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Analytical and Bioanalytical Chemistry (v.388, #1)

Quality assurance challenge 5 by M. Reichenbächer; J. W. Einax (pp. 1-1).

Solution to the neurochemical challenge by Michael L. Heien; Jonathan V. Sweedler (pp. 3-3).

Vibrational spectroscopy at surfaces and interfaces by Masatoshi Osawa; Takeshi Hasegawa (pp. 5-6).

is a professor at the Catalysis Research Center at Hokkaido University, Japan. His research interests are surface-enhanced infrared absorption spectroscopy (SEIRAS) and its application to electrochemical surface dynamics. His time-resolved SEIRAS studies coupled to electrochemical techniques and STM have opened up a new frontier in electrochemistry. His group also uses IR–visible sum frequency generation (SFG), to characterize thin organic molecular layers. is an associate professor at the Department of Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo, Japan. His research interests comprise vibrational spectroscopy for the analysis of thin molecular adsorbates on a substrate, analytical techniques for spectra, including chemometrics, and surface chemistry. He developed a new spectroscopic technique for analyzing molecular orientation in thin films (as described in the review in this issue), which has recently been commercialized.

A new spectroscopic tool for surface layer analysis: multiple-angle incidence resolution spectrometry by Takeshi Hasegawa (pp. 7-15).

A series of application studies of infrared multiple-angle incidence resolution spectrometry (IR-MAIRS) have been reported since it was first proposed as a unique technique for analyzing molecular orientation in thin films on an optically transparent substrate, and both expected and unexpected benefits of IR-MAIRS have been revealed. In this review, cutting-edge application studies are summarized systematically, and the principle of MAIRS is explained from a new point of view.

Keywords: Infrared multiple-angle incidence resolution spectrometry (IR-MAIRS); Thin films; Molecular orientation; Molecular folding; Surface analysis; Proteome analysis

Study of the dynamics of surface molecules by time-resolved sum-frequency generation spectroscopy by Jun Kubota; Kazunari Domen (pp. 17-27).

Sum-frequency generation (SFG) is a nonlinear laser-spectroscopy technique suitable for analysis of adsorbed molecules. The sub-monolayer sensitivity of SFG spectroscopy enables vibrational spectra to be obtained with high specificity for a variety of molecules on a range of surfaces, including metals, oxides, and semiconductors. The use of ultra-short laser pulses on time-scales of picoseconds also makes time-resolved measurements possible; this can reveal ultrafast transient changes in molecular arrangements. This article reviews recent time-resolved SFG spectroscopy studies revealing site-hopping of adsorbed CO on metal surfaces and the dynamics of energy relaxation at water/metal interfaces. Time-resolved sum frequency generation spectroscopy at surfaces with non-resonant laser pulse irradiation

Keywords: Sum-frequency generation; Non-linear spectroscopy; Time-resolved spectroscopy; Vibrational spectroscopy; Surface

Raman spectroscopy on transition metals by Bin Ren; Guo-Kun Liu; Xiao-Bing Lian; Zhi-Lin Yang; Zhong-Qun Tian (pp. 29-45).

Surface-enhanced Raman spectroscopy (SERS) has developed into one of the most important tools in analytical and surface sciences since its discovery in the mid-1970s. Recent work on the SERS of transition metals concluded that transition metals, other than Cu, Ag, and Au, can also generate surface enhancement as high as 4 orders of magnitude. The present article gives an overview of recent progresses in the field of Raman spectroscopy on transition metals, including experimental, theory, and applications. Experimental considerations of how to optimize the experimental conditions and calculate the surface enhancement factor are discussed first, followed by a very brief introduction of preparation of SERS-active transition metal substrates, including massive transition metal surfaces, aluminum-supported transition metal electrodes, and pure transition metal nanoparticle assembled electrodes. The advantages of using SERS in investigating surface bonding and reaction are illustrated for the adsorption and reaction of benzene on Pt and Rh electrodes. The electromagnetic enhancement, mainly lightning-rod effect, plays an essential role in the SERS of transition metals, and that the charge-transfer effect is also operative in some specific metal–molecule systems. An outlook for the field of Raman spectroscopy of transition metals is given in the last section, including the preparation of well-ordered or well-defined nanostructures, and core-shell nanoparticles for investigating species with extremely weak SERS signals, as well as some new emerging techniques, including tip-enhanced Raman spectroscopy and an in situ measuring technique. Figure Electric-field enhancement of a SERS-active Rh surface decorated with small nanohemispheres

Keywords: Interface/surface analysis; Raman spectroscopy; Nanoparticles/nanotechnology; Hydrocarbons; Kinetics; Metals

Biochemical applications of surface-enhanced infrared absorption spectroscopy by Kenichi Ataka; Joachim Heberle (pp. 47-54).

An overview is presented on the application of surface-enhanced infrared absorption (SEIRA) spectroscopy to biochemical problems. Use of SEIRA results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude and has the potential to enable new studies, from fundamental aspects to applied sciences. This report surveys studies of DNA and nucleic acid adsorption to gold surfaces, development of immunoassays, electron transfer between metal electrodes and proteins, and protein–protein interactions. Because signal enhancement in SEIRA uses surface properties of the nano-structured metal, the biomaterial must be tethered to the metal without hampering its functionality. Because many biochemical reactions proceed vectorially, their functionality depends on proper orientation of the biomaterial. Thus, surface-modification techniques are addressed that enable control of the proper orientation of proteins on the metal surface. Figure Surface enhanced infrared absorption spectroscopy (SEIRAS) on the studies of tethered protein monolayer (cytochrome c oxidase and cytochrome c) on gold substrate (left), and its potential induced surface enhanced infrared difference absorption (SEIDA) spectrum

Keywords: FTIR; Protein; Membrane; Electron transfer; Self-assembled monolayer

Monolayers at solid–solid interfaces probed with infrared spectroscopy by T. Lummerstorfer; J. Kattner; H. Hoffmann (pp. 55-64).

The sensitivities of infrared spectra of thin adsorbate layers measured in either transmission, internal reflection or external reflection can be greatly increased if a light incidence medium with a high refractive index such as an IR-transparent solid material is used. This increase in sensitivity is due to the strong enhancement of the perpendicular electric field in a thin layer of low refractive index sandwiched between two high refractive index materials. Based on model calculations of a hypothetical sample layer, the influence and optimization of experimental parameters such as incidence angle, sample layer thickness and optical contact between layers are investigated. Under optimized conditions, this enhancement can exceed a factor of 100 when compared to conventional surface IR techniques. In addition, the spectra of sandwiched sample layers are governed by a uniform surface selection rule, such that only the perpendicular vibrational components are enhanced, and they permit a straightforward, substrate-independent analysis of surface orientations. Experimental examples of monolayer spectra of long-chain hydrocarbon compounds adsorbed onto gold and silicon substrates and contacted with a germanium crystal used as the incidence medium demonstrate the simple experimental realization and unprecedented sensitivity of this sandwich technique, and they offer novel insights into the chemistry and structure of monolayers confined and compressed between two solid surfaces. Figure IR reflection spectrum of a monolayer of a fatty acid methyl ester sandwiched between silicon and germanium.

Keywords: Solid-solid interfaces; Self-assembled monolayers; Surface selection rules; Compression effects in monolayer films

Ordered adsorption of coagulation factor XII on negatively charged polymer surfaces probed by sum frequency generation vibrational spectroscopy by Xiaoyun Chen; Jie Wang; Zoltan Paszti; Fulin Wang; Joel N. Schrauben; Volodymyr V. Tarabara; Alvin H. Schmaier; Zhan Chen (pp. 65-72).

Electrostatic interactions between negatively charged polymer surfaces and factor XII (FXII), a blood coagulation factor, were investigated by sum frequency generation (SFG) vibrational spectroscopy, supplemented by several analytical techniques including attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), quartz crystal microbalance (QCM), ζ-potential measurement, and chromogenic assay. A series of sulfonated polystyrenes (sPS) with different sulfonation levels were synthesized as model surfaces with different surface charge densities. SFG spectra collected from FXII adsorbed onto PS and sPS surfaces with different surface charge densities showed remarkable differences in spectral features and especially in spectral intensity. Chromogenic assay experiments showed that highly charged sPS surfaces induced FXII autoactivation. ATR-FTIR and QCM results indicated that adsorption amounts on the PS and sPS surfaces were similar even though the surface charge densities were different. No significant conformational change was observed from FXII adsorbed onto surfaces studied. Using theoretical calculations, the possible contribution from the third-order nonlinear optical effect induced by the surface electric field was evaluated, and it was found to be unable to yield the SFG signal enhancement observed. Therefore it was concluded that the adsorbed FXII orientation and ordering were the main reasons for the remarkable SFG amide I signal increase on sPS surfaces. These investigations indicate that negatively charged surfaces facilitate or induce FXII autoactivation on the molecular level by imposing specific orientation and ordering on the adsorbed protein molecules.

Keywords: Sum frequency generation; Factor XII; Electrostatic interactions; Interfacial protein structure

Sum frequency generation spectroscopic study of the condensation effect of cholesterol on a lipid monolayer by C. Ohe; T. Sasaki; M. Noi; Y. Goto; K. Itoh (pp. 73-79).

Sum frequency generation (SFG) spectra and surface pressure–molecular area (π–A) isotherms have been obtained for mixed cholesterol–DPPC monolayers with cholesterol mole fractions, x(chol.), from 0 to 1.0, at the air–water interface, under same conditions, at 22 °C. Analysis of the spectra indicated that incorporation of cholesterol into the monolayers at 3 mN m⁻¹ greatly increases the conformational and orientational order of the alkyl chains of DPPC, maximizing these properties at x(chol.)=0.4. Analysis also indicated that order in the mixed monolayers at 15 and 35 mN m⁻¹ is not affected by incorporation of cholesterol. The π–A isotherms measured at 3 mN m⁻¹ for the mixed monolayer with x(chol.)=0.4 have the largest negative deviation of the molecular area relative to those of ideal mixtures (the so-called “condensation effect” of cholesterol), indicating the most thermodynamically stable state. Comparison of results from SFG spectra and π–A isotherms explicitly proved that the condensation effect can be interpreted in terms of conformational and orientational ordering of the alkyl chains of DPPC.

Keywords: Sum frequency generation spectroscopy; Cholesterol; Lipids; DPPC; Monolayers; Air-water interface

Silver-particle-based surface-enhanced resonance Raman scattering spectroscopy for biomolecular sensing and recognition by Kwan Kim; Hyun Sook Lee; Nam Hoon Kim (pp. 81-88).

We demonstrate in this work that 2-μm-sized Ag (μAg) powders can be used as a core material for constructing biomolecular sensing/recognition units operating via surface-enhanced resonance Raman scattering (SERRS). This is possible because μAg powders are very efficient substrates for both the diffuse reflectance IR and the surface-enhanced Raman scattering–SERRS spectroscopic characterization of molecular adsorbates prepared in a similar manner on silver surfaces. Besides, the agglomeration of μAg particles in a buffer solution can be prevented by the layer-by-layer deposition of cationic and anionic polyelectrolytes such as poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). In this particular study, we used rhodamine B isothiocyanate (RhBITC) as a SERRS marker molecule, and μAg powders adsorbed consecutively with RhBITC and PAH–PAA bilayers were finally derivatized with biotinylated poly(l-lysine). On the basis of the nature of the SERRS peaks of RhBITC, those μAg powders were confirmed to selectively recognize streptavidin molecules down to concentrations of 10⁻¹⁰ g mL⁻¹. Since a number of different molecules can be used as SERS–SERRS marker molecules, the present method proves to be an invaluable tool for multiplex biomolecular sensing/recognition via SERS and SERRS.

Keywords: Surface-enhanced Raman scattering; Surface-enhanced resonance Raman scattering; Silver powder; Molecular sensing/recognition; Biotin–streptavidin; Rhodamine B isothiocyanate

Electromagnetic and chemical interaction between Ag nanoparticles and adsorbed rhodamine molecules in surface-enhanced Raman scattering by M. Futamata; Y. Maruyama (pp. 89-102).

The critical importance of the junction between touching or closely adjacent Ag nanoparticles associated with single-molecule sensitivity (SMS) in surface-enhanced Raman scattering (SERS) was confirmed via the following observations: (1) an additional peak is observed in elastic scattering only for the SERS-active state, which originated from absorption of adsorbates, (2) local- and far-field evaluation using a finite difference time domain method could reproduce this extra peak and anticipate the significantly enhanced field even inside the adsorbates sitting at the junction through an increased coupling of the localized surface plasmons, and (3) in addition to enhanced fluorescence of adsorbed dye, an inelastic scattering peak was observed and attributed to the metal surface electron. Concerning the chemical enhancement in SERS, Cl⁻ anions activate the Ag-Cl-R6G (rhodamine) samples by inducing intrinsic electronic interaction between Ag and R6G molecules. This electronic interaction is irreversibly quenched by the addition of thiosulfate anions which dissolve Ag⁺ cations while the electromagnetic (EM) effect remains intact.

Keywords: Nanoparticles; Spectroscopy; IR spectroscopy; Raman spectroscopy; Surface analysis

Characteristics of the Raman spectra of single-walled carbon nanotube bundles under electrochemical potential control by Norihiko Takeda; Kei Murakoshi (pp. 103-108).

Resonant Raman scattering spectra of single-walled carbon nanotube–sodium dodecyl sulfate (SWNT–SDS) bundles adsorbed on Au electrodes have been investigated in aqueous electrolytes. Raman intensities of the radial breathing mode (RBM) with 785-nm laser excitation were monitored at different electrode potentials between −0.5 and +0.8 V relative to the SCE. Six resolved RBM peaks assignable to different diameter tubes all decreased in intensity when the electrode was positively biased, because of depletion of valence-band electrons associated with resonant excitation. The attenuation occurred at more positive potentials for narrower-diameter tubes with higher RBM frequencies consistent with their larger bandgaps. The results suggest the Fermi level is equilibrated in bundled SWNTs in contrast with the large Fermi-level shifts reported for isolated SWNTs.

Keywords: Single-walled carbon nanotube; Resonant Raman spectroscopy; Electrochemical potential; Oxidative doping; Fermi level

Preparation and characterization by surface-enhanced infrared absorption spectroscopy of silver nanoparticles formed on germanium substrates by electroless displacement by Jyisy Yang; Peter R. Griffiths (pp. 109-119).

In this paper, the feasibility of applying electroless displacement to prepare silver nanoparticles (AgNPs) on the surface of germanium (Ge) substrate is demonstrated, and the performances of surfaces prepared in this manner for surface-enhanced infrared absorption (SEIRA) spectroscopy are reported. The process used to produce suitable AgNPs for SEIRA by electroless deposition is simple and effective, requiring only pretreatment of the germanium surface with hot air, immersion of the substrate in a dilute solution of silver nitrate, and washing of the resulting plate. To quantify the behavior of AgNPs on a Ge substrate and to optimize the conditions for the preparation of AgNPs on Ge substrates, a monolayer of p-nitrothiophenol (PNTP) was bonded to the surface of the AgNPs by immersion of the plate in a dilute solution of PNTP and measurement of the transmission spectrum. The factors that influenced the formation of AgNPs, and hence the SEIRA signals, included the concentration of AgNO₃, the reaction time and the temperature. Results indicated that stronger absorption bands in the SEIRA spectrum of a monolayer of PNTP were obtained if the reaction rate for the displacement of silver ions by Ge was slow. This condition was achieved by keeping the concentration of AgNO₃ and the reaction temperature low. Under the optimal conditions found in this work, an enhancement factor of approximately 100 was achieved for commonly used probe molecules in SEIRA measurements.

Keywords: Surface enhancement; SEIRA; Infrared spectroscopy; Silver nanoparticles; Electroless displacement

Adsorption and self-assembly of aromatic carboxylic acids on Au/electrolyte interfaces by Bo Han; Zhihai Li; Thomas Wandlowski (pp. 121-129).

The adsorption and self-assembly of benzoic acid (BA), isophthalic acid (IA), and trimesic acid (TMA) on Au(111) single crystals and on Au(111-25 nm) quasi-single crystalline film electrodes have been investigated in 0.1 M HClO₄ by combining in situ surface-enhanced infrared reflection absorption spectroscopy (SEIRAS) and scanning tunneling microscopy (STM) with cyclic voltammetry. All three acids are physisorbed on the electrode surface in a planar orientation at negative charge densities. Excursion to positive charge densities (or more positive potentials) causes an orientation change from planar to perpendicular. Chemisorbed structures are formed through the coordination of a deprotonated carboxyl group to the positively charged electrode surface. The three acid molecules assemble in different ordered patterns, which are controlled by π-stacking (BA) or intermolecular hydrogen bonds between COOH groups (IA, TMA). A detailed analysis of the potential and time dependencies of the ν_(C=O), ν_s(OCO), and ν_(C–OH) vibration modes shows that the strength of lateral interactions increases upon chemisorption with an increasing number of COOH groups in the sequence of BA−. In addition, an “indirect” electron donation to the COOH groups takes place via the conjugated molecular skeleton superimposed on the intermolecular hydrogen bonding. Figure In-situ STM images of the physisorbed and chemisorbed adlayers of isophthalic acid on Au(111)-(1 × 1), the corresponding cyclic voltammogram and principle of the ATR-SEIRAS set-up

Keywords: Aromatic carboxylic acid; Adsorption; Self-assembly; Electrochemistry; SEIRAS; STM

In situ Raman spectroelectrochemistry of azobenzene monolayers on glassy carbon by Takashi Itoh; Richard L. McCreery (pp. 131-134).

In situ Raman spectra of chemisorbed azobenzene (AB) monolayers on glassy carbon (GC) electrodes were observed under potentiostatic conditions in acetonitrile (ACN) with tetrabutyl-ammonium tetrafluoroborate (TBA-BF₄). The Raman intensities of these spectra were high below −1000 mV, and this is attributed to the change in absorbance of AB on GC. In this paper, we describe chemisorbed AB molecules on GC electrode surfaces under potentiostatic conditions.

Keywords: IR spectroscopy/Raman spectroscopy; Electroanalytical methods; Nanoparticles/nanotechnology

Enhanced thermal stability and structural ordering in short chain n-alkanethiol monolayers on gold probed by vibrational spectroscopy and EQCM by S. Subramanian; S. Sampath (pp. 135-145).

Monolayers of alkanethiols with varied chain lengths, CH₃(CH₂)_nSH where n = 3, 5, and 7, on gold substrates have been prepared by adsorption from (1) neat thiol, (2) millimolar thiol solution in alcohol (conventional method), and (3) potential-controlled adsorption. Reflection absorption infrared spectroscopy (RAIRS) and electrochemical quartz crystal microbalance (EQCM) have been used to characterize the integrity of the monolayers. Methylene and methyl stretching modes along with C–S stretching modes have been used as benchmarks to follow the order–disorder transitions in the monolayer structure, in the temperature range from 25 to 175 °C. Monolayers adsorbed from neat thiol show superior quality in terms of stability and structural arrangement. Short chain thiols with n = 3, 5, and 7 do show substantial stability. The possibility of multilayer formation is ruled out by EQCM studies comparing the frequency and mass change associated with the monolayer desorption. Self-assembled monolayers (SAMs) adsorbed under potential control behave very similarly to the monolayers adsorbed from neat thiol as far as stability and structural orientation are concerned, irrespective of the chain length. The adsorption from neat thiol gets rid of the solvent-induced defects and arrests the propagation of defects under temperature constraints.

Keywords: RAIRS; Self-assembled monolayer; Thermal stability; EQCM; Short chain thiol

Modelling of toluene solid-phase microextraction for indoor air sampling by Pierre Mocho; Virginie Larroque; Valérie Desauziers (pp. 147-156).

Solid-phase microextraction (SPME) is a convenient and efficient sampling technique recently applied to indoor air analysis. We propose here a theoretical model of the adsorption kinetics of toluene on SPME fibre under static extraction conditions. We discuss the effects of sampling volume and initial concentration of analyte on the adsorption kinetics. This model is used to estimate the limits of detection taking into account operating conditions and to calculate theoretical calibration curves. Results of comparison with experimental data are encouraging: only 11% difference for calibration curves and 30% for the estimation of the limit of detection. On the basis of this kinetics model, the solid concentration gradient in the Carboxen coating was modelled with Fick’s second law of diffusion in unsteady-state mass-transfer mode. Mass diffusion from the gas sample to the SPME fibre was also investigated. It was shown that diffusion is the limiting step of the mass-transfer process in the static mode. Thus, the model developed, allows a better understanding of adsorption on Carboxen fibre and in the future could be a useful tool for cheap and time-saving development of SPME methods and the estimation of sampling performance. Figure PDMS/Carboxen SPME fibre (scanning electron microscopy – magnification x 220)

Keywords: Indoor air; Solid-phase microextraction; Poly(dimethylsiloxane)/Carboxen; Adsorption modelling; Toluene

DNA purification on a lab-on-valve system incorporating a renewable microcolumn with in situ monitoring by laser-induced fluorescence by Xu-Wei Chen; Zhang-Run Xu; Bai-Yan Qu; Ye-Feng Wu; Jing Zhou; Hui-Dan Zhang; Jin Fang; Jian-Hua Wang (pp. 157-163).

Bead injection in a lab-on-valve (LOV) system was adopted for DNA purification via micro solid-phase extraction (SPE) with a renewable silica microcolumn packed in a channel of the LOV unit. The complex matrix components in human whole blood, including proteins, were well eliminated by choosing properly the sample loading and elution media. The DNA purification process was monitored on-line by using laser-induced fluorescence in a demountable side part of the LOV unit incorporating optical fibers. The practical applicability of the entire system was demonstrated by separation/purification of λ-DNA in a simulated matrix and human blood genetic DNA by performing SPE, in situ monitoring of the purified products, and postcolumn PCR amplification. When DNAs in a simulated matrix (10.0 ng μl⁻¹ λ-DNA, 50 ng μl⁻¹ bovine serum albumin, 1.0% Triton X-100) were processed in the present system and laser-induced fluorescence was monitored at 610 nm, an overall extraction/collection efficiency of 70% was achieved by employing identical sample loading and an elution flow rate of 0.5 μl s⁻¹, along with a precision of 3.8% relative standard deviation. DNA separation and purification from human whole-blood samples were performed under similar conditions. Figure Lab-on-valve mesofluidic system employed for DNA separation and purification integrating a demountable fluorescence flow cell for in-situ laser induced fluorescence detection

Keywords: Lab-on-valve; Solid-phase extraction; DNA separation/purification; PCR amplification; Human whole blood

An accurate QSRR model for the prediction of the GC×GC–TOFMS retention time of polychlorinated biphenyl (PCB) congeners by Yueying Ren; Huanxiang Liu; Xiaojun Yao; Mancang Liu (pp. 165-172).

Quantitative structure–retention relationship (QSRR) models were constructed for the GC×GC–TOFMS retention time of 209 polychlorinated biphenyl (PCB) congeners. Principal component analysis (PCA) was used to recognize groups of samples with similar behavior and assist the separation of the data into training and test sets. The best multi-linear regression (BMLR) method was used for the systematic development of multi-linear regression equations; the best regression model involved four descriptors which were related to GC×GC–TOFMS chromatographic retention of PCBs. The obtained model has good predictive ability. For the test set, it gave a predictive correlation coefficient (R) of 0.988 and an average absolute relative deviation (AARD) of 3.08%. Results of a six-fold cross-validation procedure, which were in accordance with those from validation of training and test sets, demonstrated that this model was reliable. Additionally, this paper provides a simple, practical, and effective method for analytical chemists to predict the retention times of PCBs in GC.

Keywords: PCBs; QSRR; Best multi-linear regression

Analysis of glycans in glycoproteins by diffusion-ordered nuclear magnetic resonance spectroscopy by Karin Ortner; Veeramuthu N. Sivanandam; Wolfgang Buchberger; Norbert Müller (pp. 173-177).

Enzymatically cleaved glycans from sub-milligram quantities of erythropoietin (EPO) and ovalbumin have been analyzed, without further purification, by two-dimensional diffusion-ordered nuclear magnetic resonance spectroscopy. At NMR sample concentrations below 50 μmol L⁻¹ the major components of the oligosaccharide fractions could be distinguished by their anomeric proton chemical shift and their size-dependent diffusion coefficients. Figure ¹H NMR diffusion decay curves of anomeric protons in the EPO glycan fraction

Keywords: Nuclear magnetic resonance spectroscopy; Glycoproteins; Glycans; Diffusion

Vibrational overtone combination spectroscopy (VOCSY)—a new way of using IR and NIR data by Erik Alm; Rasmus Bro; Søren B. Engelsen; Bo Karlberg; Ralf J. O. Torgrip (pp. 179-188).

This work explores a novel method for rearranging 1st order (one-way) infra-red (IR) and/or near infra-red (NIR) ordinary spectra into a representation suitable for multi-way modelling and analysis. The method is based on the fact that the fundamental IR absorption and the first, second, and consecutive overtones of NIR absorptions represent identical chemical information. It is therefore possible to rearrange these overtone regions of the vectors comprising an IR and NIR spectrum into a matrix where the fundamental, 1st, 2nd, and consecutive overtones of the spectrum are arranged as either rows or columns in a matrix, resulting in a true three-way tensor of data for several samples. This tensorization facilitates explorative analysis and modelling with multi-way methods, for example parallel factor analysis (PARAFAC), N-way partial least squares (N-PLS), and Tucker models. The vibrational overtone combination spectroscopy (VOCSY) arrangement is shown to benefit from the “order advantage”, producing more robust, stable, and interpretable models than, for example, the traditional PLS modelling method. The proposed method also opens the field of NIR for true peak decomposition—a feature unique to the method because the latent factors acquired using PARAFAC can represent pure spectral components whereas latent factors in principal component analysis (PCA) and PLS usually do not.

Keywords: Near-infrared; Infrared; Calibration; PARAFAC; Multi-way; Second-order advantage

Mechanical ion gate for electrospray-ionization ion-mobility spectrometry by Li Zhou; David C. Collins; Edgar D. Lee; Milton L. Lee (pp. 189-194).

A novel ion gate for electrospray-ionization atmospheric-pressure ion-mobility spectrometry (ESI-IMS) has been constructed and evaluated. The ion gate consisted of a chopper wheel with two windows—one for periodic ion passage from the ESI source into the drift region and the other for timing and synchronization purposes. The instrument contained a 45.0 cm long drift tube comprising 78 stainless steel rings (0.12 cm thick, 4.90 cm o.d., 2.55 cm i.d.). The rings were connected together in series with 3.34-MΩ resistors. The interface plate and the back plate were also connected with the first and the last rings, respectively, of the drift tube with 3.34-MΩ resistors. A potential of −20.0 kV was applied to the back plate and the interface plate was grounded. The drift tube was maintained at an electric field strength of ∼400 V cm⁻¹. An aperture grid was attached to the last ring in front of a Faraday plate detector, center-to-center. Several sample solutions were electrosprayed at +5.0 kV with +500 V applied to the ion gate. Baseline separations of selected benzodiazepines, antidepressants, and antibiotics were observed with moderate experimental resolution of ∼70.

Keywords: Ion gate; Electrospray ionization; Ion-mobility spectrometry; Resolution

Mechanical ion gate for electrospray-ionization ion-mobility spectrometry by Li Zhou; David C. Collins; Edgar D. Lee; Milton L. Lee (pp. 189-194).

Keywords: Ion gate; Electrospray ionization; Ion-mobility spectrometry; Resolution

Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection by M. H. M. van de Meent; G. J. de Jong (pp. 195-200).

Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum albumin digest with an LC–UV–mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak capacities were determined from UV chromatograms and MS/MS data were used for Mascot database searching. Analyses with a similar gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively, and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also higher for the long column, 641 and 750 for the 3- and 15-min gradients with the short column and 1,376 and 993 for the 15- and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and increased reliability of protein identification.

Keywords: Peak capacity; Monoliths; Long columns; Gradient liquid chromatography; Protein identification

Identification of proteinaceous binders used in artworks by MALDI-TOF mass spectrometry by Stepanka Kuckova; Radovan Hynek; Milan Kodicek (pp. 201-206).

Proper identification of proteinaceous binders in artworks is essential for specification of the painting technique and thus also for selection of the restoration method; moreover, it might be helpful for the authentication of the artwork. This paper is concerned with the optimisation of analysis of the proteinaceous binders contained in the colour layers of artworks. Within this study, we worked out a method for the preparation and analysis of solid samples from artworks using tryptic cleavage and subsequent analysis of the acquired peptide mixture by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. To make this approach rational and efficient, we created a database of commonly used binders (egg yolk, egg white, casein, milk, curd, whey, gelatine, and various types of animal glues); certain peaks in the mass spectra of these binders, formed by rich protein mixtures, were matched to amino acid sequences of the individual proteins that were found in the Internet database ExPASy; their cleavage was simulated by the program Mass-2.0-alpha4. The method developed was tested on model samples of ground layers prepared by an independent laboratory and then successfully applied to a real sample originating from a painting by Edvard Munch.

Keywords: Matrix-assisted laser desorption/ionisation time of flight mass spectrometry; Artworks; Proteinaceous binders; Database

Part per trillion determination of atrazine in natural water samples by a surface plasmon resonance immunosensor by Marinella Farré; Elena Martínez; Javier Ramón; Alicia Navarro; Jelena Radjenovic; Elba Mauriz; Laura Lechuga; M. Pilar Marco; Damià Barceló (pp. 207-214).

A new immunoassay for continuously monitoring atrazine in water has been developed. It uses a portable biosensor platform based on surface plasmon resonance (SPR) technology. This immunoassay is based on the binding inhibition format with purified polyclonal antibodies, with the analyte derivative covalently immobilized on a gold sensor surface. An alkanethiol self-assembled monolayer (SAM) was formed on the gold-coated sensor surface in order to obtain a reusable sensing surface. The low detection limit for the optimized assay, calculated as the concentration that produces a 10% decrease in the blank signal, is 20 ng/L. A complete assay cycle, including regeneration, is accomplished in 25 min. Additionally, a study of the matrix effects of different types of wastewater was performed. All measurements were carried out with the SPR sensor system (β-SPR) commercialised by the company Sensia, S.L. (Spain). The small size and low response time of the β-SPR platform would allow it to be used in real contaminated locations. The immunosensor was evaluated and validated by measuring the atrazine content of 26 natural samples collected from Ebro River. Solid-phase extraction followed by gas chromatography coupled to mass spectrometric detection (SPE–GC–MS) was used to validate the new immunoassay.

Keywords: Biosensors; SPR immunosensor; Atrazine

Resolution of overlapping signals in spectrometry using a wavelet packet transform and an Elman recurrent neural network by Shouxin Ren; Ling Gao (pp. 215-225).

A novel method named a wavelet packet transform based Elman recurrent neural network (WPTERNN) was proposed for the simultaneous UV–visible spectrometric determination of Cu(II), Cd(II) and Zn(II). This method combined wavelet packet denoising with an Elman recurrent neural network. A wavelet packet transform was applied to perform data compression, to extract relevant information, and to eliminate noise and collinearity. An Elman recurrent network was applied for nonlinear multivariate calibration. In this case, using trials, the kind of wavelet function, the decomposition level, and the number of hidden nodes for the WPTERNN method were selected as Daubechies 14, 3, and 8, respectively. A program (PWPTERNN) was designed that could perform the simultaneous determination of Cu(II), Cd(II) and Zn(II). The relative standard errors of prediction (RSEP) obtained for all components using WPTERNN, a Elman recurrent neural network (ERNN), partial least squares (PLS), principal component regression (PCR), Fourier transform based PCR (FTPCR), and multivariate linear regression (MLR) were compared. Experimental results demonstrated that the WPTERRN method was successful even where there was severe overlap of spectra. The results obtained from an additional test case also demonstrated that the WPTERNN method performed very well. Figure The part of WP coefficients obtained by wavelet packet transforms

Keywords: Wavelet packet transform; Elman recurrent neural network; Overlapping signals; Spectrometry; Multicomponent analysis

Molecularly imprinted polymer solid-phase extraction coupled to square wave voltammetry at carbon fibre microelectrodes for the determination of fenbendazole in beef liver by A. Guzmán-Vázquez de Prada; Oscar A. Loaiza; B. Serra; D. Morales; P. Martínez-Ruiz; A. J. Reviejo; J. M. Pingarrón (pp. 227-234).

A molecularly imprinted polymer was developed and used for solid-phase extraction (MISPE) of the antihelmintic fenbendazole in beef liver samples. Detection of the analyte was accomplished using square wave voltammetry (SWV) at a cylindrical carbon fibre microelectrode (CFME). A mixture of MeOH/HAc (9:1) was employed both as eluent in the MISPE system and as working medium for electrochemical detection of fenbendazole. The limit of detection was 1.9 × 10⁻⁷ mol L⁻¹ (57 μg L⁻¹), which was appropriate for the determination of fenbendazole at the maximum residue level permitted by the European Commission (500 μg kg⁻¹ in liver). Given that the SW voltammetric analysis could not be directly performed in the sample extract as a consequence of interference from some sample components, a sample clean-up with a MIP for selectively retaining fenbendazole was performed. The MIP was synthesized using a 1:8:22 template/methacrylic acid/ethylene glycol dimethacrylate ratio. A Britton–Robinson Buffer of pH 9.0 was selected for retaining fenbendazole in the MIP cartridges, and an eluent volume of 5.0 mL at a flow rate of 2.0 mL min⁻¹ was chosen in the elution step. Cross-reactivity with the MIP was observed for other benzimidazoles. The synthesized MIP exhibited a good selectivity for benzimidazoles with respect to other veterinary drugs. The applicability of the MISPE-SWV method was tested with beef liver samples, spiked with fenbendazole at 5,000 and 500 μg kg⁻¹. Results obtained for ten different liver samples yielded mean recoveries of (95 ± 12)% and (96 ± 11)% for the upper and lower concentration level, respectively.

Keywords: Benzimidazoles; Fenbendazole; Molecularly imprinted polymer; Square wave voltammetry; Beef liver

Simultaneous analysis of classical neuroleptics, atypical antipsychotics and their metabolites in human plasma by Laura Mercolini; Maria Grillo; Claudio Bartoletti; Giancarlo Boncompagni; Maria Augusta Raggi (pp. 235-243).

A high-performance liquid chromatographic method has been developed for the simultaneous determination of classical neuroleptics (chlorpromazine, haloperidol, loxapine and clotiapine), atypical antipsychotics (clozapine, quetiapine and risperidone) and their active metabolites (N-desmethylclozapine, clozapine N-oxide and 9-hydroxyrisperidone) in human plasma. Separation was obtained by using a C8 reversed-phase column and a mobile phase composed of 70% aqueous phosphate buffer containing triethylamine at pH 3.0 and 30% acetonitrile. The UV detector was set at 238 nm and amitriptyline was used as the internal standard. A careful pre-treatment procedure of plasma samples was developed, using solid-phase extraction with cyanopropyl cartridges, which gives high extraction yields (≥93%). The limits of quantitation (LOQ) were always lower than 2.6 ng mL⁻¹ and the limits of detection (LOD) were always lower than 0.9 ng mL⁻¹ for all analytes. The method was applied with success to plasma samples from schizophrenic patients undergoing polypharmacy with two or more different antipsychotics. Precision data and accuracy results were satisfactory and no interference from other central nervous system (CNS) drugs was found. Hence the method is suitable for the therapeutic drug monitoring (TDM) of the analytes in psychotic patients’ plasma.

Keywords: Antipsychotic drugs; HPLC; Polypharmacy; Human plasma; Solid-phase extraction

Integrated microfluidic device with an electroplated palladium decoupler for more sensitive amperometric detection of the 8-hydroxy-deoxyguanosine (8-OH-dG) DNA adduct by Abdulilah A. Dawoud; Toshikazo Kawaguchi; Ryszard Jankowiak (pp. 245-252).

8-Hydroxy-deoxyguanosine (8-OH-dG) DNA adduct is one of the most frequently used biomarkers reporting on the oxidative stress that leads to DNA damage. More sensitive and reliable microfluidic devices are needed for the detection of these biomarkers of interest. We have developed a capillary electrophoresis (CE)-based microfluidic device with an electroplated palladium decoupler that provides significantly improved detection limit, separation efficiency, and resolving power. The poly(dimethylsiloxane) (PDMS)/glass hybrid device has fully integrated gold microelectrodes covered in situ with palladium nanoparticles using an electroplating technique. The performance and coverage of the electrodes electroplated with palladium particles were evaluated electrochemically and via scanning electron microscope (SEM) imaging, respectively. The performance of the device was tested and evaluated with different buffer systems, pH values, and electric field strengths. The results showed that this device has significantly improved resolving power, even at separation electric field strengths as high as 600 V cm⁻¹. The detection limit for the 8-OH-dG adduct is about 20 attomoles; the concentration limit is on the order of 100 nM (S/N = 3). A linear response is reported for both 8-OH-dG and dG in the range from 100 nM to 150 μM (≈100 pA μM⁻¹) with separation efficiencies of approximately 120,000–170,000 plates m⁻¹.

Keywords: Microfluidic device; DNA adducts; Biomarkers; Electroplating; Nanoparticles; Electrochemistry

Detection of antibiotics in food: Extraction of fluoroquinolones by DNA by Limin Cao; Hong Lin; Vladimir M. Mirsky (pp. 253-258).

The ability of DNA to extract fluoroquinolones from model solutions and real probes of food was demonstrated and investigated quantitatively. The interaction between fluoroquinolones and different types of DNA was studied by equilibrium dialysis. The first application of this direct approach allowed us to determine binding constants and binding stoichiometries in different conditions. The binding of enrofloxacin to heat-denatured DNA (d-DNA) from herring sperm is pH- and magnesium-dependent; the highest fraction of bound drugs was found at pH 7 and a magnesium concentration of 0.5–1 mM. Results for three types of DNA: d-DNA, double-stranded DNA and single-stranded DNA were compared. The unwound DNA showed almost doubled binding constants and stoichiometries, thus indicating preferable interaction of enrofloxacin with single-strand regions of DNA. The binding of other fluoroquinolones (lomefloxacin, ciprofloxacin, norfloxacin, danofloxacin and sarafloxacin) with d-DNA is very similar to that of enrofloxacin: the binding constants are in the range from 0.94 × 10⁵ to 2.40 × 10⁵ M⁻¹, and the stoichiometries range from 4.1 to 6.9 fluoroquinolone molecules per 100 DNA bases. The binding properties were quantitatively the same for extraction of fluoroquinolones from model aqueous solutions and from liquid food (milk). The results indicate the efficiency of DNA for selective extraction of fluoroquinolones from real samples for further analysis. This selective binding also allows us to consider DNA as a natural receptor for development of analytical techniques for fluoroquinolones.

Keywords: Fluoroquinolone; DNA; Extraction; Binding constant; Binding stoichiometry

Label-free voltammetric detection of single-nucleotide mismatches recognized by the protein MutS by Michal Masařík; Kateřina Cahová; Rene Kizek; Emil Paleček; Miroslav Fojta (pp. 259-270).

MutS, a protein involved in DNA mismatch repair, recognizes mispaired and unpaired bases in duplex DNA. We have previously used MutS in an electrochemical double-surface technique (DST) for in-vitro detection of point mutations in DNA. The DST involved binding of unlabeled MutS to DNA heteroduplexes at the surface of magnetic beads followed by a highly sensitive electrochemical determination of the protein by measurement of a catalytic protein signal (peak H) at mercury electrodes. Detection of MutS using a peak resulting from oxidation of tyrosine and tryptophan residues of the protein at a carbon-paste electrode (CPE) was also possible but was approximately three orders of magnitude less sensitive. In this work we present an optimized technique for ex-situ voltammetric determination of MutS at a CPE. Choice of optimum experimental conditions (pH of supporting electrolyte, square-wave voltammetry settings, etc.) resulted in substantial improvement of the sensitivity of the assay, enabling detection of approximately 140 pg (1.6 fmol protein monomer) MutS in a 5-μL sample. The sensitivity was increased further by acid hydrolysis of the protein before measurement. The hydrolyzed protein was detectable down to 5 pg (approx. 56 amol) MutS in 5 μL solution. By using the DST combined with determination of the bound unlabeled MutS at the CPE we demonstrated selective interactions of the protein with single-base mismatches and discrimination among different base mispairs in 30-mer or 95-mer DNA duplexes. In agreement with previous studies, binding of the protein to the 30-mer substrates followed the trend G:T>>C:A>A:A>C:T>homoduplex. The electrochemical data were confirmed by use of an independent technique—a quartz-crystal microbalance for real-time monitoring of MutS interactions with DNA duplexes containing different base mispairs. By using the electrochemical DST a G:T mismatch was detectable in up to 1000-fold excess of homoduplex DNA.

Keywords: Single-nucleotide polymorphism; Point mutation; Base mismatches; MutS; Magnetic beads; Protein electrochemistry; Quartz-crystal microbalance

Practical evaluation of universal conditions for four-plex quantitative PCR by Tsuyoshi Ishii; Hiroshi Sootome; Keizo Yamashita (pp. 271-278).

Multiplexing quantitative polymerase chain reaction (qPCR) is a powerful way to substantially increase the number of genes that can be analyzed, while also reducing sample requirements, time, and cost. However, little previous work has been done to show its feasibility for multiple gene targets. Here, we determined optimal conditions for four-color multiplex qPCR. On the basis of amplification curves, we first established that the concentration of probe-primers should be about tenfold lower than that for conventional qPCR. This condition was evaluated using four sets of probe-primers labeled with FAM, CAL Fluor Orange, TAMRA, and Quasar670, respectively. To simulate the condition that different genes have different levels of transcript abundance, a series of test samples was prepared by mixing a constant amount of two kinds of vector together with different amounts of two other vectors in a four-plex qPCR format. The PCR efficiency of the constant genes was minimally affected by the presence of the spiked vectors, and the slope factors of standard curves for the two spiked genes were sufficient for the accurate quantification. We demonstrated here that qPCR in a four-plexed format is feasible for cost-effective practical use through a combination of lower concentrations of probe-primers, an appropriate reagent, and a detection instrument. Figure Eye-catching image

Keywords: Nucleic acids (DNA|RNA); Pharmaceuticals; PCR; Bioanalytical methods

Antibody-functionalized magnetic nanoparticles for the detection of carcinoembryonic antigen using a flow-injection electrochemical device by Jin Pan; Qingwei Yang (pp. 279-286).

A magnetocontrolled immunosensing strategy based on flow-injection electrochemical impedance spectroscopy (EIS) was developed for the determination of carcinoembryonic antigen (CEA) in human serum. The immunosensor was fabricated by immobilizing anti-CEA on epoxysilane-modified core–shell magnetic Fe₃O₄/SiO₂ nanoparticles. The detection principle is based on the difference between the resistances measured before and after the antigen–antibody interaction. The performance of the immunosensor and factors influencing this performance were also proposed. The resistance response depended linearly on the CEA concentration over the range 1.5–60 ng/ml, and the immunosensor gave a detection limit of 0.5 ng/ml (S/N = 3). Coefficients of variance (CVs) of <9.8% were obtained for the intra- and interassay precisions. The method was successfully applied to the analysis of CEA in human serum. The recoveries obtained by spiking CEA standards into normal serum were 87–113%. The performance of the immunosensor was compared with a commercially available CEA ELISA. Satisfactory results were obtained according to a paired t-test method (t value < t _critical at the 95% confidence level). Importantly, the proposed immobilization protocol could be further developed to immobilize other antigens or biocompounds. Figure This study introduced a magnetocontrolled electrochemical immunosensing strategy based on antibody-functionalized magnetic core–shell Fe₃O₄/SiO₂ nanoparticles for the determination of carcinoembryonic antigen in human serum

Keywords: Carcinoembryonic antigen; Magnetocontrolled immunoassay; Magnetic composite nanoparticles; Electrochemical impedance spectroscopy

A novel microbial biosensor based on cells of Gluconobacter oxydans for the selective determination of 1,3-propanediol in the presence of glycerol and its application to bioprocess monitoring by Jaroslav Katrlík; Igor Voštiar; Jana Šefčovičová; Ján Tkáč; Vladimír Mastihuba; Milan Valach; Vladimír Štefuca; Peter Gemeiner (pp. 287-295).

Novel and selective microbial amperometric biosensors that use Gluconobacter oxydans cells to monitor the bacterial bioconversion of glycerol (Gly) to 1,3-propanediol (1,3-PD) are described. Two different mediators, ferricyanide and flexible polyvinylimidazole osmium functionalized polymer (Os-polymer), were employed to prepare two different microbial biosensors, both of which gave high detection performance. The good operational stabilities of both types of biosensor were underlined by the ability to detect 1,3-PD throughout 140 h of continuous operation. Both microbial biosensor systems showed excellent selectivity for 1,3-PD in the presence of a high excess of glycerol [selectivity ratios (1,3-PD/Gly) of 118 or 245 for the ferricyanide and Os-polymer systems, respectively]. Further, the robustness of each microbial biosensor was highlighted by the high reliability of 1,3-PD detection achieved (average RSD of standards <2%, and well below 4% for samples). The biosensor implementing the Os-polymer mediator exhibited high selectivity towards 1,3-PD detection and allowed moderate sample throughput (up to 12 h⁻¹) when integrated into a flow system. This system was used to monitor the concentration of 1,3-PD during a real bioprocess. Results from biosensor assays of 1,3-PD in bioprocess samples taken throughout the fermentation were in a very good agreement with results obtained from reference HPLC assays (R ² = 0.999).

Keywords: 1,3-Propanediol; 3-Hydroxyl-propionaldehyde; Gluconobacter oxydans ; Clostridium sp. ; Biodiesel

Differentiation of organically and conventionally produced milk by stable isotope and fatty acid analysis by Joachim Molkentin; Anette Giesemann (pp. 297-305).

Increasing sales of organic milk mean intensified tests for authenticity are required. In addition to comprehensive documentation, analytical methods to identify organic milk, and thus to differentiate it from conventional milk, are needed for consumer protection. Because the composition of milk is fundamentally dependent on the feeding of the cows, thirty-five samples from both production systems in Germany, including farm and retail milk, were collected within 12 months, to reflect seasonal variation, and appropriate properties were analysed. Fatty acid analysis enabled organic and conventional milk to be completely distinguished, because of the higher α-linolenic acid (C18:3ω3) and eicosapentaenoic acid (C20:5ω3) content of the former. Organic milk fat contained at least 0.56% C18:3ω3 whereas the maximum in conventional milk was 0.53%. Because of the parallel seasonal course of the C18:3ω3 content of organic and conventional retail samples, however, time-resolved comparison at the five sampling dates resulted in a clearer difference of 0.34 ± 0.06% on average. Analysis of stable carbon isotopes (δ¹³C) also enabled complete distinction of both types of milk; this can be explained by the different amounts of maize in the feed. For conventional milk fat δ¹³C values were −26.6‰ or higher whereas for organic milk fat values were always lower, with a maximum of −28.0‰. The time-resolved average difference was 4.5 ± 1.0‰. A strong negative correlation (r = −0.92) was found between C18:3ω3 and δ¹³C. Analysis of a larger number of samples is required to check the preliminary variation ranges obtained in this pilot study and, probably, to adjust the limits. Stable isotopes of nitrogen (δ¹⁵N) or sulfur (δ³⁴S) did not enable assignment of the origin of the milk; in cases of ambiguity, however, some trends observed might be useful in combination with other properties. Figure Correlation (r = −0.92) between δ¹³C value and C18:3ω3 content of milk fat from different production systems

Keywords: Stable isotopes; Fatty acids; Differentiation; Identification; Organic milk production; Conventional milk production

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Analytical and Bioanalytical Chemistry (v.388, #1)