Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mu m-diameter fibers was chemically etched to yield microweIls and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1 mu m microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-mu L sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed highdensity DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.