A highly reproducible and facile method for formation of ordered 2 dimensional arrays of CTAB protected 50 nm gold nanoparticles bonded to silicon wafers is described. The silicon wafers have been chemically modified with long-chain silanes terminated with thiol that penetrate the CTAB bilayer and chemically bind to the underlying gold nanoparticle. The silicon wafer provides a reproducibly smooth, chemically functionalizable and non-fluorescent substrate with a silicon phonon mode which may provide a convenient internal frequency and intensity calibration for vibrational spectroscopy. The CTAB bilayer provides a potentially biomimetic environment foranalyte, yet allows a sufficiently small nanoparticle separation to achieve a significant electric field enhancement. The arrays have been characterized using SEM and Raman spectroscopy. These studies reveal that the reproducibility of the arrays is excellent both between batches (< 10% RSD) and across a single batch (< 5% RSD). The arrays also exhibit good stability, and the effect of temperature on the arrays was also investigated. The interaction of protein and amino acid with the nanoparticle arrays was investigated using Raman microscopy to investigate their potential in bio-SERS spectroscopy. Raman of phenylalanine and the protein bovine pancreatic trypsin inhibitor, BPTI were studied using 785 nm excitation, coincident with the surface plasmon absorbance of the array. The arrays exhibit SERS enhancements of the order of 2.6 x 10 4 for phenylalanine, the standard deviation on the relative intensity of the 1555 cm -1 mode of phenylalanine is less than 10% for 100 randomly distributed locations across a single substrate and less than 20% between different substrates. Significantly, comparisons of the Raman spectra of the protein and phenlyalanine in solution and immobilized on the nanoparticle arrays indicates that the protein is non-randomly orientated on the arrays. Selective SERS enhancements suggest that aromatic residues penetrate through the bilayer inducing conformational changes in the protein.3

Optical microscopy, involving both fluorescence imaging and confocal Raman microspectroscopy, was used to visualize single, isolated, electrically active heart muscle cells. For example, short-term, dynamic changes in Raman bands during the contraction cycle, as well as persistent band changes during structural remodeling (microscopic rearrangements of cellular structures) in culture over longer periods of time, were obtained from the cellular content (sarcoplasm) of the heart cells. The results of the short-term studies, collected during electrical stimulation, showed dynamic changes in the Raman amide I band intensity, which occurred in phase with changes in cell length during cardiomyocyte contraction. The longer term studies of quiescent cardiomyocytes in culture over 3 days revealed a progressive and sustained increase in the intensity of the amide I band. Over the same period of culture, a decrease in the number of t-tubules (invaginations of the cell membrane, sarcolemma, which ensure the spreading of the action potential into the bulk of the sarcoplasm) was observed using confocal z-sections of the fluorescently labeled sarcolemma. The ability to measure both short-term dynamic changes associated with stimulated contraction and longer term persistent remodeling in the structure of intracellular macromolecules is valuable for assessing the physiological state of the cell, in real time.

In the last few years Teflon AF has emerged as the leading material for implementing waveguiding with an aqueous core because of its low refractive index (nD = 1.29). This low index should make it possible for very low limits of detection to be achieved in Teflon AF as a result of the ability to excite with laser light over an increased area. Detection limits have remained high, however, due in part to the porosity of the material. In this communication we report a significant reduction in the permeability of Teflon AF 2400 capillary walls with the deposition and subsequent treatment of polyelectrolyte multilayers. Alternating layers of polycations and polyanions on a bare Teflon AF surface are sufficient to reduce its permeability to small molecules such as methanol and benzene. Crosslinking and deprotonation of these multilayers further reduces permeability to less than 10% of the permeability value through uncoated TAF. As a consequence, detection limits are reduced. Evidence of these results is presented with gas chromatography-mass spectrometry (GC/MS) and Raman spectroscopic measurements.

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 10 8 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.