Biological warfare agents (BWAs) pose significant threats to both military forces and civilian populations. The increased concern about bioterrorism has promoted the development of rapid, sensitive, and reliable detection systems to provide an early warning for detecting the release of BWAs. We have developed a high-density DNA array to detect BWAs in real environmental samples with fast response times and high sensitivity. An optical fiber bundle containing approximately 50,000 individual 3.1 μm diameter fibers was chemically etched to yield an array of microwells and used as the substrate for the array. 50-mer single-stranded DNA probes designed to be specific for target BWAs were covalently attached to 3.1-μm microspheres, and the microspheres were distributed into the microwells to form a randomized high-density DNA array. We demonstrated the applicability of this DNA array for the identification of Bacillus thuringiensis kurstaki, a BWA simulant, in real samples. PCR was used to amplify the sequences, introduce fluorescent labels into the target molecules, and provide a second level of specificity. After hybridization of test solutions to the array, analysis was performed by evaluating the specific responses of individual probes on the array.
A microsphere-based DNA biosensor array with high packing density and low detection limits has been previously developed. Polymeric 3.1-μm-diameter microspheres are employed as the detection elements, where each microsphere is functionalized with single-stranded oligonucleotide probe sequences. The biosensor array is fabricated by randomly distributing a stock microsphere suspension, containing various oligonucleotide-functionalized microspheres, on the distal end of a chemically etched imaging fiber bundle. By placing the microspheres into wells at the end of each individual fiber, each optical channel is connected to a single microsphere. The microspheres are encoded with a unique combination of dyes in order to determine a particular microsphere’s location in the randomized array. Specific fluorescence responses are observed after hybridization with fluorescently labeled complementary targets. Enhancement of the signal to noise ratio is possible because of intrinsic redundancy of sensing elements built into the array. This microsphere-based DNA biosensor has several major advantages over existing platforms including higher sensitivity, micron-sized features, and rapid throughput. Microsphere-based DNA arrays have been successfully applied to genomic discrimination of bacteria, gene expression analysis, and the detection of bioagents.