Surface Immobilization of Catalytic Beacons Based on Ratiometric Fluorescent DNAzyme Sensors:  A Systematic Study

Abstract

DNAzyme-based catalytic beacons have the potential for sensing a large number of relevant analytes. Thus, a systematic investigation of factors affecting their performance when immobilized into gold-coated nanocapillary array membranes (NCAMs) was undertaken. Enzyme immobilization times were varied to determine that as little as 15 min was sufficient for ratiometric detection of Pb2+-specific activity, while immobilization density saturated after 1.5 h. Immobilization of the DNAzymes into NCAMs with 600 nm pore size resulted in higher immobilization efficiency and higher enzymatic activity than that with 200 nm pore size. A poly-T linker length between the tethering thiol and first oligonucleotide, used to extend the DNAzyme above the backfilling mercaptohexanol (MCH) monolayer, had no effect on DNAzyme activity. The backfilling method of immobilization, involving backfilling followed by hybridization, was found most effective for DNAzyme activity compared to immobilization of hybridized DNAzyme complex (a 67% loss of activity) or concurrent enzyme and MCH immobilization (75% loss of activity). The backfilling MCH monolayer provided approximately 3.5 times increase in activity compared to DNAzyme assembled without MCH, and was over 5 times more active than shorter and longer backfilling molecules tested. The immobilized DNAzyme retained its optimized performance at 50 mM NaCl. Finally, the generalized immobilization and ratiometric procedure was employed for a uranyl-specific DNAzyme with 25 +/- 15 times increase in ratio observed. These findings form a firm basis on which practical applications of catalytic beacons can be realized, including sensors for both Pb2+ and UO22+ ions.

Highly Sensitive and Selective Colorimetric Sensors for Uranyl (UO22+): Development and Comparison of Labeled and Label-Free DNAzyme-Gold Nanoparticle Systems

“…A 24-mer dsDNA is reported to be able to remain hybridized for about 10 minutes in the Au colloid without NaCl while introduction of a single mismatch will decrease the stability of dsDNA and thus cause dehybridization in 5 min 52. DNAzymes, however, contain a large number of mismatches between the enzyme strand and substrate strand (Figure 5A), which causes dehybridization of the complex in seconds in the absence of NaCl 75, 76. So unlike previously reported studies, DNA solution has to be added to AuNP solution together with sufficient amount of NaCl that can keep complex hybridized.…”

Applications of Aptamers as Sensors

“…An allosteric cleavage can therefore cut apart a fluorescently tagged oligomer on a hybridizing quencher-labeled oligonucleotide substrate. In recent work, a Pb 2+ -activated deoxyribozyme was used as a model system for various optical sensing formats (43,44). UO 2+ was detected in <2 min at ambient temperature with 11 ppt (45 pM) sensitivity and >1 million-fold selectivity over other metal ions (45).…”

Molecular diagnostic and drug delivery agents based on aptamer-nanomaterial conjugates

“…This NCAM sensor also allowed regenerations by rehybridization of fresh DNA strand and storage up to 30 days without significant loss of activity [101]. Solid supported fluorescent sensors can also incorporate ratiometric fluorescence internal controls which allow to standardize the number of molecules immobilized on the surface and to perform real time detection of target molecules [102]. In addition, aptamers or DNAzymes can be incorporated into microfluidic devices which have advantages of generating low waste volume output, consuming less amount of detecting material, and having facile regeneration capabilities [103, 104].…”