Quantitative surface-enhanced Raman spectroscopy of single bases in oligodeoxynucleotides.

To address the question of whether the SERS signals of ss-DNA are simply combinations of the signals from the individual bases that comprise the sequence, SERS spectra of unmodified ss-DNA sequences were obtained using a hydroxylamine-reduced Ag colloid aggregated with MgSO4. Initially, synthetic oligodeoxynucleotides with systematic structural variations were used to investigate the effect of adding single nucleobases to the 3' terminus of 10-mer and 20-mer sequences. It was found that the resulting SERS difference spectra could be used to identify the added nucleobases since they closely matched reference spectra of the same nucleobase. Investigation of the variation in intensity of an adenine probe which was moved along a test sequence showed there was a small end effect where nucleobases near the 3' terminus gave slightly larger signals but the effect was minor (30%). More significantly, in a sample set comprising 25-mer sequences where A, T or G nucleobases were substituted either near the centres of the sequences or the 5' or 3' ends, the SERS difference spectra only matched the expected form in approximately half the cases tested. This variation appeared to be due to changes in secondary structure induced by altering the sequences since uncoiling the sequences in a thermal pre-treatment step gave difference spectra which in all cases matched the expected form. Multivariate analysis of the set of substitution data showed that 99% of the variance could be accounted for in a model with just three factors whose loadings matched the spectra of the A, T, and G nucleobases and which contained no positional information. This suggests that aside from the differences in secondary structure which can be eliminated by thermal pre-treatment, the SERS spectra of the 25-mers studied here are simply the sum of their component parts. Although this means that SERS provides very little information on the primary sequence it should be excellent for the detection of post-transcription modifications to DNA which can occur at multiple positions along a given sequence.

Infrared and Raman screening of seized novel psychoactive substances: a large scale study of >200 samples.

The potential of IR absorption and Raman spectroscopy for rapid identification of novel psychoactive substances (NPS) has been tested using a set of 221 unsorted seized samples suspected of containing NPS. Both IR and Raman spectra showed large variation between the different sub-classifications of NPS and smaller, but still distinguishable, differences between closely related compounds within the same class. In initial tests, screening the samples using spectral searching against a limited reference library allowed only 41% of the samples to be fully identified. The limiting factor in the identification was the large number of active compounds in the seized samples for which no reference vibrational data were available in the libraries rather than poor spectral quality. Therefore, when 33 of these compounds were independently identified by NMR and mass spectrometry and their spectra used to extend the libraries, the percentage of samples identified by IR and Raman screening alone increased to 76%, with only 7% of samples having no identifiable constituents. This study, which is the largest of its type ever carried out, therefore demonstrates that this approach of detecting non-matching samples and then identifying them using standard analytical methods has considerable potential in NPS screening since it allows rapid identification of the constituents of the majority of street quality samples. Only one complete feedback cycle was carried out in this study but there is clearly the potential to carry out continuous identification/updating when this system is used in operational settings.

Surface-enhanced Raman spectroscopy of novel psychoactive substances using polymer-stabilized Ag nanoparticle aggregates.

A set of seized "legal high" samples and pure novel psychoactive substances have been examined by surface-enhanced Raman spectroscopy using polymer-stabilized Ag nanoparticle (Poly-SERS) films. The films both quenched fluorescence in bulk samples and allowed identification of µg quantities of drugs collected with wet swabs from contaminated surfaces.

Isolation and structural determination of non-racemic tertiary cathinone derivatives.

The racemic tertiary cathinones N,N-dimethylcathinone (1), N,N-diethylcathinone (2) and 2-(1-pyrrolidinyl)-propiophenone (3) have been prepared in reasonable yield and characterized using NMR and mass spectroscopy. HPLC indicates that these compounds are isolated as the anticipated racemic mixture. These can then be co-crystallized with (+)-O,O'-di-p-toluoyl-D-tartaric, (+)-O,O'-dibenzoyl-D-tartaric and (−)-O,O'-dibenzoyl-L-tartaric acids giving the single enantiomers S and R respectively of 1, 2 and 3, in the presence of sodium hydroxide through a dynamic kinetic resolution. X-ray structural determination confirmed the enantioselectivity. The free amines could be obtained following basification and extraction. In methanol these are reasonably stable for the period of several hours, and their identity was confirmed by HPLC and CD spectroscopy.

Simple preparation of positively charged silver nanoparticles for detection of anions by surface-enhanced Raman spectroscopy.

Modification of citrate and hydroxylamine reduced Ag colloids with thiocholine bromide, a thiol functionalized quaternary ammonium salt, creates particles where the zeta potential is switched from the normal values of ca.-50 mV to ca. +50 mV. These colloids are stable but can be aggregated with metal salts in much the same way as the parent colloids. They are excellent SERS substrates for detection of anionic targets since their positive zeta potentials promote adsorption of negatively charged ions. This is important because the vast majority of published SERS studies involve cationic or neutral targets. Moreover, the fact that the modifier is a quaternary ammonium ion means that the negative surface charge is maintained even at alkaline pH. The modified colloids can be used to detect compounds which cannot be detected using conventional negatively-charged citrate or hydroxylamine reduced metal nanoparticles, for example the detection limit was 5.0 × 10(-5) M for perchlorate and <8.7 × 10(-7) M for tetraphenylporphine tetrasulfonic acid (TPPS). More importantly, picric acid (an explosive) and diclofenac (a non-steroidal anti-inflammatory) could also be analysed quantitatively at low concentrations, 2.5 × 10(-5) M and 1.9 × 10(-5) M, respectively. Interestingly, the correct choice of aggregating agent is important for achieving high sensitivity since the anion in the aggregating salt may compete with anionic targets for surface binding sites. Finally, since the modification procedure simply involves reaction of nanoparticles with a small alkyl thiol derivative, it can easily be adapted to other particle morphologies or metals.

Ultraviolet Resonance Raman spectroscopy used to study formulations of salmon calcitonin, a starch-peptide conjugate and TGF-ß3.

Ultraviolet Resonance Raman (UVRR) spectroscopy with excitation at 244 nm was investigated here as a possible useful tool for fast characterization of biopharmaceuticals. Studies were performed on three protein drugs: salmon calcitonin (sCT), starch-peptide conjugate, and transforming growth factor-ß3 (TGF-ß3) adsorbed onto solid granules of tricalcium phosphate (TCP). Secondary structure of sCT was investigated for solutions of 0.5mg/mL up to 200mg/mL, regardless of the turbidity or aggregation states. An increase in ß-sheet content was detected when sCT solutions aggregated. UVRR spectroscopy also detected a small amount of residual organic solvent in a starch-peptide conjugate solution containing only 40 µg/mL of peptide. UVRR spectroscopy was then used to characterize a protein, TGF-ß3, adsorbed onto solid granules of TCP at 50 and 250 µg/cm(3). This study shows that UVRR is suitable to characterize the protein formulations in a broad range of concentrations, in liquid, aggregated, and solid states.

Determination of hydrogen peroxide concentration using a handheld Raman spectrometer: Detection of an explosives precursor.

It has been shown that a handheld Raman spectrometer can be used to determine hydrogen peroxide concentration in aqueous solutions in seconds. To allow quantitative analysis, the aqueous peroxide samples were mixed 50/50 (v/v) with a 4mol/dm(3) sodium perchlorate solution which acted as the internal standard. Standard calibration using relative peak heights of the strongest perchlorate (932cm(-1)) and peroxide bands (876cm(-1)) gave an average error of 1.43% for samples in the range 5-30% peroxide. PLS regression of the same data set gave an average error of 0.98%. In addition, the concentrations of the samples were estimated by searching spectra against a library of standard spectra prepared using the same range of peroxide concentrations at 5% increments and with the same perchlorate internal standard. It was found that the library searching method classified all the test samples correctly, matching either the spectra of the same concentration, if they were present, or matching to the closest concentration if an exact match was not possible. This method thus provides a very rapid technique to allow determination of hydrogen peroxide concentrations in the field, for example at suspected improvised explosives manufacturing sites, without complex calibration procedures.

Combined antenna and localized plasmon resonance in Raman scattering from random arrays of silver-coated, vertically aligned multiwalled carbon nanotubes.

The electric field enhancement associated with detailed structure within novel optical antenna nanostructures is modeled using the surface integral equation technique in the context of surface-enhanced Raman scattering (SERS). The antennae comprise random arrays of vertically aligned, multiwalled carbon nanotubes dressed with highly granular Ag. Different types of "hot-spot" underpinning the SERS are identified, but contrasting characteristics are revealed. Those at the outer edges of the Ag grains are antenna driven with field enhancement amplified in antenna antinodes while intergrain hotspots are largely independent of antenna activity. Hot-spots between the tops of antennae leaning towards each other also appear to benefit from antenna amplification.

Recent applications of Chemical Imaging to pharmaceutical process monitoring and quality control.

Chemical Imaging (CI) is an emerging platform technology that integrates conventional imaging and spectroscopy to attain both spatial and spectral information from an object. Vibrational spectroscopic methods, such as Near Infrared (NIR) and Raman spectroscopy, combined with imaging are particularly useful for analysis of biological/pharmaceutical forms. The rapid, non-destructive and non-invasive features of CI mark its potential suitability as a process analytical tool for the pharmaceutical industry, for both process monitoring and quality control in the many stages of drug production. This paper provides an overview of CI principles, instrumentation and analysis. Recent applications of Raman and NIR-CI to pharmaceutical quality and process control are presented; challenges facing CI implementation and likely future developments in the technology are also discussed.