Fluorescence is a highly sensitive, precise, and convenient detection technique that is widely used in chemistry,
molecular biology and clinical laboratories. Fluorescence in the near-IR (700 - 900 nm) offers higher molar absorptivity
and significantly lower background signals from scatter than those generated by visible wavelength excitation. The
advantageous characteristics of near-IR fluorescence, primarily the reduced background signals, make this region of the
spectrum ideal for enhancement by metal nanostructures. Though multiple groups have successfully demonstrated metal
enhanced fluorescence, there remain several challenges in transferring this technology from the research stage to the
commercial stage. Using a LI-COR Odyssey® Infrared Imaging System, we quantitatively analyzed the effects of silver
particle geometries, including size, shape, and density of metal nanostructures, on the fluorescence enhancement of
Near-IR fluorophores. Using silver island film coated glass slides, we were able to obtain an 18-fold enhancement of
IRDye®700 and a 15-fold enhancement of IRDye®800 labeled DNA oligos over dye on plain glass. We further analyzed
the silver-coated glass surfaces for enhancement reproducibility and linearity. We demonstrated that the metal enhanced
emissions remained reproducible across a slide surface, and remained linear over several orders of magnitude. Finally,
using a highly quenched labeled protein, we were able to show an enhancement and release of the quenched
fluorescence, generating a 40-fold enhancement in the fluorescence emissions when spotted on a silver nanostructure
coated glass slide. Generating silver nanostructure coated slides that enhance fluorescence while maintaining linearity
and reproducibility will provide a class of new tools benefiting molecular biologists.
Current organic fluorophores used as labeling reagents for biomolecule conjugation have significant limitations
in photostability. This compromises their performance in applications that require a photostable fluorescent reporting
group. For example, in molecular imaging and single molecule microscopy, photostable fluorescent labels are important
for observing and tracking individual molecular events over extended period of time. We report in this paper an
extremely photostable and highly fluorescent phthalocyanine dye, IRDyeTM 700DX, as a near-infrared fluorescence
labeling reagent to conjugate with biomolecules. This novel water-soluble silicon phthalocyanine dye has an isomericly
pure chemical structure. The dye is about 45 to 128 times more photostable than current near-IR fluorophores, e.g.
Alexa Fluor"R"680, CyTM 5.5, CyTM 7 and IRDyeTM 800CW dyes; and about 27 times more photostable than
tetramethylrhodamine (TMR), one of the most photostable organic dyes. This dye also meets all the other stringent
requirements as an ideal fluorophore for biomolecules labeling such as excellent water solubility, no aggregation in high
ionic strength buffer, large extinction coefficient and high fluorescent quantum yield. Antibodies conjugated with
IRDyeTM 700DX at high D/P ratio exist as monomeric species in high ionic buffer and have bright fluorescence. The
IRDyeTM 700DX conjugated antibodies generate sensitive, highly specific detection with very low background in
Western blot and cytoblot assays.
A LI-COR Model 4000 DNA Sequencer has been modified by removing the internal scanning infrared fluorescence microscope and combining it with an external, orthogonal scanner. Due to the reduced background fluorescence and light scattering of nylon membranes in the near- infrared (8000 nm) as compared to the visible region of the optical spectrum, sensitivity of labeled DNA fragments is enhanced. Dot blots of dilution series of labeled oligonucleotides reveal a detection limit of 25 attomole (25 X 10-18 mole). DNA fragments blotted onto nylon membranes using direct transfer electrophoresis in multiplex DNA sequencing can also be detected and subsequently analyzed.
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