In order to produce the most effective Ag nanoarrays for plasmon enhanced fluorescence and Raman scattering made
using ferroelectric substrates, the optimum conditions for the creation of arrays must be identified. We study here Ag
nanopattern arrays formed using ferroelectric lithography based on periodically proton exchanged (PPE) template
methods. We examine different conditions in regard to deposition of Ag nanoparticles and analyze the plasmon enhanced
signal from the resulting nanoarray. We apply FLIM (fluorescence lifetime imaging) to assess different Ag nanoarray
preparation conditions on fluorescence emission from selected fluorphores. In addition, we apply Raman and
luminescence spectroscopy with AFM (atomic force microscopy) to study the plasmon enhancement of luminescence
and Raman from the Ag nanoarrays.
Gold nano-cavity arrays supported on polydimethylsiloxane (PDMS) have been created using colloidal lithography.
PDMS is cured on top of hexagonally close packed arrays of polystyrene spheres of diameter 820 nm resulting in a close
packed sphere imprinted polymer block. The depth of the imprints is 200 nm, indicating the whole sphere is not
entrapped in the polymer during curing. The spherical nature of the imprint can be deformed by stretching of the flexible
polymer, thus creating cuboid shaped arrays. Finally, the arrays are coated with a 100 nm gold layer, which conforms to
the polymer surface to create either spherical or cuboid shaped gold nano-cavities. Experiments show that the reflectance
properties of the arrays are critically dependent on the shape of the cavity. Spherical shaped cavity arrays display diffuse
reflectance peaks at wavelengths slightly shorter than the diameter of the templating sphere, which are absent in the
cuboid arrays. Both spherical and cuboid arrays show reflectance which is strongly dependent on the angle of incidence,
with the cuboid arrays showing differing spectra depending on the direction of the impinging light with relation to the
axis of stretching. The changes in optical behavior between the spherical and cuboid cavity arrays is discussed with
relation to the change of shape of the patterning feature at the interface.
The spectroscopic and photophysical properties of ruthenium polypyridyl polypeptide conjugates of the
type [Ru(bpy)2PIC-Argn]n+2+, where bpy is 2,2-bipyridyl (bpy), PIC is 2-(4-carboxyphenyl)imidazo[4,5-
f][1,10]phenanthroline and PIC-Argn is this ligand peptide bonded to polyarginine where n is 5 or 8, is
described. The resonance Raman spectroscopy of the peptide conjugated complex and parent are strongly
pH dependent and demonstrate a switch of lowest energy charge transfer transition between bpy and pic
ligands as s function of pH. The pKa of the imidazole ring on the complex is obtained from resonance
Raman spectroscopy as 7.8 ± 0.2. The luminescence lifetime of the complex is strongly oxygen
dependent and a Stern-Volmer plot of O2 quenching for [Ru(bpy)2(PIC-Arg8)]10+ yielded a KSV value of
2300 ± 420 M-1 which was independent of pH over the range 2 to 11. The complexes, because of their
large Stokes shifts can, uniquely, be used under identical conditions of probe concentration and excitation
wavelength for resonance Raman and luminescence cellular imaging. Cellular imaging was conducted
using SP2 myeloma cells which confirmed that the [Ru(bpy)2(PIC-Arg8)]10+ is readily taken up by
mammalian cells although the parent and pentarginine analogues are not membrane permeable.
Preliminary examples of multi-parameter imaging using these probes were presented. Resonance Raman
maps of [Ru(bpy)2(PIC-Arg8)]10+ within living myeloma cells showed on the basis of spectral
discrimination, attributed to pH, three distinct regions of the cell could be identified, ascribed to the
nucleus, the cytoplasm and the membranes. Luminescence lifetime imaging showed quite large variations
in the probe lifetime within the living cell which was tentatively ascribed to variation in O2 concentration
about the cell. Preliminary estimates of O2 concentration were made and it was found that the membranes,
both inner and outer are the most O2 rich regions of the cell. Overall, we propose that such peptide
labeled luminescent metal are potentially a valuable addition to cellular imaging by providing tools for
multiplexed analysis of the cell environment.
Raman spectroscopy is an extremely powerful analytical tool. Surface enhanced Raman scattering (SERs) enables
sample sensitivity to extend down to the single molecule level. There is presently great interest in using uniform
nanostructured surfaces to give reproducible and strong surface enhanced Raman (SER) signal. The nanocavities studied
here have spherical cap architecture and are arranged uniformly in an Au array. These structures support both localised
and delocalised plasmons. Localised surface plasmon polaritons exist inside the nanocavities and delocalised or
propagating surface plasmon polaritons exist on the flat surface of the sample (Bragg plasmons). The angle dependence
property of surface enhanced Raman is used in the present work to enable comparison between SERs caused by localised
plasmons and SERs caused by delocalised plasmons. The samples used here were modified to enable separate
investigations of the two plasmon types. The externally modified array had dye placed only on the flat top surface of the
array. The internally modified array had dye placed only on the internal walls of the cavities. Results show that the
changes in Raman intensities with respect to the incident angle depend on the location of dye on the array.
Noble metal nanoparticles arrays are well established substrates for surface enhanced Raman spectroscopy (SERS).
Their ability to enhance optical fields is based on the interaction of their surface valence electrons with incident
electromagnetic radiation. In the array configuration, noble metal nanoparticles have been used to produce SER spectral
enhancements of up to 108 orders of magnitude, making them useful for the trace analysis of physiologically relevant
analytes such as proteins and peptides. Electrostatic interactions between proteins and metal surfaces result in the
preferential adsorption of positively charged protein domains onto metal surfaces. This preferential interaction has the
effect of disrupting the native conformation of the protein fold, with a concomitant loss of protein function. A major
historic advantage of Raman microspectroscopy has been is its non-invasive nature; protein denaturation on the metal
surfaces required for SER spectroscopy renders it a much more invasive technique. Further, part of the analytical power
of Raman spectroscopy lies in its use as a secondary conformation probe. The protein structural loss which occurs on
the metal surface results in secondary conformation readings which are not true to the actual native state of the analyte.
This work presents a method for chemical fabrication of noble metal SERS arrays with surface immobilized layers
which can protect protein native conformation without excessively mitigating the electromagnetic enhancements of
spectra. Peptide analytes are used as model systems for proteins. Raman spectra of alpha lactalbumin on surfaces and
when immobilized on these novel arrays are compared. We discuss the ability of the surface layer to protect protein
structure whilst improving signal intensity.
Thin films of the metallopolymer [Os(bpy)2 (PVP)10]2+, where bpy is 2,2'-dipyridyl and PVP is poly(4-vinylpyridine), luminesce at 750±12 nm upon excitation at 355nm. The luminescence decay responses can be described by a double exponential decay model in which the limiting lifetimes are 75±14 (population fraction of 0.9) and 35±8 ns (population fraction of 0.1) for films in contact with aqueous 0.1 M H2SO4. Electrochemistry has been used to create well defined concentrations of the luminescence quencher, Os3+, within the films. Time resolved spectroscopy reveals that both dynamic and static processes contribute to luminescence quenching with a rate constant for electron transfer between the photoexcited Os2+* and the Os3+ centres of 1.3x107 M-1s-1 being observed. Stable gold nanoparticles have been created within the metallopolymer by the chemical reduction of tetrachloroaurate. These nanocomposite materials exhibit enhanced emission intensity compared to the gold free films.
Occlusion of a blood vessel due to thrombosis can reduce or completely stop blood supply to different tissues or organs with the clinical consequences of myocardial infarction or stroke. Platelets are the cellular component which initiate thrombus formation, they activate in response to a variety of signals, such as interactions with a damaged blood vessel. αIIbβ3 is a membrane bound integrin protein responsible for regulating adhesion of the activated platelet to damaged blood vessels. It exists in both activated and non-activated states displaying high and low affinity respectively for ligands such as fibrinogen. αIIbβ3 determines the "stickiness" of the blood platelet and is therefore, a logical target for therapeutic measures to control thrombus formation. During the past decade considerable progress has been made to identify the role of the αIIbβ3 complex in platelet-mediated thrombus formation and the structure of αIIbβ3 has been extrapolated from the crystal structure of related integrins. However, despite these advances, the bimolecular mechanisms underlying the activation of αIIbβ3 remain poorly understood. In this contribution, we describe methodologies of deriving surface enhanced Raman spectroscopy of αIIbβ3 on nanostructured metal surfaces, fabricated by a number of methods. We compare activation of αIIbβ3 by SERS using a range of known activation conditions including Mn(II), EDTA and dithiotheritol (DTT). By studying the behaviour of the disulfide and CS marker vibrations in the spectral region 400 to 800 cm-1 using SERS we confirm that activation results in significant conformational change in the protein, and most interestingly, that the response is not the same for every agonist. This mechanistic difference has implications for the biochemical study of this protein (and indeed for understanding the role of this integrin in response to different agonists).