Anpalagan, Karthiga K (2023) Carbon quantum dots for bioimaging cells and tissues. PhD thesis, Victoria University.

Abstract

Bioimaging is an innovative technique in research and clinical settings that has a lot of significance in today’s world. It is an optical form of biosensing used to create visual representations of biological processes in cells, tissues, and anatomy that enable more accurate diagnosis and treatment of diseases. The fluorescent probes such as organic fluorophores, which include dyes and proteins are commercially in use. For example, Rhodamine 6G is one of the better ones which exhibits a quantum yield value of 80%. The photoluminescence efficiency is termed as quantum yield, and it is defined by the ratio of the number of photons emitted to the number of photons absorbed. Although quantum yield is high, fluorescent dyes are prone to photobleaching and have less photostability. As a result, their brightness degrades with time, and they possess very low resistance to change under the influence of light. Moreover, the biocompatibility of organic fluorophores is very low, and they are toxic to live cells. These shortcomings make them inappropriate for long-term bioimaging. Therefore, research is now focused on discovering new fluorescent probes with better biocompatibility, good photostability, and low cytotoxicity. As commercial fluorescent probes are expensive, non-environment-friendly, and require professional handling, new fluorescent probes fabricated via facile, cost-effective, and green synthesis routes are fast becoming appealing alternatives. One such emerging technology platform is carbon quantum dots (CQDs), which are increasingly used in cell bioimaging. These fascinating nanoscale semiconductors possess useful characteristics, such as tunable photoluminescence, biocompatibility, solubility, chemical stability, photostability, and resistance to photobleaching, which potentially make them better fluorescent probes. Generally, CQDs are fabricated from chemical-based precursors and natural resources through various fabrication methods that involve multiple steps for neutralization, surface passivation, and doping. The long fabrication processes consume a lot of energy, time, and cost. The addition of organic and inorganic chemicals during the fabrication process is very common and this is mainly for the purpose of obtaining better quantum yield. However, the usage of chemicals curtails the biocompatibility of the CQDs and limits their application in bioimaging. In order to overcome the above-mentioned gaps, this research focused on the fabrications of biocompatible CQDs from a very common edible resource, namely bread. Bread is the third-highest food wastage contributor to climate change. In this study all the fabrications of CQDs were performed completely chemical-free. A novel thermolysis synthesis route namely the toasting method was introduced for the first time to derive CQDs from bread. Our CQDs exhibited excitation tunable emission and low cytotoxicity. These biocompatible CQDs were successfully utilized in bioimaging C2C12 mouse muscle myoblasts cell lines and differentiated myotubes. This study verified that fluorescent CQDs with bioimaging capabilities can be fabricated in the absence of chemicals. When the C2C12 myotubes were allowed to differentiate in the presence of CQDs at 1mg/mL concentration, a delay in myotube formation was observed. This effect was tracked by bioimaging the differentiation at various time points and studied using western blotting. The alteration of the transcription factors and myosin heavy chain (MHC) in the presence of CQDs confirmed the influence. The cytotoxicity was assessed prior to the bioimaging and observed more than 95 % of cell viability even at 1.5 mg/mL concentration. This concentration was nearly 1000 times higher than the concentration, required for cell labelling and generally used in cell viability assays. Hence, it was shown that the CQDs from edible precursors are safe for bioimaging even at high concentrations. However, our observation suggested further work is required to study the impact of CQDs on cellular functions prior to long-term live cell or in vivo bioimaging. The efficacy of the toasting method was compared with the standard and commonly used hydrothermal technique. The experiment was expanded in this chapter by including two more types of bread. White bread, whole meal bread, and mixed grain bread were used to produce CQDs through two fabrication routes. The CQDs fabricated from both techniques were able to cross cell membranes and were capable of bioimaging colon cancer cell lines, namely CT-26 and HT-29, derived from mice and humans, respectively. The facile, cost-effective, and time-efficient toasting method doesn’t require sophisticated equipment but produced comparable CQDs to the hydrothermal technique. Even though all the CQDs were fluorescent, CQDs derived from whole-meal bread displayed the highest quantum yield of 0.81%. This quantum yield was achieved via a sustainable and chemical-free synthesis route. To further enhance this quantum yield and improve its performance, green routes were used instead of traditional methods that include chemicals. Soybean flour and lemon juice were adequate to enhance the quantum yield up to 2.31%. It is an approximately fourfold increment that was achieved without any chemical additives. This study evidently improved the application of CQDs in bioimaging via sustainable production with a focus on green engineering.

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