Developing fluorescent probes for cellular imaging of the environment

Abstract

Cellular pH environment is a crucial for healthy physiological process, and abnormal pH regulation can inflict stress to cells and drive disease. Fluorescence imaging with selective sensors is an effective method for examining living cells, but current fluorescent pH probes have a limited sensing range. New tools are needed to cover the full pH range to advance health and disease research.

Chapter 2 details the development of novel small molecule fluorescent pH sensor with ratiometric output that can be further functionalised for specific biological studies in Chapter 3 and 4.

Chapter 3 focuses on functionalising a nanoparticle platform, carbon dot, with an small organic based ratiometric pH sensor (NpRho1). The resultant nanoprobe NpRhoDot has a fluorescence response to a widened pH range from pH 4.5 – 7.5 compared to NpRho1 that was developed in Chapter 2 (pH 4.0 – 6.0). The nanoprobe demonstrated excellent selectivity and sensitivity to pH, and was applied in different cell lines in 2D and 3D cell cultures in one- and two-excitation microscope system to visualise intracellular pH through a ratiometric response.

Chapter 4 explores a multivariate approach using multiple fluorescent pH probes. Two probes (Rho1 and Fl) were selected to best discriminate pH 4 to 8 using machine learning algorithms and co-conjugated to plasma-polymerised nanoparticle for simple, reagent-free conjugation process. Their fluorescence to pH was predicted using Gaussian process regression and could determine the pH of unknown samples. Preliminary cell experiment demonstrated internalisation of PPN-Rho1-Fl into A549 cells, showing potential for multivariate analysis of pH in microscope experiments.

The pH sensing systems developed in this thesis expanded the fluorescent tools that can be used to probe intracellular pH to better understand the roles of pH in human health and diseases.