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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 stability of freshly synthesised samples of gold nanoparticles of the two capping agents (citrate and PVP) in MDM (see section 4.5.1 for the preparation of the media and its ionic strength) of different concentrations was monitored in a period of two weeks. Hydrodynamic size measured with DLS and Surface plasma resonance (SPR) was taken continuously while at the end of the experiment at day 9 samples were imaged with TEM to detect possible aggregations.

6.2.1 Stability of citrate capped NPs in undiluted MDM media Citrate capped NPs of different sizes were prepared as explained in Chapter 4:section 4.2.2.

The capping and stabilising by the citrate is achieved through charge stabilisation mechanism. Citrate is a negatively charged organic ion and by attaching it on the surface of the NPs repulsion forces between particles are created. The same negative sign of the surface of the NPs keeps them separated and thus preventing them from forming aggregates. If the conditions of the surrounding media such as ionic strength change the thickness of the electric double layer around the single particles will change in the sense that the higher ionic strength the thinner the double layer and this will affect the stability of the NPs. The stability of citrate capped 14.8 nm core size AuNPs (hereafter called G2) was tested in three different dilutions of MDM media and the results are summarised in following sections. The detailed physicochemical properties of the synthesised NPs including G2 are tabulated in the result Table 5-1 in Chapter 5: of the thesis.

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To study the stability of the NPs, G2 NPs were added into undiluted MDM media and their surface Plasmon resonance (SPR) were continuously recorded for two days using a Uv-vis spectrophotometer. Results were presented in Figure 6-3 below. The decay rate in the table was calculated below using Equation 6-1.

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0.7 0.6 0.5 0.4 0.3 0.2 0.1

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first 60 minutes of the undiluted MDM media and disappeared completely after 24 hrs after the exposure of the NPs in the undiluted MDM media as is illustrated clearly in the above Figure 6-3. The reason for the disappearance of SPR is due to the formation of aggregates of bulk scale sizes. The maximum absorbance of each spectrum is determined and plotted against time as illustrated in Figure 6-4 below.

1 0.95

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0.85 0.8 0.75 60, 0.7244 0.7 120, 0.672 0.65

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Figure 6-4: Graph showing how the maximum SPR of citrate capped AuNPs of 14.8 nm core size in undiluted MDM changes with the time.

As highlighted in Figure 6-4, the absorbance is decreasing exponentially. The absorbance dropped from 0.9892 AU to 0.7244 AU in the first 60 minutes which corresponds with a decrease of -0.2648 AU. In the following hour the absorbance has fallen to 0.672 AU which corresponds with decrease of just -0.0524 AU. The maximum absorbance has completely

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aggregates. The colours of the NPs changed from ruby red to black after 30 minutes as illustrated in Figure 6-5 below and after 24 hrs of exposure, huge aggregates of the NPs were visible at the bottom of the container.

Figure 6-5: The characteristic red colour of the freshly synthesised AuNPs has changed into black within the first 30 minutes in the full strength of the Mimimal Davis Media (MDM) DLS measurements of the above citrate capped AuNPs were performed after 30 minutes of exposing NPs in undiluted MDM and the monomodial narrow peek with z-average of 18.3 ±

0.4 with Polydispersity index (PDI) of 0.19 changed into a mixture of different peeks as illustrated in Figure 6-6 a and b respectively.

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Figure 6-6: DLS graphs showing aggregation of the citrate capped AuNPs (G2) in undiluted MDM media. a) monomodal size distribution by intensity measured with DLS of freshly synthesised 14.8 nm core size citrate capped AuNPs b) same NPs in undiluted media. Measurements were performed about 30 minutes after exposure.

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nanoscale in Figure 6-6 b which indicated that aggregations of the NPs have taken place in the media within a period of only 30 minutes.

After 9 days in the media, samples were prepared by the ultracentrigation method described in Chapter 4: section 4.4.1. Then, the grid was washed with milli-q and dried. Images in Figure 6-7 were taken with Jeol1200 TEM (see Chapter 3: section 3.1.2 for the theory of the TEM techniques).

Figure 6-7: TEM images showing aggregates of different sizes of citrate capped AuNPS in undiluted MDM media.

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citrate capped AuNPs in the undiluted MDM media supporting the disappearing SPR spectrum illustrated in Figure 6-3 and the DLS data presented in Figure 6-6.b. The sizes of aggregates larger than 1000 nm were recorded. The results from different techniques presented above for the citrate capped AuNPs in undiluted MDM media clearly revealed that this type of NPs are unstable in this strength of the media. The undiluted media has ionic strength of 19.1 mM as calculated with equation Equation 4-2. It is a well-known phenomenon that charge stabilised NPs aggregate in high ionic strength media (Badawy et al., 2010, Cumberland and Lead, 2009). Consequently, the behaviour of the citrate capped NPs in diluted MDM media was investigated and results are presented in the following sections.





6.2.1.2 AuNPs capped with citrate in 4x diluted MDM media The behaviour of G2 NPs in 4x diluted MDM media was further studied and characterised with uv-vis, DLS and TEM. The surface Plasmon resonance of the NPs was recorded for 14 days and the data were presented in Figure 6-8 below. SPR of the particles remained unchanged throughout this period of time indicating that the AuNPs particles behaved in a quite stable manner in this 4x diluted MDM media.

129 1.2

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Figure 6-8: Surface plasmon resonance measured with Uv-vis of 14.8 nm core size citrate capped AuNPs in 4x diluted MDM media measured in a period of 14 days.

For further analysis of the citrate capped AuNPs in 4x diluted MDM media, DLS measurements were taken in different days and the results were presented in Figure 6-9, Figure 6-10 Figure 6-11 and Figure 6-11 below.

13 18.17, 12.3 11

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Figure 6-9: Size distribution by intensity measured with DLS of 14.8 nm core size citrate capped AuNPs (G2) in 4x diluted MDM media measured day 1 of the exposure.

130 13 18.17, 11.6 11

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Figure 6-10: Size distribution by intensity measured with DlS of 14.8 nm core size citrate capped AuNPs after 5 days in 4x diluted MDM media.

14 12 10

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Figure 6-11: size distribution by intensity measured with DLs of 14.8 nm core size citrate capped AuNPs in 4x diluted MDM media. Measurement was taken on day 9 of the exposure.

The above DLS measurements which are spread over a period of 9 days have demonstrated that the size distribution by intensity of the citrate capped AuNPs remained monomodial with comparable polydispersity indices. In all three measurements there were no indications of aggregations taking place in the media since the intensity did not change and there are no peeks in the microscale area. Samples of the citrate capped AuNPs were prepared using ultra

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images of the NPs were illustrated in Figure 6-12.

Figure 6-12: TEM images showing stabile single particles of citrate capped AuNPs (G2) after 9 days of exposure in 4x diluted MDM media.

In contrast with the full strength of the MDM media where complete aggregation of the NPs was recorded within 30 minutes (see section 6.2.1) the original spherical shape and size of the AuNPs were preserved (Figure 6-12) which specify the stability of the particles in this strength of the media ( ionic strength is 4.78 mM).

6.2.1.3 AuNPs capped with citrate in 10x diluted MDM media One further step of the dilution of the media was carried out and the stability of the citrate capped AuNPs were tested and characterised. The Uv-vis measurements of the SPR of the particles and TEM images recorded and presented in Figure 6-13 a and b respectively.

Since the citrate capped AuNPs have shown stability in 4x diluted media similar results were expected in the 10x diluted media which was clearly confirmed by both the TEM and Uv-vis measurements.

132 1.2

–  –  –

Figure 6-13: a) SPR of citrate capped NPs in 10x diluted MDM media monitored ten days. b) : TEM images of citrate capped NPs after 9 days in 10x diluted media.

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The stability of the synthesised NPs is mainly determined by their surface chemistry which is the type of coating agents used to stabilise the NPs. While citrate stabilises through repulsion of the same charge on the surface of the NPs, PVP stabilisation is caused by sterisch effect.

The bulk molecule of the PVP polymer separates the NPs and keeps them in the media. The stability of PVP coated NPs was investigated in three different concentration of the MDM media. TEM, DLS and UV-vis analytical techniques were used to study their behaviour in the media and the results are given in the sections below.

6.2.2.1 AuNPs capped with PVP in undiluted MDM media The stability of PVP capped nanoparticles were studied in a period of 12 days. The surface plasma resonance of the PVP capped samples in undiluted media were recorded and presented in Figure 6-14.

133 0.6

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Figure 6-14: The SPR measured with uv-vis of 10 nm core size PVP capped NPs in undiluted MDM media monitored in a period of 12 days.

Unlike citrate capped NPs presented in section 6.2.1.1 where the SPR disappeared within 24 hours in the full strength media the SPR of the PVP stabilised gold NPs remains unchanged for the measured period of 12 days. There is neither shift nor loss of the intensity which showed that PVP capped AuNPs are stable in size and shape in fully concentrated Minimal Davis media (MDM) in the monitored period. For further analysis the TEM samples were prepared on the 9th day in media. It was expected that PVPNPs will stay stable in the diluted media since they are stable in the undiluted media. TEM images of 10 nm core size gold nanoparticles stabilised with PVP in three different strengths of PVP media were illustrated in Figure 6-15 below.

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Figure 6-15: TEM images of gold nanoparticles capped with PVP in undiluted MDM media. Image a is in undiluted media, b in 4x diluted media and c in 10 diluted media. Samples were placed on the TEM grid after 9 days in the media.

Figure 6-15 above clearly put on view that gold NPs capped with PVP are stable in all three concentrations of the media including undiluted media. The individual nanoparticles are clearly visible on the grid with no sign of aggregation. This shows that PVP capped NPs are more stable in MDM media and other media with similar ionic strength than citrate capped gold NPs.

After a period of trial and error to decide what strength of the MDM media can be used to maintain both the stability of the NPs and the growth of the bacteria, it was found out that on one hand NPs especially citrate capped NPs are not stable in the full strength of the MDM media (see section 6.2.1 above) while on the other hand bacterial growth in 10 diluted MDM media was practically both time-consuming and incomplete. So 4x diluted media provide enough nutrients for bacterial growth and both types of the NPs (citrate capped and PVP capped) remained quite stable. Therefore, for the remaining studies which are aimed to investigate the bacterial growth inhibition caused by the AuNPs the 4x diluted MDM media will be used.

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Pseudomonas fluorescens was grown in 4x diluted MDM media and placed in a shaking incubator rotating with 120 rpm at 25oC. Optical density at wavelength of 595 nm was recorded using UV-vis for a period of 52 hrs to scan the different growth phases of the bacteria and the results are illustrated in Figure 6-16 below.

0.018 0.016 0.014

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Figure 6-16: Typical growth curve of the psoudomonas flourescencs strain SBW25 in 4x diluted MDM media.

This growth curve is helpful to show the difference phases of bacterial growth. It shows a short lack phase of roughly 3 hours. It clearly displays an exponential phase starting after few hours and ending around 24 hours after the inoculation of the bacteria, which is when the bacteria are most vulnerable to toxicants. It is worth noting that the death phase is not very obvious here since both the dead cells and living cells scatter the light and Uv-vis measures both scatterings.

6.4 Testing the Effect of AuNPs on planktonic bacteria Generally, the growth of bacteria can be measured directly by counting cells using a cell

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bacteria cells in the media. The higher the number of cells the cloudier the suspension and more scattered the light. In this investigation, turbidity measurement was used to investigate possible bacterial growth inhibition caused by different types of AuNPs used by taking the optical density (OD) measurements of the bacteria suspension at a wave length of 595 nm.

Though the OD method is a quick, non-destructive and easier to carry out than the counting method, one inherent problem of the turbidity measurement method is that the death phase of the curve is less obvious since the dead cells still physically scatter light. The theory and the practical application of the used Uv-vis instruments were given in sections 3.1.6 and 4.4.5 respectively.

High quality gold nanoparticles of different sizes and different capping agents which were synthesised using methods described in Chapter 4: section 4.2 and their stability in different strength of MDM were thoroughly tested and described in the above sections were exposed on Pseudomonas fluorescens bacteria growing in a liquid MDM media.The overall aim was to study the interaction of the nanoparticles with the bacteria and monitor the effect of the

NPs on the growth of the bacteria. The experiment was divided into two stages which are:



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