ORIGINAL PAPER 5-Color Multiplexed Microwave-Accelerated Metal-Enhanced Fluorescence: Detection and Analysis of Multiple DNA Sequences from within one Sample Well within a Few Seconds Anatoliy Dragan & Chris D Geddes Received: 6 March 2013 /Accepted: 15 September 2014 # Springer Science+Business Media New York 2014 Abstract We present a potentially highly sensitive and selec- tive bio-assay for the potential detection of any five different DNA sequences from one sample in one well. The assay is based on a DNA “rapid catch and signal” (DNA-RCS) tech- nology developed for the detection of different DNA se- quences from a sample well area. Our signal amplification utilizes the metal-enhanced fluorescence (MEF) of dyes at- tached to the probe-DNAs, which hybridizes with the pre- formed mixture of anchor-DNA scaffolds on silver island films (SiFs). Low-power microwave irradiation accelerates both the formation of the anchor-DNA scaffold on the SiF- surface and anchor/probe DNA hybridization, i.e. “rapid catch” of target DNAs from a bulk solution, decreasing the assay run time from hours to only a few seconds. Localization of signaling dye-labels close to the SiFs make them extremely photostable, which allows for collecting/integrating the signal over a long time period. To demonstrate a 5 color DNA assay (5-plex) we have used a range of readily available Alexa™ dyes. Advantages and perspectives of the RCS-technologies ability to detect 5 different DNA sequences from within one plate-well are discussed. Keywords Metal-EnhancedFluorescence . SurfaceEnhanced Fluorescence . Plasmon Enhanced Fluorescence . Plasmon Controlled fluorescence . DNA assays . Point-of Care Diagnostics . 20 s Assays . Pathogen Detection . Bio-agent Detection Abbreviations MEF Metal-Enhanced Fluorescence NP Nanoparticle SiF Silver Island Film ssDNAsingle stranded DNA dsDNA double stranded DNA PCR Polymerase Chain Reaction RCS Rapid Catch and Signal technology LoD Limit of Detection FRET Fluorescence Resonance Energy Transfer Introduction Detection of small quantities of DNA of a certain sequence in biological fluids is a problem of the up-most importance to the life sciences. DNA sequences contain information about the origin of cells and can serve as markers for pathogens, sources of infection in humans, mutated states of genes, analysis of bacterial diversity and for many other issues concerning living organisms. Success in solving this problem, finding ap- proaches that can register small amounts of DNA sequences will pave the way for the creation of new ways of thinking of how we implement medical care. Until recently the PCR (polymerase chain reaction) tech- nique was exclusively employed for the purpose of detection of small quantities of specific DNA sequences in solution [1–4]. The power of PCR is in the ability to amplify DNA fragments to the level where simple, usually a fluorescent dye (e.g. PicoGreen, SYBR Green) [2] binding approaches, could easily register the presence of large amounts of amplified DNA. In the last few years the MEF phenomena has become one of the most promising approaches for lower copy number DNA detection. MEF of fluorophores is a consequence of their coupling with the plasmons of metal nanoparticles (NP), which occurs due to the close proximity of a dye to A. Dragan : C. D. Geddes (*) Institute of Fluorescence and Department of Chemistry and Biochemistry, UMBC, 701 East Pratt Street, Baltimore, MD 21202, USA e-mail: geddes@umbc.edu J Fluoresc DOI 10.1007/s10895-014-1458-0