(102f) An Integrated Electrokinetic Chip Platform for Point-of-Care Nucleic Acid Diagnostics

We present a reliable, sensitive, specific and disposable portable diagnostic biochip platform for DNA/RNA detection,  that is based on a variety of modern electrokinetic phenomena/technology and can be powered by a low-voltage DC battery. This turn-key platform integrates a few indepenent units on a glass slide sized chip and is fully automated such that a sequence of multi-plex analysis is carried out without external interference. All units are economically fabricated with low-cost polymeric materials, including a microfluidic chip consisting of multiple layers of polycarbonate film, such that the overall material cost is less than one dollar per disposable chip. Despite its low cost, the multi-plex chip involves a sequence of intricate but robust operations that are connected by a continuous flow, with pumping providied by an on-chip pump, such that microfluidic flow can be used to process a large volume of real physiological sample, separate debris, eliminte diffusion transport limitation and provide shear-enhanced specificity.  These units include: (i) a pretreatment unit with an electrophoretic gel loading component for extraction/separting RNA/DNA molecules from real samples with no or minimum upstream pretreatment(blood, saliva, urine etc), (ii) a depletion-zone preconcentration unit for localizing the DNA/RNA molecules next to the sensor, (iii) an ion exchange particle/membrane-based sensor, and (iv) an electroosmotic pump with a set of reservoirs for dosing the necessary buffers.

Chemically lysed samples are pipetted into the sample reservoir of the platform from which negatively charged biomolecules are loaded into the main channel through a thin agar gel by a DC electrical field. This loading process based on the principle of gel electrophoresis efficiently separates molecules of interests from cell debris and positively charged molecules. Loaded molecules are then electro-osmotically transported to the sensor/preconcentration unit which accumulates the negatively charged molecules at the ion exchange particle/membrane sensor, thus allowing specific target molecules to hybridize with probes on the sensor surface. The accumulation of negatively charged molecules in the high continuous flow (~10 microliter/min) at the sensor is achieved with a preconcentration membrane whose concentraton polarization effect introduces a depletion zone in the flow channel, which then acts as a filterless sieve for the target molecules.

The core of the diagnostic platform is a single, positively charged ion exchange particle/membrane used as a sensor. Ionic current through the ion exchange particle/membrane is sensitive to the presence of physisorbed DNA and RNA molecules due to surface charge inversion phenomenon caused by the negative charge of DNA/RNA phosphate-sugar backbone. This rather nonspecific detection mechanism of nucleic acids is rendered specific with covalent attachment of ssDNA oligoprobes complementary to the DNA/RNA targets. The ion current allows qunatification of the hybridized targets which are preferentiall retained with the non-targets being sheared away by the strong continuous flow. Current limit of detection is 1000 RNA molecules in a 10 microliter sample of raw sample, with a dynamic range from microM to fentoM or 10³ to 10⁹ targets and a specificity of two mismatched pairs for a 20b RNA.

We will present optimization/fabrication details for each unit of the detection platform and show results for the integrated platform for the detection of long single stranded mRNA and microRNA for  E. Colli in water and oral cancer cells in saliva. We will also discuss future development of multi-target sensing chips.