Biomedical Engineering Reference
In-Depth Information
Multichannel performance.
This would be required for high-throughput screening
and detection for new pharmaceuticals. Advances have already been made in
this area, with the introduction of a four-channel chip that can be rotated by 90º,
effectively providing 16 channels. Myszka and Rich (49) have more recently
described a prototype micro array chip with 64 individual immobilization sites
in a single flow cell.
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Development of advanced recognition elements for applications involving complex real-
istic samples (e.g., blood).
Stable receptor matrices that allow sensor responses and
nonspecific background effects to be resolved will also have to be developed.
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All these developments may eventually lead to miniaturized, integrated, compact, and
rugged sensing elements which would fulfill a number of the requirements of an ideal
biosensor.
20.3
Aptamers
Aptamers have been defined by James (50) in the
Encyclopaedia of Analytical Chemistry
as:
“…artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins
and other molecules. They are isolated from complex libraries of synthetic nucleic acid by
an iterative process of adsorption, recovery and reamplification. They have potential
applications in analytical devices, including biosensors, and as therapeutic agents.” In
essence, they are synthetic oligonucleotide sequences which are able to bind a wide array
of molecules (ligands) with high affinity and specificity (51; 52). Their name is derived
from the Latin word “
aptus
”, meaning “to fit” (52).
Importantly, the ligand-binding capacity of aptamers is based on their three-dimensional
conformation and not on nucleotide base pair complementarity (53). Indeed, upon associa-
tion with their molecular targets, aptamers fold into molecular structures in which the target
becomes an intrinsic part of the nucleic acid structure (54). They are therefore able to bind pro-
teins and other molecules that would not normally interact with DNA or ribonucleic acid
(RNA), making them extremely powerful as screening tools (53). They were initially reported,
in 1990, quasi simultaneously by three independent laboratories: those of Robertson and
Joyce (55), Ellington and Szostak (56), and Tuerk and Gold (57), and are obtained using an in
vitro selection and purification technique, now known as systematic evolution of ligands by
exponential enrichment (SELEX), as described in Section 20.3.2 (51).
20.3.1
Aptamer Libraries
Before the SELEX process is undertaken, an aptamer library has first to be created. Part of
the enormous potential of aptamers lies precisely in the fact that libraries with vast num-
bers of potential ligands can be created and screened within a few days. Typically, aptamer
libraries consist of 10
13
-10
18
random oligonucleotide sequences (51) and can be screened
within a number of days. This is all the more impressive when compared to conventional
libraries of potential drugs which consist generally of no more than 10
6
different molecules
and may take months to screen (53).
When creating a library however, the following factors need to be taken into account (53):
The aptamer library complexity
: Given random manufacture of aptamers, the
library complexity can be calculated relatively easily. For example, a library of
oligonucleotides of
N
nucleotides in length generated from
y
different
nucleotides (not solely limited to the four naturally occurring nucleotides) will
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