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.