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«By Yusuf Nur A thesis submitted to The University of Birmingham for the Degree of DOCTOR OF PHILOSOPHY School of Geography, Earth and Environmental ...»

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The calculated shape factor values of the synthesised particles were near 1 and higher than 0.8 for all samples as listed in the result Table 5-1 above. Figure 5-10 presents the shapes of the typical PVP capped AuNPs. It is clear from these images that the synthesised AuNPs are of high quality, monodisperse and spherical in shape. This monodispersity in shape of the nanoparticles is an essential for the interpretation, analysis and comparison of any effect-property data caused by another property of the particles such as size, surface charge,etc.

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For the quantification of the shapes of the synthesised gold nanoparticles, from each sample at least 100 particles were randomly chosen throughout the surface of the TEM copper grid and shape factor distribution diagrams of the particles were plotted and illustrated in Figure 5-11 below. It can be seen from the shape factor distribution diagrams that for all samples 95% of the particles have a shape factor value better than 0.8 which clearly confirms the sphericity of the synthesised AuNPs since this value is not far from the perfect sphere shape factor which is 1.

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Figure 5-11: Shape factor distribution diagrams of AuNPs calculated from TEM images. G1 and G2 are coated by citrate. G5 and G6 are coated by PVP polymer. Relevant physicochemical properties of the NPs presented in this graph are summarised in table

5.1 above.

5.2.2.4 Surface chemistry Surface chemistry of NPs is mainly determined by the type of coating agents stabilised with the NPs. Coating agents can have effect on a number of measureable surface properties of NPs such as pH, zeta potential and surface charge. The surface charge of the particles may arbitrate either attraction or repulsion between NPs and other components in the relevant media such as environmental present chemicals, organic materials and organisms. Steric polymerisation may on the other hand affect drastically the size of the coated particles and subsequent transport in the environment. Therefore, surface chemistry is a key property for the ultimate fate and behaviour of the NPs in the environmental relevant conditions including ionic strength, amount of naturally occurring organic matters and microorganisms. For the sake of identifying the surface properties of the NPs, pH, zeta potential and surface charge of the synthesised NPs were measured and presented in the result Table 5-3 below.

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Zeta potential is a measurement of the overall charge of the particles in media and it indicates the stability of the particles in the sense that the higher the zeta potential the more stable the particles are. It is calculated from electrophoretic mobility of the particles in an applied electric field. From the zeta potential data in Table 5-3 can be seen that the citrate capped particles have higher values of the zeta potential than the PVP capped NPs. Among the PVP stabilised particles, their zeta potential are the same magnitude regardless of the synthesis method (hot or cold) used during the synthesis of the particles which indicates that the zeta potential is mainly determined by the surface coating. The pH of citrate capped NPs are around 6 while the PVP capped samples which were reduced by NaOH have apparently high pH. This can be associated with the fact that NaOH is strong alkaline and thus it raises the pH of the solution.

5.2.2.5 Dissolved and nanoparticles fractions of gold The synthesis process of the nanoparticles begins with the reduction of gold ions into gold atoms.

To quantify the fraction of gold ions which is changed into NPs and the dissolved fraction, the concentrations of both total gold and dissolved gold have been measured using ICP-MS. The

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between total and dissolved fraction of gold. Table 5-4 shows that the percentage dissolved was less than 2.5% for all samples. In addition to that, it demonstrates that there are relatively more dissolved gold ions in the case of the PVP capped samples than the citrate capped particles.

Table 5-4: Concentration of the total and dissolved gold in the samples as prepared and measured with ICP-MS.

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5.2.3 Stability of the AuNPs Nanoparticles prepared through chemical reaction where metallic ions are reduced to atoms need to be stabilised by suitable coating agents. Lack of appropriate stabilising agents will cause continuous growth of the particles and subsequent aggregation of the NPs. The stability of the synthesised gold nanoparticles has been monitored. The z-average of the hydrodynamic diameter of the NPs was measured using DLS for a period of six months and results are presented in Figure 5-12 below. The three measurements did not reveal any significant size variations in the period studied).

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Figure 5-12: Stability of two samples of AuNPs coated with either citrate or PVP is monitored over a period of six months from the synthesis date.

The surface Plasmon resonance (SPR) of the samples was also recorded for a period of six months using Uv-vis spectrophotometer. The results presented in Figure 5-13 showed consistency of the absorption peak in that period and did not reveal any significant change in size which would be

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115 2.5 0.3

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0.5 0.05

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0.8 0.6 0.4 0.2

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Apart from the stability of the NPs over time, the effect of the ionic strength of the media on the size of both citrates capped and PVP capped NPs were investigated. Testing NPs in a range of ionic strength is essential for the understanding of the behaviour of the NPs in the environment since environmental relevant conditions may vary in ionic strength. For instance fresh surface water normally contains low concentration of dissolved ions while seawater has relatively higher ionic strength. The variation of the particles sizes as function of the ionic strength was illustrated in Figure 5-14 below. As manifested by the steep gradient of the graph in Figure 5-14 a citrate capped particles are more vulnerable to the ionic strength than the PVP capped ones.

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Figure 5-14: The correlation curve of the z-average values of citrate capped NPs and the ionic strength of the media. The media used for this purpose was monovalent electrolyte which is NaNO3. Section a represents citrate capped NP. Section b is PVP capped NP.

The increase in size of the nanoparticles indicates the formation of aggregates in the media. It is worth noting that the PVP coated NPs were tested in ionic strength as high as 100 mM and the increase in size is insignificant since the difference between the sizes is in the error

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aggregation of the citrate particles caused by ionic strength.

Recently published data has investigated the effect of a) single monovalent and divalent elctrolytes of cations, b) a mixture of electrolytes and c) the anions on aggregation of citrate capped AgNPs Nanoparticles (Baalousha et al., 2013). This research has compared the effect of the above mentions electrolytes and concluded that divalent cations (Ca(SO4), Ca(NO3)2, MgCl2 and MgSO4 have stronger influence on aggregation as compared to monvalent cations NaCl, NaNO3, Na2SO4. Regarding to the anions, sulphates and nitrates did not manifest any specific ion effect while the addition of chloride ions has enhanced the aggreagation of the NPs. Similarly, El Badawy et al have investigated the effect of monovalent cations NaNO 3 and divalent cations Ca(NO3)2 on Silver nanoparticles of different coating agents (Badawy et al., 2010). Naked silver NPs, Citrate capped silver NPs and Borate capped silver NPs have aggregated at higher ionic strength (100 mM) while the presence of Ca2+ cations have resulted in enhanced apparent aggregation at ionic strength as low as 10 mM. Investigation on the aggregations of silver nanoparticles coated with either PVP and citrate has revealed that divalent electrolytes were more efficient in destabilising the citrate coated NPs as indicated by the considerably lower critical coagulation concentrations (2.1 mM CaCl2 and

2.7 mM MgCl2 vs 47.6 mM NaCl) (Huynh and Chen, 2011).

118 7 6 5

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Figure 5-15: DLS diagram of citrate capped NPs in 16 mM NaNO 3 solution showing that citrate capped AuNPs particles clearly aggregate at this ionic strength and higher as shown by the mixture of peaks.

Figure 5-16 shows lack of aggregation of PVP coated NPs in high ionic strength solution. The ionic strength of the solution is as high as 80 mM.

17 15 13

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Figure 5-16: DLS diagram of PVP capped NPs in 80 mM NaNO3 solution showing that there is no sign of NPs aggregations in this high ionic strength solutions.

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Prior to any exposure experiment of NPs on the environment, their physicochemical properties need to be studied so that the fate and behaviour of the NPs after exposure can be compared to their original properties. It is the aim of this chapter to synthesise and fully characterise good quality gold NPs of different sizes and different surface chemistries.

Gold nanoparticles were synthesised using wet chemistry methods. Two types of coating agents (citrate and PVP) were used to stabilise freshly synthesised gold nanoparticles. During the synthesis, the first sign for the production of the NPs was manifested in colour change from yellow to ruby red. This was confirmed by the characteristic surface Plasmon resonance peak for gold which is around 520 nm. Multi - method-approach was used to fully characterise the synthesised NPs. Here, a range of different but complementing analytical and imaging techniques helped to measure relevant physicochemical properties of the NPs. In brief, stable, monodisperse, spherical AuNPs with two coating agents and a range of sizes were synthesised using one-step method approach. The NPs are homogenous in size and spherical in shape. Furthermore, the stability of the NPs was monitored and the results showed their stability even after one year from the synthesis date. The core size of citrate capped varies from 7 nm to 32 nm while PVP particles have core sizes ranging from 11nm to 85nm. The shape factors of all samples were near to 1 and better than 0.8. From the ICP_MS measurements, it can be seen that for all samples 97.5% of gold ions were converted into NPs and just a very small fraction (2.5%) is dissolved in the solution. Between different capping agents, the PVP capped NPs have more dissolved fraction than the citrate capped particles.

Since the size, shape and monodispersity of stabilised gold NPs are fundamental features for their toxicity studies, these fully characterised gold NPs can form a reliable basis for potential

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certain variables. The range of sizes and coating agents will provide more versatility of these potential reference materials for the study of property-effect relationship in (eco) toxicology studies.

These fully characterised, high quality AuNPs will be used in the following chapters to study their effect/interaction with the planktonic bacteria Pseudomonas fluorescens.

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6.1 Introduction Nanomaterials behave differently in the different compartments of the environment (Praetorius et al., 2012). Bacterial biomass forms an integral and important part of the environmental living organisms and the interaction between nanoparticles (NPs) and bacteria is an inevitable process(Pelletier et al., 2010). The effect of this interaction on both bacteria and on the behaviour of the nanomaterials is a matter of investigation and available studies remain far from being conclusive (Tong et al., 2007, Johansen et al., 2008, Kumar et al., 2011).

The main task of this chapter is threefold. First the stability and behaviour of the NPs in relevant bacteria growth media will be studied and monitored. The nanoparticles will be fully characterised in the growth media through measuring their physicochemical properties such as sizes, degree of aggregation, concentration, surface plasma and pH of the media which are important parameters for the study of the effect of the nanoparticles on any organisms. If the NPs change their properties in the full strength of the media, the media will be further diluted to preserve the original properties of NPs and special care will be taken so that the diluted media is still able to provide enough nutrients to guarantee the growth of bacteria without being starved. Secondly, the growth inhibition of environmental planktonic bacteria caused by different types of gold NPs will be investigated. Finally the behaviour of NPs after bacteria exposure will also be monitored. The bacteria chosen for this purpose is the environmental bacteria Pseudomonas fluorescens because of its widespread colonising in soil, plant and water. The strain used (SBW25) was received from Professor Christopher

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of the Pseudomonas fluorescens in general and the above mentioned strain in particular was given in Chapter 2: sections 2.3.3. In brief, pseudomonas flourescens is a rod shape, motile, non pathogenic, gram-negative aerobic bacteria with flagella as a medium for movement (see Figure 6-1.

Figure 6-1: TEM images Pseudomonas fluorescens showing its rodshape body and its flagelles(Silby, 2006).

Agar plates were prepared biweekly and bacteria samples were spread on a fresh agar plates to keep them alive (Figure 6-2). As mentioned earlier, prior to the investigation of the effect of gold NPs on bacteria their stability in a liquid bacterial growth media need to be tested.

Minimal Davis Media is used for this purpose.

Figure 6-2: Pseudomonas fluorescens growing on freshly prepared agar base plate.

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