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Heart rates during ischaemia and reperfusion are possible determinants of reperfusion arrhythmias. We used ivabradine, a selective If current inhibitor, to assess the effects of heart rate reduction (HRR) during ischaemia–reperfusion on reperfusion ventricular arrhythmias and assessed potential anti-arrhythmic mechanisms by optical mapping. Five groups of rat hearts were subjected to regional ischaemia by left anterior descending artery occlusion for 8 min followed by 10 min of reperfusion: (1) Control n = 10; (2) 1 μM of ivabradine perfusion n = 10; (3) 1 μM of ivabradine + 5 Hz atrial pacing throughout ischaemia–reperfusion n = 5; (4) 1 μM of ivabradine + 5 Hz pacing only at reperfusion; (5) 100 μM of ivabradine was used as a 1 ml bolus upon reperfusion. For optical mapping, 10 hearts (ivabradine n = 5; 5 Hz pacing n = 5) were subjected to global ischaemia whilst transmembrane voltage transients were recorded. Epicardial activation was mapped, and the rate of development of ischaemia-induced electrophysiological changes was assessed. HRR observed in the ivabradine group during both ischaemia (195 ± 11 bpm vs. control 272 ± 14 bpm, p, Highlights ► Ivabradine treatment reduced the incidence of reperfusion ventricular arrhythmia. ► The anti-arrhythmic effects of ivabradine were due to heart rate reduction. ► Mean heart rate during ischaemia is a determinant of reperfusion arrhythmias. ► Heart rate reduction delayed onset of ischaemia-induced electrophysiological changes. ► Selective heart rate lowering is a potential anti-arrhythmic therapeutic strategy.

Infrared (IR) and Raman spectra were obtained for N,N′ dicyclohexylcarbodiimide (DCC) in the solid state and in CHCl3 solution. Structures and vibrational spectra of isolated, gas-phase DCC molecules with C2 and Ci symmetries, computed at the B3-LYP/cc-pVTZ level, show that the IR and Raman spectra provide convincing evidence for a C2 structure in both the solid state and in CHCl3 solution. Using a scaled quantum-chemical force field, these density functional theory calculations have provided detailed assignments of the observed IR and Raman bands in terms of potential energy distributions. Comparison of solid-state and solution spectra, together with a Raman study of the melting behaviour of DCC, revealed that no solid-state effects were evident in the spectra.

Sudan-1 has been used for coloring food. However, recent alarms worldwide about the carcinogenic and mutagenic properties of azo-compounds have led to concerns over their human consumption. In the U.K. in 2005, over 570 products were found to be contaminated with the azo dye Sudan-1 and this and the health risks associated with this dye resulted in the subsequent international ban of this additive in all foodstuff, at all levels, relating to human consumption. These incidents have also necessitated the need for high throughput low cost reliable approaches for the detection and quantification of food contaminated by such azo compounds. While there are a small number of analytical techniques that can be considered portable, many lack sensitivity. By contrast, we show that employing a portable Raman spectrometer, using surface enhanced Raman scattering (SERS), can provide good sensitivity, such that Sudan-1 can be quantified in a complex food matrix reliably over the range of 10−3 to 10−4 mol L−1. We also de...

Monitoring enzyme kinetics is an important aspect of biochemistry and is essential when studying metabolism. In this study we demonstrate that succinate dehydrogenase activity of mitochondria can be analyzed quantitatively by surface enhanced Raman scattering (SERS). We used the artificial electron acceptor reporter molecule 2,6-dichlorophenolindophenol (DCPIP) that when oxidized is SERS active. On reduction this redox dye changes from blue to colorless and is SERS inactive. This color change allows UV/vis spectrophotometry to be used as a standard reference measurement as well as SERS. SERS analysis incorporated kinetic time course measurements and employed a portable laser Raman spectrometer using an excitation wavelength of 785 nm in conjunction with gold colloids. Good correlation coefficients and quantitative data were observed with the additional advantage that analysis by SERS also provided good fingerprint spectral information from vibrational band frequencies thus allowing the potential of multiplexing enzyme reactions. We believe that this is the first demonstration of monitoring succinate dehydrogenase activity with SERS and that SERS has considerable potential for being applied to the analysis of metabolic processes.

Surface-enhanced resonance Raman scattering (SERRS) spectra of aqueous solutions of the triphenylmethane dye methyl green have been obtained for the first time by use of citrate-reduced silver colloids and a laser excitation wavelength of 632.8 nm. Given the highly fluorescent nature of the analyte, which precluded collection of normal Raman spectra of the dye in solution and powdered state, it was highly encouraging that SERRS spectra showed no fluorescence due to quenching by the silver sol. The pH conditions for SERRS were optimised over the pH range 0.5-10 and the biggest enhancement for SERRS of this charged dye was found to be at pH 2.02, thus this condition was used for quantitative analysis. SERRS was found to be highly sensitive and enabled quantitative determination of methyl green over the range 10(-9) to 10(-7) mol dm(-3). Good fits to correlation coefficients were obtained over this range using the areas under the vibrational bands at 1615 and 737 cm(-1). Finally, a limit of detection of 83 ppb was calculated, demonstrating the sensitivity of the technique.

Surface-enhanced resonance Raman spectroscopy (SERRS) was used for the semi-quantitative analysis of the anthraquinone dyes alizarin and purpurin, using aggregated citrate-reduced silver colloids. Laser excitation wavelengths of 514.5 and 632.8 nm were employed. The limits of detection for the analysis of alizarin, by SERRS, were 4 ppm (632.8 nm excitation) using the vibrational band at 1265 cm−1 and 39 ppm (514.5 nm excitation) for the vibrational band at 1334 cm−1 and those for the analysis of purpurin, by SERRS, were 42 ppb (514.5 nm excitation) using the vibrational band at 1330 cm−1 and 8 ppm (632.8 nm excitation) for the vibrational band at 1070 cm−1. For the first time, the ability to differentiate between purpurin and alizarin using SERRS is reported. Multiple molecular species of both dyes were detected, as evidenced by several isosbestic points in a SERRS pH study using both laser-exciting wavelengths of 514.5 and 632.8 nm. Unusually, for dye molecules, equally intense SERRS signals can be obtained for alizarin at both acidic and alkaline pH values. Copyright © 2004 John Wiley & Sons, Ltd.

In this paper we report for the first time semi-quantitative analysis of indigo using surface enhanced Raman spectroscopy (SERS) and surface enhance resonance Raman spectroscopy (SERRS). Indigo, a dye widely used today in the textile industry, has been used, historically, both as a dye and as a pigment; the latter in both paintings and in printed material. The molecule is uncharged and largely insoluble in most solvents. The application of SERS/SERRS to the semi-quantitative analysis of indigo has been examined using aggregated citrate-reduced silver colloids with appropriate modifications to experimental protocols to both obtain and maximise SERRS signal intensities. Good linear correlations are observed for the dependence of the intensities of the SERRS band at 1151 cm −1 using laser exciting wavelengths of 514.5 nm ( R =0.9985) and 632.8 nm ( R =0.9963) on the indigo concentration over the range 10 −7 –10 −5 and 10 −8 –10 −5 mol dm −3 , respectively. Band intensities were normalised against an internal standard (silver sol band at 243 cm −1 ). Resonance Raman spectra (RRS) of aqueous solutions of indigo could not be collected because of its low solubility and the presence of strong fluorescence. It was, however, possible to obtain RS and RRS spectra of the solid at each laser excitation wavelength. The limits of detection (L.O.D.) of indigo by SERS and SERRS using 514.5 and 632.8 nm were 9 ppm at both exciting wavelengths. Signal enhancement by SERS and SERRS was highly pH dependent due to the formation of singly protonated and possibly doubly protonated forms of the molecule at acidic pH. The SERS and SERRS data provide evidence to suggest that an excess of monolayer coverage of the dye at the surface of silver colloids is observed at concentrations greater than 7.85×10 −6 mol dm −3 for each exciting wavelength. The data reported herein also strongly suggest the presence of multiple species of the indigo molecule.

The dye nuclear fast red has been detected and determined semi-quantitatively by means of surface enhanced resonance Raman scattering (SERRS) and surface enhanced Raman scattering (SERS), using laser exciting wavelengths of 514.5 and 632.8 nm, respectively, by employing a citrate-reduced silver colloid. A good linear correlation is observed for the dependence of the intensities of the SERRS bands at 989 cm−1 (R=0.9897) and 1278 cm−1 (R=0.9872) on dye concentration over the range 10−9 to 10−7 M, when using an exciting wavelength of 514.5 nm. At dye concentrations above 10−7 M, the concentration dependence of the SERRS signals is non-linear. This is almost certainly due to the coverage of the colloidal silver particles being in excess of a full monolayer of the dye. A linear correlation is also observed for the dependence of the intensities of the SERS bands at 989 cm−1 (R=0.9739) and 1278 cm−1 (R=0.9838) on the dye concentration over the range 10−8 to 10−6 M when using an exciting wavelength of 632.8 nm. Strong fluorescence prevented collection of resonance Raman scattering (RRS) spectra from powdered samples or aqueous solutions of the dye using an exciting wavelength of 514.5 nm, but weak bands were observed in the spectra obtained from both powdered and aqueous samples of the dye using an exciting wavelength of 632.8 nm. A study of the pH dependence of SERRS/SERS and UV–VIS absorption spectra revealed the presence of different ionisation states of the dye. The limits of detection for nuclear fast red by SERRS (514.5 nm), SERS (632.8 nm) and visible spectroscopy (535 nm) are 9, 89 and 1000 ng ml−1, respectively.

The viability of the application of surface-enhanced resonance Raman spectroscopy (SERRS) to the quantitative analysis of the dye alcian blue 8GX has been examined with the use of citrate- and borohydride-reduced silver colloids. A good linear correlation is observed for the dependence of the SERRS signal intensities at 1344 and 1536 cm−1 ( R = 0.992 and 0.994, respectively) on dye concentrations in the range of 10−9–10−7 M, when using the citrate-reduced silver sol. Likewise, a good linear correlation is also observed for the dependence of the SERRS signal intensities at 1344 and 1536 cm−1 ( R = 0.978 and 0.959, respectively) on dye concentrations in the range 10−10–10−9 M, when using the borohydride-reduced silver sol. At concentrations of dye above 10−7 M, the concentration dependence of the SERRS signals is nonlinear, almost certainly due to the coverage of the surface of the colloidal silver particles by the dye being in excess of a full monolayer. However, at concentrations of alcian blue greater than 10−7 M, resonance Raman spectroscopy (RRS) can be employed for quantitative analysis of the dye. When performing RRS, using methanol as an internal standard, we observed a good linear correlation for the dependence of the signal intensities at 1344 and 1536 cm−1 ( R = 0.995 for both bands) on the dye concentration in the 10−6–10−3 M range. The limits of detection of alcian blue by SERRS using a citrate-reduced silver sol, SERRS using a borohydride-reduced silver sol, RRS, and visible absorption spectroscopy are found to be 26 ppb, 203 ppt, 2 ppm, and 0.7 ppm, respectively.

While surface-enhanced Raman scattering (SERS) can increase the Raman cross-section by 4-6 orders of magnitude, for SERS to be effective it is necessary for the analyte to be either chemically bonded or within close proximity to the metal surface used. Therefore most studies investigating the biochemical constituents of microorganisms have introduced an external supply of gold or silver nanoparticles. As a consequence, the study of bacteria by SERS has to date been focused almost exclusively on the extracellular analysis of the Gram-negative outer cell membrane. Bacterial cells typically measure as little as 0.5 by 1 mum, and it is difficult to introduce a nanometer sized colloidal metal particle into this tiny environment. However, dissimilatory metal-reducing bacteria, including Shewanella and Geobacter species, can reduce a wide range of high valence metal ions, often within the cell, and for Ag(I) and Au(III) this can result in the formation of colloidal zero-valent particles. Here we report, for the first time, SERS of the bacterium Geobacter sulfurreducens facilitated by colloidal gold particles precipitated within the cell. In addition, we show SERS from the same organism following reduction of ionic silver, which results in colloidal silver depositions on the cell surface.

In a novel application SERRS has been used, by employing a silver sol, to monitor and analyse the conversion of indigo into the indigo carmine dye.

The application of surface enhanced resonance Raman spectroscopy (SERRS) to the semi-quantitative analysis of the dye, indigo carmine, has been examined using citrate-reduced silver colloids. Good linear correlations are observed for the dye band at 1580 cm(-1) in the concentration range 10(-7)-10(-5) and 10(-9)-10(-5) mol dm(-3), using laser exciting wavelengths of 514.5 [(R=0.9983)] and 632.8 nm [(R=0.9978)], respectively. At concentrations of dye above 10(-6) M the concentration dependence of the SERRS signals is non-linear due to the coverage of the surface of the colloidal particles by the dye being in excess of a full monolayer. At concentrations above 10(-6) M resonance Raman spectroscopy (RRS) can be employed for the quantitative analysis of the dye. An internal standard was used and a good linear correlation (R=0.997) was observed for the dependence of dye signal intensities at 1580 cm(-1) in the concentration range 10(-5)-10(-4) M using a laser exciting wavelength of 514.5 nm. The limits of detection of indigo carmine by SERRS (514.5 nm), SERRS (632.8 nm) and solution RRS (514.5 nm) are found to be 0.9, 1 and 38 ppm, respectively.

Background Gap junctional uncoupling is thought to contribute to arrhythmogenesis through its effects on conduction slowing and creation of heterogeneities, often in the context of action potential or structural abnormalities. However, it is uncertain as to whether moderate conduction slowing through gap junctional uncoupling on its own, in the absence of action potential or structural changes, is sufficient to be pro-arrhythmic. We used optical mapping: (1) to study the effects of carbenoxolone, a gap junction uncoupler, on conduction velocity and action potential duration, and (2) to assess the effects of moderate conduction slowing and gap junctional uncoupling on arrhythmia vulnerability in structurally normal hearts. Methods Normal rat hearts (n=12) were isolated and perfused with Krebs-Henseleit solution, then loaded with a voltage-sensitive dye (RH237) and perfused with an excitation-contraction uncoupler (10 μM blebbistatin) to eliminate motion artefact. Transmembrane voltage (V m ) transients were recorded using a 256-photodiode array and epicardial activation was mapped across the anterolateral left ventricle during ventricular pacing (stimulus 1 mA, cycle length 180 ms). Hearts were then perfused with 30 μM carbenoxolone (CBX; n=6) or control (n=6) for 30 min and epicardial activation mapped every 2 min. Subsequently, hearts were subjected to programmed ventricular stimulation with up to three extrastimuli and assigned an Inducibility Quotient depending on ease of arrhythmia inducibility. Results Carbenoxolone significantly slowed conduction across the left ventricular epicardial surface (Conduction velocity index: Baseline 100%, CBX 70% ± 2%, p Conclusions Gap junctional uncoupling with carbenoxolone produced a 30% reduction in conduction velocity without changes in action potential duration, and resulted in increased arrhythmia vulnerability in structurally normal hearts. These results suggest that moderate conduction slowing produced by gap junctional uncoupling in isolation, that is, in the absence of changes in action potential duration or structural abnormalities, is sufficient to increase susceptibility to ventricular arrhythmias, and therefore highlight the importance of gap junctional uncoupling in ventricular arrhythmogenesis.

Nuclear fast red [4-amino-1, 3-dihydroxy-anthracene-9, 10-dione-2-sulfonic acid (sodium salt); Fig. 1] is widely used as a dye for nuclei or plasma staining of histological and haematological specimens [1–2]. Its usefulness stems from the specificity of the take up of the dye. Such formulations are used to make aqueous solutions according to standard staining protocols [3].

Background Reperfusion arrhythmias can occur within seconds of restoration of blood flow to previously ischaemic myocardium. Heterogeneities and abrupt changes in both gap junctional coupling and action potentials during early reperfusion are thought to contribute to their pathophysiology. We tested the hypothesis that modulating gap junctional coupling can reduce the incidence of reperfusion arrhythmias, and used optical mapping to assess the mechanisms of any anti-arrhythmic effect. Methods Using a Langendorff apparatus, rat hearts were perfused with either 50 nM AAP10 to increase gap junctional coupling (n=10), 30 μM carbenoxolone (CBX) to reduce coupling (n=13) or control Krebs-Henseleit solution (n=10). Hearts were then subjected to regional ischaemia by occlusion of the left anterior descending artery for 8 min and then reperfused. Ventricular arrhythmia incidence was documented. For optical mapping experiments, a further eighteen hearts were loaded with a voltage-sensitive dye (RH237), perfused with an excitation-contraction uncoupler (10μM blebbistatin) and either AAP10 (n=6), carbenoxolone (n=6) or control (n=6), and then subjected to ischaemia and reperfusion as above whilst transmembrane voltage (V m ) transients were recorded. Results Both AAP10 and CBX reduced the incidence of reperfusion VF (AAP10 50%, CBX 77%, Control 90%) and delayed time to onset of reperfusion VF (AAP10 29.0±9.9 s; CBX 52.2±13.2 s; Control 21.8 s±6.4 s) (LogRank p 2 central ischaemic zone in the anterior left ventricle (LV) (Activation time: AAP10 15.3 ± 1.3ms; CBX 25.7 ±2.2 ms; Control 23.2±2.0 ms, p Conclusions Both increasing and reducing gap junctional coupling, with AAP10 and CBX respectively, reduced the rate and delayed the onset of reperfusion VF. This suggests that the partial uncoupling that occurs during early reperfusion is a determinant of reperfusion arrhythmias, and that both normalising the degree of coupling or further exacerbating uncoupling may be anti-arrhythmic. The anti-arrhythmic effect of increasing gap junctional coupling may be mediated by its dual effect of reducing action potential heterogeneity and improving conduction during reperfusion. Further uncoupling may be protective via attenuation of the abrupt changes in coupling and conduction that occur at reperfusion following ischaemia.