The preparation and analytical characteristics of novel prosthetic group loaded polymeric nanospheres for use in high-sensitivity bioaffinity assays is reported. Pyrroloquinoline quinone (PQQ), a prosthetic group for apoglucose dehydrogenase (apo-GDH), is loaded into poly(methyl methacrylate) (PMMA) nanospheres in the presence of methanol. PQQ released from the nanospheres in the presence of 40% acetonitrile is capable of reconstituting apo-GDH and triggers the enzymatic reaction with excess glucose. The two electrons generated are transferred from a reduced PQQ to a redox dye reagent, e.g., 2,6-dichloroindolphenol (DCPIP). The decrease in absorbance of DCPIP is observed visually or spectrophotometrically to assess the number of particles present. As initial model systems, this concept is applied to develop a microtiter plate assay to detect biotin (as a model for low molecular weight species) and C-reactive protein (CRP). For the CRP assay, neutravidin-coated PQQ-doped PMMA nanospheres are used to bind with a biotinylated reporter antibody directed toward CRP. Detection limits to CRP at subnanogram per milliliter levels are demonstrated. The advantage of this type of assay is that excess apoenzyme can be added, with detection capability dependent on the number of encapsulated PQQ species that can be readily released from the surface-bound nanospheres (ca. 20,000 PQQ molecules/PMMA particle).