Microarray platforms are used everywhere — from academic research centers to public health laboratories to pharmaceutical companies. By enabling analysis of thousands of DNA sequences at a time, they have dramatically increased throughput and helped reduce costs in bioanalytical investigations. Typically, with “spotted” microarrays, DNA from two samples (a control and the sample of interest) is labeled with different color reporters and hybridized to the microarray. By comparing the reporter intensities, investigators can determine, for instance, whether the sample is healthy or diseased tissue. But this is often not enough, said Jason R.E. Shepard, a researcher in the Biodefense Laboratory of the Wadsworth Center at the New York state Department of Health in Albany. With respect to cancer screening and diagnosis, for example, there are various types and stages of the disease, and it would be more useful to evaluate the sample of interest against a number of parameters. Multiplexed arrays can provide much information by probing many gene targets in parallel, but these analyses are still based on the relative responses of two samples. The multicolor microarray platform could permit higher throughput as well as increased experimental significance. The ability to measure multiple parameters against a control, rather than the single parameter afforded by two-color assays, could allow investigators to test a number of hypotheses. Shown here are four images colored to reflect the wavelengths used and indicating where hybridization has occurred (the red spot in the bottom left image, for example). “It has always struck me as odd that you’re doing an assay that has thousands of genes you can interrogate,” he added, “and, typically, you look at only two samples.” To address this, he adapted a microarray platform developed in the lab of David R. Walt at Tufts University, where Shepard did his graduate studies, so that the array could probe eight samples at a time. As described in an Analytical Chemistry paper published online March 7, this simultaneous multicolor array hybridization affords an analytical flexibility not available with standard two-color assays. Researchers have developed a multicolor microarray platform that offers an analytical flexibility not available with conventional two-color assays. Using various color reporters, a single bead in the fiber optic-based microarray can hybridize eight samples at a time. The limitations of conventional microarray platforms lie largely in the choice of excitation sources and reporters. Typically, these platforms use laser excitation. Probing more than the usual two colors would require additional laser wavelengths, which could lead to significantly higher costs and larger sizes. Furthermore, the platforms generally use standard organic fluorophores as reporters, which have limitations of their own. Emission signals can be weak when there are relatively low concentrations of the gene target. At the same time, the use of such fluorophores can result in photobleaching, especially with long exposure times. Shepard addressed these limitations using a white light source, an epifluorescence microscope and a combination of organic fluorophores and quantum dot reporters. The white light source and microscope allow excitation and detection across the visible spectrum (the source is also substantially less expensive than laser sources). The quantum dot reporters are often considerably brighter than organic fluorophores and are more resistant to photobleaching. Also, they offer narrow emission bands, which helps reduce spectral overlap of the reporters. Indeed, this represents one of the main questions addressed in the paper: How many reporters can you fit into a defined spectral window? “When I first thought of this concept, I took every type of reporter in my lab and bought a couple more,” Shepard said. He started with about a dozen, “but it was easy to see right away that I was going to run into spectral overlap.” Ten reporters still led to some bleedthrough. Finally, he determined that he could use as many as eight reporters without spectral overlap. It might be possible to increase the number of reporters, he added, by using specific filters, prisms or filterless grating-based optics. The filters discussed in the Analytical Chemistry paper were not specifically optimized for the reporters used. Shepard demonstrated the technique by analyzing eight Bacillus anthracis samples simultaneously. The system he used was based on an epifluorescence microscope made by Olympus and modified by Optical Analysis of Nashua, N.H. A white-light mercury bulb provided excitation, and a CCD camera made by the Cooke Corp. of Romulus, Mich., acquired images of the fluorescence. The excitation and emission filter wheels were made by Prior Scientific of Cambridge, UK. IPLab software from Scanalytics of Fairfax, Va., controlled the filter wheels and shutters and processed the images. The array platform was a 1-mm fiber optic bundle etched and embedded with microbead sensors. The fiber optic bundle contained approximately 50,000 3-μm array “spots,” to which labeled target nucleic acids were hybridized. One advantage of a fiber optic-based platform is that it offers uniform spot features, such as size and boundaries. These can vary considerably in other array formats, complicating analysis. The roughly 1-mm fiber optic bundle used for the microarray contains approximately 50,000 array “spots,” each of which is 3 μm in diameter. One advantage of the fiber optic-based platform is that the features of these spots, such as size and boundaries, are uniform, which is not always the case with other microarray platforms. The experiments showed that multiplexed analysis with the new platform offered a fourfold increase in throughput with respect to conventional two-color assays. The experiments included a polymerase chain reaction amplification step, without which analysis of eight samples might not be as efficient. Nonetheless, having more colors would still allow higher throughput as well as improved experimental significance. Investigators could use the platform to explore a number of hypotheses, measuring multiple parameters against the control instead of the single parameter offered by conventional two-color assays. Or they could assign multiple parameters as controls to circumscribe the unknown response. The platform could serve a variety of applications including pathogen diagnostics. “Particularly in the area of biodefense,” Shepard said, “I view arrays as necessary for rapid typing of unknown or emerging (mutating) pathogen detection. The natural propensity for horizontal gene transfer and the ability to engineer organisms could very well preclude detection by an assay directed at identifying a single gene target. Or even something like the possibility of multiple infectious agents turning up together could be problematic for conventional assays.” In the meantime, Shepard plans to extend the technique. “We’re going to start dabbling with different assays in which we can do multicolor analysis,” he said. “Not just DNA microarrays, but some live-cell imaging as well.” The technique is especially well suited to this because the photostability of the quantum dots will allow the researchers to track cells for hours at a time.