Nucleic acid aptamers can exhibit high binding affinities for a wide variety of targets and have received much attention
as molecular recognition elements for enhanced biosensor performance. These aptamers recognize target molecules
through a combination of conformational dependent non-covalent interactions in aqueous media which can be
investigated using capillary electrophoresis-based methods. In this paper we report on the results of our studies of the
relative binding affinity of Campylobacter jejuni aptamers using a capillary electrophoretic immunoassay. Our results
show preferential binding to C. jejuni over other common food pathogen bacteria.
Capillary electrophoresis can also be used to develop new aptamer recognition elements using an in vitro selection
process known as systematic evolution of ligand by exponential enrichment (SELEX). Recently, this process has been
adapted to use capillary electrophoresis in an attempt to shorten the overall selection process. This smart selection of
nucleic acid aptamers from a large diversity of a combinatorial DNA library is under optimization for the development
of aptamers which bind to Army-relevant targets. This paper will include a discussion of the establishment of CE-SELEX
methods for the future development of smart aptamer probes.
Immunochromatography is a rapid, reliable, and cost effective method of detecting biowarfare agents. The format is
similar to that of an over-the-counter pregnancy test. A sample is applied to one end of a cassette and then a control line,
and possibly a sample line, are visualized at the other end of the cassette. The test is based upon a sandwich assay. For
the control, a line of Protein A is immobilized on the membrane. Gold nanoparticle bound IgG flows through the
membrane and binds the Protein A, creating a visible line on the membrane. For the sample, one epitope is immobilized
on the membrane and another epitope is attached to gold nanoparticles. The sample binds gold bound epitope, travels
through the membrane, and binds membrane bound epitope. The two epitopes are not cross-reactive, therefore a sample
line is only visible if the sample is present. In order to efficiently screen for binders to a sample target, a novel,
Continuous Magnetic Activated Cell Sorter (CMACS) has been developed on a disposable, microfluidic platform. The
CMACS chip quickly sorts E. coli peptide libraries for target binders with high affinity. Peptide libraries, are composed
of approximately ten million bacteria, each displaying a different peptide on their surface. The target of interest is
conjugated to a micrometer sized magnetic particle. After the library and the target are incubated together to allow
binding, the mixture is applied to the CMACS chip. In the presence of patterned nickel and an external magnet,
separation occurs of the bead-bound bacteria from the bulk material. The bead fraction is added to bacterial growth
media where any attached E. coli grow and divide. These cells are cloned, sequenced, and the peptides are assayed for
target binding affinity. As a proof-of-principle, assays were developed for human C-reactive protein. More defense
relevant targets are currently being pursued.
A fundamental understanding of the factors which influence binding performance is critical to any technology or
methodology relying on molecular recognition of a specific target species. For the Army, there is a growing need for a
basic understanding of these interactions with traditional recognition elements (e.g., antibodies) in non-traditional
environmental conditions, such as with new and emerging threats. There is a similar need for building a base of
knowledge on non-traditional affinity ligands that are biomimetic or biosynthetic in nature.
In this paper, specific research at the Army Research Laboratory towards the development, evaluation and use of
synthetic affinity ligands for sensing applications is discussed. This includes the results of our investigations of aptamer-based
affinity ligands targeting Campylobacter jejuni. Using capillary electrophoretic techniques, the relative binding
affinities of the aptamer ligands towards the target pathogen as well as the degree of cross-reactivity with other food
borne-pathogens (i.e., Escherichia coli O157:H7 and Salmonella typhimurium) were evaluated. Current progress
towards the development of synthetic affinity ligands for sensing applications will also be discussed.
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