This chapter describes both the chemical and biological characterisation methods used throughout this thesis along with the reagents, parameters and instruments utilised.
Nuclear Magnetic Resonance (NMR) Spectroscopy
H-NMR spectra were recorded on 400 MHz Bruker spectrometer and are reported in parts per million (ppm) on the δ scale relative to CDCl3 (δ 7.26). 13C-NMR spectra were recorded on a 100 MHz Bruker spectrometer and are reported in parts per million (ppm) on the δ scale relative to CDCl3 (δ 77.16). The multiplicities of 1H signals are designated by the following abbreviations: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br = broad; dd doublet of doublets; dt = doublet of triplets; dm = doublet of multiplets. All coupling constants J are reported in hertz.
High Resolution Mass Spectroscopy (HRMS)
HRMS was conducted by the University of Auckland’s Mass Spectroscopy Centre. It was conducted on a Bruker micrOFOT-Q mass spectrometer using electrospray ionization mass spectroscopy.
Fourier Transform Infrared (FTIR) Spectroscopy
FTIR spectroscopy was conducted by utilizing two different analysis methods on a Thermo Nicolet 8700 FTIR spectrophotometer. The KBr disk method was utilized for all PANI powders. The KBr was firstly dried in an oven at 110°C before use. 150-200 mg of KBr was weighed into a mortar and 1 mg of sample was subsequently added. The mixture was ground together with a pestle and then pressed under 5 tons of pressure for 5 minutes in a pellet press. Measurements were taken immediately following pellet preparation. A blank KBr disk was also produced and used as a background to account for any moisture absorption. Absorbance spectra were taken as an average over 32 scans over a wavelength range of 550-4000 cm-1 with a resolution of 4 cman average over 32 scans over a wavelength range of 550-4000 cm-1 with a resolution of 4 cm–
UV-Visible (UV-Vis) Spectroscopy
UV-Vis spectra were collected on a Shimadzu UV-2101PC UV-Vis Scanning Spectrophotometer between 250 and 800 nm with a 1 cm quartz cuvette. N-methylpyrrolidone (NMP) was used as the solvent and the samples were filtered through a 0.45 µm PTFE syringe filter prior to analysis.
Gel Permeation Chromatography (GPC)
GPC was conducted on a system consisting of a Waters 515 HPLC pump, a Degassex DG-4400 on-line degasser connected to a TSK Gel Super AWM-H column (9 μm, 6 x 150 mm) with column guard, 0.5 μm in-line filters, a Rheodyne manual injector, and a Waters column oven. The eluent was NMP and the flow rate was 0.12 mL/min. Sample concentrations were 10 mg/mL and the injection volume was 200 μL. The solution was filtered through 0.45 μm PTFE syringe filters before injection. Polystyrene standards used were Agilent Easical GPC/SEC calibration standards. Data acquisition and processing were performed using the ASTRA 4 software (Wyatt Technologies Corporation). The detector was a Shimadzu RID-10A Differential Refractive Index detector. The columns and RI detector were maintained at 35 °C.
Cyclic voltammetry was conducted using a three-electrode setup with a glassy carbon working electrode (GCE), platinum counter electrode and Ag/AgCl reference on a BAS100B electrochemical analyser. The experiments started at -200 mV vs Ag/AgCl and increased to 1000 mV vs Ag/AgCl at a scan rate of 100 mV/s with 10 μA/V sensitivity. The solutions for the electrochemical polymerization were 0.1 M of sample in 0.5 M aqueous H2SO4 supporting electrolyte. All electropolymerization solutions were deaerated with oxygen-free nitrogen for at least 15 minutes prior to beginning.
The elemental analysis results provided in this thesis were all conducted by the Campbell Microanalytical Laboratory, the University of Otago, Dunedin, New Zealand. CHNX analysis was the method used which determines the mass fractions of elements such as carbon, hydrogen, nitrogen and certain heteroatoms (halogens, sulfur etc). Oxygen concentration is calculated by difference.
Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX) Spectroscopy
SEM scans were taken using a Phillips XL30S field emission gun with an accelerating voltage of 20 kV and a back scattered electron detector. Samples were sputter coated with platinum prior to characterization to prevent charging and increase the conductivity of the samples. The EDX detector was a SiLi (Lithium drifted) super ultra-thin window.
X-ray Photoelectron Spectroscopy (XPS)
The XPS data were collected on a Kratos Axis UltraDLD X-ray Photoelectron Spectrometer (Kratos Analytical, Manchester, UK) equipped with a hemispherical electron energy analyser. Spectra were excited using monochromatic Al Kα X-rays (1486.69 eV) with the X-ray source operating at 150W. In this case the analysis area was a 300 by 700 micron spot obtained using the hybrid magnetic and electrostatic lens and the slot aperture. Samples were secured to the sample bar using indium wire. Charge neutralisation was used to alleviate surface charge buildup, resulting in a shift of approximately 3 eV to lower binding energy. Survey scans were collected with a 160 eV pass energy, whilst core level scans were collected with a pass energy of 20 eV. The analysis chamber was at pressures in the 10-9 torr range throughout the data collection. Data analysis was performed using CasaXPS (www.casaXPS.com). Shirley backgrounds were used in the peak fitting. Quantification of survey scans utilised relative sensitivity factors supplied with the instrument. Core level data were fitted using Gaussian-Lorentzian peaks (30% Lorentzian). During curve fitting the C 1s binding energy of the adventitious hydrocarbon on the surface was used to correct for this shift, with the saturated hydrocarbon peak set to 285 eV. The spin orbit doublets of the S 2p 1/2 peak area was constrained to be exactly half the S 2p 3/2 and due to the core hole lifetime being the same the full width half maximum (FWHM) of the two peaks were constrained to be the same.
Concanavalin A binding assay of modified PANI’s
ConA is a useful, easily sourced carbohydrate binding lectin that has a strong interaction with mannose-like carbohydrate groups and a weaker interaction with glucose-like carbohydrate groups.82 There were two different ConA assays used in this thesis to measure the binding efficacy of glycopolymers. The first ConA assay is a spectroscopic method employing UV-Vis spectroscopy as described by Cairo et al..89 A buffer solution (0.1 M tris-HCl pH 7.2, 0.9 M NaCl, 1 mM CaCl2, 1 mM MnCl2) was prepared and used to dissolve all solids including ConA and the sample. The sample (4 mg) was dissolved in the above buffer solution (400 µL) and vortexed (5 minutes) to prepare a polymer stock solution. This polymer stock solution was further diluted with buffer to make the following polymer concentrations 800, 700, 600, 500, 400, 300, 200, 100, 50, and 0 µg/mL. A separate ConA stock solution was prepared by dissolving ConA (30.42 mg, 90 μM, assuming ConA tetramer with a molecular weight of 104,000 Da.) in buffer (1.63 mL) and vortexing. Each polymer sample (125 µL) was mixed with ConA solution (125 µL) and left overnight (16 hours). The next day the solution was centrifuged (5000 rpm,60 seconds), and the supernatant removed by pipette. The precipitate cake was then dissolved in methyl α-D-mannopyranoside (1 mol/L, 1 mL). Both the supernatant and pellet were measured by UV-Vis at 280 nm in a quartz cuvette over an average of 2 scans. ConA Texas Red conjugate (10 mg) was dissolved in a buffer prepared with milli-q water and the following salts: MnCl2 (10 mM), CaCl2 (10 mM) and NaCl (0.9 M). The solution was vortexed to fully dissolve the ConA and the salts, centrifuged and decanted to remove any particulates. The films were measured using fluorescent microscopy prior to the assay to get a background reading. ConA buffer solution (5 µL) was dropped onto each film and left sitting for ten minutes. The films were then washed with clean buffer (100 µL) by pipetting the solution onto the surface and using the pipette to draw the washing solution up and down multiple times to remove any unbound ConA. The films were then measured under the fluorescent microscope. The washing step was repeated five times with measurement of the fluorescence after 0, 1 and 3 washes. After the repeat washes the films were rinsed with a solution of methyl-α-Dmannopyranoside (1 mol/L, 5 µL) using the same method as the washing step and the fluorescence measured.
Biocidal efficacy of modified PANI powders – Analysis of the Minimum Lethal Concentration (MLC)
E coli ATCC® 25922™ (referred to as E coli and E coli 25922) and S aureus subsp. aureus ATCC® 6538™ (referred to as S aureus and S aureus 6538) were the strains utilised in the antimicrobial testing throughout this thesis as they are standard antibiotic strains.90-92 They are routinely used as control organisms to verify that antibiotic susceptibility results are accurate.90,93 The MLC of the powders produced in this thesis were analyzed using a method described by Jorgensen et al. and Robertson.94,95 A stock solution of sample to be tested (2 wt %) was prepared in aqueous tryptic soy broth (TSB, 30 g/L) and vortexed to suspend. Sample mixtures were ultrasonicated using a QSonica Q700 sonicator from Alphatech for 10 seconds four times. To a 96 well plate 50 µL of the stock solution was added to the top row of wells. TSB (25 µL) was added to the remaining wells in a column. 25 µL of the top well was then pipetted into the next well down creating a series of doubling dilutions. This was repeated for all the wells in the column to prepare a concentration series of polymer along with a blank containing no agent. The final 25 µL was discarded to waste. These preparations were carried out in triplicate for each sample being tested. Overnight cultures of S aureus 6538 and E coli 25922 were diluted 1:1000 in TSB and vortexed to suspend. 25 µL of each culture was added to the sample wells prepared previously. These samples were incubated for 1, 4 and 24 hours. Each well was analyzed for bactericidal activity by pipetting 5 µL to a tryptic soy agar (TSA) plate. A count of <5 colonies was taken as a lethal concentration. Determination of MLC was conducted by observing the lowest concentration required to give no growth or <5 colonies after an incubation period and a non-active concentration was considered when there was confluent growth or >5 colonies of bacterial colonies. The MLC of silver nanoparticle functionalized PANI powder 14 was assessed using the same method presented above, however, the experimental inoculum was prepared in sodium bicarbonate buffer (0.2 mmol) rather than TSB.
Biocidal efficacy of modified PANI films – Analysis of the Minimum Bactericidal Concentration
The biocidal activity of modified PANI films was assayed using a method developed in house based on the Japanese Industry Standard Z-2801. An overnight culture of the desired bacterial strain (E coli 25922 or S aureus 6538) was prepared in TSB and incubated overnight at 37 °C with 200 rpm shaking. Films to be tested were sterilized aseptically in a biosafety cabinet using successive ethanol washes, 5 minutes with 70 % ethanol and 3 minutes with 100 % ethanol. They were left in the cabinet overnight to dry fully. An ethanol sterilized hole punch was used to punch out films of 6 mm diameter in triplicate for each sample to be tested. Agarose (0.5 %) in milli-q water was prepared and heated in a microwave to liquefy. 5 µL of agarose was pipetted into the bottom of the well plate and each film section was placed on top so that it adhered to the well plate. A sterilized polypropylene film was also punched out of a stomacher bag and used as a non-biocidal cover film to sandwich the culture and prevent the culture from drying out. The experimental inoculum was prepared to a concentration of 106 CFU/mL in sodium bicarbonate buffer (0.2 mmol).
1.1. Polyaniline and Intrinsically Conducting Polymers
1.2. Click Chemistry and its Applications to Polymers
1.3. Silver Nanoparticles and Their Antimicrobial Properties
1.4. Interaction of Conducting Polymers with Lectins and Bacterial Cells
1.6. Thesis structure
3. Synthesis and Polymerization of a Modified Aniline Monomer
3.3. Results and Discussion
4. The Synthesis of a Thiolated PANI by Sulfonation and Reduction and the Subsequent Click reaction with Allyl Mannose
4.3. Results and Discussion
5. The Solvent Casting of PANI films and Surface Modification with Glucose for the Preparation of a PANI Glycopolymer
5.3. Results and Discussion
6. Polyaniline thin films incorporating Silver nanoparticles and Glucose
6.3. Results and Discussion
GET THE COMPLETE PROJECT
The Synthesis, Characterisation and Biological Interactions of Polyaniline Based Glycopolymers