Distribución de la matriz extracelular intramuscular

Prior to functionalisation, ITO glass was cleaned by washing with detergent, then ultrasonication for 15 minutes within each of acetone, water, ethanol, then dried in air. The substrate was then ultrasonicated within a 1 : 1 : 5 v/v solution of 30% hydrogen peroxide to 28% ammonia to reverse-osmosis water, then rinsed thoroughly with water, then AR grade ethanol. This base treatment was an effort to remove organic

contaminants and ensure the presence of hydroxyl groups on the surface of the ITO.23 The deposition of what may be a monolayer, or thin film of organic species, such as 4- phenylenediamine and 4-aminobenzoic acid upon ITO glass was performed in dry and dark conditions to prevent oxidation, or polymerisation. A typical solution contained 5 mM of the adsorbate in dry ethanol or 1,4-dioxane, under an inert atmosphere. The period of treatment of ITO glass varied from two days to a month at 30 to 80ºC. The slow oxidation of the aniline derivative was inferred by the gradual colour change of the soaking solution from clear and colourless, to a light apricot after two days, or a clear, blood-red after a month. The ITO slides were then rinsed with AR methanol and air- dried.

For the overgrafting of ferrocenemonocarboxaldehyde to 4-phenylenevinylene, ITO glass was first immersed for two and a half days in a 120 mM solution of 4- phenylenediamine in 1,4-dioxane, at 30°C. The ITO samples were then soaked within a 1.3 mM solution of ferrocenemonocarboxaldehyde in 2,2'-dimethoxypropane, for a day at 20 to 25°C. The treatment of ITO glass with ferrocenemonocarboxaldehyde or with N-(ferrocenylmethylidene)-4-phenylenediamine was done by using 2,2’- dimethoxypropane as a solvent and concentrations in the order of 50 mM. Samples were soaked at 20 to 40ºC for a few days, before rinsing with AR methanol and then air- dried and stored in the dark. Alternatively, species such as 2-(4- aminophenyl)ethenylferrocene were adsorbed onto ITO by immersion for 2 hours at room temperature in a 2 mM solution, using a mixed solvent of 6% v/v of 1,4-dioxane in hexane. The ITO samples were then rinsed with a 5% v/v solution of 1,4-dioxane in hexane.

UV-Vis spectra were collected using a step-size of 0.5 nm and a very slow scan speed to collect data from 250 nm to 700 nm over ten minutes. A 2 nm slit width gave a maximum illuminated area of 0.44 cm2. Before use, efforts were made to minimise the effects of the low signal-to-noise ratio by allowing a minimum of 90 minutes for the tungsten and deuterium lamps to stabilise prior to use. In general, a single baseline of air alone was collected prior to each day’s experiments and had a range of ± 0.0004 absorbance units. Typically, three such scans were collected and averaged for samples of ITO glass before and after the deposition and further treatment of species, such as 4-

phenylenediamine. Mathematical subtraction was used to extract peak position and intensities for treated samples. The positioning of the sample holder with respect to the beam has an uncertainty of ± 1° at most. This was not a concern, as there was no detectable change in absorbance position or intensity on tilting samples by up to 5° from the perpendicular, with respect to the light beam.

Voltammetry was done using 0.1 M tetrabutylammonium perchlorate (TBAP) in dry tetrahydrofuran or dry dichloromethane as the electrolyte. A minimum of three sequential scans was collected for each dye. Dry air hoods for voltammetry were not available, and despite bubbling with dry nitrogen, atmospheric moisture was absorbed into the voltammetry samples. As a consequence, the data was only collected for the dye/dye+ couple. Typical data collection parameters used a scan rate of 100 mV s-1 and a data interval of 2.5 mV was used for collection. The counter electrode was platinum mesh and the working electrode was either a platinum disc 2 mm in diameter (0.03 cm2₎

or ITO glass. A pseudo-reference electrode of Ag/AgNO₃ within 0.1 M TBAP in acetonitrile was used for non-aqueous experiments. The inner electrode contained silver wire immersed in a 1 mM solution of AgNO₃ in electrolyte, while the outer electrode had a plain electrolyte of 0.1 M TBAP in acetonitrile. A pseudo-reference electrode of Ag/AgCl in saturated potassium chloride was used for aqueous experiments. Potentials were adjusted to standard calomel electrode (SCE) values by adjusting for the difference between experimental and literature values for the ferrocene/ferrocenium redox couple. For data shown in Chapter 5, depending on the data set, a half-wave potential of either 0.115 or 0.110 VAg/Ag+ was obtained for the

ferrocene/ferrocenium redox couple. The adjustment to convert values to the SCE scale was to add either 0.043 or 0.048 V, respectively.

A calculated bracket for monolayer coverage of 2-(4-aminophenyl)ethenylferrocene (5.4) on a flat surface was done using maximum and minimum footprint areas (Fig. 2.1). The depth of 5.4 was taken as 6.6 Å, which is that of a freely-rotating ferrocenyl group.24 The width of 5.4 was estimated using Chem3D®: bound by the amine group, the minimum and maximum footprint areas, as shown in Figure 2.1, are 4.3 and 5.3 x 10-19 m2, respectively. The bracket for monolayer coverage for 5.4 is then 3.2 - 3.9 x 10-10 mol cm-2.

Figure 2.1. Chem3D® representations of 5.4 with estimates of the maximum and minimum rectangular footprint area. For clarity, only one cyclopentadiene ring of the ferrocenyl group is shown.

Experimental surface coverage estimates of 5.4 upon ITO glass were done using both voltammetry and UV-Vis spectroscopy. In order to calculate the number of moles adsorbed onto ITO by voltammetry, the current under the oxidation peak was integrated for the single-electron process, using a Gaussian peak fit of the oxidation peak. Data was taken from the second scan and the assumption was made that the current measured was due to adsorbed molecules only. The calculation of surface coverage by 5.4 from UV-Vis absorbance involved a concentration calibration plot using standard solutions. To plot the absorbance of 5.4 on ITO glass directly against the calibration data to find the number of moles in the illuminated area, in terms of mol cm-2_{, the concentration of}

the standard solutions was converted from mol L-1 _{to mol. A 2 nm slit width gave an}

illuminated area of 0.44 cm2, which combined with a 1 cm path length solution cell, gave an illuminated volume of 0.44 cm3. The number of moles of 5.4 in the light path was then the concentration of the standard multiplied by 0.44 cm3. However, the calibration is not an ideal one as there will be light losses due to scattering within the solution cell. The uncertainty in the surface coverage calculation using UV-Vis spectroscopy is ± 5%, which was derived from error in the concentration of the standard solutions, UV-Vis absorbance values and the uncertainty in the calibration plot.