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An enzyme-linked analytical nanosphere sensor (ANSor) is described, responding to enzyme-substrate turnover in the vicinity of a quantum dot (QD) due to cohnmobilized enzyme and pH sensitive ligand. QD capping by mercapto-alkanoic acids were rejected as a pH sensitive ligand, but with the use of a layer-by-layer assembly on mercaptopropionic capped QDs and an intermediate poly(allylamine hydrochloride) layer, anthraquinone sulfonate (calcium red, CaR) was introduced to modify the pH in the immobilized system > 8. QD-CaR absorption shows speciral overlap with QDroo emission at all pHs and gives a complex pH dependent fluorescence resonance energy transfer (FREL) efficiency, due to excited state proton transfer (χex = 540 nm; χem = 585 nm). In contrast QD615-CaR with spectral overlap between the QD and CaR gave a strong and reproducible pH response. QD-urease and QD-creatinine deiminase conjugates could be linked with pH changes produced by enzyme degradation of urea and crealinine, respectively. Close coupling between the pH sensitive QD and enzyme conjugate maximized signal compared with solution based assays: QD-urease and QD-CD bioconjugates were tested in model biological media (Dulbecco's modified Eagle's Medium and fetal calf serum) and in urine, showing a response in 3-4 min. [ABSTRACT FROM AUTHOR]