Plasmonic Detection of Free-Bilirubin in Neonates

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Project summary/Abstract: Over the last couple of years our laboratories have developed a new fluorescence based phenomenon, which is now having profound implications in clinical biotechnology. This new technology has led to the recent publication of over 50 peer reviewed articles, one edited hard bound volume and the launch of a new peer reviewed Springer Journal, entitled Plasmonics. This new phenomenon relies on the interactions of fluorophores with metallic nanoparticles and can increase the quantum yield of fluorescing species, provide spatially localized excitation and can even substantially improve fluorophore photostability. Our new technology clearly demonstrates that these novel effects can result in up to a million-fold more photons per fluorophore, which when applied to assay sensing platforms, provides for quick and simple high sensitivity assays. In this regard, under a recently completed NIH award to the PI, we were able to develop an enhanced assay platform for RNA target detection which has the sensitivity of PCR and ELISA, but most profoundly, without any amplification steps. In this R21 proposal we have assembled a multidisciplinary team of Spectroscopists, Biostatisticians and Clinicians alike, to develop a high sensitivity assay for the detection of free unbound Bilirubin in neonatal serum, an area of intense research and high clinical importance due to its ability to cross the blood brain barrier and participate in various neurological disorders. At present, tests for free unbound bilirubin are arduous, time consuming, and no approach has to date been standardized. The Metal-Enhanced Fluorescence (MEF) free bilirubin assay described here, offers high sensitivity detection, simple operation, can be made disposable and has the potential to even be standardized in laboratories around the world. The MEF assay functions as follows: Human Serum Albumin (HSA), which binds and transports serum free and bound bilirubin respectively, is immobilized in a hydrophilic copolymer, itself immobilized onto a glass slide. When serum, containing serum albumin predominantly bound bilirubin (> 99 %) is washed across the assay surface, free bilirubin diffuses into the polymer, binds to the embedded HSA and becomes intrinsically highly fluorescent due to its locality to surface silver nanostructures. We intend to optimize and determine the sensitivity of the assay using both laboratory and clinical samples and most importantly, statistically compare our results with the "UB analyzer", one of only 3 instruments in the USA designed to measure free bilirubin.