Odd-even effects in aryl-substituted alkanethiolate SAMs: nonsymmetrical attachment of aryl unit and its impact on the SAM structure.

Aryl-substituted alkanethiolate (AT) self-assembled monolayers (SAMs) exhibit typically so-called odd-even effects, viz. systematic variations in the film structure, packing density, and molecular inclination depending on the parity of the number of the methylene units in the alkyl linker, n. As an exception to this rule, ATs carrying an anthracen-2-yl group (Ant-n) as tail group were reported to have different behavior due the non-symmetric attachment of the anthracene unit to the AT linker, providing additional degree of freedom for the molecular organization and allowing for partial compensation of the odd-even effects. In this context, the structure of SAMs formed by adsorption of anthracene-substituted ATs (Ant-n; n = 1-6) at room temperature on Au(111) substrate was investigated by high-resolution scanning tunnelling microscopy (STM). Most of these SAMs exhibit a coexistence of two different ordered phases, some of which are common for either n = odd or n = even while other vary over the series, showing a broad variety of different structures. The average packing density of the Ant-n SAMs, derived from the analysis of the STM data, varies by 7.5-10% depending on the parity of n, being, as expected, higher for n = odd. The respective extent of the odd-even effects is noticeably lower than that usually observed for other aryl-substituted monolayers (∼25%), which goes in line with the previous findings and emphasizes the impact of the non-symmetric attachment of the aromatic unit.

Polymorphism and Building-Block-Resolved STM Imaging of Self-Assembled Monolayers of 4-Fluorobenzenemethanethiol on Au(111).

Self-assembled monolayers (SAMs) of 4-fluorobenzenemethanethiol (p-FBMT) on Au(111), prepared by immersion procedure (1 mM ethanolic solution; 60 °C; 18 h), were characterized by scanning tunneling microscopy (STM). The data suggest the formation of highly ordered monolayer with a commensurate structure, described by the 2 3 × 13 R 13 ∘ unit cell. The STM appearance of this cell occurs, however, in two different forms, with either well-localized individual spots or splitting of these spots in two components. These components are assigned to the tunneling through the entire molecule or sulfur docking group only. The respective spots correspond then to the terminal fluorine atom and sulfur docking group, manifesting, thus, building-block-resolving STM imaging. The accessibility of the docking group for direct tunneling is most likely related to a specific molecular organization for one of the two possible internal structures of the unit cell. The above results represent a showcase for potential of STM for imaging of upright-arranged and densely packed molecular assemblies, such as SAMs.

Development of sponge-like cellulose colorimetric swab immobilized with anthocyanin from red-cabbage for sweat monitoring.

Novel sponge-like biochromic swab was developed via immobilization of natural anthocyanin (Cy) biomolecular probe into microporous cellulose aerogel. The current biosensor is characterized with simple preparation, environmentally-friendly, biocompatibility, biodegradability, flexibility, portability and reversibility. This biochromic sponge-like aerogel detector displayed a color change from pink to green-yellow in response to the biochemical changes occurs to sweat. This could be ascribed to intramolecular charge transfer occurs to the molecular system of Cy. Thus, the anthocyanin probe displayed colorimetric variations in UV-Vis absorption spectra via a blue shifting from 620 to 529 nm when raising the pH value of the prepared mimic sweat solution. Natural pH sensitive anthocyanin spectroscopic probe was extracted from red-cabbage plant, characterized by HPLC, and encapsulated into microporous cellulose. The microporous sponge-like cellulose swab was prepared by activating wood pulp utilizing phosphoric acid, and then subjected to freeze-drying. This anthocyanin probe is highly soluble in water. Thus, it was encapsulated as a direct dye into cellulose substrate during the freeze-drying process. To allow a better fixation of this water-soluble anthocyanin probe to the cellulose substrate, potash alum was added to the freeze-dried mixture to act as a fixing agent or mordant (M) generating Cy/M coordination complex. The produced Cy/M nanoparticles (NPs) were explored by transmission electron microscopy (TEM). The morphological features of the generated aerogels were investigated by scan electron microscope (SEM), energy-dispersive X-ray (EDX) spectra, and Fourier-transform infrared spectra (FT-IR). The cytotoxicity of the prepared aerogel-based biosensor was also evaluated. The naked-eye colorimetric changes were studied by exploring color strength, UV-Vis spectra and CIE Lab colorimetric coordinates.

Annealing effect for self-assembled monolayers formed from terphenylethanethiol on Au(111).

The structure and morphology of self-assembled monolayers (SAMs) prepared on Au(111) from solutions of terphenylethanethiol (TP2) at room temperature and subsequently annealed at temperatures up to 473 K were investigated using scanning tunneling microscopy. This system is of particular interest because of its metastable character, holding potential for its tailored modification. Indeed, the data suggest the formation of several different structural phases, viz. α, ß, γ, and δ, appearing progressively for the as-prepared and annealed samples. The consecutive α → ß â†’ γ → δ phase transitions occurring with increasing annealing temperature involved a continuous reduction of the molecular packing density and significant changes in the substrate morphology. The major morphological changes were the appearance and progressive growth of monoatomic gold islands, on top of which the TP2 phases were formed, representing in all cases a single domain for a particular island and restricted only by the island size. For all the phases, inclined molecular orientation was assumed while a so-called lying-down arrangement, in which the TP2 backbones are orientated parallel to the gold surface, was not observed. A nearly complete desorption of the TP2 molecules was recorded at an annealing temperature of 473 K, accompanied by the drastic change in the surface morphology.

Rapid Detection and Quantification of Novel Psychoactive Substances (NPS) Using Raman Spectroscopy and Surface-Enhanced Raman Scattering.

With more than a million seizures of illegal drugs reported annually across Europe, the variety of psychoactive compounds available is vast and ever-growing. The multitude of risks associated with these compounds are well-known and can be life threatening. Hence the need for the development of new analytical techniques and approaches that allow for the rapid, sensitive, and specific quantitative detection and discrimination of such illicit materials, ultimately with portability for field testing, is of paramount importance. The aim of this study was to demonstrate the application of Raman spectroscopy and surface-enhanced Raman scattering (SERS) combined with chemometrics approaches, as rapid and portable techniques for the quantitative detection and discrimination of a wide range of novel psychoactive substances (methcathinone and aminoindane derivatives), both in powder form and in solution. The Raman spectra of the psychoactive compounds provided clear separation and classification of the compounds based on their core chemical structures; viz. methcathinones, aminoindanes, diphenidines, and synthetic cannabinoids. The SERS results also displayed similar clustering patterns, with improved limits of detections down to ~2 mM (0.41 g L-1). As mephedrone is currently very popular for recreational use we performed multiplexed quantitative detection of mephedrone (4-methylmethcathinone), and its two major metabolites (nor-mephedrone and 4-methylephedrine), as tertiary mixtures in water and healthy human urine. These findings readily illustrate the potential application of SERS for simultaneous detection of multiple NPS as mixtures without the need for lengthy prior chromatographic separation or enrichment methods.

Quantitative Online Liquid Chromatography-Surface-Enhanced Raman Scattering (LC-SERS) of Methotrexate and its Major Metabolites.

The application of Raman spectroscopy as a detection method coupled with liquid chromatography (LC) has recently attracted considerable interest, although this has currently been limited to isocratic elution. The combination of LC with rapidly advancing Raman techniques, such as surface-enhanced Raman scattering (SERS), allows for rapid separation, identification and quantification, leading to quantitative discrimination of closely eluting analytes. This study has demonstrated the utility of SERS in conjunction with reversed-phase liquid chromatography (RP-LC), for the detection and quantification of the therapeutically relevant drug molecule methotrexate (MTX) and its metabolites 7-hydroxy methotrexate (7-OH MTX) and 2,4-diamino-N(10)-methylpteroic acid (DAMPA) in pure solutions and mixtures, including spikes into human urine from a healthy individual and patients under medication. While the RP-LC analysis developed employed gradient elution, where the chemical constituents of the mobile phase were modified stepwise during analysis, this did not overtly interfere with the SERS signals. In addition, the practicability and clinical utility of this approach has also been demonstrated using authentic patients' urine samples. Here, the identification of MTX, 7-OH MTX and DAMPA are based on their unique SERS spectra, providing limits of detection of 2.36, 1.84, and 3.26 µM respectively. Although these analytes are amenable to LC and LC-MS detection an additional major benefit of the SERS approach is its applicability toward the detection of analytes that do not show UV absorption or are not ionised for mass spectrometry (MS)-based detection. The results of this study clearly demonstrate the potential application of online LC-SERS analysis for real-time high-throughput detection of drugs and their related metabolites in human biofluids.

Quantitative detection of codeine in human plasma using surface-enhanced Raman scattering via adaptation of the isotopic labelling principle.

In this study surface enhanced Raman scattering (SERS) combined with the isotopic labelling (IL) principle has been used for the quantification of codeine spiked into both water and human plasma. Multivariate statistical approaches were employed for the analysis of these SERS spectral data, particularly partial least squares regression (PLSR) which was used to generate models using the full SERS spectral data for quantification of codeine with, and without, an internal isotopic labelled standard. The PLSR models provided accurate codeine quantification in water and human plasma with high prediction accuracy (Q2). In addition, the employment of codeine-d6 as the internal standard further improved the accuracy of the model, by increasing the Q2 from 0.89 to 0.94 and decreasing the low root-mean-square error of predictions (RMSEP) from 11.36 to 8.44. Using the peak area at 1281 cm-1 assigned to C-N stretching, C-H wagging and ring breathing, the limit of detection was calculated in both water and human plasma to be 0.7 µM (209.55 ng mL-1) and 1.39 µM (416.12 ng mL-1), respectively. Due to a lack of definitive codeine vibrational assignments, density functional theory (DFT) calculations have also been used to assign the spectral bands with their corresponding vibrational modes, which were in excellent agreement with our experimental Raman and SERS findings. Thus, we have successfully demonstrated the application of SERS with isotope labelling for the absolute quantification of codeine in human plasma for the first time with a high degree of accuracy and reproducibility. The use of the IL principle which employs an isotopolog (that is to say, a molecule which is only different by the substitution of atoms by isotopes) improves quantification and reproducibility because the competition of the codeine and codeine-d6 for the metal surface used for SERS is equal and this will offset any difference in the number of particles under analysis or any fluctuations in laser fluence. It is our belief that this may open up new exciting opportunities for testing SERS in real-world samples and applications which would be an area of potential future studies.

Rapid, Accurate, and Quantitative Detection of Propranolol in Multiple Human Biofluids via Surface-Enhanced Raman Scattering.

There has been an increasing demand for rapid and sensitive techniques for the identification and quantification of pharmaceutical compounds in human biofluids during the past few decades, and surface-enhanced Raman scattering (SERS) is one of a number of physicochemical techniques with the potential to meet these demands. In this study we have developed a SERS-based analytical approach for the assessment of human biofluids in combination with chemometrics. This novel approach has enabled the detection and quantification of the ß-blocker propranolol spiked into human serum, plasma, and urine at physiologically relevant concentrations. A range of multivariate statistical analysis techniques, including principal component analysis (PCA), principal component-discriminant function analysis (PC-DFA) and partial least-squares regression (PLSR) were employed to investigate the relationship between the full SERS spectral data and the level of propranolol. The SERS spectra when combined with PCA and PC-DFA demonstrated clear differentiation of neat biofluids and biofluids spiked with varying concentrations of propranolol ranging from 0 to 120 µM, and clear trends in ordination scores space could be correlated with the level of propranolol. Since PCA and PC-DFA are categorical classifiers, PLSR modeling was subsequently used to provide accurate propranolol quantification within all biofluids with high prediction accuracy (expressed as root-mean-square error of predictions) of 0.58, 9.68, and 1.69 for serum, plasma, and urine respectively, and these models also had excellent linearity for the training and test sets between 0 and 120 µM. The limit of detection as calculated from the area under the naphthalene ring vibration from propranolol was 133.1 ng/mL (0.45 µM), 156.8 ng/mL (0.53 µM), and 168.6 ng/mL (0.57 µM) for serum, plasma, and urine, respectively. This result shows a consistent signal irrespective of biofluid, and all are well within the expected physiological level of this drug during therapy. The results of this study demonstrate the potential of SERS application as a diagnostic screening method, following further validation and optimization to improve detection of pharmaceutical compounds and quantification in human biofluids, which may open up new exciting opportunities for future use in various biomedical and forensic applications.

Rapid, accurate, and comparative differentiation of clinically and industrially relevant microorganisms via multiple vibrational spectroscopic fingerprinting.

Despite the fact that various microorganisms (e.g., bacteria, fungi, viruses, etc.) have been linked with infectious diseases, their crucial role towards sustaining life on Earth is undeniable. The huge biodiversity, combined with the wide range of biochemical capabilities of these organisms, have always been the driving force behind their large number of current, and, as of yet, undiscovered future applications. The presence of such diversity could be said to expedite the need for the development of rapid, accurate and sensitive techniques which allow for the detection, differentiation, identification and classification of such organisms. In this study, we employed Fourier transform infrared (FT-IR), Raman, and surface enhanced Raman scattering (SERS) spectroscopies, as molecular whole-organism fingerprinting techniques, combined with multivariate statistical analysis approaches for the classification of a range of industrial, environmental or clinically relevant bacteria (P. aeruginosa, P. putida, E. coli, E. faecium, S. lividans, B. subtilis, B. cereus) and yeast (S. cerevisiae). Principal components-discriminant function analysis (PC-DFA) scores plots of the spectral data collected from all three techniques allowed for the clear differentiation of all the samples down to sub-species level. The partial least squares-discriminant analysis (PLS-DA) models generated using the SERS spectral data displayed lower accuracy (74.9%) when compared to those obtained from conventional Raman (97.8%) and FT-IR (96.2%) analyses. In addition, whilst background fluorescence was detected in Raman spectra for S. cerevisiae, this fluorescence was quenched when applying SERS to the same species, and conversely SERS appeared to introduce strong fluorescence when analysing P. putida. It is also worth noting that FT-IR analysis provided spectral data of high quality and reproducibility for the whole sample set, suggesting its applicability to a wider range of samples, and perhaps the most suitable for the analysis of mixed cultures in future studies. Furthermore, our results suggest that while each of these spectroscopic approaches may favour different organisms (sample types), when combined, they would provide complementary and more in-depth knowledge (structural and/or metabolic state) of biological systems. To the best of our knowledge, this is the first time that such a comparative and combined spectroscopic study (using FT-IR, Raman and SERS) has been carried out on microbial samples.

Chicken, beams, and Campylobacter: rapid differentiation of foodborne bacteria via vibrational spectroscopy and MALDI-mass spectrometry.

Campylobacter species are one of the main causes of food poisoning worldwide. Despite the availability of established culturing and molecular techniques, due to the fastidious nature of these microorganisms, simultaneous detection and species differentiation still remains challenging. This study focused on the differentiation of eleven Campylobacter strains from six species, using Fourier transform infrared (FT-IR) and Raman spectroscopies, together with matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS), as physicochemical approaches for generating biochemical fingerprints. Cluster analysis of data from each of the three analytical approaches provided clear differentiation of each Campylobacter species, which was generally in agreement with a phylogenetic tree based on 16S rRNA gene sequences. Notably, although C. fetus subspecies fetus and venerealis are phylogenetically very closely related, using FT-IR and MALDI-TOF-MS data these subspecies were readily differentiated based on differences in the lipid (2920 and 2851 cm(-1)) and fingerprint regions (1500-500 cm(-1)) of the FT-IR spectra, and the 500-2000 m/z region of the MALDI-TOF-MS data. A finding that was further investigated with targeted lipidomics using liquid chromatography-mass spectrometry (LC-MS). Our results demonstrate that such metabolomics approaches combined with molecular biology techniques may provide critical information and knowledge related to the risk factors, virulence, and understanding of the distribution and transmission routes associated with different strains of foodborne Campylobacter spp.

Combining Raman and FT-IR spectroscopy with quantitative isotopic labeling for differentiation of E. coli cells at community and single cell levels.

There is no doubt that the contribution of microbially mediated bioprocesses toward maintenance of life on earth is vital. However, understanding these microbes in situ is currently a bottleneck, as most methods require culturing these microorganisms to suitable biomass levels so that their phenotype can be measured. The development of new culture-independent strategies such as stable isotope probing (SIP) coupled with molecular biology has been a breakthrough toward linking gene to function, while circumventing in vitro culturing. In this study, for the first time we have combined Raman spectroscopy and Fourier transform infrared (FT-IR) spectroscopy, as metabolic fingerprinting approaches, with SIP to demonstrate the quantitative labeling and differentiation of Escherichia coli cells. E. coli cells were grown in minimal medium with fixed final concentrations of carbon and nitrogen supply, but with different ratios and combinations of (13)C/(12)C glucose and (15)N/(14)N ammonium chloride, as the sole carbon and nitrogen sources, respectively. The cells were collected at stationary phase and examined by Raman and FT-IR spectroscopies. The multivariate analysis investigation of FT-IR and Raman data illustrated unique clustering patterns resulting from specific spectral shifts upon the incorporation of different isotopes, which were directly correlated with the ratio of the isotopically labeled content of the medium. Multivariate analysis results of single-cell Raman spectra followed the same trend, exhibiting a separation between E. coli cells labeled with different isotopes and multiple isotope levels of C and N.