We are in a thrilling period defined by the rapid integration of many research disciplines and groundbreaking technical advancements fundamentally transforming our diagnostic and clinical procedures. The fusion of micro-nanotechnology and biotechnology is driving our technical capabilities to unprecedented levels. Despite notable advancements in sensitivity and multiplexing capability over the past few decades, there remains ample opportunity for innovation to enhance performance as a practical instrument for real-world applications. In order to accomplish this objective, numerous complex issues pertaining to the engineering, plasmonics, electrochemistry, and biomedical applications of bead-based assays must be addressed. To achieve this, I present a novel method in assay development, plasmonic and electrochemical sensing techniques, and automation in this dissertation. The first chapter introduces reagents used in the diagnostic assays, and current diagnostic methods such as ELISA, PCR, and bead-based assays. We then discuss three examples of widely used detection techniques. These techniques are plasmonic sensing, electrochemical sensing, and fluorescence sensing.The second chapter introduces a novel sandwich bioassay called Biomarker-to-surrogate conversion (B2S). We develop this assay to overcome the one challenge that has been neglected over the past decades: the need for solutions in the current diagnostic world when it comes to amplifying signals for protein detection. We convert target proteins (antigens) into surrogate beads that are much heavier and larger than antigens. That enables us to detect them much more reliably at attomolar-level concentrations. This remarkable noble assay enables us to have background free surrogate particles as final product of the assay. Hence, it opens us many doors to develop and execute different detection setups. The third chapter aims to develop a novel signal collection method to generate signal from surrogate products of B2S described in Chapter 2. In this chapter, we overcome the mass transport problem that has been prevented most research from going below pg/mL detection ranges, by introducing a flow-through setup for processing the final buffer obtained through our B2S assay. We introduce plasmonic nanohole arrays (NHA) and their implementation in surrogate particle sensing. We employ Extraordinary Optical Transmission (EOT) with the manufactured NHA chips, combining nanofluidics and nanoplasmonics into a single unit. We measure the peak shift in the spectrum of the transmitted light due to the resonance wavelength, and hence detect the target molecule in our sample. This setup also enables us power-free and naked-eye detection of the samples, by utilizing merely sun light, at femtomolar levels. The fourth chapter is about automating the novel assay, B2S, described in Chapter 2, with the novel electrochemical sensing technique we have developed and explained in this chapter. By using a low-cost and open-source liquid handling platform called OT-2, we are able to automate the B2S assay procedure which significantly reduces the workload on the scientist. Also, we designed a printed circuit board (PCB) to control multiple SPEs, allowing us to do electrophoretic deposition simultaneously at eight different chips. Then, by combining this PCB with a commercial potentiostat, we can extract the readout data of that automated and high-throughput diagnostic setup. The finalized setup is automated from start to finish and can process 8 samples at a time, with the possibility of increasing the number.

Abstract

A Surrogate Based Characterization Technique

For Antibody Coupling Efficiency

by

Mustafa Mutlu

The immunoprecipitation protocol is of key importance to antigen purification, to downstream protein analysis, and to clinical research. The success of an immunoprecipitation experiment depends on many variables of which antibody immobilization is the most important. Antibodies must be coupled with specific solid surfaces, such as agarose beads or superparamagnetic beads, during immunoprecipitation. Although antibody coupling is crucial for the protocol, there is not an easy-to-apply, low cost, or fast method to maximize and characterize the immuno-binding capacity. In this work we propose a characterization method which gives a clear result of the coupling efficiency, which does not require complicated experimental steps or expensive laboratory equipment. By forming a two-particle complex that sandwiches an antibody, the total amount of antibody coupled to the given number of magnetic beads can be readily translated into the number of surrogate beads that are attached to the magnetic beads through the antibodies. This method doesn’t require additional biological reagents. The surrogate bead alone suffices to quantify immobilization efficiency. This method requires only fluorescence imaging, which can be done with any portable fluorescence platform. It has multiplexing capability, provides high-throughput and high-yield, is cost-effective and time-efficient. Researchers can easily track the shelf-life, quality, degradation, and deterioration of the immobilized beads. This research has characterized a couple of variables, as well as tested automating the protocol. Our design also allows other researchers to explore more variables and automate their protocols.