Transforming Microtubules into Microshuttles for Advanced Immunoassay Applications.

Abstract

There is significant evidence that microfluidic approaches to analyze arrays of biomarkers are effective in early disease detection, and point-of-care diagnostics relying on proteomic sample analyses would be beneficial in improving global health. Much progress has been made in engineering the kinesin-microtubule system for future implementation in lab on chip systems. Specifically, microtubules have been transformed into microshuttles carrying various cargos, and microfluidic platforms have been engineered to support the kinesin-microtubule system. The goal of this work is to develop a kinesin-driven enzyme-linked immunosorbent assay (ELISA) platform that reduces typical ELISA incubation times, eliminates laborious rinse steps, and challenges the standard ELISA detection limit. This dissertation is a culmination of advances made to further develop microtubules into microshuttles for future kinesin-driven advanced immunoassay applications. First, a strategy to functionalize microtubules with antibodies that retains motility characteristics while binding high densities of antibody (>100 antibody μL-1 of microtubule) on the microtubule surface was developed. These microtubules allow for the specific adsorption of small amounts of antigen on the microtubule surface in small (nL) sample volumes, ideal for low detection limits in a microtubule-based ELISA. Second, the potential and limitations of exposing the kinesin-microtubule shuttle system to body fluids, including human blood plasma, saliva, and urine that are commonly used in ELISA diagnostic tests, were characterized. The effects these medically relevant samples have on kinesin motility and the ability for specific cargos to bind to the microtubule surface were analyzed. Finally, future directions for kinesin-driven immunoassay platforms are discussed. In conclusion, the ability for microtubules to be functionalized with high densities of antibodies and maintain the ability to bind cargos in medically relevant samples without sacrificing their gliding assay motility properties corroborates that integrating the developed antibody-functionalized microtubules into advanced micro- or nanofluidic platforms holds great potential in developing a kinesin-driven ELISA platform that can function in body fluids, significantly reduce the assay time, reduce the invasiveness of diagnostic tests, and challenge the detection limits of standard ELISA.