Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Diazonium Salt-Based Surface-Enhanced Raman Spectroscopy Nanosensor: Detection and Quantitation of Aromatic Hydrocarbons in Water Samples.

Here, we present a surface-enhanced Raman spectroscopy (SERS) nanosensor for environmental pollutants detection. This study was conducted on three polycyclic aromatic hydrocarbons (PAHs): benzo[a]pyrene (BaP), fluoranthene (FL), and naphthalene (NAP). SERS substrates were chemically functionalized using 4-dodecyl benzenediazonium-tetrafluoroborate and SERS analyses were conducted to detect the pollutants alone and in mixtures. Compounds were first measured in water-methanol (9:1 volume ratio) samples. Investigation on solutions containing concentrations ranging from 10-6 g L-1 to 10-3 g L-1 provided data to plot calibration curves and to determine the performance of the sensor. The calculated limit of detection (LOD) was 0.026 mg L-1 (10-7 mol L-1) for BaP, 0.064 mg L-1 (3.2 × 10-7 mol L-1) for FL, and 3.94 mg L-1 (3.1 × 10-5 mol L-1) for NAP, respectively. The correlation between the calculated LOD values and the octanol-water partition coefficient (Kow) of the investigated PAHs suggests that the developed nanosensor is particularly suitable for detecting highly non-polar PAH compounds. Measurements conducted on a mixture of the three analytes (i) demonstrated the ability of the developed technology to detect and identify the three analytes in the mixture; (ii) provided the exact quantitation of pollutants in a mixture. Moreover, we optimized the surface regeneration step for the nanosensor.

Organometallic nanoprobe to enhance optical response on the polycyclic aromatic hydrocarbon benzo[a]pyrene immunoassay using SERS technology.

We demonstrated the use of a new organometallic nanoprobe for competitive surface-enhanced Raman scattering (SERS) immunoassay devoted to the detection of polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP) in seawater. The nanoprobes are gold nanoparticles (GNPs) labeled by a Raman reporter, the 5,5'-dithiobis(succinimidyl-2-nitrobenzoate) (DSNB) and functionalized with monoclonal antibodies anti-BaP. The antibodies are bound with a high specificity to the analyte while the GNPs enhanced the Raman scattering of the DSNB. This type of immunoassay involved the grafting of BaP onto a sensing surface. Thus, NH2-terminated self-assembled monolayer is formed on the surface of gold substrate using cysteamine. Amines finally reacted with 6-formylbenzo[a]pyrene. So, this SERS detection involves four steps: (i) the nanoprobes are incubated with the sample; (ii) a drop of the mixture is then put onto the substrate; (iii) the surface is rinsed; and (iv) the surface is analyzed by Raman spectroscopy. To synthesize the nanoprobes, firstly, we prepared GNPs according to Frens' method. Then, GNPs were spontaneously labeled by the DSNB Raman reporter, thanks to a strong gold-sulfur interaction. Thereafter, BaP antibodies were cross-linked to the DSNB labeled GNPs by reaction of proteins primary amino groups with N-hydroxyl succinimide (NHS). Before use in SERS detection, their activity was controlled by surface plasmon resonance technique. The present method allows us to detect BaP at trace concentration (2 nmol/L). The results demonstrate that the proposed method has a great potential for application in the monitoring of seawater.

Pseudo-Foster Kennedy Syndrome - a case report.

Objective: To report a case of Pseudo-Foster Kennedy (PFK) syndrome and describe its clinical and paraclinical particularities, as well as the diagnostic difficulties and established treatment. Methods: The case of a 60-year-old male patient with sudden, painless visual impairment in the left eye (LE), and a medical history of old optic nerve atrophy in his right eye (RE) was described. Results: The diagnosis of nonarteritic anterior ischemic optic neuropathy (NAION) was established based on the medical history, local and general clinical and paraclinical examination, and temporal artery biopsy. Conclusions: Although there is no current generally accepted treatment for NAION, a correct diagnosis and supportive treatment may contribute to the improvement in visual acuity (VA), improvement that in this case remained stable for 6 months after the onset. The patient is still being monitored and no relapses have been noted.

SERS detection of biomolecules using lithographed nanoparticles towards a reproducible SERS biosensor.

In this paper we highlight the accurate spectral detection of bovine serum albumin and ribonuclease-A using a surface-enhanced Raman scattering (SERS) substrate based on gold nanocylinders obtained by electron-beam lithography (EBL). The nanocylinders have diameters from 100 to 180 nm with a gap of 200 nm. We demonstrate that optimizing the size and the shape of the lithographed gold nanocylinders, we can obtain SERS spectra of proteins at low concentration. This SERS study enabled us to estimate high enhancement factors (10(5) for BSA and 10(7) for RNase-A) of important bands in the protein Raman spectrum measured for 1 mM concentration. We demonstrate that, to reach the highest enhancement, it is necessary to optimize the SERS signal and that the main parameter of optimization is the LSPR position. The LSPR have to be suitably located between the laser excitation wavelength, which is 632.8 nm, and the position of the considered Raman band. Our study underlines the efficiency of gold nanocylinder arrays in the spectral detection of proteins.

Free energy calculations on disulfide bridges reduction in proteins by combining ab initio and molecular mechanics methods.

Free energy profiles were calculated for the reduction of the four disulfide bridges in lysozyme by tris(2-carboxyethyl)phosphine (TCEP). The computational method combines high-precision density functional theory (DFT) calculations performed on the core of the reactant system with classical mechanical free energy evaluations based on the sampling of the configuration space of reaction environment. The predicted reaction energy barriers are in satisfactory agreement with experimental data, proving that the present method provides a reliable description of the mechanism of reaction. The role of the protein environment in this mechanism is further emphasized by analyzing the different contributions to the free energy profiles. It is shown that the protein environment affects the reaction by three factors: polarizability, steric hindrance of the reactant site, and S-S bridge distortion due to structural constraints. The corresponding effects are quantitatively evaluated, and the results are discussed in connection with the current two-step reaction model for the reduction of S-S bridges in proteins.

Protein S-S bridge reduction: a Raman and computational study of lysozyme interaction with TCEP.

The role of protein structure in the reactivity of the four disulfide (S-S) bridges of lysozyme was studied using Raman spectroscopy and molecular modelling. The experimental kinetics of S-S bridge reduction by tris-2-carboxyethyl phosphine (TCEP) was obtained by monitoring the protein S-S Raman bands. The kinetics are heterogeneous and were fitted using two apparent reaction rate constants. Kinetic measurements performed at different pH values indicate only moderate charge effects. The two intrinsic reaction rate constants derived for the neutral TCEP species were 0.45 and 0.052 mol(-1) s(-1), respectively. The molecular dynamics simulation of the reactants encounter shows that the accessibility of the lysozyme S-S bridges by TCEP decreases in the following order: cys30-cys115 > cys6-cys127 > cys64-cys80 > cys76-cys94. This simulation also illustrates the reaction mechanism which consists of a local unfolding followed by the reduction of the exposed S-S bridge. The Gibbs free energy for local unfolding was evaluated by comparing the actual reaction rate constant with that of a model system containing a fully exposed S-S bridge (oxidized glutathione). These values corresponding to the fast- and slow-reaction rate-constants were 8.5 and 13.8 kJ mol(-1), respectively. On the other hand, Raman measurements, as well as the molecular dynamics simulations, strongly suggest that the protein global unfolding following S-S bridge cleavage has only limited effects in stabilizing the reaction products.

Reductive unfolding of serum albumins uncovered by Raman spectroscopy.

The reductive unfolding of bovine serum albumin (BSA) and human serum albumin (HSA) induced by dithiothreitol (DTT) is investigated using Raman spectroscopy. The resolution of the S-S Raman band into both protein and oxidized DTT contributions provides a reliable basis for directly monitoring the S-S bridge exchange reaction. The related changes in the protein secondary structure are identified by analyzing the protein amide I Raman band. For the reduction of one S-S bridge of BSA, a mean Gibbs free energy of -7 kJ mol(-1) is derived by studying the reaction equilibrium. The corresponding value for the HSA S-S bridge reduction is -2 kJ mol(-1). The reaction kinetics observed via the S-S or amide I Raman bands are identical giving a reaction rate constant of (1.02 +/- 0.11) M(-1) s(-1) for BSA. The contribution of the conformational Gibbs free energy to the overall Gibbs free energy of reaction is further estimated by combining experimental data with ab initio calculations.