2002 Progress Report: Synthesis and Characterization of an Electrochemical Peptide Nucleic Acid Probe

EPA Grant Number: R827072C015
Subproject: this is subproject number 015 , established and managed by the Center Director under grant R827072
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Alabama Center For Estuarine Studies (ACES)
Center Director: Shipp, Robert L.
Title: Synthesis and Characterization of an Electrochemical Peptide Nucleic Acid Probe
Investigators: Campbell, Naomi F.
Institution: University of South Alabama
EPA Project Officer: Packard, Benjamin H
Project Period: July 1, 2000 through December 31, 2003
Project Period Covered by this Report: July 1, 2001 through December 31, 2002
RFA: Alabama Center For Estuarine Studies (ACES) (1999) RFA Text |  Recipients Lists
Research Category: Exploratory Research - Human Health , Targeted Research

Objective:

The objective of this research project is to develop an electrochemical nucleic acid hybridization assay that is sensitive (picomole to femtomole detection level), stable with storage (6 months), portable, and easy to use in a simple assay format.

Progress Summary:

Synthesis and Characterization of Ferrocenyl Peptide Nucleic Acid (PNA) Product. An electroactive molecule, ferrocene, was covalently attached to a PNA strand (base sequence 3'-GCT-TTT-GTG-TAC) using a slight modification of the poly (A) procedures. The ferrocenyl PNA was purified using gel permeation chromatography (Sephadex G-15; fractionation range of the gel is less than 1,500 Da and the molar mass of the PNA strand is 3,267 Da; the PNA strand eluted in the void volume). The BCA (bicinchoninic acid) assay was performed on all the fractions that eluted from the column to test for the presence of peptide bonds (present in the PNA strands). The fractions that tested positive using the BCA assay were pooled and characterized using cyclic voltammetry and UV-visible spectroscopy. Absorption at a wavelength of 450 nm (lambdamax for ferrocene), and voltammetry analysis of the ferrocenyl PNA sample was used to confirm the incorporation of the ferrocene into the PNA strand.

Synthesis and Immobilization of Azido-Tailed Oligonucleotide. Another aspect of the project is the synthesis and immobilization of azido-tailed poly (A). As reported in the previous annual report, the azido-tailed poly (A) was prepared using poly (A) polymerase. However, the immobilization of the azido-tailed poly (A) was not successful because of the large size to azido label ratio that occurs with the poly (A) molecule. The procedure was repeated using an oligonucleotide (20 bases total; RNA 12 bases + [rA]8). The azido label was incorporated using poly (A) polymerase and 8-azidoadenosine 5'-triphosphate. The oligonucleotide was purified using gel permeation chromatography (Sephadex G-15), and the fractions were analyzed at wavelengths of 260 nm and 280 nm. The incorporation of the azido groups into the oligonucleotide strand was confirmed by the absorption spectrum (200 nm to 400 nm) before and after irradiation of a sample with 254 nm light. The irradiated sample showed a decrease in absorption spectrum as compared to the nonirradiated sample. Next, the azido-tailed oligonucleotide complement and azido-tailed oligonucleotide control were placed in polystyrene containers (n = 6 for each sample), and the solvent evaporated overnight at room temperature. All of the samples were irradiated for 30 seconds with light at a wavelength of 254 nm. The containers were rinsed to remove any unattached oligonucleotide strands. Ultraviolet spectroscopy of the rinses was used to determine percent immobilization.

Hybridization and Electrochemical Analysis. The ferrocenyl PNA (3'-GCT-TTT-GTG-TAC) was hybridized to its immobilized oligonucleotide complement (5'-CGA-AAA-CAC-AUC; 1 base mismatch) and its immobilized oligonucleotide control (5'-CGA-AUU-GUC-AUC; 5 bases mismatch). Cyclic voltammetry were performed on each complement and control hybridization sample. The current for the control sample was 23.691 µA, and the current for the complement sample was 64.675 µA. These results showed an increased current reading for the complement hybridization versus the control sample.

Relevance and Practical Application. The development of a portable and simple-to-use hybridization assay with good sensitivity (picomole and lower detection level) is important in the monitoring of seafood safety. Food-borne illnesses can occur from eating fish and shellfish contaminated with pathogenic bacteria. Current detection methods are not suitable for rapid onsite monitoring of seafood samples for pathogenic bacterial contamination. Using an electrochemical hybridization assay format can be as portable and as simple as taking a pH reading.

Future Activities:

We will continue to develop an electrochemical nucleic acid hybridization assay that is sensitive, stable with storage, portable, and easy to use in a simple assay format.

Journal Articles:

No journal articles submitted with this report: View all 1 publications for this subproject

Supplemental Keywords:

peptide nucleic acid probe, electrochemical hybridization assay, ribosomal RNA, ferrocene nucleic acid probe, electrochemical nucleic acid probe, Alabama, AL, aquatic ecosystems, artificial reef design, coastal ecosystem, coastal environments, ecosystems, estuaries, estuarine research, fishery sampling, human modifications, land use, water use., RFA, Scientific Discipline, Geographic Area, ECOSYSTEMS, Water, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Chemical Engineering, estuarine research, Ecology, Environmental Chemistry, Ecosystem/Assessment/Indicators, State, Chemistry, Restoration, Aquatic Ecosystem, Ecological Effects - Environmental Exposure & Risk, Aquatic Ecosystems, Ecological Effects - Human Health, Terrestrial Ecosystems, Ecology and Ecosystems, Aquatic Ecosystem Restoration, Ecological Indicators, coastal ecosystem, water use, estuaries, watersheds, Alabama (AL), coastal environments, diagnostic indicators, ecosystem, water quality, electrochemical nucleic acid hybridization

Progress and Final Reports:

Original Abstract
  • 2001
  • 2003
  • Final

  • Main Center Abstract and Reports:

    R827072    Alabama Center For Estuarine Studies (ACES)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827072C001 Fluorescent Whitening Agents As Facile Pollution Markers In Shellfishing Waters
    R827072C002 Red Snapper Demographics on Artificial Reefs: The Effect of Nearest-Neighbor Dynamics
    R827072C003 Stabilization of Eroding Shorelines in Estuarine Wave Eliminates with Constructed Fringe Wetlands Incorporating Offshore Breakwaters
    R827072C004 Interaction Between Water Column Structure and Reproduction in Jellyfish Populations Of Mobile Bay (SGER)
    R827072C005 Effects of Variation in River Discharge and Wind-Driven Resuspension on Higher Trophic Levels in the Mobile Bay Ecosystem
    R827072C006 Results of Zooplankton Component
    R827072C007 Benthic Study Component
    R827072C008 A Preliminary Survey of Macroalgal and Aquatic Plant Distribution in the Mobile Tensaw Delta
    R827072C009 Fisheries-induced changes in the structure and function of shallow water "nursery habitats": an experimental assessment
    R827072C010 Effects Of Variation in River Discharge and Wind-Driven Resuspension on Lower Trophic Levels of the Mobile Bay Ecosystem
    R827072C011 Evaluation of Alabama Estuaries as Developmental Habitat for Juvenile Sea Turtles
    R827072C012 Effects of Salinity Stress on Natural and Anthropogenically-Derived Bacteria in Estuarine Environments
    R827072C013 The Role of Land-Use/Land-Cover and Sub-estuarine Ecosystem Nitrogen Cycling in the Regulation of Nitrogen Delivery to a River Dominated Estuary; Mobile Bay, Alabama
    R827072C014 Environmental Attitudes of Alabama Coastal Residents: Public Opinion Polls and Environmental Policy
    R827072C015 Synthesis and Characterization of an Electrochemical Peptide Nucleic Acid Probe
    R827072C016 Determinants of Small-Scale Variation in the Abundance of the Blue Crab Callinectes Sapidus
    R827072C017 Effects of Estrogen Pollution on the Reproductive Fitness of the Gulf Pipefish, Syngnathus scovelli
    R827072C019 A Model for Genetic Diversity Aquatic Insects of the Mobile/Tensaw River Delta
    R827072C020 Evaluating Trophic Processes as Indicators of Anthropogenic Eutrophication in Coastal Ecosystems: An Exploratory Analysis
    R827072C021 Effects of Anthropogenic Eutrophication on the Magnitude and Trophic Fate of Microphytobenthic Production in Estuaries
    R827072C022 Characteristics of Ship Waves and Wind Waves in Mobile Bay
    R827072C023 Methods Comparison Between Stripping Voltammetry and Plasma Emission Spectroscopy for Metals in Mobile Bay
    R827072C024 Changes in Water Conditions and Sedimentation Rates Associated With Construction of the Mobile Bay Causeway
    R827072C025 Cold-Induced Hibernation of Marine Vibrios in the Gulf of Mexico: A Study of Cell-Cell Communication and Dormancy in Vibrio vulnificus
    R827072C026 Holocene Sedimentary History of Weeks Bay, AL: Human and Natural Impacts on Deposition in a Gulf Coast Estuary
    R827072C027 Shelter Bottlenecks and Self-Regulation in Blue Crab Populations: Assessing the Roles of Nursery Habitats and Juvenile Interactions for Shelter Dependent Organisms
    R827072C028 Predicting Seagrass Survival in Nutrient Enriched Waters: Toward a New View of an Existing Paradigm
    R827072C029 DMSP and its Role as an Antioxidant in the Salt Marsh Macrophyte Spartina alterniflora
    R827072C030 A Preliminary Survey of Aerial and Ground-Dwelling Insects of the Mobile/Tensaw Delta
    R827072C031 Natural Biogeochemical Tags of Striped Mullet, Mugil cephalus, Estuarine Nursery Areas in the North Central Gulf of Mexico
    R827072C032 Resolution of Sedimentation Rates in Impacted Coastal Environments Using 137Cs and 210Pb Markers: Dog River and Fowl River Embayments
    R827072C033 Investigation of the Use of Pulse Amplitude Modulated (PAM) Fluorometry as an Indicator of Submerged Aquatic Vegetation Health in Mobile Bay
    R827072C034 Influence of Invasive Plant Species in Determining Diversity of Aquatic Vegetation in the Mobile-Tensaw Delta
    R827072C035 The Influence of Shallow Water Hydrodynamics on the Importance of Seagrass Detritus in Estuarine Food Webs
    R827072C036 Food Web Interactions, Spatial Subsidies and the Flow of Energy Between the Mobile Bay Delta and Offshore Waters: A SGER Proposal to the Alabama Center for Estuarine Studies
    R830651C001 Meteorological Modeling of Hurricanes and Coastal Interactions: A Stability Study For Vertical Pressure Levels
    R830651C002 Characterization of Glycoprotein Cues Used by the Parasitic Rhizocephalan Barnacle Loxothylacus texanus To Identify Its Blue Crab Host, Callinectes sapidus
    R830651C003 Survey of Diamondback Terrapin Populations in Alabama Estuaries
    R830651C004 An Assessment of Environmental Contaminant Levels in Water and Dragonfly Larvae Tissues from the Mobile/Tensaw Delta