Triple functional DNA-protein conjugates: Signal probes for Pb(2+) using evanescent wave-induced emission

We describe here a Pb(2+)-dependent DNAzyme-based evanescent wave-induced emission (EWIE) biosensing platform using triple functional DNA-protein conjugates as signal probes for Pb(2+) detection. Upon reaction with Pb(2+), the substrate strand is cleaved, releasing an invasion fragment, which is then hybridized with the complementary DNA strand immobilized on magnetic beads, while dissociating of the original hybridized signal probes. The signal probes, consisting of a streptavidin moiety and a Cy5.5 labeled DNA moiety, act simultaneously as signal conversion, signal recognition and signal report elements. Detection of the signal probes is accomplished by first adsorbing to the desthiobiotin-modified optical fiber, followed by fluorescence emission induced by an evanescent field. A linear calibration was obtained from 20 nM to 800 nM with a detection limit of 1 nM. The optical fiber system is robust enough for 250 sensing cycles and can be stored at room temperature over one month. These results demonstrate that application of DNA-streptavidin conjugates has been extended to DNAzyme-based biosensors, maintaining activity, specificity, regeneration and long-term storage ability.