| Antibody-based biodetection is central to numerous vital technologies for biological research and medical products. One critical aspect of antibody-based biodetection is the reporter strategy by which the antibody probe is visualized. Current reporter strategies are hindered by insufficient sensitivity in numerous applications, hazards related to radioactivity, and limitations associated with the use of enzymes. Identification of novel, non-enzymatic signal amplification methods that achieve improved sensitivity, simplicity and robustness compared to currently available approaches would greatly enhance antibody-based biodetection technologies.;This thesis focuses on employing photopolymerization to develop a non-enzymatic signal amplification technique that enables sensitive, specific, quantitative, and inexpensive antigenic detection of proteins. Specifically, photoinitiators are coupled to antibody probes such that in the presence of monomer and light, polymer films form specifically in regions of the surface where the probe has bound. In this manner, biorecognition is evidenced by the formation of a macroscopic polymer film that is readily visible without instrumentation. Moreover, to achieve a more easily quantified response, fluorescent nanoparticles are added to the monomer and become entrapped in the developing polymer network, thereby generating highly fluorescent films.;A variety of monomer formulations were evaluated for polymerization-based signal amplification (PBA), resulting in the identification of an acrylamide-based formulation that yields four to eight-fold thicker films and a five-fold improvement in sensitivity as compared to the previously optimal poly(ethylene glycol) diacrylate (PEGDA)-based formulation. Also, investigation into the eosin photoinitiation mechanism revealed that eosin is capable of overcoming 100- to 1000-fold excess polymerization inhibitors, likely contributing to its success as a PBA initiator.;Fluorescent PBA (FPBA) was evaluated for biodetection in antibody microarrays and was found to enable detection of as few as 0.16 biotin-labeled target molecules per mm2, constituting a 100-fold improvement in sensitivity compared to using a traditional fluor-labeled probe. Additionally, FPBA was demonstrated for fluorescent immunostaining of cells, yielding similar signal intensities and sensitivity as the highly sensitive enzymatic tyramide signal amplification method, yet with the advantages of enabling better signal localization and not being impacted by endogenous cellular enzymes. These results suggest that PBA would be also advantageous in other antibody-based and cell-based detection applications. |
