Biomedical Engineering Reference
In-Depth Information
1 Introduction
Proteins are involved in all functions of organisms and they show a much greater
variety and complexity compared to genes. For years and decades scientists have
been committed to the investigation of proteins, its structure and function. In life
sciences and especially in medicine and diagnostics a better knowledge of protein-
protein-interactions may lead to a better understanding of diseases, the develop-
ment of new diagnostics, targeted drugs and therapy monitoring tools. Substantial
progress in technology developments demonstrated that microarrays have become
an important tool for investigating proteins. However, major challenges have to be
dealt with when investigating protein-protein-interactions [
1
]. Proteins are a
heterogeneous group of molecules and it is difficult to preserve their full func-
tionality in experimental environments. This challenge refers to both: the physical
handling of proteins with pipettes or automated arrayers and to experimental
conditions such as appropriate buffers or washing procedures [
2
]. High resolution
detection devices and corresponding labelling methods are needed [
3
]. Sophisti-
cated analysis software tools are essential to receive distinctive results. Finally,
investigations should be time saving and as cost effective as possible to be
successful in the market.
Protein Microarrays have become an important high throughput tool to investi-
gate protein-protein interactions because they provide a parallel, fast and straight-
forward analysis of as many proteins as can be potentially immobilized on a given
surface [
4
-
7
]. For diagnostic applications typically a chosen subset of capture agents
is deposited on planar substrates, to allow the parallel analysis of two or more
interactions in a single reaction [
8
,
9
]. Today, typical applications include autoim-
mune diseases, infectious diseases or cytokine analysis and a growing number of
further applications or specifications are under development. In screening experi-
ments of disease markers not only the expression and/or the amount of the proteins
may be of interest but also their post-translational modifications e.g. phosphoryla-
tions or glycosylations [
10
].
2 Substrates for Protein Binding
Depending on the actual sample format and the diagnostic application, various
routes can potentially be taken to generate, incubate and read out any protein array.
For the majority of diagnostic applications one key requirement is to produce the
multiplexed test format as cost effective as possible—a requirement that already has
to be considered during research and development. While planar protein arrays have
historically been developed using inexpensive membrane formats [
5
,
6
] a significant
number of publications described the use of glass slides for capture probe immo-
bilisation. The latter offer the advantage of fluorescent detection schemes, which
provide an improved signal to noise ratio when compared to auto-fluorescent
