Transglutaminases (protein-glutamine:amine y-glutamyl- transferase, EC 2.3.2.13) are a family of calcium-dependent enzymes which catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When lysine acts as the acyl-acceptor substrate, α-glutamyl lysine isopeptide bond is formed. Isopeptide catalyzation results in protein cross-linkage which is prevalent throughout biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond. Furthermore, mTGs promiscuity in donor substrate preference highlights its biocatalytic potential. To realize the potential of the enzyme, a high-reactivity tag was necessary for protein labelling. To address this, an enzyme-coupled assay was developed to characterize peptides in the hopes of developing orthogonal substrates to facilitate mTG-mediated labelling and biocatalysis. The discovery of high-reactivity peptide tags allowed the realization of in vitro protein labelling- facilitated by mTG. The 7M48 peptide was fused to a test protein, where it was subsequently propargylated with propargyl amine to fluorescently label or immobilize a test protein. Although there are endless possibilities for in vitro bio-conjugation through mTG, proteolytic activation limits any in-cell labelling strategies with this enzyme. To circumvent this issue, development of an alternative bacterial enzyme, Bacillus subtilis transglutaminase (bTG), was chosen to replace mTG. bTG maintains the advantages associated with mTG but is expressed in its active form. Unlike mTG, there is limited preliminary research associated with the enzyme or its substrate scope. To better understanding substrate reactivity, a FRET-based assay was developed allows for the discovery of new high-reactivity peptides for bTG. These peptides were then used in labelling strategies to demonstrate the potential bTG-mediated bioconjugation. This strategy includes the added advantage of potential for in-cellulo labelling.

Unconventional nanofabrication techniques; both those which have been newly developed and those under development, had brought inexpensive, facile, yet high quality means to fabricate nanostructures that have feature sizes of less than 100 nm in industry and academia. This dissertation focuses on developing unconventional fabrication techniques, building studying platforms, and studying the mechanisms behind them. The studies are divided into two main facets and four chapters. The first facet, in Chapter II and Chapter III, deals with the research and development of different nanofabrication techniques and nanostructures. These techniques include litho-synthesis, colloidal lithography, and photolithography. The nanostructures that were fabricated by these techniques include the metal nanoparticle arrays, and the self-assembled CdSe nanoring arrays. At the same time, the dissertation provides mechanisms and models to describe the physical and chemical nature of these techniques. The second area of this study, in Chapter III to Chapter V, presents the applications of these nanostructures in fundamental studies, i.e. the mechanisms of plasmon enhanced fluorescence and photo-oxidation kinetics of CdSe quantum dots, and applications such as molecular sensing and material fabrication. More specifically, these applications include tuning the optical properties of CdSe quantum dots, biomodification of CdSe quantum dots, and copper ion detection using plasmon and photo enhanced CdSe quantum dots. We have successfully accomplished our research goals in this dissertation. Firstly, we were able to tune the emission wavelength of quantum dots, blue-shifted for up to 45 nm, and their surface functionalization with photo-oxidation. A kinetic model to calculate the photo-oxidation rates was established. Secondly, we established a simple mathematical model to explain the mechanism of plasmon enhanced fluoresce of quantum dots. Our calculation and experimental data support the fluorescence resonance energy transfer (FRET) mechanism between quantum dots and the metal nanoparticles. Thirdly, we successfully pattered the CdSe quantum dots (diameter ~4 nm) into nanorings with tunable diameters and annular sizes on different substrates. We also established a physical model to quantitatively explain the mechanism with the forces that involved in the formation of the nanorings.

The work presented in this thesis describes methodologies developed for integration and accurate interpretation of barcoded DNA, to empower large-scale-omics analysis. The objectives mainly aim at enabling multiplexed proteomic measurements in high-throughput format through DNA barcoding and massive parallel sequencing. The thesis is based on four scientific papers that focus on three main criteria; (i) to prepare reagents for large-scale affinity-proteomics, (ii) to present technical advances in barcoding systems for parallel protein detection, and (iii) address challenges in complex sequencing data analysis. In the first part, bio-conjugation of antibodies is assessed at significantly downscaled reagent quantities. This allows for selection of affinity binders without restrictions to accessibility in large amounts and purity from amine-containing buffers or stabilizer materials (Paper I). This is followed by DNA barcoding of antibodies using minimal reagent quantities. The procedure additionally enables efficient purification of barcoded antibodies from free remaining DNA residues to improve sensitivity and accuracy of the subsequent measurements (Paper II). By utilizing a solid-phase approach on magnetic beads, a high-throughput set-up is ready to be facilitated by automation. Subsequently, the applicability of prepared bio-conjugates for parallel protein detection is demonstrated in different types of standard immunoassays (Papers I and II). As the second part, the method immuno-sequencing (I-Seq) is presented for DNAmediated protein detection using barcoded antibodies. I-Seq achieved the detection of clinically relevant proteins in human blood plasma by parallel DNA readout (Paper II). The methodology is further developed to track antibody-antigen interaction events on suspension bead arrays, while being encapsulated in barcoded emulsion droplets (Paper III). The method, denoted compartmentalized immuno-sequencing (cI-Seq), is potent to perform specific detections with paired antibodies and can provide information on details of joint recognition events. Recent progress in technical developments of DNA sequencing has increased the interest in large-scale studies to analyze higher number of samples in parallel. The third part of this thesis focuses on addressing challenges of large-scale sequencing analysis. Decoding of a huge DNA-barcoded data is presented, aiming at phase-defined sequence investigation of canine MHC loci in over 3000 samples (Paper IV). The analysis revealed new single nucleotide variations and a notable number of novel haplotypes for the 2nd exon of DLA DRB1. Taken together, this thesis demonstrates emerging applications of barcoded sequencing in protein and DNA detection. Improvements through the barcoding systems for assay parallelization, de-convolution of antigen-antibody interactions, sequence variant analysis, as well as large-scale data interpretation would aid biomedical studies to achieve a deeper understanding of biological processes. The future perspectives of the developed methodologies may therefore stem for advancing large-scale omics investigations, particularly in the promising field of DNA-mediated proteomics, for highly multiplex studies of numerous samples at a notably improved molecular resolution. / <p>QC 20150203</p>