The research described in this proposal details a procedure for detecting and identifying bilirubin using a novel method of surface bound diazonium salts and surface enhanced Raman scattering. Bilirubin is known for its detrimental neurological effects on infants suffering from hyper- bilirubinemia. Current methods for detecting and monitoring bilirubin suffer from poor selectivity due to the presence of hemoglobin and related heme species that absorb at the same wavelengths as bilirubin. Solution diazonium methods move the absorption away from the wavelengths associated with heme groups and are often used in clinical labs. These two suffer from poor selectivity with respect to biliverdin, free bilirubin and bilirubin conjugated with sugars. Raman spectroscopy offers the advantage of high molecular specificity in contrast to UV-Vis absorption spectroscopy. CC Technology proposes synthesizing a diazonium salt that contains a thiol functionality that will anchor the salt to a silver substrate that provides a strong surface enhanced Raman signal and which when reacted with bilirubin, will form a dye which absorbs at the laser wavelength. Two laser sources will be considered: a 633 nm HeNe laser source and an 852 nm DBR diode laser source. These represent commercially available, low cost Raman systems that could be used in a marketable instrument. The ability to excite where the diazotized bilirubin absorbs will provide additional sensitivity and selectivity through a resonance enhancement of the Raman scattering. A surface bound species also offers the potential advantage of washing the sample after reaction to remove interferences in the Raman spectroscopy. Preliminary studies with diazotized p-aminothiphenol showed excellent sensitivity (250 micromolar bilirubin) and selectivity through several bilirubin vibrational bands in the surface Raman spectrum.
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