The SERS Experiment

Similar behavior of other aromatic disulfides and thiols on gold electrodes has been described based on the SERS experiments [167]. Adsorption of benzenethiol, benzenemethanethiol, p-cyanobenzenemethanethiol, diphenyl sulfide, and dibenzyl sulfide was studied on the roughened gold electrode. All these species adsorb dissociatively as the corresponding thiolates. Monolayers formed from symmetric disulfides were exactly like those formed from the corresponding thiols. These monolayers were stable in a wide potential window from -1-800 to —1000 mV (versus SCE), which was limited by the oxidation of the Au surface from the positive side and hydrogen evolution at —1000 to —1200 mV at the negative side. 

Another point which is important to remember when evaluating the LFE is its sensitivity to geometrical factors of the metal bumps. For example, Gersten and Nitzan show that for a silver ellipsoid with an adsorbed molecule positioned along the major axis, 1 nm away from the surface, the enhancement changes from —200 for both axes of 50 nm to about 8 x 10" for a major axis of 50 nm and an aspect ratio of 5 1. In most of the SERS experiments, the roughness is not uniform. Therefore, averaging over the distribution of protusions should be carried out, which certainly decreases the expected LFE enhancement even further. Another... 

SERS data indicate for both complexes an adsorbate-substrate interaction between the complex and the colloidal Ag surface. From the SERS experiments it has been also concluded that the complexes are oriented face-on to the surface being the number of Cu macrocycles oriented face-on to the surface larger than that of the Ni macrocycles. The organization of the complexes on Au film is different to that found on Ag colloids and rather random. Complexes are better organized on the Ag surface. The phenyl groups avoid a better organization of the Ni complex on the surface. 

A study that combined cyclic voltammetry with a time-resolved, surface-enhanced Raman spectroscopy (SERS) examination of the electroreduction of p-nitrobenzoic acid (PNBA) provided a more complete description of this complex mechanism. The reductive cyclic voltammogram to — 1.4 V is shown in Figure 3-21 (pH = 11), for smooth and roughened Ag electrodes. The use of roughened electrodes is necessary for the SERS experiment. While an adsorption prepeak was indicated on the roughened electrodes, the overall cyclic voltammetric responses for both electrodes were remarkably similar. In such cases, adsorption equilibrium is probably obtained, and qualitative comparisons between SERS studies on roughened electrodes and CV studies on smooth electrodes are possible. 

The substrate is, of course, a necessary component of any SERS experiment. A wide variety of substrate surfaces have been prepared for SERS studies by an equally wide range of teclmiques [ ]. Two important substrates are electrocheniically prepared electrodes and colloidal surfaces (either deposited or in solution). 

The first SERS experiments were performed with electrochemically roughened electrodes and metal colloids, and many other types of suitable SERS substrates are known - e.g. metal island films, metal films over nanoparticles (see Fig. 4.58, below) or rough substrates, gratings, and sputter-deposited metal particles. 

The bearings must be adequate to handle the shaft loads without excessive wear, provided lubrication is maintained. Usually this is not a point of real question provided the manufacturer has had experience in the type of loads imposed by the ser ice conditions, and the responsibility for adequate design must be his. 

All SERS experiments were conducted with a polycrystalline silver working electrode prepared by press-fitting a 6 mm diameter cylinder of silver into one end of a 0.375 inch diameter Teflon rod through which a 6 nm diameter concentric hole had been drilled. Electrical contact was made via a copper wire soldered to the silver. The geometric area of the silver disk was 0.28 cm2. 

The SERS cell was built following the design of Brandt (16). Prior to the experiment, the silver electrode was polished with 5,... 

The electrode potential was controlled with an EG G Princeton Applied Research (PAR) model 173 potentiostat/galvanostat and is referenced to a saturated calomel electrode (SCE). A PAR model 276 current-to-voltage converter allowed monitoring of current during the ORC and SERS experiments and it also provided for positive feedback iR compensation for accurate potential control. 

The catalytic center is formed by residues from both lobes. Sequence comparisons, mutation experiments and biochemical studies indicate an essential fimction in catalysis of phosphate transfer for the conserved amino acids Lys72, Aspl66 and Aspl84 (numbering of PKA). However, the catalytic mechanism of phosphate transfer is not definitely established. It is generally assumed that Aspl66, which is invariant in all protein kinases, serves as a catalytic base for activation of the Ser/Thr hydroxyl and that the reaction takes place by an in-line attack of the Ser-OH at the y-phosphate. 

The members of the protein kinase C family are central signal proteins and as such, are involved in the regulation of a multitude of cellular processes. A problem in the identification of substrates of protein kinase C is its low substrate specificity which often cannot be differentiated from that of protein kinase A, particularly in in vitro experiments. The consensus sequence of the phosphorylation sites in substrate proteins are similar to those of protein kinase A, in that basic amino acids are required in the neighborhood of the Ser/Thr residue to be phosphorylated. The following consensus sequences may be formulated for phosphorylation by protein kinase C ( = phosphorylation site) S /T XK/R K/RXXS /T K/RXXS /T XK/R K/RXS /T K/RXS /T XK/R (Pearson and Kemp, 1991). 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

For a SERS experiment, the water-soluble cellular fraction from pollen can be obtained as shown in the schematic in Fig. 4.8. About 0.2 mg of freeze-dried pollen, which correspond to a few hundred pollen grains, was suspended in 100 dl water and incubated at room temperature. When water is added to intact pollen, the cellular interior bearing the allergens, but also other proteins, carbohydrates, pigments and nucleic acids is released [91] and hence should be contained in the aqueous supernatant. About 2 pi of the supernatant, which should contain the amount of soluble content of a few individual pollen grains can then be added to 20 pi of a solution of gold nanoparticles. The gold nanoparticles were prepared as described by Lee and Meisel [92]. In the control experiments, the volume of the nanoparticle suspension is replaced by pure water. 

Fig. 4.8. Schematic of SERS experiment on pollen cellular fraction. Freeze-dried pollen was incubated with water, the supernatant was probed by SERS by adding a small amount to a solution of gold nanoparticles. The Raman experiments were carried out using a water immersion objective...

Rules of Thumb Since in most circumstances packing HETP is sensitive to only few variables, and due to the unreliability of even the best mass-transfer model, it has been the authors experience that rules of thumb for HETP are more accurate and more reliable than mass-transfer models. A similar conclusion was reached by Porter and Jenkins (IChemE Symp. Ser. 56, Summary paper, London, 1979). 

These modification are indicative of a chemical mechanism in the SERS process in particular in very thin films for which chemical reactions are optimised. These experiments show in addition that metallic tubes are mainly involved, evidenced by the decrease of the Breit-Wigner-Fano component, as due to a possible direct interaction between nanotubes and C6o leading to the formation of SWNTs+C6o complexes [19]. 

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