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Analytical and Bioanalytical Chemistry (v.394, #7)

Surface-enhanced Raman spectroscopy by Jürgen Popp; Thomas Mayerhöfer (pp. 1717-1718).

is a full professor at the Friedrich Schiller University of Jena, Germany, where he holds a chair of physical chemistry. In June 2006 he also became Scientific Director at the Institute of Photonic Technology. His work has been awarded the Faculty Prize of Chemistry (1995), by the “Bayerischer Habilitationsförderpreis” (1997), the “Förderpreis der Würzburger Korporationen” (2001), and the Kirchhoff–Bunsen Award (2002). J. Popp’s research interests are mainly centred around the development and application of frequency, time, and spatially resolved innovative laser spectroscopic methods and techniques ranging from the UV to the NIR region for derivation of structure-activity or dynamic relationships. This type of investigative approach is essential for resolving important questions in biology, medicine, pharmacy, and astronomy, and in the environmental and materials sciences. In particular, his expertise in Raman spectroscopy and in the development of innovative Raman techniques should be emphasized. Results obtained by J. Popp have been published in more than 200 scientific articles in premier, peer-reviewed journals. He has patented five inventions in spectroscopic instrumentation. Since 2008 he has been coordinator of the Network of Excellence for Biophotonics “Photonics4Life”. has been at the Institute of Photonic Technology since 2007. He is a Network Support Officer for the Network of Excellence for Biophotonics “Photonics4Life” which is coordinated by Jürgen Popp. His research interests are focused on optical modelling and dispersion analysis of low-symmetry crystals, and polycrystalline and layered materials.

Surface-enhanced Raman scattering for protein detection by Xiao X. Han; Bing Zhao; Yukihiro Ozaki (pp. 1719-1727).

Proteins are essential components of organisms and they participate in every process within cells. The key characteristic of proteins that allows their diverse functions is their ability to bind other molecules specifically and tightly. With the development of proteomics, exploring high-efficiency detection methods for large-scale proteins is increasingly important. In recent years, rapid development of surface-enhanced Raman scattering (SERS)-based biosensors leads to the SERS realm of applications from chemical analysis to nanostructure characterization and biomedical applications. For proteins, early studies focused on investigating SERS spectra of individual proteins, and the successful design of nanoparticle probes has promoted great progress of SERS-based immunoassays. In this review we outline the development of SERS-based methods for proteins with particular focus on our proposed protein-mediated SERS-active substrates and their applications in label-free and Raman dye-labeled protein detection. Figure Protein-mediated SERS-active substrates for protein detection

Keywords: SERS; Protein detection; SERS-active substrate; Label-free detection; Raman dye-labeled detection

Surface-enhanced Raman spectroscopy: substrate-related issues by Xiu-Mei Lin; Yan Cui; Yan-Hui Xu; Bin Ren; Zhong-Qun Tian (pp. 1729-1745).

After over 30 years of development, surface-enhanced Raman spectroscopy (SERS) is now facing a very important stage in its history. The explosive development of nanoscience and nanotechnology has assisted the rapid development of SERS, especially during the last 5 years. Further development of surface-enhanced Raman spectroscopy is mainly limited by the reproducible preparation of clean and highly surface enhanced Raman scattering (SERS) active substrates. This review deals with some substrate-related issues. Various methods will be introduced for preparing SERS substrates of Ag and Au for analytical purposes, from SERS substrates prepared by electrochemical or vacuum methods, to well-dispersed Au or Ag nanoparticle sols, to nanoparticle thin film substrates, and finally to ordered nanostructured substrates. Emphasis is placed on the analysis of the advantages and weaknesses of different methods in preparing SERS substrates. Closely related to the application of SERS in the analysis of trace sample and unknown systems, the existing cleaning methods for SERS substrates are analyzed and a combined chemical adsorption and electrochemical oxidation method is proposed to eliminate the interference of contaminants. A defocusing method is proposed to deal with the laser-induced sample decomposition problem frequently met in SERS measurement to obtain strong signals. The existing methods to estimate the surface enhancement factor, a criterion to characterize the SERS activity of a substrate, are analyzed and some guidelines are proposed to obtain the correct enhancement factor.

Keywords: Silver; Gold; Substrate preparation; Substrate cleaning; Photodecomposition; Surface enhancement factor

Surface-enhanced Raman scattering: realization of localized surface plasmon resonance using unique substrates and methods by Mohammad Kamal Hossain; Yasutaka Kitahama; Genin Gary Huang; Xiaoxia Han; Yukihiro Ozaki (pp. 1747-1760).

Surface-enhanced Raman scattering (SERS) enhancement and the reproducibility of the SERS signal strongly reflect the quality and nature of the SERS substrates because of diverse localized surface plasmon resonance (LSPR) excitations excited at interstitials or sharp edges. LSPR excitations are the most important ingredients for achieving huge enhancements in the SERS process. In this report, we introduce several gold and silver nanoparticle-based SERS-active substrates developed solely by us and use these substrates to investigate the influence of LSPR excitations on SERS. SERS-active gold substrates were fabricated by immobilizing colloidal gold nanoparticles on glass slides without using any surfactants or electrolytes, whereas most of the SERS-active substrates that use colloidal gold/silver nanoparticles are not free of surfactant. Isolated aggregates, chain-like elongated aggregates and two-dimensional (2D) nanostructures were found to consist mostly of monolayers rather than agglomerations. With reference to correlated LSPR and SERS, combined experiments were carried out on a single platform at the same spatial position. The isolated aggregates mostly show a broadened and shifted SPR peak, whereas a weak blue-shifted peak is observed near 430 nm in addition to broadened peaks centered at 635 and 720 nm in the red spectral region in the chain-like elongated aggregates. In the case of 2D nanostructures, several SPR peaks are observed in diverse frequency regions. The characteristics of LSPR and SERS for the same gold nanoaggregates lead to a good correlation between SPR and SERS images. The elongated gold nanostructures show a higher enhancement of the Raman signal than the the isolated and 2D samples. In the case of SERS-active silver substrates for protein detection, a new approach has been adopted, in contrast to the conventional fabrication method. Colloidal silver nanoparticles are immobilized on the protein functionalized glass slides, and further SERS measurements are carried out based on LSPR excitations. A new strategy for the detection of biomolecules, particularly glutathione, under aqueous conditions is proposed. Finally, supramolecular J-aggregates of ionic dyes incorporated with silver colloidal aggregates are characterized by SERS measurements and correlated to finite-difference time-domain analysis with reference to LSPR excitations. Figure SPR and SERS images for isolated, elongated and two-dimensional gold nanostructures

Keywords: Surface-enhanced Raman scattering; Localized surface plasmon resonances; SERS-active substrates; Nanoparticles; Nanoaggregates; Biomolecules

Optical fibre SERS sensors by P. R. Stoddart; D. J. White (pp. 1761-1774).

Surface-enhanced Raman scattering (SERS) has established itself as an important analytical technique. However, efforts to transfer the technology from the laboratory to the production line, clinic or field have been frustrated by the lack of robust affordable substrates and the complexity of interfacing between sample and spectrometer. Prompted by the success of optical fibre systems for implementing normal Raman scattering spectroscopy in remote locations and biomedical applications, attention has now shifted to the development of SERS-active optical fibres. Other workers have attempted to develop SERS probes with extended interaction lengths and both far-field and near-field SERS imaging techniques for high-resolution chemical mapping of surfaces. This review discusses the development of these technologies and presents the current state of the art. Although recent developments show great promise, some outstanding challenges and opportunities remain to be addressed.

Keywords: Optical fibre sensors; Surface-enhanced raman scattering; Scanning near-field optical microscopy; Tip-enhanced Raman spectroscopy

Tip-enhanced Raman spectroscopy for nanoscale strain characterization by Alvarado Tarun; Norihiko Hayazawa; Satoshi Kawata (pp. 1775-1785).

Tip-enhanced Raman spectroscopy (TERS), which utilizes the strong localized optical field generated at the apex of a metallic tip when illuminated, has been shown to successfully probe the vibrational spectrum of today’s and tomorrow’s state-of-the-art silicon and next-generation semiconductor devices, such as quantum dots. Collecting and analyzing the vibrational spectrum not only aids in material identification but also provides insight into strain distributions in semiconductors. Here, the potential of TERS for nanoscale characterization of strain in silicon devices is reviewed. Emphasis will be placed on the key challenges of obtaining spectroscopic images of strain in actual strained silicon devices. Figure Figure Concept of Tip Enhanced Raman Spectroscopy (TERS), which utilizes the strong localized optical field generated at the apex of a metallic tip when illuminated. TERS has been demonstrated to successfully probe the vibrational spectrum of today’s and tomorrow’s state-of-the-art silicon and next generation semiconductor devices

Keywords: IR spectroscopy; Raman spectroscopy; Laser spectroscopy; Nanoparticles/nanotechnology; Spectroscopy/instrumentation; UV/VIS

Tip-enhanced near-field optical microscopy of carbon nanotubes by A. Hartschuh; H. Qian; C. Georgi; M. Böhmler; L. Novotny (pp. 1787-1795).

We review recent experimental studies on single-walled carbon nanotubes on substrates using tip-enhanced near-field optical microscopy (TENOM). High-resolution optical and topographic imaging with sub 15 nm spatial resolution is shown to provide novel insights into the spectroscopic properties of these nanoscale materials. In the case of semiconducting nanotubes, the simultaneous observation of Raman scattering and photoluminescence (PL) is possible, enabling a direct correlation between vibrational and electronic properties on the nanoscale. So far, applications of TENOM have focused on the spectroscopy of localized phonon modes, local band energy renormalizations induced by charge carrier doping, the environmental sensitivity of nanotube PL, and inter-nanotube energy transfer. At the end of this review we discuss the remaining limitations and challenges in this field. Figure Tip-enhanced Raman scattering and photoluminescence spectroscopy with sub 15 nm spatial resolution provides novel insights into the electronic and vibronic properties of single-walled carbon nanotubes.

Keywords: Near-field optical microscopy; TERS; Carbon nanotubes

Surface-enhanced Raman scattering as a tool to probe cytochrome P450-catalysed substrate oxidation by Elena Bailo; Ljiljana Fruk; Christof M. Niemeyer; Volker Deckert (pp. 1797-1801).

Surface-enhanced Raman scattering was used as a spectroscopic tool to investigate the changes brought upon cytochrome P450BSß after fatty acid binding. Differences in the spectra of substrate-free and substrate-bound enzyme were observed indicating the potential for this method to be used in the screening of P450 substrates. In particular, the binding characteristics of myristic acid, an inherent substrate, and hydroxylauric acid, a product of fatty acid oxidation, towards P450BSß in the presence of H₂O₂ were investigated. Specific spectral changes could be assigned to changes in the heme environment only for myristic acid, indicating an occurrence of oxidation process characteristic for the enzymatic substrate.

Keywords: Surface-enhanced Raman spectroscopy; Bioanalytical methods; Protein binding

Imaging the cell wall of living single yeast cells using surface-enhanced Raman spectroscopy by Athiyanathil Sujith; Tamitake Itoh; Hiroko Abe; Ken-ichi Yoshida; Manikantan S. Kiran; Vasudevanpillai Biju; Misturu Ishikawa (pp. 1803-1809).

The surface of a living yeast cell (Saccharomyces cerevisiae strain W303-1A) has been labeled with silver (Ag) nanoparticles that can form nanoaggregates which have been shown to have surface-enhanced Raman scattering (SERS) activity. The cell wall of a single living yeast cell has been imaged by use of a Raman microspectroscope. The SERS spectra measured from different Ag nanoaggregates were found to be different. This can be explained on the basis of detailed spectral interpretation. The SERS spectral response originates from mannoproteins which cover the outermost regions of the yeast cell wall. Analysis of SERS spectra from the cell wall and the extracted mannoproteins from the yeast has been performed for the clarification of variation in SERS spectra.

Keywords: Surface-enhanced Raman spectroscopy; Living cell analysis; Raman spectroscopic imaging; Yeast cell wall; Spectroscopy/Theory; Laser spectroscopy; Cell systems/Single cell analysis

Ultrafast plasmon dynamics and evanescent field distribution of reproducible surface-enhanced Raman-scattering substrates by Dana Cialla; Ronald Siebert; Uwe Hübner; Robert Möller; Henrik Schneidewind; Roland Mattheis; Jörg Petschulat; Andreas Tünnermann; Thomas Pertsch; Benjamin Dietzek; Jürgen Popp (pp. 1811-1818).

Surface-enhanced Raman scattering (SERS) is a potent tool in bioanalytical science because the technique combines high sensitivity with molecular specificity. However, the widespread and routine use of SERS in quantitative biomedical diagnostics is limited by tight requirements on the reproducibility of the noble metal substrates used. To solve this problem, we recently introduced a novel approach to reproducible SERS substrates. In this contribution, we apply ultrafast time-resolved spectroscopy to investigate the photo-induced collective charge-carrier dynamics in such substrates, which represents the fundamental origin of the SERS mechanism. The ultrafast experiments are accompanied by scanning-near field optical microscopy and SERS experiments to correlate the appearance of plasmon dynamics with the resultant evanescent field distribution and the analytically relevant SERS enhancement. Figure Ultrafast time-resolved differential absorption spectroscopy combined with scanning near-field optical microscopy (left) and atomic force microscopy (right) yields insight into the photoinduced charge-carrier dynamics in innovative reproducible SERS-substrates

Keywords: Nanostructured gold surfaces; Surface-enhanced Raman scattering (SERS); Reproducible SERS substrates; Femtosecond time-resolved dynamics; Scanning near-field optical microscopy

Gold nanoparticle dimer plasmonics: finite element method calculations of the electromagnetic enhancement to surface-enhanced Raman spectroscopy by Jeffrey M. McMahon; Anne-Isabelle Henry; Kristin L. Wustholz; Michael J. Natan; R. Griffith Freeman; Richard P. Van Duyne; George C. Schatz (pp. 1819-1825).

Finite element method calculations were carried out to determine extinction spectra and the electromagnetic (EM) contributions to surface-enhanced Raman spectroscopy (SERS) for 90-nm Au nanoparticle dimers modeled after experimental nanotags. The calculations revealed that the EM properties depend significantly on the junction region, specifically the distance between the nanoparticles for spacings of less than 1 nm. For extinction spectra, spacings below 1 nm lead to maxima that are strongly red-shifted from the 600-nm plasmon maximum associated with an isolated nanoparticle. This result agrees qualitatively well with experimental transmission electron microscopy images and localized surface plasmon resonance spectra that are also presented. The calculations further revealed that spacings below 0.5 nm, and especially a slight fusing of the nanoparticles to give tiny crevices, leads to EM enhancements of 10¹⁰ or greater. Assuming a uniform coating of SERS molecules around both nanoparticles, we determined that regardless of the separation, the highest EM fields always dominate the SERS signal. In addition, we determined that for small separations less than 3% of the molecules always contribute to greater than 90% of the signal.

Keywords: Finite element method; Surface-enhanced Raman spectroscopy; Electromagnetic field enhancement; Nanoparticle dimer

Surface-enhanced Raman scattering in nanoliter droplets: towards high-sensitivity detection of mercury (II) ions by Guoqing Wang; Chaesung Lim; Lingxin Chen; Hyangah Chon; Jaebum Choo; Jongin Hong; Andrew J. deMello (pp. 1827-1832).

We report a new method for the trace analysis of mercury (II) ions in water. The approach involves the use of droplet-based microfluidics combined with surface-enhanced Raman scattering (SERS) detection. This novel combination provides both fast and sensitive detection of mercury (II) ions in water. Specifically, mercury (II) ion detection is performed by using the strong affinity between gold nanoparticles and mercury (II) ions. This interaction causes a change in the SERS signal of the reporter molecule rhodamine B that is a function of mercury (II) ion concentration. To allow both reproducible and quantitative analysis, aqueous samples are encapsulated within nanoliter-sized droplets. Manipulation of such droplets through winding microchannels affords rapid and efficient mixing of the contents. Additionally, memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, are removed since the aqueous droplets are completely isolated by a continuous oil phase. Quantitative analysis of mercury (II) ions was performed by calculating spectral peak area of rhodamine B at 1,647 cm⁻¹. Using this approach, the calculated concentration limit of detection was estimated to be between 100 and 500 ppt. Compared with fluorescence-based methods for the trace analysis of mercury (II) ions, the detection sensitivities were enhanced by approximately one order of magnitude. The proposed analytical method offers a rapid and reproducible trace detection capability for mercury (II) ions in water.

Keywords: Surface-enhanced Raman scattering; Microdroplet; Lab on a chip; Mercury detection; Trace analysis

Quantitative analysis of methyl green using surface-enhanced resonance Raman scattering by Iqbal T. Shadi; William Cheung; Royston Goodacre (pp. 1833-1838).

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.

Keywords: pH; Methyl green; Silver sol; Surface-enhanced resonance Raman spectroscopy (SERRS)

Multiplexing with SERS labels using mixed SAMs of Raman reporter molecules by Magdalena Gellner; Karsten Kömpe; Sebastian Schlücker (pp. 1839-1844).

Surface-enhanced Raman scattering (SERS) offers a tremendous multiplexing capacity for the selective detection of biomolecules in targeted research. SERS labels comprising self-assembled monolayers (SAMs) of Raman reporter molecules on the surface of metal nanoparticles are sensitive and robust probes. Advantages of a SAM include maximum sensitivity, minimal unwanted co-adsorption of molecules from the surroundings, and reproducible SERS spectra with only few dominant Raman bands—all of these independent of a particular SERS substrate. We demonstrate experimentally how to increase the multiplexing capacity of SERS labels by using mixed SAMs with up to three different Raman reporter molecules on the surface of the metal colloid. Type and stoichiometry of a particular Raman label in a multi-component SAM are additional parameters compared with one-component SAMs. All one-, two-, and three-component SAMs on gold nanospheres can be easily discriminated, either by their original SERS spectra or the corresponding bar codes.

Keywords: Gold; Nanoparticle; Multiplexing; Self-assembled monolayer; Surface-enhanced Raman scattering

A bioinformatics approach to the development of immunoassays for specified risk material in canned meat products by Paul Reece; Monique Bremer; Robert Stones; Christopher Danks; Sabine Baumgartner; Victoria Tomkies; Claudia Hemetsberger; Nathalie Smits; Walter Lubbe (pp. 1845-1851).

A bioinformatics approach to developing antibodies to specific proteins has been evaluated for the production of antibodies to heat-processed specified risk tissues from ruminants (brain and eye tissue). The approach involved the identification of proteins specific to ruminant tissues by interrogation of the annotation fields within the Swissprot database. These protein sequences were then interrogated for peptide sequences that were unique to the protein. Peptides were selected that met these criteria as close as possible and that were also theoretically resistant to either pepsin or trypsin. The selected peptides were synthesised and used as immunogens to raise monoclonal antibodies. Antibodies specific for the synthetic peptides were raised to half of the selected peptides. These antibodies have each been incorporated into a competitive enzyme-linked immunosorbent assay (ELISA) and shown to be able to detect the heat-processed parent protein after digestion with either pepsin or trypsin. One antibody, specific for alpha crystallin peptide (from bovine eye tissue), was able to detect the peptide in canned meat products spiked with 10% eye tissue. These results, although preliminary in nature, show that bioinformatics in conjunction with enzyme digestion can be used to develop ELISA for proteins in high-temperature processed foods and demonstrate that the approach is worth further study.

Keywords: Specified risk material; Bioinformatics; Immunoassay; Heat-processed proteins; Antibody

Ion mobility-mass spectrometry analysis of isomeric carbohydrate precursor ions by Maolei Zhu; Brad Bendiak; Brian Clowers; Herbert H. Hill Jr (pp. 1853-1867).

The rapid separation of isomeric precursor ions of oligosaccharides prior to their analysis by mass spectrometry to the nth power (MS n) was demonstrated using an ambient pressure ion mobility spectrometer (IMS) interfaced with a quadrupole ion trap. Separations were not limited to specific types of isomers; representative isomers differing solely in the stereochemistry of sugars, in their anomeric configurations, and in their overall branching patterns and linkage positions could be resolved in the millisecond time frame. Physical separation of precursor ions permitted independent mass spectra of individual oligosaccharide isomers to be acquired to at least MS³, the number of stages of dissociation limited only practically by the abundance of specific product ions. IMS–MS n analysis was particularly valuable in the evaluation of isomeric oligosaccharides that yielded identical sets of product ions in tandem mass spectrometry experiments, revealing pairs of isomers that would otherwise not be known to be present in a mixture if evaluated solely by MS dissociation methods alone. A practical example of IMS–MSⁿ analysis of a set of isomers included within a single high-performance liquid chromatography fraction of oligosaccharides released from bovine submaxillary mucin is described.

Keywords: Carbohydrate isomers; Separation; Precursor ions; Tandem mass spectrometry; Ion mobility spectrometry

Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited by Michael Schleeger; Christoph Wagner; Michiel J. Vellekoop; Bernhard Lendl; Joachim Heberle (pp. 1869-1877).

Fourier-transform infrared (FT-IR) difference spectroscopy has been proven to be a significant tool in biospectroscopy. In particular, the step-scan technique monitors structural and electronic changes at time resolutions down to a few nanoseconds retaining the multiplex advantage of FT-IR. For the elucidation of the functional mechanisms of proteins, this technique is currently limited to repetitive systems undergoing a rapid photocycle. To overcome this obstacle, we developed a flow-flash experiment in a miniaturised flow channel which was integrated into a step-scan FT-IR spectroscopic setup. As a proof of principle, we studied the rebinding reaction of CO to myoglobin after photodissociation. The use of microfluidics reduced the sample consumption drastically such that a typical step-scan experiment takes only a few 10 ml of a millimolar sample solution, making this method particularly interesting for the investigation of biological samples that are only available in small quantities. Moreover, the flow cell provides the unique opportunity to assess the reaction mechanism of proteins that cycle slowly or react irreversibly. We infer that this novel approach will help in the elucidation of molecular reactions as complex as those of vectorial ion transfer in membrane proteins. The potential application to the oxygen splitting reaction of cytochrome c oxidase is discussed.

Keywords: Carbonmonoxymyoglobin; Cytochrome c oxidase; Microfluidics; Step-scan spectroscopy; Vibrational spectroscopy

Determination of protein surface excess on a liquid/solid interface by single-molecule counting by Nan Li; Hui Tang; Hongwei Gai; Xiuling Dong; Qi Wang; Edward S. Yeung (pp. 1879-1885).

Determination of protein surface excess is an important way of evaluating the properties of biomaterials and the characteristics of biosensors. A single-molecule counting method is presented that uses a standard fluorescence microscope to measure coverage of a liquid/solid interface by adsorbed proteins. The extremely low surface excess of lysozyme and bovine serum albumin (BSA), in a bulk concentration range from 0.3 nmol L⁻¹ (0.02 μg mL⁻¹) to 3 nmol L⁻¹ (0.2 μg mL⁻¹), were measured by recording the counts of spatially isolated single molecules on either hydrophilic (glass) or hydrophobic (polydimethylsiloxane, PDMS) surfaces at different pH. The differences observed in amounts of adsorbed proteins under different experimental conditions can be qualitatively explained by the combined interactions of electrostatic and hydrophobic forces. This, in turn, implies that single-molecule counting is an effective way of measuring surface coverage at a liquid/solid interface. Figure Adsorption fraction of proteins on different surfaces changed with pH.

Keywords: Single-molecule imaging; Protein adsorption; Hydrophilic/hydrophobic surface; Surface excess

An SEC/MALS study of alternan degradation during size-exclusion chromatographic analysis by André M. Striegel; Samantha L. Isenberg; Gregory L. Côté (pp. 1887-1893).

Ultrahigh-molar-mass (M) polymers such as DNA, cellulose, and polyolefins are routinely analyzed using size-exclusion chromatography (SEC) to obtain molar mass averages, distributions, and architectural information. It has long been contended that high-M polymers can degrade during SEC analysis; if true, the inaccurate molar mass information obtained can adversely affect decisions regarding processing and end-use properties of the macromolecules. However, most evidence to the effect of degradation has been circumstantial and open to alternative interpretation. For example, the shift in SEC elution volume as a function of increased chromatographic flow rate, observed using only a concentration-sensitive detector, may be the result of degradation or of elution via a nondegradatory slalom chromatography mechanism. Here, using both concentration-sensitive and multiangle static light-scattering detection, we provide unambiguous evidence that the polysaccharide alternan actually degrades during SEC analysis. The decrease in molar mass and size of alternan with increasing flow rate, measured using light scattering, allows ruling out an SC mode of elution and can only be interpreted as due to degradation. These findings demonstrate the extreme fragility of ultrahigh-M polymers and the care that must be taken for accurate characterization. Figure Scission of alternan chains in liquid chromatography.

Keywords: Alternan; Size-exclusion chromatography; On-column flow-induced degradation; Multiangle light scattering

Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography-tandem mass spectrometry following solid-phase extraction by Fabien Lamoureux; Jean-michel Gaulier; François-Ludovic Sauvage; Magali Mercerolle; Christine Vallejo; Gérard Lachâtre (pp. 1895-1901).

The detection of ethyl-β-d-6-glucuronide (EtG), a stable phase II metabolite of ethanol, is of interest in both clinical and forensic contexts with the aim of monitoring alcohol abuse. We present a liquid chromatography-electrospray ionisation-tandem mass spectrometry method for the detection and quantification of EtG in hair. Thirty milligrams of washed and cut hair were cleaned up using solid-phase extraction graphite cartridges. Separation was then performed using an Uptisphere-3SI column, and the detection was operated in the negative mode. After validation, the method was applied to hair samples taken from four fatalities (F) with documented excessive drinking habits, 12 heavy drinkers (HD) and seven social drinkers (SD). The method exhibits limits of detection and quantification of 4 and 10 pg/mg, respectively. Intra- and inter-assay standard deviation and relative bias were less than 20% over the calibrating range (10 to 3,000 pg/mg). EtG hair concentrations in SD were <10 pg/mg and >50 pg/mg for F and HD (range, 54 to 497 pg/mg). The present assay appears convenient to carry out owing to the very small quantity of hair samples required to determine an effective heavy alcohol consumption (EtG hair concentration >50 pg/mg).

Keywords: Ethyl-glucuronide; Hair analysis; Liquid chromatography-tandem mass spectrometry; Solid-phase extraction; Heavy drinking

Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase by Zhen-Dan Shi; Omid Motabar; Ehud Goldin; Ke Liu; Noel Southall; Ellen Sidransky; Christopher P. Austin; Gary L. Griffiths; Wei Zheng (pp. 1903-1909).

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-d-galactopyranoside, for a new α-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK _a of resorufin (~6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease.

Keywords: Alpha-galactosidase; Enzyme assay; Assay optimization; Assay miniaturization; Fabry disease

Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis by Bo Xu; Xiaojun Feng; Youzhi Xu; Wei Du; Qingming Luo; Bi-Feng Liu (pp. 1911-1917).

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated. Figure

Keywords: Microfluidic chip; Two-dimensional electrophoresis; Micellar electrokinetic chromatography; Capillary zone electrophoresis

Fast log P determination by ultra-high-pressure liquid chromatography coupled with UV and mass spectrometry detections by Yveline Henchoz; Davy Guillarme; Sophie Martel; Serge Rudaz; Jean-Luc Veuthey; Pierre-Alain Carrupt (pp. 1919-1930).

Ultra-high-pressure liquid chromatography (UHPLC) systems able to work with columns packed with sub-2 μm particles offer very fast methods to determine the lipophilicity of new chemical entities. The careful development of the most suitable experimental conditions presented here will help medicinal chemists for high-throughput screening (HTS) log P _oct measurements. The approach was optimized using a well-balanced set of 38 model compounds and a series of 28 basic compounds such as β-blockers, local anesthetics, piperazines, clonidine, and derivatives. Different organic modifiers and hybrid stationary phases packed with 1.7-μm particles were evaluated in isocratic as well as gradient modes, and the advantages and limitations of tested conditions pointed out. The UHPLC approach offered a significant enhancement over the classical HPLC methods, by a factor 50 in the lipophilicity determination throughput. The hyphenation of UHPLC with MS detection allowed a further increase in the throughput. Data and results reported herein prove that the UHPLC-MS method can represent a progress in the HTS-measurement of lipophilicity due to its speed (at least a factor of 500 with respect to HPLC approaches) and to an extended field of application. Figure The UHPLC approach described here greatly enhanced the time required for log P determination (5' min by compound using UV detection) and, at least, 8 compounds measured in a 5' run when Mass Spectrometry detection in used. These developments offer to medicinal chemists a high-throughput method to estimate the lipophilicity of NCEs

Keywords: Lipophilicity; UHPLC; Reversed-phase liquid chromatography; Partition coefficients; Physicochemical profiling

Identification of γ-ray irradiated medicinal herbs using pulsed photostimulated luminescence, thermoluminescence, and electron spin resonance spectroscopy by Sukdeb Pal; Byeong Keun Kim; Won Young Kim; Min Jung Kim; Hyeon A. Ki; Kyeong-Hee Lee; Woo Suk Kang; In Ho Kang; Shin Jung Kang; Joon Myong Song (pp. 1931-1945).

Dried herbal samples consisting of root, rhizome, cortex, fruit, peel, flower, spike, ramulus, folium, and whole plant of 20 different medicinal herbs were investigated using pulsed photostimulated luminescence (PPSL), thermoluminescence (TL), and electron spin resonance spectroscopy (ESR) to identify γ-ray irradiation treatment. Samples were irradiated at 0–50 kGy using a 60Co irradiator. PPSL measurement was applied as a rapid screening method. Control samples of 19 different herbs had photon counts less than the lower threshold value (700 counts 60 s−1). The photon counts of non-irradiated clematidis radix and irradiated evodia and gardenia fruits were between the lower and upper threshold values (700–5,000 counts 60 s−1). TL ratios, i.e., integrated areas of the first glow (TL1)/the second glow (TL2), were found to be less than 0.1 in all non-irradiated samples and higher than 0.1 in irradiated ones providing definite proof of radiation treatment. ESR spectroscopy was applied as an alternative rapid method. In most of the irradiated samples, mainly radiation-induced cellulosic, sugar, and relatively complicated carbohydrate radical ESR signals were detected. No radiation-specific ESR signal, except one intense singlet, was observed for irradiated scrophularia and scutellaria root and artemisiae argyi folium. Figure PPSL can be used as a rapid simple preliminary screening method and a combination of ESR and TL tests for a definite proof of gamma irradiation treatment of medicinal herbs.

Keywords: Medicinal herbs; γ-Ray irradiation; Pulsed photostimulated luminescence (PPSL); Thermoluminescence (TL); Electron spin resonance (ESR)

A microsystem of low-voltage-driven electrophoresis on microchip with array electrode pairs for the separation of amino acids by Yi Xu; Xiaoguo Hu; Jing Liang; Jianxin Sun; Wenwen Gu; Tianming Zhao; Zhiyu Wen (pp. 1947-1953).

In this paper, a new approach for the separation of amino acids on the electrophoresis chip-based low-voltage-driven electrophoresis was reported in detail. This low-voltage-driven electrophoresis process could be realized by powering directly the arrayed electrode pairs with low direct current (DC) voltage to generate a moving electric field along the separation microchannel, which could maintain enough electric field strength for electrophoresis. The proposed microfluidic electrophoresis chip was bonded directly with silicon-on-insulator (SOI) substrate and polydimethylsiloxane (PDMS) cover plate at room temperature. The microfluidic channels and the arrayed electrodes were etched on SOI wafer by silicon microelectromechanical system technology. A specially integrated circuit was proposed to power a 30–60-V DC voltage to particular sets of these electrode pairs in a controlled sequence such that the moving electric field could be formed, and the low-voltage-driven electrophoresis could be realized in the microchannel. In the experiments, with 10⁻⁴ mol/L phenylalanine and lysine as analytes, the separation of amino acids on the low-voltage-driven electrophoresis microchip was conducted by homemade integrated control circuit; a method for separating amino acids was well established. It was also shown that the phenylalanine and lysine mixture was effectively separated in less than 7 min and with a resolution of 2.0. To the best of our knowledge, the low-voltage-driven microchip electrophoresis device could be of potential prospective in the fields of integrated and miniaturized biochemical analysis system.

Keywords: Electrophoresis driven by low-voltage on microchip; Microelectrode array; Integrated circuit; Separation of amino acids; Microelectromechanical systems technology

Electrochemical time of flight method for determination of diffusion coefficients of glucose in solutions and gels by Ágnes Varga; Gergely Gyetvai; Lívia Nagy; Géza Nagy (pp. 1955-1963).

The diffusion coefficient of glucose in different media is an important parameter in life sciences, as well as in biotechnology and microbiology. In this work a simple, fast method is proposed that is based on the electrochemical time of flight principle. In most of the earlier time of flight experiments performed, a constant flight distance was applied. In the present work a scanning electrochemical microscope (SECM) was applied as a measuring tool. With use of the SECM, the flying distance could be changed with high precision, making measurements with several flight distances more accurate and reliable values could be obtained for solutions as well as for gels. The conventional voltammetric methods are not applicable for glucose detection. In our work electrocatalytic copper oxide coated copper microelectrodes and micro-sized amperometric enzyme sensors were used as detectors, while microdroplet-ejecting pneumatically driven micropipettes were used as a source. Figure Experimental set up for SCEM-TOF diffusion coefficient measurements

Keywords: Glucose diffusion coefficient; Scanning electrochemical microscopy; Electrochemical time of flight method; Copper electrode; Enzyme electrode; Microelectrodes

Multivariate standard addition method solved by net analyte signal calculation and rank annihilation factor analysis by Bahram Hemmateenejad; Saeed Yousefinejad (pp. 1965-1975).

This article describes the use of the net analyte signal (NAS) concept and rank annihilation factor analysis (RAFA) for building two different multivariate standard addition models called “SANAS” and “SARAF.” In the former, by the definition of a new subspace, the NAS vector of the analyte of interest in an unknown sample as well as the NAS vectors of samples spiked with various amounts of the standard solutions are calculated and then their Euclidean norms are plotted against the concentration of added standard. In this way, a simple linear standard addition graph similar to that in univariate calibration is obtained, from which the concentration of the analyte in the unknown sample and the analytical figures of merit are readily calculated. In the SARAF method, the concentration of the analyte in the unknown sample is varied iteratively until the contribution of the analyte in the response data matrix is completely annihilated. The proposed methods were evaluated by analyzing simulated absorbance data as well as by the analysis of two indicators in synthetic matrices as experimental data. The resultant predicted concentrations of unknown samples showed that the SANAS and SARAF methods both produced accurate results with relative errors of prediction lower than 5% in most cases.

Keywords: Standard addition; Multivariate calibration; Net analyte signal; Rank annihilation factor analysis

Use of ¹⁸O water and ESI-MS detection in subsite characterisation and investigation of the hydrolytic action of an endoglucanase by Herje Schagerlöf; Carina Nilsson; Lo Gorton; Folke Tjerneld; Henrik Stålbrand; Arieh Cohen (pp. 1977-1984).

We present a novel method for investigating subsite-substrate interactions of glycoside hydrolases and the determination of the oligosaccharide cleavage point based on the analysis of the hydrolysis products produced in the presence of ¹⁸O-labelled water. Conventional techniques for such determination of the hydrolysis pattern call for the chemical modification of the substrate, whereas the method presented makes it possible to use natural substrates, utilising the selectivity and sensitivity of mass spectrometry. This method is very useful for the detection and analysis of enzyme-catalysed hydrolysis, provided that the conditions are chosen where ¹⁸O incorporation without the presence of the enzyme is absent or undetectable. Such conditions were found and used in incubations of cellopentaose with the well-characterised endoglucanase Cel5A from Bacillus agaradhaerens. We were able to confirm that the preferred glycoside bond to be hydrolysed is the third one counting from the non-reducing end of the cellopentaose. Thus, cellopentaose prefers to bind from the –3 to the +2 subsites, which is in accordance with published crystallographic data. The main advantage of the method presented is that there is no need for a priori chemical modification/labelling of oligosaccharide substrates, which are processes that can disturb the enzyme–substrate interaction. From ¹⁸O incorporation we could demonstrate that the enzyme also has an oxygen-exchange activity on cellotriose and cellobiose. This is in agreement with the mechanism for transglycosylation and indicates that it is possible for the enzyme to perform such reactions.

Keywords: Enzyme hydrolytic action; Endoglucanase; Subsites; Bacillus agaradhaerens Cel5A; Mass spectrometry; ¹⁸O

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Analytical and Bioanalytical Chemistry (v.394, #7)

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Analytical and Bioanalytical Chemistry (v.394, #7)