Fiber Optic Microarray Detection of Freshwater Cyanobacteria. Shonda Gaylord PhD Candidate TCSVM Walt Laboratory Woods Hole Oceanographic Institute
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1 Fiber Optic Microarray Detection of Freshwater Cyanobacteria Shonda Gaylord PhD Candidate TCSVM Walt Laboratory Woods Hole Oceanographic Institute
2 Why Detection Matters The release of cyanotoxins into waters used for drinking and recreational activities Cyanotoxins: neurotoxins, cytotoxins or hepatoxins The LD 50 for such toxins respectively is 20ug/kg and 50ug/kg compared to Cobra venom which has an LD 50 of 185ug/kg in murine studies WHO drinking water guidance value for safe consumption is 1µg/L The potential toxicity of these organisms demonstrates the need to safely and effectively monitor influxes of large amounts of cyanobacteria in freshwater systems for public health measures and environmental integrity
3 Cyanobacteria In collaboration with WHOI, Tufts Chemistry Department and Ahura Technologies the overall project goal is to adapt and validate a rapid and accurate optical fiber-based technology for CyanoHAB cell detection and enumeration in both laboratory and field settings.
4 Capture & Signal Probes Base pair mismatches for capture probe development are highlighted in yellow Microcystis probe #1 S. elongates C. raciborskii A. flos-aquae C A C C G A T G T T C T T C C C A A T C C G C T G G T G T T C T T C A G A A T A C A G A C C C T T T A C G C C C A A T C C G G A C C C T T T A C G C C C A A T C Cylindro probe #2 A. cylindrica M. aeruginosa A. flos-aquae Signal probe #1 M. aeruginosa C. raciborskii A. flos-aquae C A G C A G A C T T T C A G T T C C C A G C A G A C T T A C A T G G C C C A G C C A C A C C T T C C G G T A C A G C A G A C T T A C A A T G C C C T G A G A C A C G G C C C A G A C C T G A G A C A C G G C C C A G A C C T G A G A C A C G G C C C A G A C C T G A G A C A C G G C C C A G A C
5 Fluorescent In Situ Hybridization Cylindrospermopsis Microcystis Cylindro probe 1 Micro probe 11 20x: Few Dozen 20x: ~100
6 + = false positives - = false negatives FISH Microarray
7 Fiber Optic Bundles Individual Cladded Optical Fibers Silica Jacket Epstein, J. R. and Walt, D. R., Chem. Soc. Rev., 2003, 32,
8 Microspheres in Etched Wells Wet etching with HCl Pantano, P.; Walt, D.R. Chem. Mater. 1996, 8,
9 Base Pairing A A C G T G A T T G C A C T
10 Microsphere Array Probe A Probe B Probe C 1) Different DNA probes are first attached to beads 2) Probes are combined into a library CCD chip Fiber bundle Microwells etched on distal fiber face 3) Probe bead library is distributed randomly into wells on fiber array surface
11 Microarray Assay Encoding Sandwich hybridization assay Direct hybridization Cy5 Background Cy3 Direct hybridization o o
12 Flourescence, a.u. Cylindrospermopsis Net Signal Intensities Cy3 o um 1 um 100 nm [C-probe-Cy3]
13 Detection Arrays of both Microcystis and Cylindrospermopsis are able to detect 1 nm of target within 10 minutes and are currently being tested for detection of pm target concentrations. Microarray platforms are capable of detecting multiple analytes simultaneously. Goal is to provide an early warning system utilizing laboratory-based flow-through systems, or remote, moored instruments capable of detecting and providing early warning of organisms that threaten public and ecosystem health.
14 Ahura 28x Magnifications Field of view: 350 x 280 micron Over 2000 three-µm beads in field of view CCD camera Touch screen display NA: 0.55 actuators Depth of focus: ~1 micron Two-color measurement, Cy3 and Cy5 dyes Folded to reduce height and width Barcode scanner computer
15 Acknowledgements SAC Committee Dr. Saul Tzipori Dr. David Walt Dr. Dan Milner Dr. Don Anderson Groups Walt Lab WSSS Program Funding NIH EPA STAR
16 Neurotoxins Anatoxin a Anatoxin a(s) ZACCARONI et al, 2008
17 Microarray Probes Probes successfully tested for cross-reactivity are then transitioned to fiberoptic microarray format and tested against synthetic target and cell lysates from target species. Probe Type Target Organism Length (bp) Sequence (5 3 ) Capture Probe Cylindrospermopsis 18 CAG CAG ACT TTC AGT TCC Synthetic Target Cy3 Labeled Cylindrospermopsis 18 GGA ACT GAA AGT CTG CTG Capture Probe Microcystis 24 CCG CCT TTA GGT CGT TAA GC Synthetic Target Cy3 Labeled Microcystis 24 GGT TGC TTA ACG ACC TAA AGG C
18 Instrumentation 75 W Xenon Arc Lamp Excitation Fliter Wheel and Shutter Control Z Positioner X-Y Positioner G3 Macintosh w/ IP LAB 20X 10X 5X CCD Camera Sensor Array Dichroic Housing Emission Filter Wheel and Shutter Control
19 Imaging System
20 Analytical Measurements Using the software in IPLab overlay the ROI obtained form the Eu image onto the background and hybridization images Using the Measurements/ROI option measure the mean intensities of the beads and the standard deviation of the signals
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Application Note GENOMICS INFORMATICS PROTEOMICS METABOLOMICS A T C T GATCCTTC T G AAC GGAAC T AATTTC AA G AATCTGATCCTTG AACTACCTTCCAAGGTG New Stringent Two-Color Gene Expression Workflow Enables More