(283a) Microfluidic / Nanofluidic Sensors Using Catalytic DNA for Heavy Metal Detection

A microchip-based lead sensor was developed that employs lead-specific catalytic DNA as the recognition element. Lead-specific catalytic DNA (DNAzyme) cleaves its complementary substrate DNA strand in the presence of only cationic lead (Pb2+). Fluorescent tags on the substrate DNA transduce the Pb2+ concentration to a measurable, optical signal. The DNAzyme responds sensitively and selectively to Pb2+. The nano/microfluidic system has a hybrid three-dimensional fluidic structure that incorporates a nuclear track-etched nanocapillary membrane between two crossed, spatially separated, poly(dimethylsiloxane) (PDMS) microfluidic channels. This allows reliable sample injection, electrophoretic separation, and isolated delivery of Pb2+ to the DNAzyme in a spatially confined detection window where the fluorescent substrate fragments are interrogated using laser-induced fluorescence (LIF). Data illustrate that the detection of Pb2+ is rapid, selective, and sensitive; reaching drinking water standards within seconds without sample preparation.

Microfluidic devices are also being fabricated using PMMA to create a chip that could inject a sample analyte plug containing many metallic species in various molecular forms (e.g. copper, lead, manganese, cadmium, etc.) through a molecular gate (i.e. NCAM membrane) and into a microfluidic separation channel. As the analyte plug would move along the separation channel, it would separate into various bands and a detector (i.e. two parallel plate capacitive sensors in a modified Wheatstone bridge arrangement probed at the resonant frequency) would sense each band as it passed. The device consists of three separate microfluidic layers that contains one or more microfluidic channels per layer. These layers are separated in the vertical direction by NCAMs (nano-capillary array membranes) that serve as molecular gates across which sample plugs could be injected and separated bands could be collected.

Various strategies are being explored to immobilize DNAzyme on PMMA and glass, such as biotin-streptavidin interaction and amine-aldehyde chemistry. To form the enclosed chip, a PDMS microchannel structure is aligned onto the glass so that the microchannels bisect the predeposited DNA sites. The fluorescence in the channel is quenched upon hybridization with the quencher strand, while the fluorescence that is sealed beneath PDMS continues to act as an internal reference. In the presence of lead, a fluorescence increase is observed with the reaction of Pb2+ with the complex. The sensing chemistry can be regenerated by hybridization of DNAzyme with more substrate strand in the channel. This device is an important step toward detecting multiple heavy metal ions with a single injection by introducing different DNAzymes at different locations on a single molecular gated device. Efforts to immobilize Pb-specific DNAzyme in the PMMA microfluidic devices and to study the Pb detection activity in the microchannel using fluorescence microscopy will be described.