| The present dissertation work focused on the design and development of a novel signal amplification system based on the reconstitution of an apo-enzyme (apo-glucose dehydrogenase, apo-GDH, and apo-horseradish peroxidase, apo-HRP) with their corresponding prosthetic groups, e.g., pyrroloquinoline quinone (PQQ) and hemin, respectively. The remarkable signal generation ability of these two enzyme systems enables the development of ultra-sensitive measurement methods, such as immunoassays and non-immunological binding assays.;An ultra-sensitive detection system was designed by combining the reconstitution and the enzymatic amplification effects observed by employing PQQ loaded polymeric (polymethylmethacrylate, PMMA) nanospheres as tracers for immunoassays. The PMMA nanospheres were formulated with a lipophilic version of a PQQ salt, and characterized with UV-Vis spectrometry, SEM, and the GDH-PQQ reconstitution assay. The binding capacity of the nanospheres functionalized with NeutrAvidin was further evaluated with a biotinlyated solid surface and the modified particles were finally employed as an antibody label in a sandwich immunoassay for C-reactive protein, that exhibited a detection limit of 220 pg/mL.;A novel homogeneous competitive binding assay for biotin was also developed based on the GDH-PQQ reconstitution assay. The biotinylated apo-GDH was fully characterized with respect to the mole ratio of biotin to apo-GDH, residual activity, and avidin binding effects (incubation time and dose-dependence). In the presence of avidin, the biotin-apo-GDH competes with free biotin for the limited binding sites of the avidin. Thus, the concentration of free biotin is inversely proportional to the enzymatic signal. A detection limit of less than 10 nM biotin was demonstrated in this homogeneous assay system.;Finally, the GDH and HRP enzyme systems were employed to develop a protease assay and a homogeneous competitive binding assay using a suitable biological substrate probe, where PQQ or hemin was synthetically derivatized with small molecules (such as alanine methyl ester and thyroxine) or oligopeptides. These prosthetic group derivatives were investigated and evaluated in the reconstitution assays with their respective apo-enzymes. The PQQ- or hemin-small drug molecule conjugates, when successfully prepared, showed potential in further developing an ultrasensitive homogeneous assay with fast and direct signal transduction. |
