Surface-enhanced Raman scattering (SERS) pH sensors for cancer and cystic fibrosis in vitro disease models Surface-enhanced Raman scattering (SERS) pH sensors for cancer and cystic fibrosis in vitro disease models

Abstract

The development of pH sensing tools for in vitro cell cultures is imperative to maximize the pathophysiological knowledge the scientific community can extract from disease models. Surface-enhanced Raman scattering (SERS) has emerged as a method well suited to pH measurements in cell cultures because SERS optical sensors are readily miniaturised and biocompatible. SERS probes use the protonation state of a reporter molecule adsorbed to a plasmonic surface to measure pH and can be delivered to the intracellular or extracellular space of cells for sensing applications.

This thesis aimed to improve the understanding of the pH sensitivity of 4-mercaptobenzoic acid (MBA) SERS probes; to create extracellular SERS substrates for integration into advanced cell culture platforms; and to develop user-friendly SERS probes and assays to complement state of the art cell culture models. Through the course of addressing these aims, three SERS substrates were explored and evaluated for in vitro pH sensing applications: gold nanoparticles (AuNPs), gold nanofibres (AuNF), and gold nanoparticle decorated microspheres (SERS-MS).

In Chapter 2, the pH sensitivity of SERS nanoprobes functionalised with MBA (MBA-AuNPs) was explored using lab-based and computational methods. The pH sensitive carboxylate peak in the MBA-AuNPs SERS spectrum is typically used for pH sensing and its sensitivity is widely attributed to the protonation state of MBA molecules at the nanoparticle surface. This thesis demonstrated the intensity of the carboxylate peak in MBA-AuNPs pH calibration curves increases beyond what is expected for a response solely originating from the protonation state of MBA molecules. Chapter 2 demonstrated that, for MBA-AuNPs, the carboxylate SERS peak experiences a pH-dependent increase in SERS enhancement. The potential of MBA-AuNPs as intracellular pH probes were then explore in hepatocellular carcinoma cell line HepG2/C3A. Transmission electron microscopy and SERS pH measurements showed the majority of intracellular MBA-AuNPs were in the lysosome; a pH environment too acidic for SERS sensing. This chapter yielded new insight into the pH sensitivity of MBA SERS probes and examined the limitations of their use to measure intracellular pH in a commonly used hepatocellular carcinoma cell line.

In Chapter 3, a SERS-active nanofibre mesh (AuNF) was developed to collect spatially localized extracellular pH measurements in the cellular microenvironment of hepatocellular carcinoma cell line HepG2/C3A cells grown in 2D. AuNF were fabricated by coating electrospun polyurethane nanofibres with gold and functionalised with MBA to make MBA-AuNF. HepG2/C3A were cultured on the surface of MBA-AuNF to facilitate pH measurements in close proximity to the cellular membrane. This method could be used to detect extracellular pH changes following the exposure of HepG2/C3A to staurosporine, an apoptosis-inducing drug. This work demonstrated the integration of a novel pH sensor into 2D cell culture for non-invasive extracellular pH measurements. Finally, Chapter 4 and Chapter 5, developed the application of SERS microsensors (SERS-MS) for the study of airway-surface liquid (ASL) pH in 2D and 3D airway models grown from patient-derived basal epithelial cells. The ASL pH of people with cystic fibrosis is a matter of scientific debate and SERS pH sensors for in vitro epithelial models have the potential to provide insight into airway pH regulation. SERS-MS were fabricated by adhering MBA functionalised AuNPs to polymer microparticles. Using SERS-MS, ASL pH was measured in CF and non-CF 2D epithelium models grown from donor-derived basal epithelial cells. This work demonstrated a ‘use friendly’ SERS pH assay where pH measurements were collected minutes after administering the probes. SERS-MS were also integrated into 3D airway organoids for pH sensing in the lumen and extracellular environment of organoids. This work demonstrated organoids could be cultured in the presence of SERS-MS for several weeks and Raman spectroscopy used to measure pH in the lumen and extracellular matrix surrounding organoids.