Enhancing the speed of morpholino-DNA biosensor by electrokinetic concentration of DNA in a microfluidic chip

Electrokinetic methods that conveniently concentrate charged analytes by orders of magnitude are highly attractive for nucleic acid assays where they can bypass the complexity and costs of enzyme-based amplification. The present study demonstrates an electrokinetic concentration device incorporating charge-neutral morpholino (MO) probes: as DNA analyte is concentrated in a microfluidic channel using ion concentration polarization (ICP) it is simultaneously hybridized to spots of complementary MO probes immobilized on the channel floor. This approach is uniquely favored by the match between the optimum buffer ionic strength of approximately 10 mM for both MO-DNA surface hybridization and electrokinetic concentration. The simple and easily scalable poly(dimethylsiloxane) (PDMS) microfluidic device was fabricated using soft lithography and contact printing of a conductive polymer, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOT:PSS) as a cation-selective membrane material. Using the microfluidic concentrator, we could increase the concentration of DNA by three orders of magnitude in less than 5 min. at an electric field of 75 V cm−1. The 1000-fold increase in concentration of DNA led to an increase in the speed of MO-DNA hybridization by two orders of magnitude and enabled a detection sensitivity of ~1 nM within 15 minutes of concentration. Using the proposed microfluidic concentrator, we also demonstrated a rapid hybridization with a binary DNA mixture, containing a fully complementary and a non-complementary sequence to mimic molecular backgrounds present in real DNA samples.