Absorbance enhancement in microplate wells for improved-sensitivity biosensors

A genericopticalbiosensingstrategywasdevelopedthatreliesontheabsorbanceenhancementphenom- enon occurringinamultiplescatteringmatrix.Experimentally,insertsmadeofglass fiber membranewere placedintomicroplatewellsinordertosignificantlylengthenthetrajectoryoftheincidentlightthroughthe sampleandthereforeincreasethecorrespondingabsorbance.Enhancementfactorwascalculatedby comparingtheabsorbancevaluesmeasuredforagivenamountofdyewithandwithouttheabsorbance- enhancinginsertsinthewells.Moreover,thedilutionofdyeinsolutionswithdifferentrefractiveindices(RI) clearlyrevealedthattheenhancementfactorincreasedwiththe ΔRI betweenthemembraneandthe surroundingmedium,reachingamaximumvalue(EF425)whenthemembranesweredried.Onthisbasis, two H2O2-biosensingsystemsweredevelopedbasedonthebiofunctionalizationoftheglass fiberinserts either withcytochrome c or horseradishperoxidase(HRP)andthe analyticalperformancesweresystem- aticallycomparedwiththecorrespondingbioassayinsolution.Theefficiencyoftheabsorbance-enhancement approachwasparticularlyclearinthecaseofthecytochrome c-basedbiosensorwithasensitivitygainof40 folds andwiderdynamicrange.Therefore,thedevelopedstrategyrepresentsapromisingwaytoconvert standardcolorimetricbioassaysintoopticalbiosensorswithimprovedsensitivity.