Optimisation of cadmium selenide quantum dot biosynthesis in Saccharomyces cerevisiae and the role of glutathione By Jordan Brooks A thesis submitted to the Graduate Program in Biology in conformity with the requirements for the degree of Master of Science Queen’s University Kingston, Ontario, Canada September 2015 Copyright © Jordan Brooks, 2015 Abstract The biosynthesis of quantum dots has been explored as an alternative to traditional physicochemical methods; however, relatively few studies have determined optimal synthesis parameters. Saccharomyces cerevisiae sequentially treated with sodium selenite and cadmium chloride synthesized CdSe quantum dots in the cytoplasm. These nanoparticles displayed a prominent yellow fluorescence, with an emission maximum of approximately 540 nm. Investigations into the optimisation of the biosynthetic method revealed that quantum dots were produced more efficiently when stationary phase cultures were treated directly with 1 mM sodium selenite for 6 hours, followed by incubation with 3 mM cadmium chloride in fresh growth medium. Synthesis of quantum dots reached a maximum after approximately 84 hours of reaction time. The influence of glutathione to the biosynthetic mechanism was explored through the use of 1-chloro-2,4-dinitrobenzene and buthionine sulfoximine to deplete intracellular glutathione content. The synthesis of CdSe quantum dots was significantly inhibited in most cases by 1-chloro-2,4-dinitrobenzene and buthionine sulfoximine treatment, suggesting that glutathione plays an important role in the biosynthetic process, particularly following the addition of cadmium. The possible mechanism for CdSe quantum dot formation is discussed. ii Acknowledgements Firstly, I would like to thank my supervisor Dr. Daniel D. Lefebvre. His constant support and helpful advice were invaluable throughout the course of my thesis project. As well, thank you to Dr. Yuxiang Wang, Dr. William Plaxton, Dr. Wayne Snedden, Dr. Ian Chin-Sang, and Dr. Chris Moyes for the use of their lab equipment and supplies. Finally, I would like to thank my family—particularly Robert Driver, and Jennifer and Ken Brooks—for their unwavering love and support. iii Table of Contents Abstract......................................................................................................................................... ii Acknowledgments ....................................................................................................................... iii Table of Contents ........................................................................................................................ iv List of Figures............................................................................................................................... vi List of Abbreviations ................................................................................................................ viii Chapter 1: General Introduction and Review............................................................................ 1 1.1 Quantum dots ......................................................................................................................... 1 1.2 Quantum confinement effect.................................................................................................. 2 1.3 Types of QDs........................................................................................................................... 3 1.4 Applications of QDs................................................................................................................ 7 1.5 Traditional physicochemical production methods............................................................... 8 1.6 QD biosynthesis....................................................................................................................... 8 1.7 Optimisation of CdSe QD biosynthesis in Saccharomyces cerevisiae.............................. 16 Chapter 2: Materials and Methods .......................................................................................... 18 Chapter 3: Results....................................................................................................................... 22 Chapter 4: Discussion................................................................................................................. 42 iv Summary...................................................................................................................................... 55 Literature Cited ......................................................................................................................... 56 Appendix...................................................................................................................................... 69 v List of Figures Figure 1. Emission spectra of S. cerevisiae culture at various stages of the CdSe QD biosynthetic procedure............................................................................................................... 29 Figure 2. Fluorescence microscopy images of S. cerevisiae cells............................................ 29 Figure 3. Intracellular glutathione content of S. cerevisiae cells at various stages of growth.......................................................................................................................................... 30 Figure 4. The production of CdSe QDs in S. cerevisiae cultures grown for various time periods................................................................................................................................. 31 Figure 5. The production of CdSe QDs in stationary phase S. cerevisiae cultures exposed to selenium through various methods......................................................................... 32 Figure 6. The production of CdSe QDs in stationary phase S. cerevisiae cultures exposed to various concentrations of selenite for 24 hours..................................................... 33 Figure 7. The production of CdSe QDs in stationary phase S. cerevisiae cultures exposed to 1 mM selenium for various time periods............................................................... 34 Figure 8. The production of CdSe QDs in selenium-exposed S. cerevisiae cultures treated with cadmium chloride by various methods............................................................... 35 Figure 9. The production of CdSe QDs in selenium-exposed S. cerevisiae cultures treated with various concentrations of cadmium chloride...................................................... 36 Figure 10. The production of CdSe QDs in S. cerevisiae cultures at various time points following the addition of cadmium chloride................................................................. 37 vi Figure 11. The production of CdSe QDs in S. cerevisiae cultures following various biosynthetic procedures.............................................................................................................. 38 Figure 12. Characterisation of CdSe QDs produced through the optimised biosynthetic method.................................................................................................................... 39 Figure 13. The production of CdSe QDs in S. cerevisiae cultures treated with CDNB at various stages of the biosynthetic method............................................................................ 40 Figure 14. The production of CdSe QDs in S. cerevisiae cultures treated with BSO at various stages of the biosynthetic method............................................................................ 41 vii List of Abbreviations ATP……………………………………………………………………...Adenosine triphosphate BSO………………………………………………………………………Buthionine sulfoximine CDNB……………………………………………………………….1-chloro-2,4-dinitrobenzene CS………………………………………………………………………………Cysteine synthase ddH2O…………………………………………………………………….Double-distilled water DTNB..................................................5,5'-dithiobis-(2-nitrobenzoic acid) or Ellman’s reagent EDTA………………………………………………………….Ethylenediaminetetraacetic acid FTIR…………………………………………………Fourier transform infrared spectroscopy GR…………………………………………………………………………Glutathione reductase GSH…………………………………………………………………………Reduced glutathione GS-Cd-SG…………………………………………………………..Bis-glutathionato-cadmium GS-Se-SG……………………………………………………………………Selenodiglutathione GS-Se-…………………………………………………………………………...Selenopersulfide HMT1………………………....Heterogeneous nuclear ribonucleoproteins methyltransferase HSe-…………………………………………………………………………...Hydrogen selenide kDa..……………………………………………………………………………………Kilodalton viii NADH……………………………………………………….Nicotinamide adenine dinucleotide NADPH…………………………………………Nicotinamide adenine dinucleotide phosphate PBS……………………………………………………………………Phosphate buffered saline QD…………………………………………………………………………………..Quantum dot RFU…………………………………………………………………...Relative fluorescence unit SSA…………………………………………………………………………...5-sulfosalicylic acid TR………………………………………………………………………...Thioredoxin reductase UV……………………………………………………………………………………...Ultraviolet Ycf1………………………………………………………………………..Yeast cadmium factor YPD……………………………………………………………...Yeast extract peptone dextrose Zrt1………………………………………………………………......Zinc-regulated transporter ix Chapter 1: General Introduction and Review 1.1 — Quantum dots A quantum dot (QD) is a semiconductor nanoparticle, typically in the size range of 1 – 100 nm (roughly 10 – 50 atoms in diameter). At the nanoscale, QDs possess several desirable traits, with physical and optoelectronic properties differing significantly from bulk materials or discrete molecules.