Improved Proximity Assays for Protein Quantitation

Abstract

The work presented in this dissertation is focused on the development of proximity assay formats for biosensor applications in medical diagnostics, biological research, and point-of-care testing. An electrochemical proximity assay (ECPA) was developed for direct detection of a wide variety of target proteins. The detection limit of antibody-based reusable ECPA for insulin was 20 fM, which is lower than commercially available ELISAs, with a dynamic range through 6.25 nM, more than two orders of magnitude wider than the ELISAs. These improvements come with the added benefit of direct electrochemical readout. The measurement of hormone secretion from endocrine tissue via reusable ECPA and characterization by surface plasmon resonance (SPR) permit ECPA to be utilized in secretion sampling or high-throughput screening applications. Chapter 1 introduces basic concepts and common types of biosensors, the origin of the concept of proximity assay which plays a crucial role in ECPA, the reason of choosing DNA for the development of ECPA model systems, and the properties of apamers and antibodies as bioreceptors in biosensor technologies. In chapter 2, a simple and rapid automated microchip electrophoresis method was developed for measurement of dissociation constants of DNA aptamers against proteins and small molecules. Chapter 3 is mainly focused on the DNA model systems used for the development of asymmetric proximity ligation assay (PLA) and ECPA. Predictions from these model systems greatly promoted improvement of assays for detection of human thrombin, insulin and other biomolecules. The electrochemical proximity assay is presented in Chapter 4, where the first generation of ECPA is composed of two thrombin aptamers/insulin antibody-oligonucleotide conjugates which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. A faster, easier and reusable ECPA is then introduced in Chapter 5, where regeneration of the base DNA monolayer was accomplished enzymatically. Uracils were incorporated into selected probe strands, and these strands were enzymatically cleaved using a uracil-DNA excision mix. This allows measurement time to be reduced 30-fold for aptamer-based assay, from 90 min to 3 min, without significant loss of sensitivity. The measurement of hormones in blood serum and from secretions by pancreatic islets shows that ECPA could be a powerful tool for fundamental biochemistry, clinical analysis, or high-throughput screening applications. These works are not meant to be final, and proposals to include ECPA into an electronic device or to integrate ECPA with other detection technologies are outlined in Chapter 6.