Steven E. J. Bell

Quantitative surface-enhanced Raman spectroscopy

The purpose of this tutorial review is to show how surface-enhanced Raman (SERS) and resonance Raman (SERRS) spectroscopy have evolved to the stage where they can be used as a quantitative analytical technique. SER(R)S has enormous potential for a range of applications where high sensitivity needs to be combined with good discrimination between molecular targets, particularly since low cost, compact spectrometers can read the high signal levels that SER(R)S typically provides. These advantages over conventional Raman measurements come at the cost of increased complexity and this review discusses the factors that need to be controlled to generate stable and reproducible SER(R)S calibrations.

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.

SERS enhancement by aggregated Au colloids: effect of particle size

Aggregated Au colloids have been widely used as SERS enhancing media for many years but to date there has been no systematic investigation of the effect of the particle size on the enhancements given by simple aggregated Au colloid solutions. Previous systematic studies on isolated particles in solution or multiple particles deposited onto surfaces reported widely different optimum particle sizes for the same excitation wavelength and also disagreed on the extent to which surface plasmon absorption spectra were a good predictor of enhancement factors. In this work the spectroscopic properties of a range of samples of monodisperse Au colloids with diameters ranging from 21 to 146 nm have been investigated in solution. The UV/visible absorption spectra of the colloids show complex changes as a function of aggregating salt (MgSO(4)) concentration which diminish when the colloid is fully aggregated. Under these conditions, the relative SERS enhancements provided by the variously sized colloids vary very significantly across the size range. The largest signals in the raw data are observed for 46 nm colloids but correction for the total surface area available to generate enhancement shows that particles with 74 nm diameter give the largest enhancement per unit surface area. The observed enhancements do not correlate with absorbance at the excitation wavelength but the large differences between differently sized colloids demonstrate that even in the randomly aggregated particle assemblies studied here, inhomogeneous broadening does not mask the underlying changes due to differences in particle diameter.

Label-Free Detection of Single-Base Mismatches in DNA by Surface-Enhanced Raman Spectroscopy†

Detection of specific DNA sequences and identification of single-nucleotide polymorphisms (SNPs) are both important for diagnostic purposes, since alterations in the DNA sequence are the cause of the most common inherited disorders.1 Methods for label-free identification of short DNA sequences are currently relatively limited. The most obvious choice is MALDI-TOF mass spectrometry, which is extensively used in SNP genotyping methods and has high sensitivity.2 However, MALDI-TOF equipment is expensive, which partly offsets the significant advantage of being able to avoid labeling steps in the analysis. Surface-enhanced Raman spectroscopy (SERS) is an obvious alternative method, which has the potential to combine high sensitivity with low cost.3 However, SERS studies of DNA most often involve detection of SERS labels;4 even those that do not are typically carried out on sequences that have been thiolated to promote surface binding.5 This approach not only increases the cost of the assay, but also the specific binding through a sulfur group gives the DNA a more tilted orientation that may prevent all the bases from interacting sufficiently with the substrate to provide good Raman signals. Indeed, it has been reported that the SERS spectra of thiolated DNA are completely dominated by adenine signals.5 A recent approach utilized TERS (tip-enhanced Raman spectroscopy)to record the spectra of an unthiolated single poly C strand, in order to work towards a method of obtaining sequence information by moving the TERS probe in intervals of one base-to-base distance.6 This approach, although promising, requires complex instrumentation, and, additionally, acquisition of Raman signals with single-base sensitivity is extremely challenging. Herein we show that it is possible to obtain distinctive SERS spectra of unthiolated DNA sequences, which can be used to detect a single-nucleotide mismatch. This detection can be achieved by adsorbing the sequences nonspecifically through the nucleotide side chains onto the enhancing surface of silver colloids, where the sequences adopt a flattened configuration. To this end, the colloids were aggregated with an electrolyte that does not bind strongly in the silver surface (MgSO4 in this case),7 and therefore allowed the DNA sequence to bind through its constituent bases. Previously, we demonstrated that silver colloids can provide very large SERS enhancements for the negatively charged mononucleotides when colloids were aggregated with MgSO4.7 We applied the same principle for the detection of single-base mismatches in short DNA sequences. Initially, parallel studies were carried out using both thiolated and unthiolated 30-mer DNA-1 sequences (Table ​(Table1),1), which were either probed directly or after thermal pretreatment, as previously described.5 All the samples gave acceptable SERS signals, as shown in Figure ​Figure1,1, thus demonstrating that thiolation is not necessary to obtain high-quality SERS spectra of DNA. In fact the unthiolated samples gave signals that were at least as large as the thiolated analogues. The spectra of the thermally treated thiolated sample strongly resemble the spectra of adenine,8 which agrees with previous data obtained on Au substrates and was attributed to adenine having a stronger scattering cross-section than the other bases.5 Interestingly, the SERS spectra of the unthiolated and thiolated form with no thermal pretreatment are different from the thermally treated sample, but similar to each other. This result suggests that the untreated thiolated sample adopts a similar adsorption configuration to the unthiolated sample, which is believed to lie flat with respect to the surface because of strong nonspecific binding between the bases and the metal surface. Similar binding interactions presumably also occur with thiolated strands9 if they are not heat-treated. However, the most important point is that the spectra of simple unthiolated sequences are not dominated by the adenine modes, but show bands that arise from all the bases. Figure 1 SERS spectra of 10−5 m solutions of a) thermally treated thiolated HS-DNA-1, b) untreated, thiolated HS-DNA-1, and c) unthiolated DNA-1. All spectra were recorded on citrate-reduced Ag colloids and aggregated with 0.1 m MgSO4. Table 1 DNA sequences used in this study. To aid the analysis of the DNA spectra, the SERS spectra of the three nucleobases poly A, poly C, and poly T were recorded and used as reference spectra. The SERS spectrum of dGMP is shown instead of poly G because poly G forms secondary structures through Hoogsteen base pairing.10 We have found that these structures are not sufficiently flexible to allow the nucleobases to directly adsorb to the surfaces of the Ag nanoparticles, and they therefore give only weakly enhanced Raman signals. For this work, hydroxylamine-reduced Ag colloids were used as the enhancing media because they are easier to synthesize than citrate-reduced colloids, and the low concentration of Cl− ions introduced during their synthesis does not affect DNA binding. Indeed the SERS spectra (Figure S1 in the Supporting Information) show that when DNA is added to colloid aggregated with MgSO4, it displaces the surface chloride layer and the 244 cm−1 Ag–Cl band disappears. However, if the colloid is aggregated by using a high concentration of NaCl, no DNA Raman signals are observed because the DNA cannot compete with the high concentration of NaCl (0.1 m) for surface sites. Irrespective of these considerations, the main point is that by using suitable conditions, the SERS spectra of unthiolated homopolymers or mononucletides can be obtained (Figure ​(Figure2);2); these spectra give characteristic bands that can be used for the identification of each nucleotide in a DNA sequence. For example, in the spectrum of the unthiolated form of DNA-1 (Figure 1 c), the bands at 737 and 682 cm−1 can be assigned to the ring breathing modes of poly A and poly G, respectively. The band at 792 cm−1 can be assigned to the ring breathing modes of poly C and poly T, which overlap. The band at 1635 cm−1 is assigned to the carbonyl stretching mode of poly C, while the intense 1576 cm−1 poly G band is observed as a shoulder of the adenine band at 1559 cm−1. Figure 2 SERS spectra of 10−6 m solutions of the 24-mers a) poly A, b) 10−5 dGMP, c) poly C, and d) poly T on hydroxylamine-reduced Ag colloid aggregated with 0.1 m MgSO4. Given that the SERS spectra of the unthiolated DNA strands show features associated with all the constituent bases, in principle it should be possible to detect even relatively small changes in the DNA sequence. We have tested the ability to detect A→G and C→A polymorphisms in 25-mer and 23-mer DNA sequences. Figure ​Figure33 shows the SERS spectra of DNA-2 and DNA-3, where the adenosine nucleotide in DNA-2 is replaced by a guanosine nucleotide in DNA-3. Most of the features appear unchanged but the band at 1559 cm−1 in the DNA-2 spectrum is broader and shifts to 1569 cm−1 in the DNA-3 spectrum. This observation is consistent with the strongest dGMP band, which lies at 1576 cm−1 and increases in intensity in DNA-3. Figure 3 SERS spectra of a) DNA-2 and b) DNA-3. c) Simple difference spectrum of DNA-3 minus DNA-2 ((b)−(a)) shown with its intensity expanded three times. d) Model difference spectrum, dGMP minus poly A. All spectra are for hydroxylamine-reduced Ag colloids ... The best way to highlight the changes in the spectra is to digitally subtract them, which should remove the contributions from unchanged nucleotides to give difference spectra that contain positive and negative features corresponding to the exchanged nucleotides. Figure 3 c shows the experimental difference spectrum. Negative bands that can be assigned to poly A vibrations are observed at 1334 cm−1 and 737 cm−1. Positive G bands are observed at 1574 and 1509 cm−1, the expected increased band at 1332 cm−1 is cancelled by the stronger negative band at 1334 cm−1, which corresponds to adenine. More strikingly, when the experimental difference spectrum is compared to a model spectrum created by subtracting the spectra of poly A from dGMP (Figure 3 d), the agreement between the experimental and predicted pattern of band intensity changes is excellent. This result provides clear evidence that the changes in the spectra are due to the A→G polymorphism. Each polymorphism would be expected to give a different pattern of band changes. For example, Figure ​Figure44 shows SERS data for DNA-4 and DNA-5, which correspond to C→A polymorphism. Again, small changes can be observed at the expected parts of the raw spectra, for example, the ratio of the intensity of the C band at 792 cm−1 to that of the A band at 737 cm−1 is slightly lower in the spectrum of ssDNA-5. However, the best evidence again comes from the difference spectra where the experimental data can be compared to the model obtained by subtracting the spectrum of poly C from that of poly A. Again there is excellent agreement between the expected and observed changes. Figure 4 SERS spectra of a) DNA-4 and b) DNA-5. c) Simple difference spectrum of DNA-5 minus DNA-4 ((b)−(a)) shown with its intensity expanded eight times. d) Model difference spectrum, poly A minus poly C. All spectra are for hydroxylamine-reduced Ag ... This work demonstrates that the SERS spectra of simple unthiolated DNA can be obtained under appropriate experimental conditions. These spectra show bands that are characteristic of the constituent bases and are sufficiently reproducible that differences arising from single-base mismatch can be identified in short DNA strands.

Rapid analysis of ecstasy and related phenethylamines in seized tablets by Raman spectroscopy

Raman spectroscopy with far-red excitation has been used to study seized, tableted samples of MDMA (N-methyl-3,4-methylenedioxyamphetamine) and related compounds (MDA, MDEA, MBDB, 2C-B and amphetamine sulfate), as well as pure standards of these drugs. We have found that by using far-red (785 nm) excitation the level of fluorescence background even in untreated seized samples is sufficiently low that there is little difficulty in obtaining good quality data with moderate 2 min data accumulation times. The spectra can be used to distinguish between even chemically-similar substances, such as the geometrical isomers MDEA and MBDB, and between different polymorphic/hydrated forms of the same drug. Moreover, these differences can be found even in directly recorded spectra of seized samples which have been bulked with other materials, giving a rapid and non-destructive method for drug identification. The spectra can be processed to give unambiguous identification of both drug and excipients (even when more than one compound has been used as the bulking agent) and the relative intensities of drug and excipient bands can be used for quantitative or at least semi-quantitative analysis. Finally, the simple nature of the measurements lends itself to automatic sample handling so that sample throughputs of 20 samples per hour can be achieved with no real difficulty.

Composition profiling of seized ecstasy tablets by Raman spectroscopy.

Raman spectroscopy with far-red excitation has been investigated as a simple and rapid technique for composition profiling of seized ecstasy (MDMA, N-methyl-3,4-methylenedioxyamphetamine) tablets. The spectra obtained are rich in vibrational bands and allow the active drug and excipient used to bulk the tablets to be identified. Relative band heights can be used to determine drug/excipient ratios and the degree of hydration of the drug while the fact that 50 tablets per hour can be analysed allows large numbers of spectra to be recorded. The ability of Raman spectroscopy to distinguish between ecstasy tablets on the basis of their chemical composition is illustrated here by a sample set of 400 tablets taken from a large seizure of > 50,000 tablets that were found in eight large bags. The tablets are all similar in appearance and carry the same logo. Conventional analysis by GC-MS showed they contained MDMA. Initial Raman studies of samples from each of the eight bags showed that despite some tablet-to-tablet variation within each bag the contents could be classified on the basis of the excipients used. The tablets in five of the bags were sorbitol-based, two were cellulose-based and one bag contained tablets with a glucose excipient. More extensive analysis of 50 tablets from each of a representative series of sample bags have distribution profiles that showed the contents of each bag were approximately normally distributed about a mean value, rather than being mixtures of several discrete types. Two of the sorbitol-containing sample sets were indistinguishable while a third was similar but not identical to these, in that it contained the same excipient and MDMA with the same degree of hydration but had a slightly different MDMA/sorbitol ratio. The cellulose-based samples were badly manufactured and showed considerable tablet-to-tablet variation in their drug/excipient ratio while the glucose-based tablets had a tight distribution in their drug/excipient ratios. The degree of hydration in the MDMA feedstocks used to manufacture the cellulose-, glucose- and sorbitol-based tablets were all different from each other. This study, because it centres on a single seizure of physically similar tablets with the same active drug, highlights the fact that simple physical descriptions coupled with active drug content do not in themselves fully characterize the nature of the seized materials. There is considerable variation in the composition of the tablets within this single seizure and the fact that this variation can be detected from Raman spectra demonstrates that the potential benefits of obtaining highly detailed spectra can indeed translate into information that is not readily available from other methods but would be useful for tracing of drug distribution networks.

Reduced-size polarized basis sets for calculations of molecular electric properties. I. The basis set generation

Following the recent studies of basis sets explicitly dependent on oscillatory external electric field we have investigated the possibility of some further truncation of the so‐called polarized basis sets without any major deterioration of the computed data for molecular dipole moments, dipole polarizabilities, and related electric properties of molecules. It has been found that basis sets of contracted Gaussian functions of the form [3s1p] for H and [4s3p1d] for the first‐row atoms can satisfy this requirement with particular choice of contractions in their polarization part. With m denoting the number of primitive GTOs in the contracted polarization function, the basis sets devised in this article will be referred to as the ZmPol sets. In comparison with earlier, medium‐size polarized basis sets (PolX), these new ZmPol basis sets are reduced by 2/3 in their size and lead to the order of magnitude computing time savings for large molecules. Simultaneously, the dipole moment and polarizability data remain at almost the same level of accuracy as in the case of the PolX sets. Among a variety of possible applications in computational chemistry, the ZmPolX are also to be used for calculations of frequencies and intensities in the Raman spectra of large organic molecules (see Part II, this issue).

A critical evaluation of Raman spectroscopy for the analysis of lipids: fatty acid methyl esters.

The work presented here is aimed at determining the potential and limitations of Raman spectroscopy for fat analysis by carrying out a systematic investigation of C4−C24 FAME. These provide a simple, well-characterized set of compounds in which the effect of making incremental changes can be studied over a wide range of chain lengths and degrees of unsaturation. The effect of temperature on the spectra was investigated over much larger ranges than would normally be encountered in real analytical measurements. It was found that for liquid FAME the best internal standard band was the carbonyl stretching vibration ρ(C=O), whose position is affected by changes in sample chain length and physical state; in the samples studied here, it was found to lie between 1729 and 1748 cm−1. Further, molar unsaturation could be correlated with the ratio of the ρ(C=O) to either ρ(C=C) or δ(H−C=) with R2>0.995. Chain length was correlated with the δ(CH2)tw/ρ(C=O) ratio, (where “tw” indicates twisting) but separate plots for odd- and even-numbered carbon chains were necessary to obtain R2>0.99 for liquid samples. Combining the odd- and even-numbered carbon chain data in a single plot reduced the correlation to R2=0.94–0.96, depending on the band ratios used. For molal unsaturation the band ratio that correlated linearly with unsaturation (R2>0.99) was ρ(C=C)/δ(CH2)sc (where “sc” indicates scissoring). Other band ratios show much more complex behavior with changes in chemical and physical structure. This complex behavior results from the fact that the bands do not arise from simple vibrations of small, discrete regions of the molecules but are due to complex motions of large sections of the FAME so that making incremental changes in structure does not necessarily lead to simple incremental changes in spectra.

Controlling Assembly of Mixed Thiol Monolayers on Silver Nanoparticles to Tune Their Surface Properties

Modifying the surfaces of metal nanoparticles with self-assembled monolayers of functionalized thiols provides a simple and direct method to alter their surface properties. Mixed self-assembled monolayers can extend this approach since, in principle, the surfaces can be tuned by altering the proportion of each modifier that is adsorbed. However, this works best if the composition and microstructure of the monolayers can be controlled. Here, we have modified preprepared silver colloids with binary mixtures of thiols at varying concentrations and modifier ratios. Surface-enhanced Raman spectroscopy was then used to determine the effect of altering these parameters on the composition of the resulting mixed monolayers. The data could be explained using a new model based on a modified competitive Langmuir approach. It was found that the composition of the mixed monolayer only reflected the ratio of modifiers in the feedstock when the total amount of modifier was sufficient for approximately one monolayer coverage. At higher modifier concentrations the thermodynamically favored modifier dominated, but working at near monolayer concentrations allowed the surface composition to be controlled by changing the ratios of modifiers. Finally, a positively charged porphyrin probe molecule was used to investigate the microstructure of the mixed monolayers, i.e., homogeneous versus domains. In this case the modifier domains were found to be <2 nm.

Prediction of adipose tissue composition using Raman spectroscopy: average properties and individual fatty acids.

Raman spectroscopy has been used for the first time to predict the FA composition of unextracted adipose tissue of pork, beef, lamb, and chicken. It was found that the bulk unsaturation parameters could be predicted successfully [R2=0.97, root mean square error of prediction (RMSEP)=4.6% of 4 δ], with cis unsaturation, which accounted for the majority of the unsaturation, giving similar correlations. The combined abundance of all measured PUFA (≥2 double bonds per chain) was also well predicted with R2=0.97 and RMSEP=4.0% of 4 δ. Trans unsaturation was not as well modeled (R2=0.52, RMSEP=18% of 4 δ); this reduced prediction ability can be attributed to the low levels of trans FA found in adipose tissue (0.035 times the cis unsaturation level). For the individual FA, the average partial least squares (PLS) regression coefficient of the 18 most abundant FA (relative abundances ranging from 0.1 to 38.6% of the total FA content) was R2=0.73; the average RMSEP=11.9% of 4 δ. Regression coefficients and prediction errors for the five most abundant FA were all better than the average value (in some cases as low as RMSEP=4.7% of 4 δ). Cross-correlation between the abundances of the minor FA and more abundant acids could be determined by principal component analysis methods, and the resulting groups of correlated compounds were also well predicted using PLS. The accuracy of the prediction of individual FA was at least as good as other spectroscopic methods, and the extremely straightforward sampling method meant that very rapid analysis of samples at ambient temperature was easily achieved. This work shows that Raman profiling of hundreds of samples per day is easily achievable with an automated sampling system.