Optimisation of Cadmium Selenide Quantum Dot Biosynthesis In

Home , Cadmium selenide, Zinc selenide

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

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.

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.

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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

Summary...... 55

Literature Cited ...... 56

Appendix...... 69

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

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

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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

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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

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. Most notably, QDs display broad absorption and strong photoluminescence, with the specific wavelength of emission dependant on the size of the nanoparticle. The size- tunable nature of QDs has made them the subject of intense research due to their potential for use in biolabelling, optoelectronics, and computing.

These nanoscale crystals can take several forms, but are most commonly produced as II-

VI chalcogenides, IV-VI chalcogenides, and III-V nanoparticles. In particular, a substantial amount of research has been dedicated to development of II-VI QDs such as CdS (Herron et al.

1990; Yang et al. 2005), CdSe (Crouch et al. 2003; Nordell et al. 2005), CdTe (Rajh et al. 1993;

Talapin et al. 2001), ZnS (Nanda et al. 2000; Li et al. 2007), ZnSe (Hines and Guyot-Sionnesltt

1998; Jun et al. 2000), ZnTe (Jun et al. 2001; Xie et al. 2005), and ZnO (Guo et al. 2000; Mädler et al. 2002). Advances in physical and chemical production methods, as well as concerns over the toxicity of cadmium, have prompted further research toward III-V QDs as alternative options, including GaN (Mićić et al. 1999), GaP (Hwang et al. 2007), GaAs (Lauth et al. 2013),

InN (Greenberg et al. 2005; Wu et al. 2005), InP (Greenberg et al. 2005), and InAs (Xie and

Peng 2008). Furthermore, increased demand for QDs with emissions tunable through the near- infrared spectrum has led to the development of IV-VI nanoparticles such as PbS (Hines and

Scholes 2003; Bakueva et al. 2004), PbSe (Lipovskii et al. 1997; Yu et al. 2004), PbTe (Murphy

1 et al. 2006), SnS (Xu et al. 2009; Deepa and Nagaraju 2012), SnSe (Li et al. 2011), SnTe

(Kovalenko et al. 2007), and SnO (Zhang and Gao 2004).

1.2 — Quantum confinement effect

The unique physical and optoelectronic properties of QDs are ultimately due to their small size. Referred to as “artificial atoms” (Kastner et al. 1994), QDs exhibit characteristics which differ significantly from that of bulk or larger aggregates of materials of the same composition. In larger, bulk semiconductors, the structure of the periodic crystal lattice is the main determinant of the electronic band structure. The allowed energy bands—the range of energies which an electron may have—as well as the bandgap—the minimum energy required to excite an electron from the valence band to the conduction band—remains constant due to a continuous energy state independent of the size and shape of the crystal (Bawendi et al. 1990).

When a photon of energy greater than the bandgap is absorbed, an excited electron will leave behind a hole. The distance between an electron-hole pairing, or exciton, is known as the exciton-Bohr radius. If the radius of the crystallite is smaller than its exciton-Bohr radius, a significant deviation from the bulk properties can be observed due to a principle known as

“quantum confinement” (Bryant 1988). At this size range, the excitons are confined in three dimensions, resulting in a transition of the band structure from continuous to discrete energy levels; consequently, the bandgap becomes dependant on the size of the particle, increasing in energy as particle size decreases. Since the recombination of an electron-hole pairing is a radiative process yielding a photon of equal energy to the bandgap, quantum confinement results in light emissions which blue-shift as particle size is reduced (Horan and Blau 1990). The range of wavelengths that manufactured QDs can emit encompasses a broad range, from the lower energy infrared region (Hines and Scholes 2003; Bakueva et al. 2004; Zheng et al. 2004),

2 through the visible light spectrum (Danek et al. 1996; Hines and Guyot-Sionnest 1996; Dabbousi et al. 1997; Gerion et al. 2001; Zheng et al. 2004; Nordell et al. 2005), and into the ultraviolet

(UV) region (Guo et al. 2000; Song and Lee 2001; Zheng et al. 2004).

1.3 — Types of QDs

1.3.1 — Core-type QDs

As described previously, QDs at their most basic form are a single bielemental material such as CdS or CdSe. These “core-type QDs” are nanoscale crystallites with a consistent internal composition, and optoelectronic properties which can be tuned through adjusting the particle size. A common feature of core-type QDs is the often problematic “trap emissions.” At this small particle size, QDs have a large proportion of surface atoms that are incompletely bonded, disturbing the periodic crystal lattice. Incompletely bonded atoms present “dangling” orbitals with their own band structure, which, if within the bandgap of the QD, can create “trap sites” for the unwanted decay of excitons (Pokrant and Whaley 1999; Smith and Nie 2010;

Veamatahau et al. 2015). As such, when a photon of sufficient energy is absorbed, there will be two competing pathways for the resultant emission (Underwood et al. 2001). First, the excited electron may recombine with the hole to produce the expected band edge emission of the QD.

However, electrons from the dangling bonds of surface atoms may also relax to radiatively combine with the photogenerated hole, producing a secondary emission. This will alter the observed fluorescence emission of QDs, producing two distinct emission peaks, one at the band edge—desirable—and another at a lower energy from the trap sites—undesirable. Quantum yield may also be significantly reduced due to excited electrons non-radiatively decaying to the trap sites. Smaller QDs will typically have more pronounced trap emissions due to their high

3 surface-area-to-volume ratios (Baker and Kamat 2010; Veamatahau et al. 2015). To alleviate unwanted trap emissions and loss of quantum yield, core-type QDs are often capped with organic ligands that coat the surface, passivating dangling orbitals and preventing the formation of trap sites (Pokrant and Whaley 1999). Alternatively, in some cases, trap emissions may be desirable.

Several examples of “white-light emitting” QDs have been produced with high surface-area-to- volume ratios that provide a relatively large number of trap sites. The contributions of the band edge emission and trap emissions together can produce a broad overall fluorescence, resulting in the output of white light (Bowers et al. 2005; Kudera et al. 2007; Dukes et al. 2010).

1.3.2 — Core/shell QDs

Despite the addition of organic ligands that coat the surface of core-type QDs, trap sites can still be prevalent due to steric hindrance preventing the full passivation of surface dangling bonds (Pokrant and Whaley 1999). To improve the quality of passivation, QDs can be coated with a shell of a second inorganic semiconductor. Overgrowth of a shell provides a number of advantages over core-type QDs including improved quantum yield and emission brightness, and can also change the optoelectronic properties of a QD (Reiss et al. 2009). Core/shell QDs can be classified as type I, reverse type I, or type II, determined by the relationship of the core and shell bandgaps.

1.3.2.1 — Type I core/shell QDs

Type I core/shell QDs have a shell with a higher energy bandgap than the core, where the valence and conduction band edges of the core lie within the bandgap of the shell. As such, excitons will remain confined to the core material, and the optoelectronic properties will be mostly preserved. Examples of type I core/shell QDs include CdSe/ZnS (Hines and Guyot-

Sionnest 1996; Dabbousi et al. 1997; Gerion et al. 2001), and CdSe/CdS (Peng et al. 1997).

Typically, the purpose of type I core/shell QDs is to enhance the existing properties of the core, improving both quantum yield and emission intensities. The shell material passivates dangling bonds of surface atoms, preventing the formation of trap sites, while also providing a physical barrier between the core and the external medium to protect the QD surface against the photodegradative effects of the environment. The original properties of the core material in type

I core/shell QDs are well maintained, though overgrowth of the shell can result in a slight red shift of the absorption and emission wavelengths due to leakage of excitons into the shell material (Chattopadhyay et al. 2012).

1.3.2.2 — Reverse type I core/shell QDs

In contrast to type I configurations, reverse type I core/shell QDs have a shell with a smaller bandgap overgrown on a core possessing a larger one, where the conduction and valence band edges of the shell lie within the bandgap of the core. In this case, excitons will be either partially or completely confined to the shell depending on its thickness. This characteristic provides a greater degree of control over QD emissions—thicker shells will have a greater proportion of excitons delocalized in the shell. Accordingly, there will be a red-shift of the emission wavelength. Reverse type I core/shell QDs include CdS/HgS (Schooss et al. 1994),

CdS/CdSe (Tian et al. 1996), and ZnSe/CdSe (Zhong et al. 2005). With the shell a contributing factor in the absorption and emission profiles, the potential for trap sites and the influence of the environment on exposed surface atoms again becomes a concern. Growth of a secondary shell can protect against these issues, improving the quantum yield of reverse type I core/shell QDs.

1.3.2.3 — Type II core/shell QDs

Type II core/shell QDs are characterized as having a shell where the conduction and valence band edge are both lower or both higher than the band edges of the core. This results in a spatial separation of exciton-pairs within the nanocrystal, and a smaller effective bandgap than either of the constituent semiconductors. Consequently, a drastic red-shift of the emission wavelength can be observed. With increasing shell thickness, fluorescence will be tuned toward lower energy emissions, allowing for wavelengths otherwise unattainable by the individual component materials. Both CdTe/CdSe (Kim et al. 2003; Yu et al. 2005) and CdSe/ZnTe (Kim et al. 2003; Cheng et al. 2005) are examples of type II core/shell QDs. Similar to that of reverse type I, the quantum yield and emission intensities of type II core/shell QDs can be improved through the overgrowth of an additional shell to passivate trap sites and provide a barrier to the external environment.

1.3.3 — Alloyed QDs

While the characteristic trait of QDs is their size-tunable fluorescence, certain applications may place strict size restrictions on the materials involved. In such cases, a variety of emission wavelengths may be desired that core-type and core/shell QDs cannot fulfill. A relatively recent solution to this challenge is the use of alloyed QDs, such as CdSeTe (Bailey and

Nie 2003), PbSeS (Maikov et al. 2010), and ZnCdSe (Zhao 2011). The emission wavelengths of these multicomponent crystallites can be tuned through adjusting their compositional ratios, resulting in a large range of possible emissions without changing overall particle size.

Remarkably, the relationship between the compositional characteristics and the absorption/emission of alloyed QDs is non-linear, allowing for novel spectral properties not

6 obtainable in the individual components (Bailey and Nie 2003). Furthermore, alloyed QDs can be designated either “homogenous” or “gradient” type. Homogenous alloyed QDs feature a consistent internal composition, regardless of particle size. In contrast, gradient alloyed QDs will have a core rich in one particular semiconductor with a steady shift toward the other in the outer regions of the crystallite. In the case of CdSeTe QDs, this difference is due to variation in reactivity of the components, with Te significantly more reactive than Se toward Cd (Bailey and

Nie 2003). Reaction under Cd-limited conditions will result in the production of homogenous alloyed QDs, as CdSe and CdTe form in a manner determined by their initial molar ratios and intrinsic reactivity. However, in Cd-rich conditions, gradient alloyed QDs will develop as CdTe forms at a faster rate in the core, with a shift toward CdSe as Te is consumed. The resulting particle will have a Te-rich core and a Se-rich shell.

1.4 — Applications of QDs

The unique optoelectronic properties and precise size-tunability of QDs make them attractive materials to a variety of industries. In particular, the electronics sector has seen rapid progress in the development of QD-based display technologies (Shirasaki et al. 2013).

Significant research has been focused on the production of light emitting diode systems which incorporate QDs (Colvin et al. 1994; Mueller et al. 2005; Stouwdam and Janssen 2008). The biological sciences have embraced QDs for a number of years due to their use in cellular labelling (Jaiswal et al. 2003; Wu et al. 2003; Sukhanova et al. 2004; Medintz et al. 2005). QDs offer several advantages over conventional organic dyes; notably, the narrow emission spectra allow for the use of several probes with less spectral overlap, the broad absorption spectra allows for many QDs to be excited simultaneously while minimizing autofluorescence from the sample, and QDs typically possess greater photochemical stability than organic dyes (Cheng et al. 2014).

Lastly, QDs are increasingly gaining importance to photovoltaics, featuring in the development of solar cells (Pattantyus-Abraham et al. 2010), photodiodes (Pal et al. 2012) and photodetectors

(Konstantatos et al. 2006).

1.5 — Traditional physicochemical production methods

Generally, the synthesis of QDs takes one of following forms: vapour-liquid phase deposition or colloidal synthesis. In vapour-liquid phase deposition, the epitaxial growth of semiconductor crystals is initiated on a solid substrate through the use of appropriate liquid or gaseous precursors. In contrast, colloidal synthesis involves the production of QDs from precursors dissolved in solution, along with surfactants responsible for limiting the growth and aggregation of the nanoparticles (Jacob et al. 2015). Unfortunately, these synthesis routes have a significant environmental impact, involving the use of toxic, combustible, and explosive reagents, as well as requiring high temperatures. Alternative, environmentally-friendly methods for the effective synthesis of QDs are highly sought after.

1.6 — QD biosynthesis

It is well documented that many living organisms possess the intrinsic ability to synthesize a variety of inorganic materials of precise shape and size (Mandal et al. 2006). For example, unicellular organisms such as magnetotactic bacteria and diatoms produce magnetite nanoparticles (Dickson 1999) and silica-based materials (Kröger et al. 1999), respectively, while multicellular organisms regularly synthesize materials such as bones, teeth, and shells

(Lowenstam 1981). This ability to accurately regulate the synthesis of inorganic compounds has led to the exploitation of living organisms for the production of several useful materials (Mandal et al. 2006). Recently, the production of QDs through biological means has been explored as an

8 environmentally-friendly alternative to traditional physicochemical synthesis—nearly all published instances of QD biosynthesis occur at ambient temperatures and atmospheric pressure, requiring considerably less energy inputs than physicochemical methods. Microbes, in particular, have shown a propensity for the biosynthesis of QDs. The first reports of QD biosynthesis were by the unicellular yeasts Candida glabrata and Schizosaccharomyces pombe, which produce CdS nanoparticles in response to challenges with cadmium salts (Dameron et al.

1989a; Dameron et al. 1989b). Since then, a variety of organisms have been shown to be capable of producing QDs, including yeast, non-yeast fungi, non-photosynthetic bacteria, photosynthetic bacteria, plants, and annelids. Biosynthetic processes offer several advantages over physical and chemical methods, though there are limitations to the complexity of produced

QDs which have yet to be overcome. To date, there have been no reports of the biosynthesis of core/shell or alloyed QDs, though several core-type QDs have been produced. These include

CdS, CdSe, CdTe, ZnS, ZnO, and PbS. For a complete list of reports of QD biosynthesis, see

Table 1.

Type of Location of Type Species Reference Organism biosynthesis of QD (Sweeney et al. 2004; Mi et CdS al. 2011) Intracellular Escherichia coli CdSe (Yan et al. 2014) CdTe (Monrás et al. 2012) Extracellular CdTe (Bao et al. 2010b) Non- Serratia (Malarkodi and Annadurai photosynthetic Extracellular ZnS nematodiphila 2012) bacteria Brevibacterium Intracellular CdS (Pandian et al. 2011) casei Lactobacillus (Selvarajan and Extracellular ZnO plantarum Mohanasrinivasan 2013) Lactobacillus sp. Extracellular CdS (Prasad and Jha 2010)

Klebsiella (Holmes et al. 1997; Smith et Extracellular CdS pneumoniae al. 1998) Klebsiella aerogenes Extracellular CdS (Holmes et al. 1995a) Pseudomonas sp. Intracellular CdSe (Ayano et al. 2014) Rhodobacter ZnS (Bai et al. 2006) Extracellular Photosynthetic sphaeroides PbS (Bai and Zhang 2009) bacteria Rhodopseudomonas Intracellular CdS (Bai et al. 2009) palustris (Cui et al. 2009; Li et al. CdSe 2013) Intracellular Saccharomyces (Sandana Mala and Rose ZnS cerevisiae 2014) CdS (Prasad and Jha 2010) Extracellular CdTe (Bao et al. 2010a) (Dameron et al. 1989a; Yeast Williams et al. 1996; Schizosaccharomyces Intracellular CdS Williams et al. 2002; pombe Kowshik et al. 2002a; Krumov et al. 2007) (Dameron et al. 1989a; Candida glabrata Intracellular CdS Dameron et al. 1989b; Krumov et al. 2007) Torulopsis sp. Intracellular PbS (Kowshik et al. 2002b) CdS (Ahmad et al. 2002) CdSe (Kumar et al. 2007) Fusarium oxysporum Extracellular CdTe (Syed and Ahmad 2013) ZnS (Mirzadeh et al. 2013) Non-yeast Coriolus versicolor Extracellular CdS (Sanghi and Verma 2009) fungi Phanerochaete Extracellular CdS (Chen et al. 2014) chrysosporium Helminthosporum Extracellular CdSe (Suresh 2014) solani Alternaria alternata Extracellular ZnO (Sarkar et al. 2013) Lycopersicon Plants Intracellular CdS (Reese et al. 1992) esculentum Annelids Lumbricus rubellus Intracellular CdTe (Stürzenbaum et al. 2013)

Table 1. Biosynthesis of QDs by living organisms.

1.6.1 — QD biosynthesis in bacteria

There appears to be large variation between bacterial species with regards to the detoxification of heavy metals and subsequent formation of nanoparticles, having been shown to

10 produce QDs adhered to the cell surface, in the extracellular medium, and retained intracellularly. The first published instance of QD biosynthesis in bacteria was reported in

Klebsiella pneumoniae (Holmes et al. 1997). In this species, cadmium exposure results in the production of nanometre-sized particles deposited on the cell surface (Aiking et al. 1982). These extracellularly produced nanoparticles were confirmed to be composed of crystalline CdS and ranged in size from 5-200 nm (Holmes et al. 1995a; Smith et al. 1998). Initially found to provide photo-protection to bacterial cells through absorption of harmful UV-A radiation

(Holmes et al. 1995b), the CdS crystallites were further observed to display many absorption and fluorescence characteristics analogous to photoactive CdS QDs produced through conventional physicochemical synthesis (Holmes et al. 1997; Smith et al. 1998).

Bacteria have also been demonstrated to produce QDs extracellularly (Bao et al. 2010b;

Malarkodi and Annadurai 2012). When Escherichia coli K12 was incubated with sodium tellurite and cadmium chloride, extracellular CdTe nanoparticles were produced. These QDs were confirmed to be coated by a protein capping layer and were successfully used in the imaging of cervical cancer cells in vitro. It was reported that the biosynthesis of the CdTe QDs was directly dependant on proteins secreted by E. coli, likely through a role in capping and controlling growth, and that no QDs were produced in the absence of bacterial cells or bacterially-secreted proteins (Bao et al. 2010b). The same group reported similar results for the extracellular biosynthesis of CdSe QDs in Saccharomyces cerevisiae (Bao et al. 2010a).

However, in both cases, the addition of a strong reducing agent (sodium borohydride), as well as capping ligands (mercaptosuccinic acid and citrate), to the reaction mixture calls the results into question. In fact, Monrás et al. (2012) found that highly fluorescent CdTe QDs could be produced under the same conditions without the presence of bacterial cells. Perhaps more

11 convincing evidence for the extracellular biosynthesis of QDs with bacteria comes in the form of

ZnS production in Serratia nematodiphila (Malarkodi and Annadurai 2012). In this strain, incubation with zinc sulfate resulted in the synthesis of crystalline ZnS nanoparticles approximately 80 nm in diameter. These QDs were found to be coated with an unidentified protein. Further research is required to determine the precise role that bacterially-secreted proteins play in the extracellular biosynthesis of QDs.

In contrast to K. pneumonia, E. coli and Brevibacterium casei will synthesize CdS intracellularly when incubated with cadmium chloride and sodium sulfide (Sweeney et al. 2004;

Pandian et al. 2011). In both cases, nanocrystal formation was found to be significantly affected by the growth phase of the cells. Cultures which were grown to stationary phase prior to incubation with QD precursors produced significantly more CdS than those in logarithmic phase

(Sweeney et al. 2004; Pandian et al. 2011). In the case of E. coli, this resulted in a 20-fold increase compared to cells in the late logarithmic phase. Similarly, CdSe is produced intracellularly in E. coli when grown in the presence of cadmium chloride and sodium selenite.

The CdSe crystallites had a protein capping layer and were synthesized in significantly larger amounts in stationary phase cells (Yan et al. 2014). Several species have been reported to either produce QDs exclusively or in larger quantities at stationary phase (Holmes et al. 1995a;

Sweeney et al. 2004; Bai et al. 2009; Cui et al. 2009; Pandian et al. 2011; Li et al. 2013; Yan et al. 2014). Sweeney and colleagues (2004) correlated higher content of intracellular thiols to the growth phase-dependent synthesis of CdS crystallites, though thiol levels alone could not account fully for the discrepancy.

In E. coli, it was found that while strains ABLE C and TG1 could produce CdS QDs, strains RI89 and DH10B could not (Sweeney et al. 2004). This suggested that genetic

12 differences between strains of the same species can greatly affect the ability to synthesize QDs, opening the door for genetic engineering toward nanoparticle production. E. coli strain JM109 was co-transformed with SpPCS—the phytochelatin synthase of S. pombe—as well as a feedback-desensitized gshA—responsible for the production of glutamate-cysteine ligase, the first and rate-limiting of two enzymes responsible for glutathione production—to dramatically increase the production of phytochelatin (Kang et al. 2008). These alterations allowed the synthesis of intracellular CdS crystallites in non-stationary phase cells. As well, following genetic engineering, strain RI89 was found capable of producing CdS (Kang et al. 2008), despite having been previously reported incapable (Sweeney et al. 2004). Likewise, E. coli overexpressing gshA synthesized intracellular glutathione-capped CdTe QDs when exposed to cadmium chloride and sodium tellurite, while wild-type cells did not produce CdTe under the same conditions (Monrás et al. 2012).

1.6.2 — QD biosynthesis in non-yeast fungi

Fungal species seem particularly suited to the extracellular biosynthesis of QDs. Much of the research on QD biosynthesis in fungi has focused on the use of Fusarium oxysporum, shown to produce extracellular CdS (Ahmad et al. 2002), CdSe (Kumar et al. 2007), CdTe (Syed and

Ahmad 2013), and ZnS (Mirzadeh et al. 2013). The earliest report of fungal biosynthesis noted that F. oxysporum, when incubated in aqueous cadmium sulfate, changed the solution colour from clear to yellow. This colour change was due to the production of CdS nanoparticles in the size range of 5-20 nm (Ahmad et al. 2002). The exact mechanism behind extracellular QD biosynthesis in F. oxysporum is still a mystery, though some progress has been made towards revealing the process. The absorption spectrum of the aqueous CdS solution indicated the presence of proteins (Ahmad et al. 2002). Similar results were found in the extracellular

13 synthesis of CdSe and CdTe by F. oxysporum, where further Fourier transform infrared spectroscopy (FTIR) analysis confirmed that the QDs were capped by proteins (Kumar et al.

2007; Syed and Ahmad 2013). This suggested the secretion of proteins by the fungus into the reaction solution. Aliquots of this protein solution were capable of producing CdS when added to cadmium sulfate. However, incubation with cadmium sulfate after the protein solution had been dialysed to remove low molecular weight compounds did not result in CdS synthesis. The ability to produce QDs was recovered upon addition of ATP and NADH to the dialysed solution.

Furthermore, when incubated with cadmium nitrate, no extracellular CdS was formed by F. oxysporum. The evidence indicated the secretion of a sulfate reductase enzyme into the reaction solution, initiating the synthesis of CdS QDs (Ahmad et al. 2002).

Apart from F. oxysporum, Coriolus versicolor and Phanerochaete chrysosporium have been found to synthesize CdS extracellularly (Sanghi and Verma 2009; Chen et al. 2014). Both studies focused heavily on FTIR analysis to determine the nature of the capping agents. From a mechanistic standpoint, Chen et al. (2014) proposed that cysteine may be “secreted” onto the fungal cell surface, where cadmium ions are captured via chelation with thiol groups.

Simultaneously, sulfide ions in solution may combine with remaining cadmium ions to form CdS nuclei. The cadmium-thiolate complexes may then covalently bind to the CdS nuclei to form passivation layers, while other secreted biomolecules such as proteins may assemble on the surface of the CdS nanocrystals to play a role in the capping of the QDs.

1.6.3 — QD biosynthesis in yeast

Much of the pioneering research into QD biosynthesis has occurred in yeast species. In fact, the first report of QD biosynthesis utilized the unicellular yeasts C. glabrata and S. pombe

(Dameron et al. 1989a). It was found that these species produced intracellular CdS when incubated with cadmium salts. However, depending on the species and the growth media used, variation in the capping ligand was observed. When grown in a rich medium, CdS synthesized in C. glabrata were primarily coated by glutathione and γ-glutamylcysteine, as opposed to growth in minimal medium which resulted in crystallites coated with phytochelatin analogues.

In the case of S. pombe, phytochelatins coated CdS regardless of the media used (Dameron et al.

1989b). Kowshik et al. (2002a) proposed that CdS may form in S. pombe due to the activation of phytochelatin synthase during cadmium exposure. This results in increased levels of intracellular phytochelatin, which chelate cadmium ions, with the subsequent cadmium- phytochelatin complex moving into a vacuole through HMT1, an ATP binding cassette-type protein. In the vacuole, sulfur can be incorporated into the complex to form the CdS nanocrystal.

Interestingly, S. pombe was found to produce maximal CdS during the mid-exponential phase, with stationary phase cells producing no significant amount of QDs (Williams et al. 1996).

Recently, research into yeast-mediated QD biosynthesis has shifted toward

Saccharomyces cerevisiae. The usefulness of this species to QD biosynthesis was once thought limited, having been reported incapable of producing CdS (Joho et al. 1986). However, this observation has since been shown false, with S. cerevisiae producing intracellular CdS crystallites when grown in the presence of cadmium chloride (Prasad and Jha 2010; Huang et al.

2012). Cells cultured to stationary phase prior to addition of cadmium chloride produced significantly more CdS (Huang et al. 2012). Likewise, S. cerevisiae MTCC 2918 exposed to zinc sulfate produced intracellular ZnS crystallites (Sandana Mala and Rose 2014). A particularly noteworthy example of QD biosynthesis in S. cerevisiae is the production of intracellular CdSe when strain BY4742 was sequentially cultured with sodium selenite and

15 cadmium chloride. Interestingly, it was reported that the size of CdSe crystallites could be tuned from green to yellow emissions simply through increasing the incubation time with cadmium chloride (Cui et al. 2009). The designed biosynthetic process is unique in that it couples unrelated biochemical reactions to achieve the production of CdSe. Upon treatment with selenite, cells reduced selenite into various organoselenium compounds such as selenocystine and selenomethionine, as determined by high-performance liquid chromatography with inductively coupled plasma mass spectrometry. It was suggested that subsequent addition of cadmium chloride initiated the production of CdSe through the interaction of reduced selenium species with glutathione-bound cadmium, prior to its transport into the vacuole (Cui et al. 2009).

The role of glutathione metabolism to the biosynthetic mechanism was explored through the deletion of the GSH1 gene, responsible for glutamate-cysteine ligase in S. cerevisiae. Mutants with deletions of this gene are unable to synthesize GSH, and were found to display significantly lower fluorescence intensity from CdSe QDs than wild-type, indicating the production of a lesser quantity of the QDs. Additionally, GSH1 was also found to be upregulated during CdSe synthesis in wild- type cells, with nanoparticle production increasing with elevated levels of intracellular glutathione. Mutants bearing a deletion of GSH2— which encodes glutathione synthetase, the second and final enzyme in GSH biosynthetic pathway—cannot produce GSH but will accumulate γ-glutamylcysteine. When used for the biosynthesis of CdSe QDs, these mutants displayed a significant reduction in fluorescence intensity compared to wild-type, suggesting that γ-glutamylcysteine was not an effective replacement for GSH (Li et al. 2013).

1.7 — Optimisation of CdSe QD biosynthesis in Saccharomyces cerevisiae

For the biologically-mediated synthesis of QDs to become a viable alternative to physicochemical production methods, several critical challenges must be overcome. In

16 particular, current physicochemical procedures output comparatively large quantities of QDs.

Thus far, most of the studies which report the biosynthesis of QDs with living organisms have focused primarily on the characterization of the nanoparticles, while only relatively few studies have investigated the optimisation of the biosynthetic method, in terms of maximised QD output, to any significant degree (Pandian et al. 2011; Mi et al. 2011; Sandana Mala and Rose 2014; Yan et al. 2014). As well, despite recent progress toward elucidating the biosynthetic mechanism of

CdSe in S. cerevisiae, particularly with regards to the involvement of GSH (Li et al. 2013), there is much to be done before a complete understanding can be established. In the present study, the biosynthesis and characterisation of CdSe QDs produced in S. cerevisiae is reported.

Furthermore, optimisation of the production method to maximise QD synthesis was explored, as well as the provision of substantial evidence for the involvement of GSH in the biosynthetic mechanism, primarily following cadmium treatment.

Chapter 2: Materials and Methods

Yeast strain and culture conditions

Saccharomyces cerevisiae BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) was obtained from the American Type Culture Collection (Manassas, VA). Cultures were grown from an initial density of 0.1% biomass in sterilized YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose) at 30 °C with reciprocal shaking (180 rpm). Culture volumes were 20% of the total flask volume. To maintain healthy stock cultures, stocks were renewed to 0.1% biomass every 72 hours by centrifuging an appropriate amount of culture at 2000 x g (Beckman Coulter Allegra

6R. GH-3.8A) for 5 minutes and resuspending in sterilized YPD medium.

Biosynthesis of CdSe quantum dots

The basic technique for CdSe quantum dot biosynthesis was performed as described by Cui et al.

(2009) and Li et al. (2013), with slight modifications. Cells were grown in YPD for 24 hours before the addition of 5 mM sodium selenite (Na2SeO3) (Sigma, Mississauga, ON) directly to the growth medium. After 24 hours of exposure to Na2SeO3, CdSe quantum dot biosynthesis was initiated when selenium-exposed cells were harvested by centrifugation at 2000 x g (Beckman

Coulter Allegra 6R. GH-3.8A) for 5 minutes, and transferred to an equal volume of fresh YPD with 1 mM cadmium chloride (CdCl2) (Sigma, Mississauga, ON).

Cell imaging

To prepare cells for imaging, a 1 mL aliquot of experimental culture was centrifuged at 16,000 x g (IEC MicroMax microfuge) for 5 minutes, then washed three times with 1 mL ddH2O. A 5 μL sample was examined under 200 x magnification, or 400 x magnification using oil immersion,

18 with a Zeiss Axioplan 2 microscope equipped with U-MWU filters (330-385/400/420 nm).

Images were captured with a Zeiss AxioCam Hrm camera mounted to the microscope, and the imaging software used was AxioVision V4.8.2.0.

Fluorescence spectroscopy

Prior to spectroscopic analysis, 1 mL samples of experimental culture were centrifuged at 16,000 x g (IEC MicroMax microfuge) for 5 minutes, then washed three times with 1 mL ddH2O. The emission spectra of 200 μL aliquots were measured in an opaque white 96 well fluorescence plate (Sigma, Mississauga, ON) using a SpectraMax Gemini XS spectrofluorometer. Emissions were measured at 5 nm intervals between 400 – 750 nm, with an excitation wavelength of 385 nm.

Extraction of CdSe quantum dots from yeast cells

An appropriate amount of experimental yeast cells containing intracellular CdSe quantum dots were harvested through centrifugation, washed 3 times with ddH2O, and resuspended in a lysis buffer (0.2% sodium dodecyl sulfate (SDS), 10 mM Tris-Cl, pH 7.5) at 25% of the buffer volume in a 1.5 mL centrifuge tube. Compared to the cell volume, an equal volume of 0.5 mm zirconium oxide beads were added. The mixture was homogenized with a bead mill homogenizer (Next Advance) for two runs of three minutes. Cellular debris was removed through centrifugation at 16,000 x g (IEC MicroMax microfuge) for 5 minutes. The supernatant containing CdSe quantum dots was removed and filtered with a centrifugal filter unit (Amicon

Ultra-15, 100 kDa) to remove compounds smaller than 100 kDa.

UV-Visible absorption spectroscopy

A SpectraMax Plus 384 UV-Visible Spectrophotometer was used for the measurement of optical density and absorbance spectra. For an estimation of cell concentration, 200 μL aliquots of the culture were placed in clear 96 well UV plates (Sigma, Mississauga, ON) and examined at a wavelength of 600 nm. Samples (1 mL) of control and experimental cultures were centrifuged at

16,000 x g (IEC MicroMax microfuge) for 5 minutes, then washed three times with 1 mL ddH2O. The absorption spectra of 200 μL aliquots were analyzed from 200 – 750 nm, at a resolution of 1 nm.

Measurement of intracellular glutathione content

Sample preparation was modified from Li et al. (2013). The cell density of all samples were standardised to OD600 = 1.0 prior to analysis. One mL of culture was centrifuged at 16,000 x g

(IEC MicroMax microfuge) for 5 minutes, and the subsequent pellet washed three times with 1 x phosphate buffered saline (PBS). Washed cell pellets were resuspended in 1.3% 5-sulfosalicylic acid, then homogenized with zirconium oxide beads (0.5 mm) in a bead mill homogenizer (Next

Advance) for three minutes. Beads and cell debris were removed by centrifugation at 16,000 x g

(IEC MicroMax microfuge) for 5 minutes, and the supernatant removed for measurement of glutathione content. Total reduced glutathione content in the sample was determined as outlined by Vandeputte et al. (1994). Prior to the assay, several stock solutions were prepared: stock buffer (143 mM NaH2PO4, 6.3 mM EDTA, pH 7.4), 10 mM 5,5'-dithiobis-(2-nitrobenzoic acid)

(DTNB) in stock buffer, 2 mM NADPH in stock buffer, and 8.5 IU/mL glutathione reductase

(GR) in stock buffer. Lastly, the assay reagent was prepared by adding 1 : 1.7 : 7.3 parts of

DTNB stock, NADPH stock, and stock buffer, respectively. Twenty μL aliquots of the

20 experimental sample and blanks (containing only stock buffer) were added to a clear 96 well UV plate, followed by 20 μL of stock buffer. Two hundred μL of assay reagent was then added to each well, and the plate allowed to sit at room temperature for 5 minutes. To initiate the reaction, 40 μL of GR stock was added to each well, then immediately the plate was transferred to a SpectraMax Plus 384 UV-Visible Spectrophotometer. The reaction was measured kinetically for 2 minutes, at a wavelength of 415 nm.

Standard solutions of glutathione (80, 40, 20, 10, 5, 2.5, 1.25 nmol/mL) were prepared by diluting a 500 mM GSH stock into 10mM HCl with 1.3% 5-sulfosalicylic acid (SSA). GSH standards and blanks (containing only 10 mM HCl with 1.3% SSA) were processed through the enzymatic glutathione assay outlined above. The standard curve was produced using replicates of 4 determinations, as shown in the figure Appendix I.

Depletion of intracellular glutathione

Cultures treated with 1-chloro-2,4-dinitrobenzene (CDNB) or buthionine sulfoximine (BSO) were centrifuged at 2000 x g (Beckman Coulter Allegra 6R. GH-3.8A) for 5 minutes, then resuspended in ddH2O with 0.5 mM CDNB or 1 mM BSO for 1 hour. Subsequently, cells were centrifuged once again and the biosynthetic procedure continued.

Statistical analysis

Means, standard error, ANOVA, and Tukey’s test were performed using Graphpad Prism 6.

Data presented graphically are means and standard errors. Two sample, one-tailed student t-tests with unequal variance were used on data where appropriate. For comparison between fluorescence spectra, the integrated fluorescence intensity—the sum of the emission intensities for each measured wavelength—was calculated.

Chapter 3: Results

Biosynthesis of CdSe QDs

The general procedure for the biosynthesis of CdSe QDs in S. cerevisiae is shown in terms of the culture emission spectrum, following excitation at 385 nm, at various stages of the process in one representative culture (Fig. 1). The addition of selenium to a culture (sodium selenite) put the cells into an appropriate state to interact with cadmium and form CdSe QDs. Prior to selenite exposure, S. cerevisiae cells displayed a prominent autofluorescence maximum at approximately

460 nm. Following selenite treatment, autofluorescence was significantly diminished over time, until minimal fluorescence is detected. Subsequent incubation with cadmium chloride initiates the synthesis of CdSe QDs, with a strong fluorescence emission maximum developing at between 525 – 600 nm over time. Fluorescence microscopy images of S. cerevisiae cells following the biosynthetic procedure revealed a strong intracellular fluorescence in treated cells

(Fig. 2), primarily localized in the cytoplasm (Fig. 2 inset). Cells treated only with selenite displayed minimal fluorescence (Fig. 2).

Glutathione content at growth phases of Saccharomyces cerevisiae

The effect of growth phase on intracellular GSH content was investigated (Fig. 3). Population growth of S. cerevisiae was assessed through the measurement of optical density at 600 nm. The growth curve could be differentiated into several distinct phases: lag phase (0 – 2 hours), early exponential phase (2 – 5 hours), mid-exponential phase (5 – 7 hours), late exponential phase (7 –

10 hours), and stationary phase (10 – 24 hours) (Fig. 3A). The concentration of intracellular glutathione at various growth times was measured by comparing data from culture samples to a standard curve generated from known quantities of reduced glutathione (Appendix I). Cultures

22 in stationary phase contained nearly three-fold higher intracellular glutathione concentrations than cells in exponential phase (Fig. 3B). Glutathione levels reached a maximum of 115.0 ± 4.76 nmol/mL after cultures were grown for 12 hours, in contrast to cultures grown for 4, 6, 8, or 10 hours with glutathione levels of 25.43 ± 1.17, 30.67 ± 1.88, 34.95 ± 3.42, and 55.07 ± 4.57 nmol/mL, respectively. Glutathione content in the stationary phase (12 hours of growth) was significantly greater than cultures in the exponential phase (4, 6, 8, or 10 hours of growth; P <

0.05).

Optimisation of quantum dot production

The QD synthesis protocol described by Cui et al. (2009) and Li et al. (2013) was employed as the basic technique for all investigations. The optimisation of each variable was investigated in an effort to increase CdSe QD biosynthesis. An overview of the general synthesis protocol and the variables targeted for optimisation are shown in Appendix II. Variables considered were the growth phase of the cells used, selenite incubation methods, concentration of selenite, duration of selenium exposure, cadmium incubation methods, cadmium concentration, and the duration of the reaction. The basic protocol began with the addition of 5 mM sodium selenite directly to a culture of S. cerevisiae. After 24 hours of selenium exposure, cells were harvested and incubated with 1 mM cadmium chloride in fresh YPD medium. The fluorescence emission spectra of S. cerevisiae culture samples at 24 hours subsequent to cadmium exposure were used to assess the production of CdSe QDs under various conditions. In all cases, samples were centrifuged and resuspended in water to prevent light emission interference from constituents of the medium during spectral analysis. For all investigations into the optimisation of the biosynthetic procedure, integrated fluorescence intensity` was calculated from spectra and used to compare between treatments.

Growth phase

To explore the influence of growth phase on the production of CdSe QDs, cultures grown for 4,

8, 12, or 24 hours were standardised to the typical cell density of stationary phase cultures

(OD600 = 1.3), and subjected to the biosynthetic procedure. Emission intensity was found to increase with longer growth periods, reaching a maximum in cultures grown for 12 hours (Fig.

4A). Integrated fluorescence intensity reached a maximum of 14,636 ± 277 RFU in cultures grown for 12 hours, while those grown for 4 or 6 hours reached maximum integrated fluorescence intensities of 7,553 ± 163, and 9,188 ± 355 RFU, respectively (Fig. 4B). This indicates that cultures grown for 12 hours produced significantly more CdSe QDs than those grown for 4 or 6 hours (P < 0.05). Cultures grown for 48 hours experienced a significant decrease in integrated fluorescence intensity (data not shown; P < 0.05).

Selenite incubation conditions

Cultures grown 12 hours to stationary phase were incubated with 5 mM sodium selenite for 24 hours, either through the direct addition of selenite to the culture or through resuspension of cells in fresh growth medium with selenite added. The emission intensity of cultures exposed to selenium by the addition of selenite directly to the culture was greater than those in fresh growth media (Fig. 5A). The integrated fluorescence intensity of cultures exposed to selenium by direct selenite addition was 15,032 ± 634 RFU. In contrast, exposure to selenium through resuspension of cells in fresh selenite-containing medium resulted in an integrated fluorescence intensity of

6,429 ± 647 RFU. The difference between the two selenium exposure methods was found to be significant (P < 0.05), indicating that more CdSe QDs had been produced through direct selenite addition (Fig. 5B).

Selenium concentration

To investigate the influence of selenium concentrations on the biosynthesis of CdSe QDs, 0.1,

0.5, 1, 3, 5, or 10 mM sodium selenite was added directly to stationary phase (12 hours) cultures during the biosynthetic procedure. The greatest emission intensity was obtained from the addition of 1 mM sodium selenite (Fig. 6A). Integrated fluorescence intensity of the spectra reached a maximum of 14,731 ± 349 RFU from the addition of 1 mM sodium selenite (Fig. 6B).

The integrated fluorescence intensity produced by the addition of 1 mM sodium selenite was found to be significantly greater than the integrated fluorescence intensities of all other sodium selenite concentrations (P < 0.05).

Selenium exposure time

Stationary phase (12 hours) cultures were incubated with directly added 1 mM sodium selenite for various durations of selenium exposure. The greatest emission intensity of subsequently synthesized QDs was observed after cells were exposed for 6 hours (Fig. 7A), with a corresponding integrated fluorescence intensity of 16,037 ± 306 RFU (Fig. 7B). The integrated fluorescence intensity of cultures exposed for 6 hours was significantly greater than cultures exposed for a lesser duration (0, 1, 2, or 4 hours) (P < 0.05).

Cadmium incubation conditions

Cultures grown to stationary phase (12 hours) were incubated with 1 mM sodium selenite for 6 hours through the direct addition of selenite to the culture. Subsequently, 1 mM cadmium chloride was added either directly to the culture or through resuspension of cells in fresh growth media containing the cadmium chloride. When added with fresh media, the emission intensity of cultures was greater than when cadmium was added directly to the culture (Fig. 8A), with

25 integrated fluorescence intensities of 18,442 ± 1367 and 6,355 ± 137 RFU, respectively (Fig.

8B). The integrated fluorescence intensity of cultures treated with cadmium in fresh growth media was significantly greater than cultures exposed to cadmium by direct addition to the culture (P < 0.05).

Cadmium concentration

Stationary phase (12 hours) cultures were treated with 1 mM sodium selenite through direct addition to the culture, and allowed to incubate for 6 hours. Selenium-exposed cultures were centrifuged and resuspended in fresh growth media containing 0.1, 0.5, 1, 2, 3, or 4 mM cadmium chloride. Generally, emission spectra intensities increased with cadmium concentrations (Fig. 9A). Treatment with 3 mM cadmium chloride resulted in an integrated fluorescence intensity of 18,756 ± 411 RFU (Fig. 9B). The integrated fluorescence intensity produced by cultures treated with 3 mM cadmium chloride was significantly greater than those produced by cultures treated with lesser concentrations of cadmium (P < 0.05). However, no significant difference was observed between this and concentrations higher than 3 mM (Fig. 9B).

Reaction time

Twelve-hour old stationary phase cultures that had been exposed to selenium by direct addition of 1 mM sodium selenite for 6 hours were incubated with 3 mM cadmium chloride in fresh growth media, and allowed to react. Samples were taken at various time points and the emission intensity measured to track the synthesis of CdSe QDs. Emission intensity increased steadily with longer reaction times, reaching a maximum after 84 hours (Fig. 10A), with an integrated fluorescence intensity of 82,014 ± 1,884 RFU (Fig. 10B). The integrated fluorescence intensity of cultures exposed to cadmium for 84 hours was significantly greater than all shorter reaction

26 times (P < 0.05). Reactions times longer than 84 hours did not result in significantly greater emission intensities or integrated fluorescence intensities (data not shown).

Comparison between optimised and basic protocol

Cultures treated with the basic QD protocol as described by Cui et al. (2009) and Li et al. (2013) reached maximum emission intensity approximately 60 hours following cadmium addition (data not shown). In contrast, those treated with the optimised method (shown in Appendix III) reached maximum emission intensity 84 hours following cadmium addition (Fig. 10). The maximum emission intensity of cultures treated with the optimised protocol was greater than those treated with the basic protocol (Fig. 11A). The corresponding integrated fluorescence intensity of cultures treated with the optimised protocol was significantly greater than those treated with the basic protocol (Fig. 11B; P < 0.05).

Characterisation of CdSe QDs

CdSe QDs produced through the optimised biosynthetic method were extracted from cells via homogenization with 0.5 mm zirconium oxide beads in a bead mill homogenizer, followed by washing the lysate with a centrifugal filter unit with a cut-off of 100 kDa. The purified QD solution displayed prominent yellow fluorescence when excited by 365 nm UV light (Fig. 12 inset). This QD solution was analyzed in terms of the absorption and emission spectra, revealing a broad absorption profile primarily through the UV range and a Stoke’s shifted emission maximum at approximately 540 nm (Fig. 12).

Influence of glutathione on CdSe QD biosynthesis

To investigate the importance of GSH to the CdSe QD biosynthetic mechanism, cultures were treated with either CDNB (Fig. 13) or BSO (Fig. 14) for 1 hour at various stages of the optimised biosynthetic method. Treatment with CDNB prior to selenium exposure resulted in a significant, though not complete reduction of emission intensity (Fig. 13A) and corresponding integrated fluorescence intensity (Fig. 13B). In contrast, treatment with CDNB prior to cadmium addition, or prior to both selenium exposure and cadmium addition, resulted in a near complete reduction of emission intensity (Fig. 13A) and integrated fluorescence intensity (Fig. 13B). In all cases, integrated fluorescence intensities of CDNB treated cultures were significantly less than untreated cultures (P < 0.05). Cultures treated with BSO prior to selenium exposure showed a slight, though non-significant, reduction in emission intensity. However, treatment with BSO prior to cadmium addition, or prior to both selenium exposure and cadmium addition, resulted in significantly (P < 0.05) reduced emission intensities (Fig. 14A) and integrated fluorescence intensities (Fig. 14B), though to a lesser degree than CDNB treatment.

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Figure 1. Emission spectra of S. cerevisiae culture at various stages of the CdSe QD biosynthetic procedure (385 nm excitation). ( ) Autofluorescence of cells prior to selenium exposure; ( ) Following selenium exposure using sodium selenite; ( ) Following treatment of selenium- exposed cells with cadmium chloride. Means are shown (n = 4).

Figure 2. Fluorescence microscope images (200 x magnification) of S. cerevisiae cells following 24 hours of exposure to sodium selenite (left; scale bar = 20 μm), and 24 hours following the QD biosynthetic procedure (right; scale bar = 20 μm). Inset is fluorescence microscope image (400 x magnification) of S. cerevisiae cells following the QD biosynthetic procedure (scale bar = 5 μm).

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Figure 4. The production of CdSe QDs in S. cerevisiae cultures grown for various time periods. (A) Emission spectra (385 nm excitation) and (B) corresponding integrated fluorescence intensities of S. cerevisiae cultures at the conclusion of the biosynthetic process, initiated with cells grown for 6 (orange), 8 (blue), 12 (green), and 24 (red) hours. An asterisk denotes significant deviation (P < 0.05) from largest value (12 hours). Means and SE (n = 4) are shown.

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Figure 5. The production of CdSe QDs in stationary phase S. cerevisiae cultures exposed to selenium through various methods. (A) Emission spectra (385 nm excitation) and (B) corresponding integrated fluorescence intensities of S. cerevisiae cultures at the conclusion of the biosynthetic process, with sodium selenite added directly to the growth medium (dotted line), or through incubation with fresh growth medium (solid line). An asterisk denotes significant deviation (P < 0.05) from largest value (direct addition). Means and SE (n = 4) shown.

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Figure 6. The production of CdSe QDs in stationary phase S. cerevisiae cultures exposed to various concentrations of selenite for 24 hours. (A) Emission spectra (385 nm excitation) and (B) corresponding integrated fluorescence intensities of S. cerevisiae cultures at the conclusion of the biosynthetic process, with 0.1 (red), 0.5 (orange), 1 (pink), 3 (blue), 5 (green), or 10 mM (black) selenite added directly to the growth medium. An asterisk denotes significant deviation (P < 0.05) from largest value (1 mM). Means and SE (n = 4) shown.

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Figure 10. The production of CdSe QDs in S. cerevisiae cultures at various time points following the addition of cadmium chloride. (A) Emission spectra (385 nm excitation) and (B) corresponding integrated fluorescence intensities of S. cerevisiae cultures after reaction times of 0 (orange), 2 (purple), 8 (green), 24 (red), 48 (blue), 72 (yellow), or 84 hours (black). An asterisk denotes significant deviation (P < 0.05) from largest value (84 hours). Means and SE (n = 4) shown.

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Figure 11. The production of CdSe QDs in S. cerevisiae cultures following various biosynthetic procedures. (A) Emission spectra (385 nm excitation) and (B) corresponding integrated fluorescence intensities of S. cerevisiae cultures at the conclusion of the biosynthetic process once maximum emission intensity was measured. Maximum emission intensity was achieved for the basic protocol (dotted line) after 60 hours, and for the optimised protocol (solid line) after 84 hours. An asterisk denotes significant deviation (P < 0.05) from largest value (optimised protocol). Means and SE (n = 4) are shown.

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Figure 12. Characterisation of CdSe QDs produced through the optimised biosynthetic method. UV-Visible absorption spectrum (dotted line) and emission spectrum (solid line) of purified CdSe QD solution. Inset is photograph of purified CdSe QD solution (excitation 365 nm).

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Figure 13. The production of CdSe QDs in S. cerevisiae cultures treated with CDNB at various stages of the biosynthetic method. (A) Emission spectra (385 nm excitation) and (B) corresponding integrated fluorescence intensities of S. cerevisiae cultures at the conclusion of the biosynthetic process, having received no CDNB treatment (red), or treatment with CDNB for 1 hour prior to selenium exposure (blue), prior to cadmium addition (orange), or prior to both selenium exposure and cadmium addition (black). An asterisk denotes significant deviation (P < 0.05) from cultures not treated with CDNB. Means and SE (n = 4) shown.

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Figure 14. The production of CdSe QDs in S. cerevisiae cultures treated with BSO at various stages of the biosynthetic method. (A) Emission spectra (385 nm excitation) and (B) corresponding integrated fluorescence intensities of S. cerevisiae cultures at the conclusion of the biosynthetic process, having received no BSO treatment (red), or treatment with BSO for 1 hour prior to selenium exposure (blue), prior to cadmium addition (orange), or prior to both selenium exposure and cadmium addition (black). An asterisk denotes significant deviation (P < 0.05) from cultures not treated with BSO. Means and SE (n = 4) shown.

Chapter 4: Discussion

The biosynthesis of CdSe QDs in cultures of S. cerevisiae was monitored by measuring fluorescence emissions when excited by 385 nm light. Cells which had not yet been exposed to selenite displayed an autofluorescence maximum at 460 nm (Fig. 1). This is likely due to the presence of intracellular NADPH, known to fluoresce at approximately 460 nm in S. cerevisiae

(Horvath et al. 1993). Cultures were exposed to selenium through the addition of sodium selenite, followed by treatment with cadmium chloride to initiate QD biosynthesis. At this stage, a strong emission maximum between 525 – 600 nm developed over time (Fig. 1). Such emission maxima are characteristic of CdSe QDs (Crouch et al. 2003). Furthermore, these cultures were examined using fluorescence microscopy (Fig. 2). Treated cells fluoresced bright yellow, indicating the formation of QDs through an intracellular mechanism. Analysis of isolated QD samples obtained from solutions of cell extracts revealed a broad absorption band edge, with a

Stoke’s shifted emission maximum at 540 nm (Fig. 12). The lack of a defined first excitonic absorption peak, as well as the relatively broad emission, may be due to synthesis of non- uniform sized nanoparticles. The observed spectra are similar to those reported previously in S. cerevisiae, in which transmission electron microscopy was used to visualize the synthesized particles, and energy dispersive X-ray spectroscopy confirmed them to be composed of CdSe

(Cui et al. 2009; Li et al. 2013). Control cells exposed only to sodium selenite did not exhibit notable emissions when visualized by fluorescence microscopy (Fig. 2). However, control cells incubated only with cadmium chloride led to the development of blue-emitting CdS QDs (data not shown), as previously reported by Huang et al. (2012). The CdS QDs did not appear to be produced in selenium-exposed cells treated with cadmium chloride. Based on these results, the assumption can be made that the emission and absorption spectra, as well as the yellow

42 intracellular fluorescence, which develop following completion of the biosynthetic process is the result of the intracellular biosynthesis of CdSe QDs.

Investigations were undertaken to determine the optimal parameters of the biosynthetic procedure. Firstly, cultures grown for different periods of time prior to selenium exposure were studied to explore the importance of growth phase to the biosynthesis of CdSe QDs. Stationary phase cultures, specifically those cultures grown for 12 or 24 hours, were found to produce significantly greater integrated fluorescence intensities than cultures in the exponential phase, indicating the production of more CdSe QDs (Fig. 4). This seems to be a common observation in reports on the biological synthesis of QDs, having been noted in a number of studies (Holmes et al. 1997; Sweeney et al. 2004; Bai et al. 2009; Pandian et al. 2011; Yan et al. 2014). In particular, Cui et al. (2009) and Li et al. (2013) previously reported the importance of stationary phase cultures to the biosynthesis of CdSe in S. cerevisiae. However, this trend is not fully consistent; Williams et al. (1996) found that stationary phase cells did not produce CdS in S. pombe due to limited Cd uptake and minimal inorganic sulfide in the cells. The nature of the relationship between culture growth phase and the biosynthesis of QDs warrants further investigation. In E. coli, the synthesis of CdS is greatly enhanced with the use of stationary phase cultures (Sweeney et al. 2004). It was found that intracellular GSH, free reduced thiols, and sulfur were present in significantly greater quantities in the stationary phase, indicating their possible role in the synthesis of the QDs. For the biosynthesis of CdSe in S. cerevisiae, a similar investigation has been undertaken to establish a correlation between intracellular GSH content and the influence of growth phase to the biosynthesis of QDs. Cultures in the stationary phase, grown for 12 or 24 hours, were found to contain significantly greater levels of intracellular GSH than those in the exponential phase (Fig. 3B). Taking into account the greater QD production

43 observed at stationary phase, this correlation suggests the possible involvement of GSH to the biosynthetic process. However, under the same experimental conditions, variation in GSH content could not fully account for the difference between the integrated fluorescence intensities of the stationary phase and exponential phase cultures, suggesting that other factors may play a role as well. Furthermore, selenite toxicity may factor into the increased efficiency of stationary phase cells to produce CdSe QDs. Letavayová et al. (2008) found that the toxicity of selenite was in part caused by the formation of double strand breaks in the DNA of exposed cells.

Toxicity was less severe in S. cerevisiae cells in the stationary phase as compared to the exponential phase, possibly due to decreased DNA replication preventing the induction of double strand breaks. During the biosynthesis of CdSe QDs, S. cerevisiae cells in the exponential phase may have a lower biosynthetic capacity due to being increasingly burdened by selenite toxicity.

The exact mechanism of CdSe QD biosynthesis in S. cerevisiae is not well understood, though for synthesis to occur, several fundamental criteria must be met. Importantly, both cadmium and selenium must be in an appropriate valence state to interact together. The biosynthetic procedure makes use of cadmium chloride, a Cd2+ salt, as the cadmium precursor.

As a result, selenite must be reduced from the +4 to the -2 oxidation state for it to bind cadmium and form CdSe. Localization is another important consideration, as the reduced selenium species must be positioned in the cell with cadmium in order to interact. Following exposure to sodium selenite, S. cerevisiae cells were observed to develop a slight orange colouration (data not shown), suggesting the reduction of selenite to elemental selenium, Se(0). The biological reduction of selenite to Se(0) has been observed in several organisms, and is thought to be mediated by GSH (Turner et al. 1998; Hunter and Manter 2008; Dhanjal and Cameotra 2010;

Mishra et al. 2011; Hnain et al. 2013). In the case of CdSe QD biosynthesis, a related process

44 may be responsible for the reduction of selenite into the -2 oxidation state. Uptake of selenite into S. cerevisiae seems to be through the opportunistic use of high and low affinity phosphate transporters (Lazard et al. 2010), as well as the monocarboxylate transporter Jen1p (McDermott et al. 2010). Phosphate conditions of the growth media can influence which phosphate uptake system is used, with the high affinity Pho84p being favoured for selenite uptake under low phosphate conditions, and the low affinity Pho87p, Pho90p, and Pho91p used when phosphate levels are high. Once inside the cell, selenite will undergo a series of reactions within the cytoplasm:

2- - (1) 6GSH + 3SeO3 GS-Se-SG + 2O2 + 5H2O

(2a) GS-Se-SG + GSH GS-Se- + GSSG + H+

(2b) GS-Se-SG + NADPH GR/TR GSH + GS-Se- + NADP+

(3a) GS-Se- + H+ GSH + Se(0)

(3b) GS-Se- + GSH GSSG + HSe- where GR is glutathione reductase and TR is thioredoxin reductase. Selenite spontaneously reacts with GSH to form the stable intermediate species selenodiglutathione (GS-Se-SG), as shown in Reaction 1 (Braga et al. 2004; Kessi and Hanselmann 2004; Cui et al. 2008).

Glutathione is the most abundant biological thiol in S. cerevisiae, reaching levels as high as 10 mM (Penninckx 2002). As a result, it is anticipated that in most conditions, there will be an excess of intracellular GSH available. Once incorporated within the GSH complex, selenodiglutathione can be further reduced by excess GSH to form selenopersulfide (GS-Se-), as shown in Reaction 2a (Braga et al. 2004). However, selenodiglutathione has also been

45 demonstrated as an excellent substrate for glutathione reductase (Ganther 1971), as well as for the mammalian thioredoxin and thioredoxin reductase system (Björnstedt et al. 1992), resulting in the production of selenopersulfide (Reaction 2b). It is possible that selenodiglutathione interacts in a similar manner with the S. cerevisiae thioredoxin system. Selenopersulfide is a labile intermediate species that can dismutate into reduced GSH and elemental selenium

(Reaction 3a), or produce hydrogen selenide (HSe-) through reaction with GSH (Reaction 3b).

The yeast genome does not contain any of the sequences typically associated with selenoprotein biosynthesis (Birringer et al. 2002), suggesting that S. cerevisiae is incapable of producing specific selenoproteins. Nonetheless, selenium can become incorporated non-specifically into proteins due to its close ionic similarity to sulfur (Hatfield 2012). Selenide produced through the above reactions can act as a substrate for non-specific synthesis of selenocysteine by cysteine synthase (Ng and Anderson 1978).

In contrast, the interactions of cadmium with S. cerevisiae are relatively straightforward.

Cadmium is a non-essential heavy metal, with no known biological function in yeast

(Muthukumar and Nachiappan 2010). Uptake into the cell is an active process facilitated by the high-affinity zinc transporter Zrt1, due to the similarities between cadmium and zinc (Gomes et al. 2002; Adamis et al. 2003). Cadmium ions are detoxified through interaction with GSH, binding with two molecules of GSH to form a bis-glutathionato-cadmium complex (GS-Cd-SG) in the cytoplasm (Li et al. 1997). Detoxification of cadmium reaches a conclusion when the bis- glutathionato-cadmium complex is transported into the vacuole by the Ycf1 transporter (Li et al.

1997; Gomes et al. 2002).

In terms of the biosynthesis of CdSe QDs in S. cerevisiae, substantial evidence suggests that GSH plays an important role. Stationary phase cells were found to produce significantly

46 more QDs compared to exponential phase cells (Fig. 4). As such, intracellular GSH levels were assessed to determine whether a correlation existed with the quantity of QDs synthesized at different growth phases. It was found that stationary phase cells contained significantly greater levels of GSH than those in the exponential phase (Fig. 3B), suggesting a possible link between

GSH content and efficiency of QD production. The importance of stationary phase to the biosynthetic process may be due to differences in the non-specific incorporation of selenium into organic species. Ponce de León et al. (2002) reported that when selenite was added during the exponential phase in S. cerevisiae, most selenium was incorporated as selenomethionine, a non- catalytic organic selenium species. On the other hand, stationary phase cultures produced a wider variety of organic selenium species, a larger portion of which is catalytic selenocysteine

(Bierla et al. 2013). Selenocysteine has been shown to produce fluorescent CdSe QDs when incubated with cadmium chloride in vitro (Cui et al. 2009), and may be important to the in vivo mechanism as well. Additionally, cultures treated with CDNB (Fig. 13) or BSO (Fig. 14) to deplete intracellular GSH levels produced significantly less QDs than controls, providing further evidence for the impact of GSH to the CdSe QD biosynthetic mechanism. CDNB is a substrate for GSH S-transferase, conjugating with GSH to irreversibly form 2,4-dinitrophenyl-S- glutathione. Likewise, BSO is an inhibitor of glutamate-cysteine ligase, the first and rate- limiting of two enzymes responsible for GSH production. Treatment with CDNB prior to selenium exposure results in a significant, though not total, reduction of CdSe QD synthesis (Fig.

13). Likewise, cells treated with BSO prior to selenium exposure inhibited CdSe QD synthesis, though to a lesser degree than CDNB treatment (Fig. 14). These observations are in agreement with a previous report of S. cerevisiae GSH1 knockout mutants synthesizing a limited quantity of

CdSe QDs despite being incapable of producing GSH (Li et al. 2013). In that case,

47 selenocysteine, suspected to be the precursor to CdSe QD formation, was not entirely absent from the cells. Taken as a whole, these results seem to suggest that S. cerevisiae can use an alternative method independent of GSH to synthesize the organic selenium precursors.

Conversely, treatment with CDNB prior to cadmium chloride exposure drastically inhibits QD synthesis, with only minor fluorescence detected at the end of biosynthetic procedure (Fig. 13).

Treatment with BSO prior to cadmium chloride exposure resulted in a significant reduction of

QDs produced, though, again, its effects were less substantial than CDNB (Fig. 14). The lesser impact of BSO on QD synthesis is likely due to cells maintaining an intracellular pool of previously synthesized GSH, unaffected by brief BSO treatment (Prévéral et al. 2006).

Interestingly, when cells were treated with CDNB (Fig. 13) or BSO (Fig. 14) prior to both selenium exposure and treatment with cadmium chloride, no significant difference in QD output was observed from treatment only prior to cadmium chloride addition. The lack of a cumulative impact indicates that GSH serves a crucial role in the biosynthesis of CdSe QDs, particularly following the addition of cadmium chloride. It is possible that GSH-conjugated cadmium complexes may act as the precursor to QD production. As well, GSH may function as a capping ligand, stabilizing the QDs in the cytoplasm.

Several mechanisms have been proposed for the biosynthesis of QDs in living organisms.

Cui et al. (2009) found that the in vitro synthesis of CdSe QDs could be achieved through reaction of selenocysteine with cadmium chloride. It was proposed that the mechanism for CdSe formation in S. cerevisiae depended on the interaction of cadmium chloride with this intracellular organic selenium species. A similar mechanism has been put forward for the biosynthesis of

CdSe in E. coli (Yan et al. 2014). However, several studies have shown that similar QDs can be produced without the use of selenocysteine. For instance, PbSe QDs have been synthesized in

48 vitro using only GSH, glutathione reductase, NADPH, sodium selenite and glutathione-bound lead (Cui et al. 2012), possibly through the interaction of lead with selenium intermediates produced by the GSH-mediated reduction of selenite. A similar mechanism was proposed by

Stürzenbaum et al. (2013) to explain the synthesis of CdTe QDs in the chloragogenous tissue, or liver equivalent, in earthworms. It was suggested that cadmium reacted with H2Te, produced by the reduction of sodium tellurite with GSH. Likewise, studies on the mutual detoxification of selenite and mercuric chloride in mammals have revealed the synthesis of glutathione-coated mercuric selenide, produced through the reaction of glutathione-bound mercury with the selenite metabolites selenopersulfide or hydrogen selenide (Gailer 2002). A consideration of all evidence available seems to indicate that the biosynthesis of CdSe QDs in S. cerevisiae may involve more than one mechanism. As such, the following mechanism is proposed:

2- - (1) 6GSH + 3SeO3 GS-Se-SG + 2O2 + 5H2O

(2a) GS-Se-SG + GSH GS-Se- + GSSG + H+

(2b) GS-Se-SG + NADPH GR/TR GSH + GS-Se- + NADP+

(4) GS-Se- + GS-Cd+ + H+ GS-CdSe + GSH

(3b) GS-Se- + GSH GSSG + HSe-

(5) HSe- + GS-Cd+ GS-CdSe + H+

(6) HSe- CS Selenocysteine

(7) Selenocysteine + Cd2+ CdSe-Cys

Initially, selenite in the cytoplasm will react with GSH to form selenodiglutathione (Reaction 1).

Selenodiglutathione can further react with excess GSH to form selenopersulfide (Reaction 2a).

Additionally, selenodiglutathione may act as a substrate for glutathione reductase or the thioredoxin system to form selenopersulfide (Reaction 2b). Autofluorescence in S. cerevisiae cells, presumed to be due to the presence of intracellular NADPH, was observed to decrease with selenite treatment (Fig. 1B). This may indicate the depletion of NADPH due to the actions of glutathione reductase at this stage of the reduction. Furthermore, selenopersulfide may react with glutathione-bound cadmium in the cytoplasm to form glutathione-coated CdSe (Reaction 4), or will react with excess GSH to form hydrogen selenide (Reaction 3b). Hydrogen selenide could also interact with glutathione-bound cadmium at this stage to from glutathione-coated

CdSe (Reaction 5). Lastly, hydrogen selenide, acting as a substrate for cysteine synthase (CS), can be non-specifically incorporated into selenocysteine (Reaction 6). Cysteine-coated CdSe may form when selenocysteine is exposed to cadmium ions in the cytoplasm (Reaction 7). Li et al. (2013) reported that S. cerevisiae unable to synthesize GSH were still able to produce selenocysteine, suggesting the contributions of a pathway independent of GSH. The synthesis of

CdSe QDs by way of selenocysteine formed from this GSH-independent pathway may explain why cells treated with CDNB before selenium exposure did not experience a complete reduction in QD production (Fig. 13). Intracellular fluorescence appears to be localized in the cytoplasm, outside of the vacuoles (Fig. 2 inset). This suggests that glutathione-bound cadmium interacts with selenium compounds to form CdSe prior to being transported by Ycf1 into the vacuole.

Preceding treatment with cadmium chloride to initiate QD production, S. cerevisiae cells must be exposed to selenium to put them into the appropriate state to form CdSe. Investigations undertaken to optimise this stage of the procedure revealed that more QDs were produced when

50 sodium selenite was added directly to the culture, as opposed to harvesting the cells and resuspending them in fresh media with sodium selenite (Fig. 5). While the reason for this difference is not clear, it is possible that the addition of fresh growth media causes the cells to begin dividing, making them more susceptible to selenite-induced DNA damage (Letavayová et al. 2008), or that the addition of fresh growth media results in cells transitioning out of the stationary phase and lessening their GSH content. Furthermore, a sodium selenite concentration of 1 mM was found to be ideal (Fig. 6) for a selenium exposure duration of 6 hours prior to cadmium addition (Fig. 7). While even 1 mM is approximately five times what has been reported to be inhibitory to growth (Suhajda et al. 2000), higher concentrations and longer selenium exposure times may overwhelm cellular defences against selenium toxicity too rapidly to efficiently synthesize QDs. Selenium toxicity is thought to occur due to an accumulation of oxidative stress, as the reaction between various catalytic selenium compounds with intracellular thiols results in the production of superoxide and hydrogen peroxide (Spallholz 1994). Ponce de

León et al. (2002) found that while higher selenite concentrations led to greater cell death, there was an accompanying increase in the amount of selenium non-specifically incorporated as organic selenium species. A balance between the toxicity of high selenium concentrations and the incorporation of selenium into organic compounds may be important for CdSe QD production.

Following selenium exposure, cells are treated with cadmium chloride to initiate CdSe

QD synthesis. Incubation of S. cerevisiae in fresh media (Fig. 8) with 3 mM cadmium chloride

(Fig. 9) was found to produce optimal results. Notably, treatment with concentrations greater than 3 mM did not result in a decrease of QD production. This seems to indicate that the limiting factor is the selenium exposure step involving the preparation and accumulation of intracellular

51 selenium species to react with added cadmium. The rate of QD synthesis may reach a maximum once intracellular selenium species are saturated with cadmium precursors. The optimal Se:Cd precursor ratio was determined to be 1:3 mM. The same ratio was reported in the synthesis of

CdSe in E. coli (Yan et al. 2014). An abundance of negatively charged selenium ions on the surface of CdSe QDs often results in diminished fluorescence due to the prevalence of trap sites for non-radiative recombination that selenium provides. The addition of excess cadmium can passivate the surface, electrostatically shielding charge carriers from trap sites, and increasing emission intensity (Smith and Nie 2010). This property of CdSe QDs may provide an explanation for the optimal 1:3 mM ratio, as opposed to the previously reported usage of 5:1 mM sodium selenite and cadmium chloride, respectively (Cui et al. 2009; Li et al. 2013). Finally, following cadmium chloride addition, the maximum quantity of CdSe QDs was found to be produced after approximately 84 hours of reaction time (Fig. 11). This is in good agreement with the production of CdS in B. casei, found to reach a maximum after 96 hours (Pandian et al.

2011).

In summary, this study has demonstrated the biosynthesis of CdSe QDs by S. cerevisiae treated with sodium selenite and cadmium chloride. The nanoparticles appear to be formed in the cytoplasm of the cells, and display a strong emission maximum between 525 - 600 nm. The mechanism proposed for the intracellular formation of CdSe involves the reaction of cadmium with selenopersulfide, hydrogen selenide, or selenocysteine. These selenium species may be produced through a series of abiotic and enzymatic reactions involved in the reduction of selenite, of which GSH seems to play a crucial role, particularly after cadmium treatment.

Investigations into the optimisation of the biosynthetic procedure were undertaken, revealing that maximal QDs were produced with the use of stationary phase cells treated with 1 mM sodium

52 selenite by direct addition to the culture. Following 6 hours of selenium exposure, resuspension of cells in fresh media with 3 mM cadmium chloride for approximately 84 hours gave the highest

QD production. An overview of the optimised method is shown in Appendix III.

The demonstrated method of CdSe QD production has several clear advantages over physicochemical procedures. Namely, this method of “green” synthesis is less energy-intensive, proceeding at ambient pressure and temperature, and does not require the use of potentially explosive reagents. For the first time, significant efforts have been undertaken to optimise each aspect of the CdSe biosynthetic procedure in S. cerevisiae, resulting in an increased output of

QDs (Fig. 11). However, for the biologically-mediated synthesis of QDs to be relevant from a practical standpoint, the quantity of production must compete with that of physicochemical methods. Li et al. (2013) reported that S. cerevisiae produced approximately 2.45 μmol, or roughly 469 μg, of CdSe per gram of yeast cells. Assuming a proportionate relationship between our measured maximum emission intensities of the original technique (Cui et al. 2009; Li et al.

2013) and the optimised method, and their corresponding QD biosynthetic yields, the CdSe output utilizing the optimised procedure is estimated at 800 μg per gram of yeast cells—an approximately 70% increase in achievable emission intensity. However, while optimisation has seemingly pushed the natural QD synthesis capability of S. cerevisiae to its limits, the improvement on output is marginal when compared to quantities produced through physicochemical means. Recent developments on the production of CdSe QDs using supercritical fluids have resulted in continuous synthesis rates of 200 mg per hour (Chakrabarty et al. 2015), far in excess of what is currently possible through biosynthesis. At this rate of synthesis, it would require an estimated 1275 L of S. cerevisiae batch culture to produce an equivalent quantity of CdSe QDs using the optimised method. An additional difficulty includes

53 the need to harvest the cells and extract intracellular QDs. Genetic manipulation may be the most promising route to advance biological synthesis into a competitive domain with physicochemical methods. Selenocysteine seems to be an important precursor to QD production, though the removal of GSH does not completely inhibit its production (Li et al. 2013). This suggests an alternative GSH-independent pathway being utilized for selenite reduction.

Exploration of this pathway may reveal targets for genetic manipulation for further control of

QD biosynthesis, much as (Li et al. 2013) achieved through the manipulation of GSH-related genes. Additionally, it seems plausible that QD production may be controlled through targeting various aspects of the selenite and cadmium pathway. This may include the transporters responsible for uptake of selenite and cadmium into the cell, or GSH-related genes responsible for the synthesis of GSH and its precursors.

Summary

 S. cerevisiae synthesized intracellular CdSe quantum dots localized in the

cytoplasm when sequentially treated with sodium selenite and cadmium chloride.

 Biosynthesis initiated by treating stationary phase cells with 1 mM sodium

selenite—added directly to the growth medium—for 6 hours, followed by

resuspension of cells in fresh growth medium with 3 mM cadmium chloride for

84 hours, was found to be the optimal biosynthetic protocol.

 In most cases, treatment of cells with 1-chloro-2,4-dinitrobenzene or buthionine

sulfoximine resulted in a significant reduction of quantum dot output.

 Treatment with CDNB prior to cadmium addition resulted in a near complete

removal of QD biosynthesis, suggesting that GSH plays a critical role following

cadmium treatment

 The mechanism of biosynthesis relied heavily on the presence of glutathione,

possibly due to its involvement in the metabolism of selenite into potential

quantum dot precursors such as selenodiglutathione, selenopersulfide, or

selenocysteine, which may interact with cadmium to form CdSe.

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Appendix

0 .0 0 6

y = (6.88 x 105)x + (2.27 x 105)

0 .0 0 4

l S 0 .0 0 2

0 .0 0 0 0 2 0 4 0 6 0 8 0

n m /m L G S H

Appendix I: GSH content standard curve generated from reaction of known quantities of GSH. Error bars represent standard error (n = 4).

Appendix II: General procedure for the biosynthesis of CdSe QDs in S. cerevisiae. Variables targeted for optimisation are shown on right.

Appendix III: Optimised procedure for the biosynthesis of CdSe QDs in S. cerevisiae. Optimised variables shown in red.