Chapter 3 – Electrochemical Synthesis of PVP-Protected Silver Nanoparticles and

Chapter 2: Experimental

2.4 Chemicals and Synthesis

2.4.1 Chapter 3 – Electrochemical Synthesis of PVP-Protected Silver Nanoparticles and

2.4.1.1 Electrochemical Synthesis of PVP-Protected Silver Nanoparticles

The electrochemical synthesis of poly(N-vinylpyrrolidone) (PVP) protected silver nanoparticles was carried out using a procedure similar to that employed by Yin and co-workers (10)_{. The voltammetric characterisation was carried out in a}

conventional three electrode cell where a 4 mm platinum disc electrode served as the working electrode and a platinum and silver wire served as the counter and reference electrode, respectively. A three electrode cell was also employed for the synthesis of the PVP-protected silver nanoparticles but with two platinum sheet electrodes, 1 x 5 cm, serving as the working and counter electrode and a silver wire as a reference electrode. The electrodes were cleaned as outlined in Section 2.2.3 before being placed in a solution containing various amounts of PVP dissolved in 10 mL of 5 x 10-3_{mol dm}-3_AgNO₃_{and 0.1 mol dm}-3_KNO₃_{. The PVP content was}

expressed in g dm-3 _{and had an average molecular weight of 40,000. The}

electrolysis was carried out using various electrochemical techniques, namely potentiostatic and galvanostatic, for either a fixed amount of time or until a certain total charge had passed. Details are outlined in the results and figure captions. The solution was under constant agitation using a stirring bead and magnetic stirring plate during the electrolysis to aid the flux of silver ions to the electrode surface and to avoid the formation of flocculates that can form in the vicinity of the cathode when the nanoparticle concentration is high.

Experimental Chapter 2 2.4.1.2 UV-Vis Spectroscopy of PVP-Protected Silver Nanoparticle Solution

The silver nanoparticle solution was placed in a 1 cm diameter quartz cuvette either undiluted or diluted with deionised water. In most cases, the solution required dilution as the absorption intensity was very high. The dilution factor is specified in the relevant result section and figure caption.

For the experiments which monitored the effect of added salts on the stability or aggregation of the PVP-protected silver nanoparticle, the suspension of nanoparticles were diluted 120 times in deionised water. To 2.85 mL of this solution 0.15 mL of either 0.1 mol dm-3_AgNO₃_{or KNO}₃_{was added to give a final}

3 mL solution containing 5 x 10-3_{mol dm}-3_{of the added salt. This solution was}

introduced into 1 cm diameter quartz cuvette and the absorbance monitored over 1 month. All solutions were stored in a sealed vial covered with tinfoil and away from light.

2.4.1.3 Immobilisation of PVP-Protected Silver Nanoparticles onto a Glassy Carbon Electrode for the Electrochemical Detection of Nitrate

A polished 4 mm diameter glassy carbon electrode was polished as outlined in Section 2.2.3. The electrode was then suspended for 12 hours in a PVP-protected silver nanoparticle solution which had been synthesised potentiostatically at an applied potential of -6 V until a charge of 180 C was reached from an aqueous solution containing 0.05 mol dm-3_AgNO₃_{, 0.1 mol dm}-3_KNO₃_{and 423.75 g dm}-3

PVP. After 12 hours, the electrode was air dried until all solvent had evaporated before being placed in a nitrate-containing solution to test for its ability to perform as an electrochemical sensor.

Experimental Chapter 2 2.4.1.4 Polyacrylamide-PVP-Protected Silver Nanoparticle Composite

2.4.1.4.1 Polyacrylamide-PVP-Protected Silver Nanoparticle Composite Synthesis The polyacrylamide-PVP (PAAm-PVP) and the polyacrylamide-PVP-protected silver nanoparticle (PAAm-PVP-Ag-np) composites were prepared by dissolving acrylamide monomer (AAm) and N,N’-methylenebisacrylamide (MBA) in 10 mL of water. The solution was purged for 10 minutes with nitrogen to remove any dissolved oxygen. To this, either 10 mL of a 423.75 g dm-3_{PVP solution in water,}

also purged for 10 minutes, was added to make the PAAm-PVP hydrogels or for the PAAm-PVP-Ag-np composites, 10 mL of the PVP-protected silver nanoparticle solution which had been synthesised potentiostatically at an applied potential of -6 V until a charge of 180 C was reached from an aqueous solution containing 0.05 mol dm-3_AgNO₃_{, 0.1 mol dm}-3_KNO₃_{and 423.75 g dm}-3_{PVP. To initiate}

polymerisation, 300-400 L of a freshly made 1% ammonium persulfate aqueous solution and 5-10 L tetramethylethylenediamine (TEMED) were added with vigorous mechanical stirring.

2.4.1.4.2 Polyacrylamide-PVP-Protected Silver Nanoparticle Composite as an Electrode The hydrogel electrodes were prepared by casting the gel solution around a 1 mm diameter polished platinum wire. This was achieved by suspending the wire in a 6 mm diameter standard household straw 5 cm in length with a plastic plug at one end. The gel solution was poured into the straw where gelation occurred. Once set the plug was removed and the gel slid from the straw easily. The electrodes were suspended in a large beaker of deionised water for 1 day to extract any unreacted reagents.

2.4.1.4.3 Polyacrylamide-PVP-Protected Silver Nanoparticle Composite for Use as a Heterogeneous Catalyst for the Reduction of 4-Nitrophenol

The components of the gels, once combined, were pipetted into a glass vial 5 cm in height and 1 cm in diameter. This served as the mould for the gels to set at room temperature. Typical gelation occurred within 10 minutes but the composites were left in mould for at least 2 hours to allow complete polymerisation. To remove the

Experimental Chapter 2 gels from the glass vials the bottom of the glass tube was cut with a grinding wheel allowing the gel cylinder to be easily slide out to the tube intact. The gel cylinder was then cut using a sharp razor blade into uniform discs 3 mm in length. The discs once rinsed, were ready for use in subsequent experiments.

4-Nitrophenol and NaBH4 solutions were freshly made for each experiment and

degassed with N2 for 10 minutes prior to initiating the reduction reaction. The

number of discs per volume of solution was kept constant for each run and magnetically stirred at the same RPM as this affected the rate of the reaction. To monitor the rate of the reduction reaction three fully swollen discs were added to a solution containing 3 mL of 1.2 x 10-4_{mol dm}-3_{4-nitrophenol and 6 mL of}

0.1 mol dm-3_NaBH₄_{, in other words 3 discs in 9 mL of 4 x 10}-5_{mol dm}-3

4-nitrophenol and 6.7 x 10-2_{mol dm}-3_NaBH₄_.

2.4.1.5 Biological Preparation for Anti-Bacterial Testing

The potential anti-bacterial activity of the PAAm-PVP and PAAm-PVP-Ag-np composites were tested against five different strains of bacteria, namely (a)

Staphylococcus aureus (S. aureus), (b) methicillin-resistant Staphylococcus. aureus

(MRSA), (c) Pseudomonas aeruginosa (ATCC 27853), (d) Pseudomonas aeruginosa (ATCC 10145) and (e) Escherichia coli (E.coli). All anti-bacterial testing was carried out in the Medical Mycology Lab, Department of Biology, Maynooth University under the direction of Dr. Kevin Kavanagh.

2.4.1.5.1 Chemicals for Bacterial Biological Preparations

Chemicals were purchased from commercial sources and were used without further purification. Deionised water was used to prepare all media. The nutrient broth medium containing peptone, yeast extract and nutrient agar were purchased from Scharlau Microbiology and prepared following the manufacturer’s instructions in sterilised water (13 g in 1 L of deionised water). E.coli was supplied as a clinical isolate by the Clinical Microbiology Laboratory, St. James’ Hospital, Dublin, Ireland and were originally isolated from a gastro-intestinal tract infection.

Experimental Chapter 2 St. James’ Hospital, Dublin, Ireland and were originally isolated from a urinary tract infection. MRSA was obtained as a clinical isolate from Microbiologics, North St. Cloud Mn, USA and was originally isolated from a wound infection. P. aeruginosa 27853 and 10145 were both obtained from the American Type Culture Collection (ATCC) Marasses, VA, USA.

2.4.1.5.2 Sterilisation

Sterilisation of the microbiological equipment and media was carried out in a Dixons ST2228 autoclave at 121 ± 2˚ C and 124 kPa for 20 minutes. All worktops and benches were sterilised by washing with 70 % (v/v) ethanol/water prior to use. Agar plates were prepared in a laminar flow hood and the plates were streaked in an aseptic area around a Bunsen burner.

2.4.1.5.3 Cell Density

Bacterial cell density was recorded at an optical density of 600 nm (OD600) using an Eppendorf BioPhotometer.

2.4.1.5.4 Procedure for Bacterial Biological Preparations

The in vitro antimicrobial activity of the PAAm-PVP-Ag-np composite was investigated against both Gram-positive and Gram-negative bacteria. All bacteria were grown on nutrient broth agar plates at 37 ± 1˚C and maintained at 4 ± 1˚C for short term storage. Cultures were routinely sub-cultured every 4-6 weeks. The five different bacteria were grown overnight to the stationary phase in nutrient broth at 37 ± 1˚C and 2000 rpm. The cells were diluted to give an OD600 = 0.05 and streaked onto the agar plates. The plates were incubated for 1 hour at 37 ± 1˚C. The hydrogel discs were then placed on the plates and incubated for a further 24 hours at 37 ± 1˚C before the zones of inhibition around the discs were measured. 2.4.1.5.5 Measuring the Zones of Inhibition

To determine the zone of inhibition (ZOI), the diameter of the disc and the diameter of the disc plus the zone itself were measured in mm. Then, using the area of a circle, A = πr2_{, the area of the disc was subtracted from the total area of}

Experimental Chapter 2

2.4.2 Chapter 4 – Silver Particles Electrochemically Deposited on a

In document Electrochemical Synthesis of Silver Nanoparticles for Applications in Nitrate Detection, Catalysis and Antibacterial Activity (Page 73-78)