High-resolution near-infrared imaging of DNA microarrays with time-resolved acquisition of fluorescence lifetimes

Ultrasensitive, near-infrared (NIR), time-resolved fluorescence is evaluate d as a detection method for reading DNA hybridization events on solid surfa ces for microarray applications. In addition, the potential of mulitiplexed analyses using time-resolved identification protocols is described. To car ry out this work, a NIR time-resolved confocal imager was constructed to re ad fluorescence signatures from the arrays. The device utilized a 780-nm pu lsed diode laser, a single-photon avalanche diode (SPAD), and a high-numeri cal-aperture microscope objective mounted in an epi-illumination format. Du e to the small size of the components that are required to con; struct this imager, the entire detector could easily be mounted on high-resolution tra nslational stages and scanned over the stationary arrays. The instrument re sponse function of the device was determined to be 275 ps (fwhm), which is adequate for measuring fluorophores with subnanosecond lifetimes, To charac terize the system, NIR dyes were deposited directly on different substrate materials typically used for DNA microarrays, and the fluorescence lifetime s of two representative dyes were measured. The fluorescence lifetime for a luminum tetrasulfonated naphthalocyanine was found to be 1.92 ns, and a val ue of 1.21 ns was determined for the tricarbocyanine dye, IRD800, when it w as deposited onto poly(methyl methacrylate) (PMMA) and measured in the dry state. Finally, the imager was used to monitor hybridization events using p robe oligonucleotides chemically tethered to a PMMA substrate via a glutard ialdehyde linkage to an aminated-PMMA surface. The limit of detection for o ligonucleotides containing a NIR fluorescent reporter was determined to be 0.38 molecules/mum(2), with this detection limit improving by a factor of 1 0 when a time-gate was implemented. Fluorescence lifetime analysis of the h ybridization events on PMMA indicated a lifetime value of 1.23 ns for the N IR-labeled oligonucleotides when using maximum-likelihood estimators.