Plastic Antibodies - Designing Receptors for the Next Generation of Biosensors - page 106

1.2 Aptamers
Nucleic acids ligands (aptamers) can also be exploited as affinity tools. The term
“aptamer” derives from the Latin word “aptus,” which means “fitting,” and the
Greek word “meros,” which means “particle.” Aptamers are short (15-60
nucleotides) single-stranded nucleic acid (DNA or RNA) oligomers with a specific
and complex three-dimensional shape, which allows them to recognize a variety of
targets ranging from small organic molecules to large protein complexes [ 6 , 25 ].
Aptamers can exhibit affinities down to the nanomolar range, but in contrast to
mAbs they are produced entirely in vitro through the generation of combinatorial
libraries (10 14 -10 15 synthetic nucleic acid sequences) and the subsequent stringent
selection process with the immobilized target. The selected sequences are amplified
by polymerase chain reaction (PCR) and used in several selection/amplification
cycles (6-20) with increasingly stringent selection conditions in a process called
SELEX (systemic evolution of ligands by exponential enrichment). Eventually, these
aptamers are cloned, sequenced, and tested for the intended application [ 6 , 25 ].
Aptamers with molecular weight 5-20 kDa are smaller than antibodies and can
be used in high-density arrays. Furthermore, thanks to their robustness, aptamers
can be chemically modified by, e.g., through biotinylation or by addition of
fluorescent labels. They are exploited in ELISA assays or as detection elements
in biosensors. Target binding may significantly alter the structure of an aptamer in a
reversible way and such an event could be exploited to detect molecules of interest,
either fluorescently or electrochemically. Moreover, unlike antibodies, aptamers
are easy to regenerate, either at high temperature or high salt concentration which
can be used in affinity purification of proteins. Another important advantage is that
aptamers can be generated for virtually any target, even those for which antibodies
cannot easily be raised (such as toxins or poorly immunogenic molecules). The
SELEX process can also be performed under conditions similar to those used in the
assay for which the aptamer has been developed. In this way it can be ensured that
the oligonucleotide will retain its structure and recognition ability in the final
process for which it was intended [ 26 ].
SELEX processes are, however, quite lengthy and labor intensive [ 27 ]. In
addition attempts to automate SELEX procedure have so far proved to be unsuc-
cessful. Moreover, despite their claimed robustness, aptamers are prone to degra-
dation. In addition, commercialization of aptamer technology has been hindered by
exclusive ownership of IP by a small number of companies [ 6 , 28 ].
1.3 Molecularly Imprinted Polymers
The process of molecular imprinting is schematically represented in Fig. 1 . The
synthesis of MIPs involves monomers which possess functional groups capable
of interacting with the target molecule (template), either covalently or through