Thesis
Small molecule pH sensors for biological raman imaging
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2021
- Thesis identifier
- T16136
- Person Identifier (Local)
- 201781344
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Intracellular pH (pHi) homeostasis in eukaryotic cells is intertwined with a myriad of normal cellular behaviours as well as pathological processes. It is strictly regulated, at an energy cost, in different microenvironments to enable the optimal structure and function of the proteins and nucleic acids contained within. For example, mitochondrial pH (pHmito) is typically alkaline (ca. pH 8.0), providing an electrochemical gradient to drive adenosine triphosphate (ATP) synthesis, while lysosomal pH (pHlyso) is typically acidic (ca. pH 4.5) – enabling the function of the enclosed hydrolytic enzymes for recycling cellular material. Given the importance of pHi homeostasis, it is unsurprising that numerous disease states such as cancers and neurodegenerative diseases are associated with aberrant pHi profiles. As such, tools for quantitatively measuring pHi, in live cells, in real time, are invaluable for chemical biology. The field of small molecule pHi sensors has traditionally been dominated with probes based on fluorescent scaffolds. Herein, the development of a series of bisarylbutadiyne compounds which exhibited pH sensitive alkyne stretching frequencies (νalkyne) in Raman spectroscopy is reported – representing the first small-molecule pHi sensors for Raman microscopy. These compounds were readily visualized in a cellular environment, with subcellular resolution using Raman and stimulated Raman scattering (SRS) microscopy. They proved capable of quantitative, ratiometric pH sensing, in live cells, with temporal resolution. The modular design of the bisarylbutadiyne series rendered them synthetically tractable and enabled rational tuning of properties such as pKa(H) and subcellular localization. Exploiting this modularity, sensors for the cytoplasmic, mitochondrial, and lysosomal microenvironments were developed. The cytoplasmic pH sensor was applied to the quantitative study of pHi in apoptotic prostate cancer (PC3) cells; and the mitochondrial pH sensor was applied to the study of pHmito during mitophagy, and inhibition of subunits of the electron transport chain in HeLa cells. A significant challenge in pHi sensing is the ability to apply multiple pHi sensors simultaneously, to examine links between pH regulation in different organelles. This is difficult to achieve with fluorescent pH probes as their broad absorption/emission spectra are likely to overlap. Here, progress has been made towards developing second generation sensors with νalkyne values which were spectroscopically resolvable from the original series. This work was carried out with the aim of enabling simultaneous measurement of multiple pHi values from different microenvironments simultaneously.
- Advisor / supervisor
- Tomkinson, Nicholas C. O.
- Resource Type
- Note
- This thesis was previously held under moratorium from 17th January 2022 until 17th January 2024.
- DOI
- Embargo Note
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