Development and application of a fiber optic array system for detection and enumeration of potentially toxic cyanobacteria
PIs D.M. Anderson and W.W. Carmichael
Harmful algal blooms (HABs) have become a serious threat to freshwater and marine waters worldwide, impacting humans, animals, and aquatic ecosystems. In freshwater, many cyanobacterial blooms (cyanoHABs) produce neurotoxic, hepatotoxic, dermatotoxic, or other bioactive compounds. As a result, managers need rapid, sensitive methods that can accurately identify and enumerate harmful species in a water body, yet to date, no methods exist that can be used to enumerate multiple species in the same sample and that can be deployed in small, bench-top instruments or on moorings for automated detection. This proposed project employs an innovative approach to HAB cell enumeration – fiber optic genosensors. It builds from prior studies on marine HAB species, for which this technology is well advanced. Once the technology is adapted and refined for freshwater cyanobacteria, we will test them using a portable instrument designed for clinical use in human pathogen detection.
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. Specific objectives are to:
- Design ribosomal RNA (rRNA) signal and capture probes for the three most important toxic cyanobacteria (Microcystis aeruginosa, Cylindrospermopsis raciborskii, and Anabaena flos-aquae) using published sequences
- Design and test a second probe pair for each species to incorporate redundancy into the array;
- Test these probes in the fiber-optic array format and determine detection limits, specificity, and dynamic range
- Refine hybridization conditions to reduce processing time
- Develop procedures to analyze multiple cyanoHAB species simultaneously using a single fiber bundle in a multiplexed format and validate it using mixed cultures and spiked and unspiked field samples
- Work with individuals and agencies responsible for fresh- and brackish water management to determine desired detection limits, precision, new cyanobacterial species for future probe design, and operational characteristics for the assay and instrumentation that would be developed around it
- Prepare a detailed protocol for sample handling and processing for this method
This project addresses a significant constraint to freshwater monitoring and management – the critical need for methods that accelerate and simplify enumeration of potentially toxic cyanobacterial species. In this regard, we note several significant advantages of the proposed technology – it can analyze dozens of target species on a single optical fiber bundle using encoded beads, and the arrays are reusable hundreds of times. This multiplexing ability is of obvious importance in regions where multiple freshwater HAB species co-occur. The technology can readily be adapted to target other cyanoHAB species as well as microbial pathogens and microorganisms of many types. Furthermore, it is highly amenable to automation, bringing us closer to the goal of 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.
Walt, D.R. 2000. Bead-based fiber-optic arrays. Science 287: 451-452.
Walt, D.R. and J. Epstein. 2003. Fluorescence-based fiber optic arrays: a universal platform for sensing. Chem. Soc. Rev. 32: 203-214.
Rudi, K., O.M. Skulberg, R. Skulberg, and K.S. Jakobsen. 2000. Application of sequence-specific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization. Appl. & Environ. microbial. 66(9): 4004-4011.
Ahn, S., D.M. Kulis, D.L. Erdner, D.M. Anderson and D.R. Walt. 2006. Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species. Appl. & Environ. Microbiol. 72(9):5742-5749.
Anderson, D.M., D. Kulis, D. Erdner, S. Ahn, and D. Walt. 2006. Fibre optic microarrays for the detection and enumeration of harmful algal bloom species. African J. Mar. Sci. 28(2): 231-25.
Carmichael, W.W., S.M.F.O. Azevedo, J.S. An, R.J.R. Molica, E.M. Jochimsen, S. Lau, K.L. Rinehart, G.R. Shaw, and G.K. Eaglesham. 2001. Human fatalities from cyanobacteria: Chemical and biological evidence for cyanotoxins. Environ. Health Perspectives. 109 (7): 663-668.