title: Developing Novel Micro- And Nano- Technologies To Explore The Glyco-Interactome
Creators: name: Cooper, Oren
Publisher: name: Griffith University
Year: 2022
Subjects: subject: Chemical Vapor Deposition
subject: 3(Glycidyloxypropyl)-trimethoxysilane
subject: Glycomics
subject: Microcantilever Array
subject: Micro-electromechanical Systems
subject: Self-assembled Monolayers
subject: carbon dots
subject: confocal microscopy
subject: fluorescent multivalent nanoparticles
subject: GOPTS

description: Recent advancements in the field of micro- and nano- technology has provided glycobiologists with an exciting opportunity to explore the diverse roles of glycans in an unprecedented manner. Although a variety of tools are already available to study and assign protein-glycan interactions, they rely heavily on labelling techniques that can increase heterogeneity and structural complexity. This has hindered many advancements, and as such the development of fast, easy and inexpensive label-free, biosensing tools for the analysis of carbohydrate-protein interactions has become highly desirable. Of these emerging technologies, microelectromechanical systems (MEMs), microplasmonics and nanoparticle platforms represent promising pathways for high-resolution, high-throughput and label free detection (LFD) of biological and chemical analytes. This PhD project investigates the design and fabrication of novel label free micro- and nano- glyco-platforms which can be used to explore complex biological interactions within the context of glycobiology. The major focus of this thesis examines various self-assembled monolayers (SAMs) for the biofunctionalization of both two-dimensional and zero-dimensional materials. Given this bifurcation, this work has been divided into two major parts. Part I (Chapter 1-3) explores the use of two-dimensional materials for the generation of both MEMs and plasmonics platforms for the detection of carbohydrate-lectin interactions. Part II (Chapter 4-6) describes the glycan functionalization of zero-dimensional nanoparticles known as carbon dots (CDs) and their advantages for biosensing and drug delivery. Taken together, this work, presents the framework for the design of next generation platforms for label free and quantitative detection of the glyco-interactome. Part I explores the recent progress in the area of LFD using two dimensional materials such as silicon (Si), silicon carbide (SiC) and gold (Au). Additionally, the fabrication and biological validation of these LFD platforms from the perspective of carbohydrate-lectin interactions is explored. A published review (Chapter one) explores novel micro- and nano- technologies that provide researchers with high-throughput, efficient drug screening and early pathogen detection platforms. In particular, the paper highlights the design and fabrication advantages of SiC for micromachining miniaturized sensing devices. From here, the paper focuses on plasmonics and MEMs based sensing in the context of molecule-specific sensing technologies including their advantages and limitations. The use of SAMs for generating high-throughput glycan microarrays on SiC is discussed in Chapter two. This published article explores the critical first steps for the biofunctionalization of inorganic surfaces. Here, the framework for chemical vapour deposition (CVD) of 3-(glycidyloxypropyl)-trimethoxysilane (GOPTS) onto SiC to create an appropriate inorganic/organic heterointerface for presentation of functionally active glycans is explored. In depth atomic force microscopy characterisation of these SiC glycan microarrays revealed a strong epoxide surface density leading to an increased sensitivity compared to commercial microarray surfaces. Additionally, specific lectin-glycan interactions were shown to be in good accordance with our in-house glycan array published previously. Finally, for the first time, a conventional piezoelectric microarray printer is shown to be suitable for biofunctionalization of SiC free-standing membranes. This work serves as proof-of-principle for the generation of MEMs and plasmonics platforms explored in Chapter three. Chapter three is comprised of two separate sections which explore the use of GOPTS as a versatile biofunctionalization route for MEMs and plasmonics devices. The first section is a submitted article entitled “Label-Free Sensing Platform For Glycomics Using Bioprinting And Microresonators.” This article provides the framework for the development of a novel Si microcantilever glycan array (MCA) that is capable of detecting carbohydrate-lectin interactions with picomolar sensitivity. The second section of this chapters explores SAMs for the generation of plasmonics devices Here, surface plasmon resonance (SPR) measurements performed on Au chips functionalized with GOPTS revealed an enhanced analyte sensitivity compared to commercial carboxymethyl dextran chips. Taken together, the results from our MCA and SPR experiments demonstrate that CVD of GOPTS provides an exceptional surface for the immobilization of various biomolecules (glycans and proteins) onto inorganic two-dimensional sensing surfaces (Au and Si). This chapter, provides glycobiologists with a suitable framework for glycan functionalization of emerging MEMs and plasmonics platforms. Part II explores glycan functionalization of zero-dimensional CDs to investigate the glycol-interactome across a range of biological events. A comprehensive literature review (Chapter four) on glyconanomaterials (GNM) and the impact they have had in the field of glycobiology provides an overview of the various nano-architectures that have been used to explore and exploit the diverse role of glycans in biology. Here, the effect of glycan multivalency is explored extensively, given that this property is central to all GNMs. Additionally, thanks to the various tuneable chemical and physical properties (such as photonic, plasmonic, electronic and magnetic), which are unique to each GNM, innovative opportunities to explore cellular, tissue and organismal interactions with unmatched sensitivity re discussed. Finally, the effect of glycans to augment the solubility, biocompatibility, toxicity and specific targeting of nanomaterials is described in the context of drug delivery, diagnostics and vaccination. Chapter five provides the framework for the functionalization of zero-dimensional CDs using self-assembled glycan monolayers (SAGM). The published article provides detailed physical and biological characterisation of CDs functionalized with lactose SAGM. Using GOPTS, a versatile method to generate high yield of lactose-conjugated CDs was developed. Lactose functionality of CDs was biologically assessed on lectin microarrays and within cellular models. Thanks to CDs inherent photoluminescence and nanometer dimensions they proved an excellent tool for exploring in vitro carbohydrate interactions. Here, flow cytometry and confocal analysis of various cell lines revealed not only differential uptake mechanisms for the lactose coated CDs but also varied intracellular fates compared to the uncoated CDs. Chapter six of this thesis explores sialic acid binding immunoglobulin-like lectins (siglecs) interactions with sialic acid (Sia) CDs. Here, SAGM of α2,3-sialyllactose (2,3-SL) and α2,6-sialyllactose (2,6-SL) were used to functionalize CDs and explore the effect that multivalency has on siglec interactions. The new class of Sia analogues have an inherently high affinity to siglec ligands due to the high surface/volume ratio of CDs. In particular the 2,3SL coated CD revealed a 15-fold increase in affinity to Siglec-1 compared to its monovalent counterpart. Further, the 2,6SL coated CDs bound CD22 with high affinity (IC50 ~ 70 μM) and demonstrated a significant cytotoxic effect to Burkitt’s Lymphoma DAUDI cells. Taken together this work provides the framework for the design of intelligent CDs capable of targeting siglecs. This thesis, attempts to provide glycobiologists a novel tool box with which to explore the complex world of carbohydrate interactions. Given the expansive improvements in the design and fabrication of micro- and nano- technology within the last decade, suitable biofunctionalization routes which complement the self-sensing capabilities of these novel platforms are of great value, especially within the field of glycobiology. To this end, the GNMs fabricated throughout this report provide insight into some of the most exciting and promising hybrid nano systems that offer a unique opportunity to mimic and detect nature’s own glyco-architecture.