Subnanoliter enzymatic assays on microarrays

Angenendt, Philipp; Lehrach, Hans; Kreutzberger, Jürgen; Glökler, Jörn

doi:10.1002/pmic.200400955

Cited by 44 publications

(30 citation statements)

References 29 publications

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“…For the time being, the preparation of proteomic arrays is carried out by the use of highprecision contact printing or ink-jet technology, using a sample of subnanolitre volume and spot size of about 100 mm [94]. Such proteomic arrays allow for the monitoring of enzymatic reactions with the amount of enzyme in the spot of about 100-1000 molecules [97]. Gyros reported on creation of the CD-microlaboratory based on a microfluidic protein array (using different affinity binders) and a combination of this CD-microlaboratory with Bruker MALDI-MS and MS/MS (http://www.gyros.com).…”

Section: Protein Arrays For Separation Of Multiprotein Mixturesmentioning

confidence: 99%

Nanotechnologies in proteomics

Ivanov

Govorun

Быков³

et al. 2006

Proteomics

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Progress in proteomic researches is largely determined by development and implementation of new methods for the revelation and identification of proteins in biological material in a wide concentration range (from 10 23 M to single molecules). The most perspective approaches to address this problem involve (i) nanotechnological physicochemical procedures for the separation of multicomponent protein mixtures; among these of particular interest are biospecific nanotechnological procedures for selection of proteins from multicomponent protein mixtures with their subsequent concentration on solid support; (ii) identification and counting of single molecules by use of molecular detectors. The prototypes of biospecific nanotechnological procedures, based on the capture of ligand biomolecules by biomolecules of immobilized ligate and the concentration of the captured ligands on appropriate surfaces, are well known; these are affinity chromatography, magnetic biobeads technology, different biosensor methods, etc. Here, we review the most promising nanotechnological approaches for selection of proteins and kinetic characterization of their complexes based on these biospecific methods with subsequent MS/MS identification of proteins and protein complexes. Two major groups of methods for the analysis and identification of individual molecules and their complexes by use of molecular detectors will be reviewed: scanning probe microscopy (SPM) (including atomic-force microscopy) and cryomassdetector technology.

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Section: Protein Arrays For Separation Of Multiprotein Mixturesmentioning

confidence: 99%

Nanotechnologies in proteomics

Ivanov

Govorun

Быков³

et al. 2006

Proteomics

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“…This approach identified novel (in vitro) substrate specificity of kinases and has provided new data on enzyme-substrate interactions at a molecular level. Recently, we have introduced multiplex enzymatic assays on microarrays using the multiple-spotting technology, and we were able to detect 35 enzyme molecules per spot 105 . This approach allows the rapid screening of thousands of samples on a single microarray, which has applications in drug screening and high-throughput enzymatics.…”

confidence: 99%

Miniaturization in functional genomics and proteomics

et al. 2005

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| Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future. SYSTEMS BIOLOGYThe study of the complex interactions that occur at all levels of biological information -from whole-genome sequence interactions to developmental and biochemical networks -and their functional relationship to organism-level phenotypes. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 465 and concomitantly reducing the complexity of genomic DNA. Although PCR can be used to amplify small numbers of DNA molecules in low microlitre or nanolitre volumes in vitro, problems are associated with the dilution of heterozygous DNA. This could result in incorrect genotyping results (homozygous genotypes) because of the preferential amplification of one of the alleles. Extreme dilution indicates that there is a sufficiently high integrity of the nucleic-acid templates to allow efficient amplification. Furthermore, amplification of low concentrations of nucleic acids requires strict quality control, as it is often associated with contamination problems, which can be problematic in high-throughput applications. In contrast to nucleic-acid manipulation, it is currently impossible to amplify proteins from a biological sample. R E V I E W S Miniaturization in functional genomicsGenome research and functional genomics have catalysed the development of high-throughput and miniaturized approaches for the analysis of biomolecules [5][6][7][8] . Figure 1 | DNA micorarrays. DNA microarrays that are based on non-porous solid supports such as glass have paved the way for highly parallel miniaturized analysis of biomolecules. Using robotic workstations, ~0.25-1 nl of DNA solution are printed on a slide, creating spots that range from 100 to 150 µm in diameter. Successive samples are then spaced to avoid contact between adjacent spots, with approximately 200 µm between each spot 7 . Currently, about 50,000 cDNAs can be robotically spotted onto a microscope slide and hybridized with a fluorescently labelled probe. Using photolithographical masking techniques, arrays that have 400,000 distinct oligonucleotides have been produced, each in its own 20-µm 2 region (today, this can be as small as ~5 µm 2 ) REF. 8. After spotting, nucleic-acid samples can be hybridized on these pre-structured microarrays. Hybridization events are monitored using fluorescence scanners. A typical fluorescence read-out of highly parallel hybridization on a cDNA microarray is shown in the bottom panel. Box 1 | Technological problems in miniaturizationEvery molecular biological method can be miniaturized from microl...

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“…In these reaction entities enzymatic assays can be conducted that catalyze the formation of unbound reporter molecules. In a first study, enzymatic activity of enzymes such as alkaline phosphatase, ß-galactosidase or cathepsin D was detected [189]. Moreover, the applicability of MIST was shown for multiplex immunosorbent assays [190,191] and for the screening of large populations of recombinant antibody fragments on a microarray [192].…”

Section: Applicationsmentioning

confidence: 99%

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

Sobek¹,

Bartscherer²,

Jacob³

et al. 2006

CCHTS

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110

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Over the last years microarray technology has become one of the principal platform technologies for the highthroughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.

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Subnanoliter enzymatic assays on microarrays

Cited by 44 publications

References 29 publications

Nanotechnologies in proteomics

Nanotechnologies in proteomics

Miniaturization in functional genomics and proteomics

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

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