Engineering Hybrid Polymer Materials for Enhanced Biosensing

Abstract

Biosensors, as a potential diagnostic tool contributing to next-generation medicine, have been continuously researched and optimized to achieve improved performance with lower cost. Among them, electrochemical sensors coupled with multi-functional polymers have attracted particular attention because of their ability to provide real-time, quantitative, and highly-sensitive analysis. For example, appropriate polymers can enhance the bioavailability of an immobilized biomolecule or recognition elements and/or facilitate lower limits-of-detection when used as a coating.

In this thesis, magnetic microgel beads and ion-conductive polymers were fabricated to serve as anti-fouling bioactive immobilization platforms. Magnetic microgel beads are routinely used in biosensing and bioseparation applications given their high surface area for immobilization (and thus high capacity to capture biomolecules) coupled with their facile separation from suspension using a magnetic field. However, current magnetic beads are typically based on silica or polystyrene and thus have relatively poor protein-repellent properties, leading to enhanced binding of non-target molecules and thus reduced signal:noise ratios. In response, magnetic microgel beads based on the highly protein-repellent polymer poly(oligo(ethylene glycol) methacrylate (POEGMA) were fabricated using a semi-batch inverse emulsion polymerization. The resulting microgel beads have a narrow size distribution centred around ~5 μm, a low level of aggregation, and high colloidal stability, all at a low cost. Effective magnetic separation can be achieved within five minutes, while the inherent protein-repellent properties of POEGMA significantly reduce non-specific protein adsorption. Upon using carbodiimide chemistry to tether a methylene blue-linked DNAzyme to the microgel bead that is selectively cleaved in the presence of E. coli, a 6.3-fold higher signal was measured upon exposure to E. coli in buffer and a 97-fold higher signal retention was achieved in clinical urine (based on the electrochemical detection of released methylene blue from capture probes) relative to that achieved with Dynabeads®, a leading commercial magnetic bead.

To reduce non-specific adsorption on gold electrodes without compromising the conductivity and thus signal:noise of the electrochemical device, three types of water-soluble polymers were synthesized and tested for their anti-fouling performance and conductivity when coated on gold electrodes. POEGMA polymer functionalized with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) failed to show sufficient conductivity, while POEGMA polymer functionalized with thiol groups that bound directly to the gold electrode maintained sufficient ion conductivity but reduced the DNA signal at the desired voltage (as generated by hybridization between cleaved probes from DNAzyme and capture probes grafted on electrodes). The possible explanation is that the capture probes were not immobilized on polymer efficiently because of the reversibility of imine bonds formed between amine labelled probes and aldehyde groups from polymer. Zwitterionic polymer poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide] (PDMAPS) functionalized with thiol groups enhanced the conductivity of the electrode (showing a lower resistance to ion conductivity even compared to the bare electrode), although further optimization is still required to realize higher DNA signals for clinical applications. Overall, both magnetic beads microgels and conducting non-fouling polymers enabled significantly improved performance of electrochemical biosensors for E.coli detection. Magnetic microgel beads show potential for commercialization in the future.