technology and bioinformatics were rapidly integrated into the development of microarray-based assay systems. Now, DNA microarrays, some of them built from tens of thousands of different oligonucleotide probes per square centimetre, are well-established highthroughput hybridization systems that generate huge sets of genomic data within a single experiment (Fig. 1). Their use for the analysis of single nucleotide polymorphisms and in expression profiling has already changed pharmaceutical research, and their use as diagnostic tools will have a big impact on medical and biological research. As known from gene expression studies, however, mRNA level and protein expression do not necessarily correlate [7–9]. Protein functionality is often dependent on post-translational processing of the precursor protein and regulation of cellular pathways frequently occurs by specific interaction between proteins and/or by reversible covalent modifications such as phosphorylation. To obtain detailed information about a complex biological system, information on the state of many proteins is required. The analysis of the proteome of a cell (i.e. the quantification of all proteins and the determination of their post-translational modifications and how these are dependent on cell-state and environmental influences) is not possible without novel experimental approaches. High-throughput protein analysis methods allowing a fast, direct and quantitative detection are needed. Efforts are underway, therefore, to expand microarray technology beyond DNA chips and establish array-based approaches to characterize proteomes (Fig. 1) [10–12].

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Cite this paper

@inproceedings{Templin2002MiniaturizedLA, title={Miniaturized ligand-binding assays: theoretical considerations}, author={Markus F. Templin and Dieter Stoll and Monika Schrenk and Petra C. Traub and Christian F. V{\"{o}hringer and Roger Ekins}, year={2002} }