Fiber-Optic Microarray for Simultaneous Detection of Multiple Harmful Algal Bloom Species

Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 m) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment. Harmful algal blooms (HABs) result from the proliferation of certain types of phytoplankton species. In some cases, accumulation of these organisms can cause a discoloration of the seawater, giving rise to the name “red tides” (3). HABs pose a serious threat to public health because many HAB species produce potent toxins, which are responsible for a variety of shellfish poisoning syndromes. Consumption of HAB-contaminated shellfish has been linked to mortalities of wild and farmed fish, seabirds, and mammals (13, 24) and can result in illness or death in humans (4, 31). In addition, HABs adversely affect the coastal economy, causing economic loss due to restrictions on seafood industries and reduced tourism (22, 37). The economic loss caused by HAB is estimated to be at least $49 million each year in the United States alone. As the frequency of HAB occurrences has increased worldwide (22), new techniques have been developed to monitor seawater for the presence of HAB species. Traditionally, phytoplankton are detected and enumerated by direct observation using light or electron microscopy of live or preserved seawater samples. Although this method can provide direct visual confirmation of target organisms, it is both time-consuming and requires expertise in phytoplankton taxonomy because of the difficulty in identifying morphologically similar species or strains (20, 36). The latter problem frequently occurs in the study of HAB species, because toxic and nontoxic phytoplankton species can coexist in the collected sample, and in most samples, the HAB species of interest is often a minor compo