Biomedical Engineering Reference
In-Depth Information
5 VideoScan Assays for the Detection of Nucleic Acids
As described so far, applications are not limited to the detection of antigens,
peptides and proteins but are also useable for nucleic acids. In the following sec-
tions we will focus on endpoint and real-time applications for the detection and
analysis of nucleic acids. In respect to the analysis of this important biomolecule,
planar surface technologies like microarrays are dominant for the simultaneous
analysis of transcriptional changes, single-nucleotide polymorphisms, and identi-
fication of genes [
52
,
53
]. Commonly the modus operandi is a direct hybridization
of the labeled target to the complementary capture molecule on a defined spot (e.g.,
microarray) [
54
]. Though this approach results in a low signal bias during
hybridization, it is well known that the planar paradigm also leads to a decreased
sensitivity [
55
]. Moreover, preparation of microarrays requires tight control of the
spotting. Spots may vary significantly and have only a little redundancy. With the
introduction of microbeads, however, the aim was to overcome these limitations
[
4
]. Capture-probe-loaded microbeads can be prepared in large bulks with opti-
mized coupling chemistry, which leads to a reduced signal variation. They can be
used at high redundancy. In contrast to planar spots, the spherical character of
microbeads leads approximately to a hybridization behavior, as in aqueous solution
[
4
]. Moreover small spot (microbead diameter) sizes are positively correlated with
hybridization efficiency of target to capture probes due to transition from mass
transfer to kinetically limiting factors [
56
,
57
]. Microbeads also offer a larger
surface area compared to planar spots of the same diameter. Conventional
microbead-based assays are used in suspension but recently the paradigm changed
to surface microbead arrays where microbeads reside on the well bottom
[
4
,
14
,
25
,
58
]. In contrast, VideoScan utilizes randomly ordered microbeads which
are spatially distributed on the surface of a well [
59
]. A disadvantage of this
approach is the reduced density of microbeads per area compared to spatial defined
positions, for example on patterned surfaces, as found elsewhere [
20
,
52
,
54
,
60
].
However, manufacturing of such test systems is more complex.
5.1 The VideoScan Multiplex Hybridization Assay
Escherichia coli (E. coli) is a common bacterium of the intestinal microflora of
mammals and birds but also an important cause of bacterial infections. E. coli can
be grouped into numerous pathovars and can cause intestinal as well as extrain-
testinal disease. Within the family of Enterobacteriaceae, E. coli shows the largest
known variability of virulence as well as the highest known number of virulence-
associated genes (VAGs). Typical VAGs in E. coli from diarrheic hosts (iVAGs)
code for adhesins and toxins. Typical VAGs in extraintestinal pathogenic
E. coli (eVAGs) code for adhesins, toxins, iron acquisition systems, lipopolysac-
charides, capsules of polysaccharides and invasins. The primary method for testing
