Surface immobilization of catalytic beacons based on ratiometric fluorescent DNAzyme sensors : a systematic study

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...

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Veröffentlicht in:Langmuir : the ACS journal of surfaces and colloids. - 1992. - 23(2007), 18 vom: 28. Aug., Seite 9513-21
1. Verfasser: Wernette, Daryl P (VerfasserIn)
Weitere Verfasser: Mead, Carolyn, Bohn, Paul W, Lu, Yi
Format: Aufsatz
Sprache:English
Veröffentlicht: 2007
Zugriff auf das übergeordnete Werk:Langmuir : the ACS journal of surfaces and colloids
Schlagworte:Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. DNA, Catalytic Enzymes, Immobilized Molecular Probes Sodium Chloride 451W47IQ8X
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245 1 0 |a Surface immobilization of catalytic beacons based on ratiometric fluorescent DNAzyme sensors  |b a systematic study 
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520 |a 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 
650 4 |a Journal Article 
650 4 |a Research Support, Non-U.S. Gov't 
650 4 |a Research Support, U.S. Gov't, Non-P.H.S. 
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650 7 |a Enzymes, Immobilized  |2 NLM 
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700 1 |a Mead, Carolyn  |e verfasserin  |4 aut 
700 1 |a Bohn, Paul W  |e verfasserin  |4 aut 
700 1 |a Lu, Yi  |e verfasserin  |4 aut 
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