HR: 11:50h
AN: B22A-06    [Abstracts]
TI: Gold-functionalized DNAzyme Nanosensors to Quantify Heavy Metal Gradients
AU: Adriaens, P
EM: adriaens@umich.edu
AF: The University of Michigan, 1351 Beal Ave/180 EWRE, Ann Arbor, MI 48109 United States
AU: * Vannela, R
EM: rvannela@engin.umich.edu
AF: The University of Michigan, 1351 Beal Ave/180 EWRE, Ann Arbor, MI 48109 United States
AB: The presence of heavy metals in environmental systems is characterized by steep gradients due to the presence of solid and soluble redox species. The capability to selectively quantify low concentrations of individual redox species continues to present a challenge for site characterization and risk assessment. Recently, DNAzymes, single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by ``in vitro selection'', have come to the fore as unique molecular tools for metal detection in aqueous environment. Here we describe the selection and characterization of an RNA-cleaving autocatalytic DNAzyme that links chemical catalysis with real-time fluorescence signaling in the single molecule. From a random pool of 40-nt templates, we have isolated Hg-active DNAzymes using in vitro selection procedures in combination with non-homologous random recombination (NRR). However, significant cleavage was seen for DNA sequences isolated in generation G5. By G8 cycle, more than 32% of the DNA construct was cleaved after 3-h incubation. After eight rounds of selection and amplification, the amount of eluted DNA after respective reaction was found to produce active sequences leading to 32 % catalysis. The introduction of nonhomologous random recombination at 9th cycle yielded drastic increase in catalysis to 55% of the total oligonucleotide pool. DNA sequences from the 9th round of selection (G9) were amplified by PCR and cloned into a vector. DNA sequencing was performed and the M-fold software revealed variety of different families of sequences in the active pool - with DNA-010 sequence being dominant in each family. Similar experiments conducted with As5+ and selection did not show any cleavage until the 8th cycle, with cleavage increasing to 18 % at 14th cycle. Since the catalytic activity can be characterized in terms of metal specificity, speciation and affinity, the catalytic DNA serves as a sensor for a variety of metal ion species (e.g. Hg2+ and As5+) These specific and sensitive nanosensors will be embedded on gold particle arrays for enhanced signal amplification, rendering them amenable to detect metal concentration gradients in situ.
DE: 1094 Instruments and techniques
DE: 1610 Atmosphere (0315, 0325)
DE: 1894 Instruments and techniques: modeling
DE: 4803 Analytical chemistry
DE: 4894 Instruments, sensors, and techniques
SC: Biogeosciences [B]
MN: Fall Meeting 2005