Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species

Abstract

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 mum) 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.

FIG. 1.

Optimizations of sandwich hybridization for the capture probe (NA1S) (A) and signal probe (AlexS) (B) using A. fundyense as a target cell. Error bars, standard deviations from three measurements. (A) Hybridization time with the capture probe was optimized with two different numbers of cells: 500 cells and 5 cells of A. fundyense. The solid curve represents the polynomial fit for 500 cells, and the dashed curve represents that for 5 cells, with R² values of 0.996 and 0.994, respectively. (B) Hybridization time with the signal probe was optimized with 50 cells of A. fundyense. The solid curve represents the polynomial fit, with an R² of 0.998. Each point represents the average of triplicate measurements.

FIG. 2.

Dynamic range of the dose-response curve from the DNA microarray. The NA1S single-probe microarray was used as a representative array, and serially diluted rRNA samples from A. fundyense were used as the target. The solid line represents the linear fit, with an R² of 0.984. Each point represents the average of triplicate measurements. Error bars, standard deviations from three measurements.

FIG. 3.

Effect of natural plankton cells and detritus present in seawater. (A) Signal intensities from target samples obtained using various numbers of A. fundyense cells spiked into 1 ml seawater concentrate, which is the equivalent of 1 liter of raw seawater. (B) Signal intensities obtained using various volumes of seawater concentrate spiked with 1,000 A. fundyense cells. The single bead-type array with the NA1S probe was used for the signal measurement. The standard deviation of the background was 15, and the threshold for a positive signal was calculated to be 45 (3 × 15). Each data point represents the average of triplicate measurements. Error bars, standard deviations from three measurements.

FIG. 4.

Simultaneous detection of single (A) or multiple (B) HAB species using a multiplexed microarray containing three probe types: NA1S, AO2, and auD1S. The standard deviation of the background was 17, and the threshold limit for a positive signal was calculated to be 51 (3 × 17). The positive threshold is shown as dashed lines. Each point represents the average of triplicate measurements. AF, A. fundyense GTCA 28; AO D2 (A) or D2 (B), A. ostenfeldii HT-240D2; AO D6 (A) or D6 (B), A. ostenfeldii HT-120D6; PN, P. australis 1BA.