(215c) Developing a Rapid, Specific and Sensitive Molecular Sensing Kit: An Electrokinetic Nanocolloid Platform

The development
of highly sensitive detection platform for genetic and peptide identification
has attracted great attention to a wide variety of fields such as genomics,
proteomics, clinical diagnosis etc.¹ Conventional lab based
technique mostly employs optical detection methods, such as in microarray
analysis and real-time PCR, which is highly sensitive and specific.²
However, these techniques require qualified professionals and elaborate
fluorescent tagging/sensing equipment/reagents, which severely limit their
portability. Furthermore, DNA hybridization reactions in microarray analyses
are slow (>hours) due to the diffusion-limiting binding kinetics, making the
technique difficult for point-of-need and high-throughput applications.³
Non-specific binding and interference are also key challenges for protein and
DNA microarrays with heterogeneous samples. Thus, there is a need to develop
extremely sensitive, rapid, specific, portable and robust DNA/RNA/peptide
detection platforms for applications ranging from genomics and proteomics to
pathogen identification.⁴ We present a new detection platform that
can meet these stringent challenges. The platform adopts an open-flow
microfluidic format that can concentrate a small number of target molecules in
a 100 microliter sample in 10 minutes, thus reducing the transport resistance. The concentration technique
involves binding onto probe-functionalized nanobeads whose induced electric
dipole reverses direction upon docking at a particular AC field frequency. This
dipole reversal upon molecular docking allows us trap the unhybridized beads in
an on-chip DEP (dielectrophoretic) trap where the target molecules from the
open-flow are trapped. The AC electric field does not impart a force on
the flowing liquid and there is hence little hydrodynamic resistance like
membrane filters. Moreover, the hybridized beads are released individually and
can be trapped and quantified downstream with a nanocolloid impedance sensor.
Alternatively, the reversed nanocolloid dipoles can be detected and quantified
with an impedance sensor at the DEP trap. As there are about 60 target
molecules per nanocolloid and as the impedance resolution is nearly one
nanocolloid, the
detection sensitivity obtained by this platform is below pM. Moreover,
hybridization in the presence of shear flow is highly specific and can distinguish
between three bases mismatches when compared with DNA from targeted species and
a closely related congeneric species having mismatches at positions 12, 21 and
22 of the 26 base tag sequences (reading from 5' to 3', respectively). This is attributed
to large hydrodynamic shear force (>10⁴ s^-1) generated
by flow that removes any non-specific binding of congener DNA onto the nanobead.
The open flow platform also minimizes the masking of the Warburg impedance
signal for electron transfer to the nanocolloids by the electrical double layer
impedance signature. This sensitive and specific electrokinetic nanocolloid
impedance assay for biomolecules hence offers a rapid and portable platform
that does not require optical sensing and fluorescent labeling. Genetic
detection of specific organisms and pathogens in real-world heterogeneous
samples will be demonstrated. 

References:

[1] E.Palecek,
Trends Biotechnol. 22 (2004) 55.

[2] J. H.
Watterson, P. A. E. Piunno, U. J. Krull, Anal. Chim. Acta 457 (2002) 29.

[3] J. Kian-Kok
Ng, H. Feng, W.T. Liu, Anal. Chim. Acta 582, (2007) 295.

[4] S. Senapati
et al. Biomicrofluidics 3 (2009) 022407.