Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Chemistry

Advisor

Adam B. Braunschweig

Committee Members

David R. Mootoo

Shana Elbaum-Garfinkle

Subject Categories

Biochemistry | Biology and Biomimetic Materials | Biomaterials | Biotechnology | Materials Chemistry | Organic Chemistry | Polymer and Organic Materials | Polymer Chemistry | Polymer Science | Structural Materials

Keywords

hypersurface photolithography, glycan, microarray, surface chemistry, polymer brush

Abstract

Hypersurface Photolithography (HP) is a printing method for fabricating structures and patterns composed of soft materials bound to solid surfaces and with ~1 micrometer resolution in the x, y, and z dimensions. This platform leverages benign, low intensity light to perform photochemical surface reactions with spatial and temporal control of irradiation, and, as a result, is particularly useful for patterning delicate organic and biological material. In particular, surface- initiated controlled radical polymerizations can be leveraged to create arbitrary polymer and block- copolymer brush patterns. Chapter 1 will review the advances in instrumentation architectures from our group that have made these hypersurfaces possible, and the investigations and development of surface-based organic chemistry and grafted-from photopolymerizations that have arisen through these investigations. Over the course of this discussion, we describe specific applications that have benefited from HP. By combining organic chemistry with the instrumentation developed, HP has ushered in a new era of surface chemistry that will lead to new fundamental science and previously unimaginable technologies.

Chapter 2 addresses the challenges involved in fabricating multiplexed microarrays— where different biological probes are spatially encoded onto a surface into spots with micrometer- scale diameters. Further miniaturization of feature diameters could increase the number of probes in a microarray, reduce the sample required for analysis, and decrease costs. Scanning probe lithography (SPL) has gained popularity for patterning delicate, biologically active materials, but no versatile SPL-based multiplexing strategy has been devised. Here, we combine microfluidics, beam pen lithography, and photochemical surface reactions to create multiplexed arrays. For proof of concept, the thiol-ene reaction was optimized, and the reaction kinetics were analyzed. Subsequently, we created several patterns containing multiple fluorescent alkenes, where each pattern was designed to demonstrate a different capability of this instrument. This patterning strategy is a powerful approach to studying and optimizing organic reactions on surfaces and creating massively multiplexed arrays and, as such, could provide an entirely new approach for miniaturizing biochips or understanding interfacial reactivity.

In Chapter 3, we show that the surface-initiated thiol-(meth)acrylate polymerization can be used to create brush polymer patterns with precise control over the feature height at each microscale pixel. The reaction was studied using a printer where a digital micromirror device controls light delivery to the surface, so multiple reaction conditions can be examined in each print. The resulting increases in experimental throughput and precision were demonstrated by studying systematically the effect of photocatalyst, photoinitiator, and light intensity on feature growth rate. In addition to demonstrating the utility of surface-initiated thiol-(meth)acrylate chemistry for creating complex brush polymer patterns, this work describes an improved and high-throughput approach for studying grafted-from photopolymerizations.

Polymer brush patterns have a central role in established and emerging research disciplines, from microarrays and smart surfaces to tissue engineering. The properties of these patterned surfaces are dependent on monomer composition, polymer height, and brush distribution across the surface. No current lithographic method, however, is capable of adjusting each of these variables independently and with micrometer-scale resolution. In Chapter 4, we report a technique termed Polymer Brush Hypersurface Photolithography, which produces polymeric pixels by combining a digital micromirror device (DMD), an air-free reaction chamber, and microfluidics to independently control monomer composition and polymer height of each pixel. The printer capabilities are demonstrated by preparing patterns from combinatorial polymer and block copolymer brushes. Images from polymeric pixels are created using the light reflected from a DMD to photochemically initiate atom-transfer radical polymerization from initiators immobilized on Si/SiO2 wafers. Patterning is combined with high-throughput analysis of grafted-from polymerization kinetics, accelerating reaction discovery, and optimization of polymer coatings.

Spatially encoded glycan microarrays promise to rapidly accelerate our understanding of glycan binding in myriad biological processes, which could lead to new therapeutics and previously unknown drug targets. In Chapter 5, we bring together a digital micromirror device, microfluidic introduction of inks, and advanced surface photochemistry to produce multiplexed glycan microarrays with reduced feature diameters, an increased number of features per array, and precise control of glycan density at each feature. The versatility of this platform was validated by printing two distinct glycan microarrays where, in the first, different glycans were immobilized to create a multiplexed array and, in another, the density of a single glycan was varied systematically to explore the effect of surface presentation on lectin−glycan binding. For lectin binding studies on these miniaturized microarrays, a microfluidic incubation chip was developed that channels multiple different protein solutions over the array. Using the multiplexed array, binding between eight lectin solutions and five different glycosides was determined, such that a single array can interrogate the binding between 40 lectin−glycan combinations. The incubation chip was then used on the array with varied glycan density to study the effects of glycan density on lectin binding. These results show that this novel printer could rapidly advance our understanding of critical unresolved questions in glycobiology, while simultaneously increasing the throughput and reducing the cost of these experiments.

Interactions between cell surface glycans and glycan binding proteins (GBPs) have a central role in immune response, pathogen-host recognition, cell-cell communication, and myriad other biological processes. Because of the weak association between GBPs and glycans in solution, multivalent and cooperative interactions in the dense glycocalyx have an outsized role in directing binding affinity and selectivity. However, a major challenge in glycobiology is that few experimental approaches exist for examining and understanding quantitatively how glycan density affects avidity with GBPs, and there is a need for new tools that can fabricate glycan arrays with the ability to vary their density controllably and systematically in each feature. Here we use thiol- ene reactions to fabricate glycan arrays using a recently developed photochemical printer that leverages a digital micromirror device and microfluidics to create multiplexed patterns of immobilized mannosides, where the density of mannosides at each feature was varied by dilution with the inert spacer allyl alcohol. Association between these immobilized glycans and FITC- labelled concanavalin A (ConA) – a tetrameric GBP that binds to mannosides multivalently – was measured by fluorescence microscopy. We observed that fluorescence decreased nonlinearly with increasing spacer concentration in the features, and we present a model that relates average mannoside-mannoside spacing to the abrupt drop-off in ConA binding. Applying these recent advances in microscale photolithography to the challenge of mimicking the architecture of the glycocalyx could lead to a rapid understanding of how information is trafficked on the cell surface.

Finally, Chapter 7 reports a novel glycan array architecture that binds the mannose-specific glycan binding protein, ConA, with sub-femtomolar avidity. A new radical photopolymerization developed specifically for this application combines the grafted-from thiol-(meth)acrylate polymerization with thiol-ene chemistry to graft glycans to the growing polymer brushes. The propagation of the brushes was studied by carrying out this grafted-to/grafted-from radical photopolymerization (GTGFRP) at >400 different conditions using hypersurface photolithography, a printing strategy that substantially accelerates reaction discovery and optimization on surfaces. The effect of brush height and the grafting density of mannosides on the binding of ConA to the brushes was studied systematically, and we found that multivalent and cooperative binding account for the unprecedented sensitivity of the GTGFRP brushes. This study further demonstrates the ease with which new chemistry can be tailored for an application as a result of the advantages of hypersurface photolithography.

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