Heavy metals are a ubiquitous and troublesome class of pollutants, and lead occupies a prominent position as a contaminant requiring constant attention due to its numerous toxicological effects over a wide range of exposure. Anthropogenic sources of lead from military operations require active monitoring to ensure environmental compliance and protection. Current testing involves sample collection followed by complex laboratory testing. There is no available, portable product that meets all requirements for in situ measurement of lead in groundwater (i.e., rugged, reliable, sensitive, selective, and remotely operable).
The objective of this project was to develop a highly selective and sensitive miniaturized sensor for lead by combining two recent advances: (1) catalytic deoxyribonucleic acid (DNA) that is reactive only to lead and can be tagged to produce fluorescence and (2) nanoscale fluidic molecular gates that can manipulate fluid flow and perform molecular separations on tiny volumes. This work built on SERDP SEED project ER-1265, which demonstrated the proof of concept for combination of these breakthroughs onto an integrated device. This project aimed to further develop the chemistry and engineering needed to create a microfluidic device for separating, sensing, and quantifying lead in a complex matrix, as well as to manipulate the sensor platform for separation and detection of other heavy metals.
Capillary electrophoresis columns were microfabricated in polydimethylsiloxane and polycarbonate to precisely control fluidic movement by applying an electric field across distal ends of the columns. A three-dimensional arrangement of these channels provides the option to pass a selectable sample volume through a novel molecular gate polymeric membrane perforated with a number of long narrow channels, typically on the order of 100 nanometers in diameter. Specific recognition elements that cause a measurable response in the presence of a particular species can be incorporated into these channels or microchannels on the opposing side of the molecular gate membrane. In either case, the device’s detection zone was chemically modified with a unique sequence of catalytic DNA that cleaves an associated strand of substrate DNA in the presence of lead. Tagging the substrate DNA with a fluorophore allows for detection of the substrate DNA fragments, thus providing a sensitive optical signal for the presence of lead. This research was concerned with all chemical aspects of design and function for a rugged miniaturized sensor capable of remote, selective, and sensitive detection of bioavailable lead. The design was targeted at waterborne lead as one of the Army’s top ten emerging contaminants.
This work produced a microchip-based lead sensor that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb2+). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb2+ concentration. The DNAzyme responds sensitively and selectively to Pb2+, and immobilizing the DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. The immobilized DNAzyme retains its Pb2+ detection activity in the microfluidic device and can be regenerated and reused. The particular DNAzyme shows no response to other common metal cations, and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. Attempts were made to incorporate a second DNAzyme with selectivity for uranium in the same microfluidic chip sensor to demonstrate multiplexing capabilities for multiple metal analytes in a single injection. Crosstalk between the lead DNAzyme and the uranium DNAzyme severely limited the utility of this multi-analytic chip.
This project successfully demonstrated the feasibility of incorporating Pb2+-specific catalytic DNA in a polymer-based microfluidic device. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. (Project Completed - 2012)