Incorporation of a DNAzyme into Au-coated nanocapillary array membranes with an internal standard for Pb(II) sensing

A Pb(II)-specific DNAzyme has been successfully incorporated into Au-coated polycarbonate track-etched (PCTE) nanocapillary array membranes (NCAMs) by thiol-gold immobilization. Incorporation of the DNAzyme into the membrane provides a substrate-bound sensor using a novel internal control methodology for fluorescence-based detection of Pb(II). A non-cleavable substrate strand, identical to the cleavable DNAzyme substrate strand except the RNA-base is replaced by the corresponding DNA-base, is used for ratiometric comparison of intensities. The cleavable substrate strand is labeled with fluorescein, and the non-cleavable strand is labeled with a red fluorophore (Cy5 or Alexa 546) for detection after release from the membrane surface. This internal standard based ratiometric method allows for real-time monitoring of Pb(II)-induced cleavage, as well as standardizing variations in substrate size, solution detection volume, and monolayer density. The result is a Pb(II)-sensing structure that can be stored in a prepared state for 30 days, regenerated after reaction, and detect Pb(II) concentrations as low as 17 nM (3.5 ppb).