Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection.
Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here 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 (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.
Dalavoy
Tulika S
TS
U.S. Army Corps of Engineers, Construction Engineering Research Laboratory, 2902 Newmark Drive, Champaign, Illinois 61822, USA.
Wernette
Daryl P
DP
Gong
Maojun
M
Sweedler
Jonathan V
JV
Lu
Yi
Y
Flachsbart
Bruce R
BR
Shannon
Mark A
MA
Bohn
Paul W
PW
Cropek
Donald M
DM
D015374:Biosensing Techniques / Q000379:methods*; D002384:Catalysis; D021881:DNA, Catalytic / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005453:Fluorescence; D007854:Lead / Q000032:analysis*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013499:Surface Properties
D021881:DNA, Catalytic; D004800:Enzymes, Immobilized; D007854:Lead; D019904:Polymethyl Methacrylate