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Analytical and Bioanalytical Chemistry (v.398, #4)

Focus on sensor interfaces by Pankaj Vadgama (pp. 1543-1544).

is currently Professor of Clinical Biochemistry and Director of the Interdisciplinary Research Centre (IRC) in Biomedical Materials at Queen Mary University of London. His research interests are polymeric membranes for sample separation, electrochemical metabolite sensors for continuous monitoring and microfluidics for bioreactors. He is currently Head of Service for Clinical Biochemistry at Barts and the London NHS Trust.

Protein immobilization at gold–thiol surfaces and potential for biosensing by Marco Frasconi; Franco Mazzei; Tommaso Ferri (pp. 1545-1564).

Self-assembled monolayers (SAMs) provide a convenient, flexible and simple system to tailor the interfacial properties of metals, metal oxides and semiconductors. Monomolecular films prepared by self-assembly are attractive for several exciting applications because of the unique possibility of making the selection of different types of terminal functional groups and as emerging tools for nanoscale observation of biological interactions. The tenability of SAMs as platforms for preparing biosurfaces is reviewed and critically discussed. The different immobilization approaches used for anchoring proteins to SAMs are considered as well as the nature of SAMs; particular emphasis is placed on the chemical specificity of protein attachment in view of preserving protein native structure necessary for its functionality. Regarding this aspect, particular attention is devoted to the relation between the immobilization process and the electrochemical response (i.e. electron transfer) of redox proteins, a field where SAMs have attracted remarkable attention as model systems for the design of electronic devices. Strategies for creating protein patterns on SAMs are also outlined, with an outlook on promising and challenging future directions for protein biochip research and applications.

Keywords: Interface/surface analysis; Biosensors; Protein immobilization; Thiols; Bioanalytical methods; Self-assembled monolayers

Mesoporous materials in sensing: morphology and functionality at the meso-interface by Brian J. Melde; Brandy J. Johnson (pp. 1565-1573).

Mesoporous materials are finding increasing utility in sensing applications. These applications can benefit from a surface area that may exceed 1,000 m² g⁻¹ and fast diffusion of analytes through a porous structure. This article reviews recent developments in mesoporous materials-based sensing and provides examples of the impact of different surface functionality, pore structure, and macro-morphology in an attempt to illustrate the contribution of these factors to the selectivity and sensitivity of a sensor response. The materials discussed include ordered mesoporous silicates synthesized with surfactants, hard templated ordered mesoporous carbons, and metal oxides with porous textures which have been applied to advantage in various detection schemes. Chemical functionalization of mesoporous materials through silane grafting, co-condensation, and adsorption are also addressed. Figure In mesoporous materials, morphological factors and functionality combine to produce unique characteristics

Keywords: Mesopore; Micropore; Morphology; Surfactant-template; Imprint; Metal oxide

The increasing importance of carbon nanotubes and nanostructured conducting polymers in biosensors by Emer Lahiff; Carol Lynam; Niamh Gilmartin; Richard O’Kennedy; Dermot Diamond (pp. 1575-1589).

The growing need for analytical devices requiring smaller sample volumes, decreased power consumption and improved performance have been driving forces behind the rapid growth in nanomaterials research. Due to their dimensions, nanostructured materials display unique properties not traditionally observed in bulk materials. Characteristics such as increased surface area along with enhanced electrical/optical properties make them suitable for numerous applications such as nanoelectronics, photovoltaics and chemical/biological sensing. In this review we examine the potential that exists to use nanostructured materials for biosensor devices. By incorporating nanomaterials, it is possible to achieve enhanced sensitivity, improved response time and smaller size. Here we report some of the success that has been achieved in this area. Many nanoparticle and nanofibre geometries are particularly relevant, but in this paper we specifically focus on organic nanostructures, reviewing conducting polymer nanostructures and carbon nanotubes. Figure

Keywords: Biosensor; Carbon nanotubes; Conducting polymers; Nanomaterials; Biomolecule immobilisation

Digital biosensors with built-in logic for biomedical applications—biosensors based on a biocomputing concept by Joseph Wang; Evgeny Katz (pp. 1591-1603).

This article reviews biomolecular logic systems for bioanalytical applications, specifically concentrating on the prospects and fundamental and practical challenges of designing digitally operating biosensors logically processing multiple biochemical signals. Such digitally processed information produces a final output in the form of a yes/no response through Boolean logic networks composed of biomolecular systems, and hence leads to a high-fidelity biosensing compared with traditional single (or parallel) sensing devices. It also allows direct coupling of the signal processing with chemical actuators to produce integrated “smart” “sense/act” (biosensor-bioactuator) systems. Unlike common biosensing devices based on a single input (analyte), devices based on biochemical logic systems require a fundamentally new approach for the sensor design and operation and careful attention to the interface of biocomputing systems and electronic transducers. As common in conventional biosensors, the success of the enzyme logic biosensor would depend, in part, on the immobilization of the biocomputing reagent layer. Such surface confinement provides a contact between the biocomputing layer and the transducing surface and combines efficiently the individual logic-gate elements. Particular attention should thus be given to the composition, preparation, and immobilization of the biocomputing surface layer, to the role of the system scalability, and to the efficient transduction of the output signals. By processing complex patterns of multiple physiological markers, such multisignal digital biosensors should have a profound impact upon the rapid diagnosis and treatment of diseases, and particularly upon the timely detection and alert of medical emergencies (along with immediate therapeutic intervention). Other fields ranging from biotechnology to homeland security would benefit from these advances in new biocomputing biosensors and the corresponding closed-loop “add/act” operation. Figure Biochemical computing and logic-gate systems based on biomolecules have the potential to revolutionize the field of biosensors. This article reviews the prospects, fundamental and practical challenges of designing digitally operating biosensors logically processing multiple biochemical signals.

Keywords: Biosensor; Biocomputing; Biomolecular computing; Logic gate; Logic network; Enzyme; Biomedical application; Electrode

Towards a fluorescent molecular switch for nucleic acid biosensing by Melissa Massey; Ulrich J. Krull (pp. 1605-1614).

A novel fluorescent molecular switch for the detection of nucleic acid hybridization has been explored in relation to the development of a structure that would be amenable for operation when immobilized for solid-phase analyses. The structure was prepared by self-assembly, and used Neutravidin as the central multivalent docking molecule, a newly synthesized biotinylated long-chain linker for intercalating dye that was modified with thiazole orange (TO) at one end, and a biotinylated probe oligonucleotide. Self-assembly of the biotinylated components on adjacent Neutravidin binding sites allowed for physical placement of an oligonucleotide probe molecule next to tethered TO. The TO located at the end of the flexible linker chain was available to intercalate, and could report if a duplex structure was formed by a probe–target interaction by means of fluorescence intensity. Subsequently, regeneration of the single-stranded probe was possible without loss of the intercalator to solution. The switch constructs were assembled in solution and subsequently immobilized onto biotin functionalized optical fibers to complete the sensor design. Solution-phase fluorescence lifetime data showed a biexponential behavior for switch constructs, suggesting intercalation as well as a significant secondary binding mode for the immobilized TO. It was found that the secondary binding mechanism for the dye to DNA could be decreased, thus shifting the dye to intercalative binding modes, by adjusting the solution conditions to a pH below the pI of Neutravidin, and by increasing the ionic strength of the buffer. Preliminary work demonstrated that it was possible to achieve up to a fivefold increase in fluorescence intensity on hybridization to the target.

Keywords: Thiazole orange; Nucleic acid; Hybridization; Molecular switch; Fluorescence

Novel multicolor fluorescently labeled silica nanoparticles for interface fluorescence resonance energy transfer to and from labeled avidin by Sayed M. Saleh; Rainer Müller; Heike S. Mader; Axel Duerkop; Otto S. Wolfbeis (pp. 1615-1623).

Fluorescent silica nanoparticles (SiNPs) were prepared by covalent attachment of fluorophores to the amino-modified surface of SiNPs with a typical diameter of 15 nm. The SiNPs are intended for use in novel kinds of fluorescence resonance energy transfer (FRET)-based affinity assays at the interface between nanoparticle and sample solution. Various labels were employed to obtain a complete set of colored SiNPs, with excitation maxima ranging from 337 to 659 nm and emission maxima ranging from 436 nm to the near infrared (710 nm). The nanoparticles were characterized in terms of size and composition using transmission electron microscopy, thermogravimetry, elemental analysis, and dynamic light scattering. The surface of the fluorescent SiNPs was biotinylated, and binding of labeled avidin to the surface was studied via FRET in two model cases. In the first, FRET occurs from the biotinylated fluorescent SiNP (the donor) to the labeled avidin (the acceptor). In the second, FRET occurs in the other direction. Aside from its use in the biotin–avidin system, such SiNPs also are believed to be generally useful fluorescent markers in various kinds of FRET assays, not the least because the fluorophore is located on the surface of the SiNPs (and thus always much closer to the second fluorophore) rather than being doped deep in its interior.

Keywords: Silica nanoparticles; Surface modification; Near-infrared label; FRET; Surface affinity assay; Biotin

Optimisation of the conditions for stripping voltammetric analysis at liquid–liquid interfaces supported at micropore arrays: a computational simulation by Jörg Strutwolf; Damien W. M. Arrigan (pp. 1625-1631).

Micropore membranes have been used to form arrays of microinterfaces between immiscible electrolyte solutions (µITIES) as a basis for the sensing of non-redox-active ions. Implementation of stripping voltammetry as a sensing method at these arrays of µITIES was applied recently to detect drugs and biomolecules at low concentrations. The present study uses computational simulation to investigate the optimum conditions for stripping voltammetric sensing at the µITIES array. In this scenario, the diffusion of ions in both the aqueous and the organic phases contributes to the sensing response. The influence of the preconcentration time, the micropore aspect ratio, the location of the microinterface within the pore, the ratio of the diffusion coefficients of the analyte ion in the organic and aqueous phases, and the pore wall angle were investigated. The simulations reveal that the accessibility of the microinterfaces during the preconcentration period should not be hampered by a recessed interface and that diffusional transport in the phase where the analyte ions are preconcentrated should be minimized. This will ensure that the ions are accumulated within the micropores close to the interface and thus be readily available for back transfer during the stripping process. On the basis of the results, an optimal combination of the examined parameters is proposed, which together improve the stripping voltammetric signal and provide an improvement in the detection limit. Figure Simulation was used to study stripping voltammetry at micro-liquid-liquid interface arrays. Optimum conditions include the minimisation of diffusion within the gelled organic phase.

Keywords: ITIES; Liquid–liquid interface; Stripping voltammetry; Computational electrochemistry; Simulation

In situ evaluation of chromium–DNA damage using a DNA-electrochemical biosensor by S. Carlos B. Oliveira; A. M. Oliveira-Brett (pp. 1633-1641).

The in situ evaluation of the direct interaction of chromium species with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine bases. The results obtained revealed the interaction with dsDNA of the Cr(IV) and Cr(V) reactive intermediates of Cr(III) oxidation by O₂ dissolved in the solution bound to dsDNA. This interaction leads to different modifications and causes oxidative damage in the B-DNA structure. Using polyhomonucleotides of guanine and adenine, it was shown that the interaction between reactive intermediates Cr(IV) and Cr(V)–DNA causes oxidative damage and preferentially takes place at guanine-rich segments, leading to the formation of 8-oxoguanine, the oxidation product of guanine residues and a biomarker of DNA oxidative damage. The interaction of Cr(VI) with dsDNA causes breaking of hydrogen bonds, conformational changes, and unfolding of the double helix, which enables easier access of other oxidative agents to interact with DNA, and the occurrence of oxidative damage to DNA.

Keywords: Chromium; Chromium intermediates–DNA interaction; DNA damage; Oxidative damage; Milli-8-Oxoguanine; Differential pulse voltammetry; Glassy carbon

Direct electrochemistry and bioelectrocatalysis of a class II non-symbiotic plant haemoglobin immobilised on screen-printed carbon electrodes by Fereshteh Chekin; Nélida Leiva; Jahan Bakhsh Raoof; Lo Gorton; Leif Bülow (pp. 1643-1649).

In this study, direct electron transfer (ET) has been achieved between an immobilised non-symbiotic plant haemoglobin class II from Beta vulgaris (nsBvHb2) and three different screen-printed carbon electrodes based on graphite (SPCE), multi-walled carbon nanotubes (MWCNT-SPCE), and single-walled carbon nanotubes (SWCNT-SPCE) without the aid of any electron mediator. The nsBvHb2 modified electrodes were studied with cyclic voltammetry (CV) and also when placed in a wall-jet flow through cell for their electrocatalytic properties for reduction of H₂O₂. The immobilised nsBvHb2 displayed a couple of stable and well-defined redox peaks with a formal potential (E°′) of −33.5 mV (vs. Ag|AgCl|3 M KCl) at pH 7.4. The ET rate constant of nsBvHb2, k _s, was also determined at the surface of the three types of electrodes in phosphate buffer solution pH 7.4, and was found to be 0.50 s⁻¹ on SPCE, 2.78 s⁻¹ on MWCNT-SPCE and 4.06 s⁻¹ on SWCNT-SPCE, respectively. The average surface coverage of electrochemically active nsBvHb2 immobilised on the SPCEs, MWCNT-SPCEs and SWCNT-SPCEs obtained was 2.85 × 10⁻¹⁰ mol cm⁻², 4.13 × 10⁻¹⁰ mol cm⁻² and 5.20 × 10⁻¹⁰ mol cm⁻². During the experiments the immobilised nsBvHb2 was stable and kept its electrochemical and catalytic activities. The nsBvHb2 modified electrodes also displayed an excellent response to the reduction of hydrogen peroxide (H₂O₂) with a linear detection range from 1 μM to 1000 μM on the surface of SPCEs, from 0.5 μM to 1000 μM on MWCNT-SPCEs, and from 0.1 μM to 1000 μM on SWCNT-SPCEs. The lower limit of detection was 0.8 μM, 0.4 μM and 0.1 μM at 3σ at the SPCEs, the MWCNT-SPCEs, and the SWCNT-SPCEs, respectively, and the apparent Michaelis–Menten constant, $$ {hbox{K}}_{ m{M}}^{ m{app}} $$ , for the H₂O₂ sensors was estimated to be 0.32 mM , 0.29 mM and 0.27 mM, respectively.

Keywords: Screen-printed carbon electrodes; Non-symbiotic plant haemoglobin; Carbon nanotube; Direct electron transfer; Hydrogen peroxide

Enzymatic electrodes nanostructured with functionalized carbon nanotubes for biofuel cell applications by E. Nazaruk; K. Sadowska; J. F. Biernat; J. Rogalski; G. Ginalska; R. Bilewicz (pp. 1651-1660).

Nanostructured bioelectrodes were designed and assembled into a biofuel cell with no separating membrane. The glassy carbon electrodes were modified with mediator-functionalized carbon nanotubes. Ferrocene (Fc) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) bound chemically to the carbon nanotubes were found useful as mediators of the enzyme catalyzed electrode processes. Glucose oxidase from Aspergillus niger AM-11 and laccase from Cerrena unicolor C-139 were incorporated in a liquid-crystalline matrix-monoolein cubic phase. The carbon nanotubes–nanostructured electrode surface was covered with the cubic phase film containing the enzyme and acted as the catalytic surface for the oxidation of glucose and reduction of oxygen. Thanks to the mediating role of derivatized nanotubes the catalysis was almost ten times more efficient than on the GCE electrodes: catalytic current of glucose oxidation was 1 mA cm⁻² and oxygen reduction current exceeded 0.6 mA cm⁻². The open circuit voltage of the biofuel cell was 0.43 V. Application of carbon nanotubes increased the maximum power output of the constructed biofuel cell to 100 μW cm⁻² without stirring of the solution which was ca. 100 times more efficient than using the same bioelectrodes without nanotubes on the electrode surface. Figure Schematic representation of biofuel cell circuit

Keywords: Biofuel cell; Laccase; Glucose oxidase; Cubic phase; Carbon nanotubes

Redox electrodeposition polymers: adaptation of the redox potential of polymer-bound Os complexes for bioanalytical applications by Dmitrii A. Guschin; John Castillo; Nina Dimcheva; Wolfgang Schuhmann (pp. 1661-1673).

The design of polymers carrying suitable ligands for coordinating Os complexes in ligand exchange reactions against labile chloro ligands is a strategy for the synthesis of redox polymers with bound Os centers which exhibit a wide variation in their redox potential. This strategy is applied to polymers with an additional variation of the properties of the polymer backbone with respect to pH-dependent solubility, monomer composition, hydrophilicity etc. A library of Os-complex-modified electrodeposition polymers was synthesized and initially tested with respect to their electron-transfer ability in combination with enzymes such as glucose oxidase, cellobiose dehydrogenase, and PQQ-dependent glucose dehydrogenase entrapped during the pH-induced deposition process. The different polymer-bound Os complexes in a library containing 50 different redox polymers allowed the statistical evaluation of the impact of an individual ligand to the overall redox potential of an Os complex. Using a simple linear regression algorithm prediction of the redox potential of Os complexes becomes feasible. Thus, a redox polymer can now be designed to optimally interact in electron-transfer reactions with a selected enzyme. Figure A library of redox electrodeposition polymers was synthesized and the formal potentials of the polymer-bound Os-complexes were adjusted through variations of the coordination shell. Optimal adaptation to the redox potentials of enzymes could be attained

Keywords: Redox polymer; Electrodeposition polymer; Biosensor; Os complex; Redox potential

Glassy carbon electrodes modified by multiwalled carbon nanotubes and poly(neutral red): A comparative study of different brands and application to electrocatalytic ascorbate determination by Ricardo C. Carvalho; Carla Gouveia-Caridade; Christopher M. A. Brett (pp. 1675-1685).

The electrochemical behaviour of glassy carbon electrodes coated with multiwalled carbon nanotubes (MWCNT) from three different sources and with different loadings has been compared, with a view to sensor applications. Additionally, poly(neutral red) (PNR) was electrosynthesised by potential cycling on bare glassy carbon and on MWCNT-modified glassy carbon electrodes, and characterised by cyclic voltammetry and scanning electron microscopy. Well-defined voltammetric responses were observed for hexacyanoferrate (II) oxidation with differences between the MWCNT types as well as from loading. The MWCNT and PNR/MWCNT-modified electrodes were applied to the oxidative determination of ascorbate, the electrocatalytic effects observed varying according to the type of nanotubes. Comparison was made with electrodes surface-modified by graphite powder. All modified electrode configurations with and without PNR were successfully employed for ascorbate oxidation at +0.05 V vs saturated calomel electrode with detection limits down to 4 μM; good operational stability and storage stability were also obtained.

Keywords: Multiwalled carbon nanotubes; Poly(neutral red); Ascorbate; Modified electrodes

Bio-sensing using recessed gold-filled capillary amperometric electrodes by A. Kacanovska; Z. Rong; M. Schmidt; P. St. J. Russell; P. Vadgama (pp. 1687-1694).

A novel recessed electrode is reported for amperometric detection of hydrogen peroxide and via glucose oxidase for the detection of glucose. The electrode utilised electrodeposited platinum over a gold wire surface, which proved to be an effective peroxide-detecting surface. Compared with a traditional exposed electrode surface, the recessed tip facilitated an extended linear range for glucose from 4 to over 14 mM. Bio-fouling, as assessed by exposure to bovine serum albumin, was also significantly reduced. Though response time at the recess was increased, it was within an acceptable range for physiological monitoring. Moreover, the recess enabled precise measurement of the hydrogen peroxide diffusion coefficient; this was based on a bipartite expression for the transient amperometric current at the recessed structure following a step change in ambient hydrogen peroxide concentration. An important aspect of the diffusion measurement was the curve fitting routine used to map on to the theoretical response curve. Figure Needle glucose electrodes for simplified monitoring

Keywords: Recessed tip electrode; Hydrogen peroxide; Glucose; Diffusion coefficient; Amperometry

Modeling the relative impact of capsular tissue effects on implanted glucose sensor time lag and signal attenuation by Matthew T. Novak; Fan Yuan; William M. Reichert (pp. 1695-1705).

Little is known mechanistically about why implanted glucose sensors lag behind blood glucose levels in both the time to peak sensor response and the magnitude of peak sensor response. A mathematical model of glucose transport from capillaries through surrounding tissue to the sensor surface was constructed to address how different aspects of the tissue affect glucose transport to an implanted sensor. Physiologically relevant values of capsule diffusion coefficient, capsule porosity, cellular glucose consumption, capsule thickness, and subcutaneous vessel density were used as inputs to create simulated sensor traces that mimic experimental instances of time lag and concentration attenuation relative to a given blood glucose profile. Using logarithmic sensitivity analysis, each parameter was analyzed to study the effect of these variables on both lag and attenuation. Results identify capsule thickness as the strongest determinant of sensor time lag, while subcutaneous vessel density and capsule porosity had the largest effects on attenuation of glucose that reaches the sensor surface. These findings provide mechanistic insight for the rational design of sensor modifications that may alleviate the deleterious consequences of tissue effects on implanted sensor performance.

Keywords: Biomaterials; Biosensors; Modeling

Raman spectroscopic imaging for in vivo detection of cerebral brain metastases by Matthias Kirsch; Gabriele Schackert; Reiner Salzer; Christoph Krafft (pp. 1707-1713).

We report for the first time a proof-of-concept experiment employing Raman spectroscopy to detect intracerebral tumors in vivo by brain surface mapping. Raman spectroscopy is a non-destructive biophotonic method which probes molecular vibrations. It provides a specific fingerprint of the biochemical composition and structure of tissue without using any labels. Here, the Raman system was coupled to a fiber-optic probe. Metastatic brain tumors were induced by injection of murine melanoma cells into the carotid artery of mice, which led to subcortical and cortical tumor growth within 14 days. Before data acquisition, the cortex was exposed by creating a bony window covered by a calcium fluoride window. Spectral contributions were assigned to proteins, lipids, blood, water, bone, and melanin. Based on the spectral information, Raman images enabled the localization of cortical and subcortical tumor cell aggregates with accuracy of roughly 250 μm. This study demonstrates the prospects of Raman spectroscopy as an intravital tool to detect cerebral pathologies and opens the field for biophotonic imaging of the living brain. Future investigations aim to reduce the exposure time from minutes to seconds and improve the lateral resolution.

Keywords: In vivo Raman spectroscopy; Brain metastases; Fiber-optic probes; Murine brain tumor model

Selective extraction of low molecular weight proteins by mesoporous silica particles with modified internal and external surfaces by Yanxia Qi; Dapeng Wu; Junying Wei; Kun Ding; Hua Wang; Yangjun Zhang; Xiaohong Qian; Yafeng Guan (pp. 1715-1722).

A new mesoporous silica material with modified external and internal surfaces (alkyl diol–vinyl-SiO₂) was prepared and applied to selectively extract low molecular weight (LMW) proteins and peptides (less than 11 kDa) from biological samples. X-ray diffraction, N₂ adsorption, transmission electron microscopy, and Fourier transform infrared spectroscopy were used to characterize the alkyl diol–vinyl-SiO₂. This material was used to extract LMW proteins (peptides) from standard proteins, a mixture of peptides, and crude human plasma. We compared the amounts of different proteins adsorbed by alkyl diol–vinyl-SiO₂ and by vinyl-SiO₂ (vinyl group on the internal surface of the mesoporous silica material). The desorption recovery of alkyl diol–vinyl-SiO₂ by different eluents was also investigated by using a standard protein (insulin) as a model LMW protein. The material could efficiently extract LMW proteins and peptides from a mixture of standard proteins and crude human plasma with good extraction efficiency and desorption recovery. Furthermore, the alkyl diol–vinyl-SiO₂ had much better extraction selectivity for LMW proteins and peptides than a commercial C₁₈ solid phase extraction material.

Keywords: Interior and external modification; Mesoporous silica; Peptides; Selective extraction

Allergen microarrays on high-sensitivity silicon slides by Marina Cretich; Daniela Breda; Francesco Damin; Marta Borghi; Laura Sola; Selim M. Unlu; Samuele E. Burastero; Marcella Chiari (pp. 1723-1733).

We have recently introduced a silicon substrate for high-sensitivity microarrays, coated with a functional polymer named copoly(DMA-NAS-MAPS). The silicon dioxide thickness has been optimized to produce a fluorescence intensification due to the optical constructive interference between the incident and reflected lights of the fluorescent radiation. The polymeric coating efficiently suppresses aspecific interaction, making the low background a distinctive feature of these slides. Here, we used the new silicon microarray substrate for allergy diagnosis, in the detection of specific IgE in serum samples of subjects with sensitizations to inhalant allergens. We compared the performance of silicon versus glass substrates. Reproducibility data were measured. Moreover, receiver-operating characteristic (ROC) curves were plotted to discriminate between the allergy and no allergy status in 30 well-characterized serum samples. We found that reproducibility of the microarray on glass supports was not different from available data on allergen arrays, whereas the reproducibility on the silicon substrate was consistently better than on glass. Moreover, silicon significantly enhanced the performance of the allergen microarray as compared to glass in accurately identifying allergic patients spanning a wide range of specific IgE titers to the considered allergens.

Keywords: Protein microarrays; Diagnosis; Fluorescence; Allergy; Sensitivity; Specificity; ROC curves

Development and validation of a sandwich ELISA for the determination of potentially allergenic sesame (Sesamum indicum) in food by Gerda Redl; Fatima T. Husain; Ines E. Bretbacher; Albert Nemes; Margit Cichna-Markl (pp. 1735-1745).

This paper presents a sandwich enzyme-linked immunosorbent assay (ELISA) that allows the determination of traces of sesame in food. Chicken anti-sesame antibodies, used as coating antibodies, and rabbit anti-sesame antibodies, used as secondary antibodies, were prepared by immunization with a protein extract of white, peeled sesame. The ELISA did not show any cross-reactivity with 19 food ingredients commonly found in sesame-containing foodstuffs such as seeds, nuts, and cereals. In whole grain bread, crisp toast, and snacks, the limit of detection (S/N = 3) was 0.5, 0.5, and 0.3 μg sesame protein/g, and the limit of quantification (S/N = 10) was 0.6, 0.8, and 1.4 μg sesame protein/g, respectively. The analysis of blank food matrices (whole grain bread, white bread, crisp toast, and snacks) spiked with sesame protein at four spike levels generally resulted in mean recoveries from 72% to 145%. In the case of spiking blank food matrices with sesame seeds, the ELISA proved to be more accurate for whole wheat cookies than for whole wheat bread. Figure Applicability of the ELISA to detect unroasted and roasted white sesame seeds

Keywords: Sesame; Sesamum indicum ; Allergen; Enzyme-linked immunosorbent assay (ELISA); Food

Automating a 96-well microtiter plate assay for identification of AGEs inhibitors or inducers: application to the screening of a small natural compounds library by Séverine Derbré; Julia Gatto; Aude Pelleray; Laurie Coulon; Denis Séraphin; Pascal Richomme (pp. 1747-1758).

Advanced glycation end-products (AGEs) are involved in the pathogenesis of numerous affections such as diabetes and neurological diseases. AGEs are also implied in various changes in tissues and organs. Therefore, compounds able to break them or inhibit their formation may be considered as potential drugs, dietary supplements, or bioactive additives. In this study, we have developed a rapid and reliable (Z′ factor calculation) anti-AGEs activity screening based on the overall fluorescence of AGEs. This method was successfully evaluated on known AGEs inhibitors and on a small library of natural compounds, yielding coherent results when compared with literature data.

Keywords: Advanced glycation end-products; Aging; Automation; Diabetes; Natural products; Biological screening

Testosterone metabolism revisited: discovery of new metabolites by Oscar J. Pozo; Josep Marcos; Rosa Ventura; Andreu Fabregat; Jordi Segura (pp. 1759-1770).

The metabolism of testosterone is revisited. Four previously unreported metabolites were detected in urine after hydrolysis with KOH using a liquid chromatography–tandem mass spectrometry method and precursor ion scan mode. The metabolites were characterized by a product ion scan obtained with accurate mass measurements. Androsta-4,6-dien-3,17-dione, androsta-1,4-dien-3,17-dione, 17-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione were proposed as feasible structures for these metabolites on the basis of the mass spectrometry data. The proposed structures were confirmed by analysis of synthetic reference compounds. Only 15-androsten-3,17-dione could not be confirmed, owing to the lack of a commercially available standard. That all four compounds are testosterone metabolites was confirmed by the qualitative analysis of several urine samples collected before and after administration of testosterone undecanoate. The metabolite androsta-1,4-dien-3,17-dione has a structure analogous to that of the exogenous anabolic steroid boldenone. Specific transitions for boldenone and its metabolite 17β-hydroxy-5β-androst-1-en-3-one were also monitored. Both compounds were also detected after KOH treatment, suggesting that this metabolic pathway is involved in the endogenous detection of boldenone previously reported by several authors. Figure Four previously unreported testosterone metabolites released after basic treatment of the urine have been characterized using mass spectrometric techniques. The structure of three of them has been confirmed by comparison with reference material.

Keywords: Testosterone; Metabolites; Mass spectrometry; Doping analysis; Boldenone

Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC–HR-MS by David Falck; Jon S. B. de Vlieger; Wilfried M. A. Niessen; Jeroen Kool; Maarten Honing; Martin Giera; Hubertus Irth (pp. 1771-1780).

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures. Figure P38 α online screening platform

Keywords: p38 MAP kinase; High-resolution screening; Fluorescence enhancement; LC–MS; Bioassays

Evaluation of standard and advanced preprocessing methods for the univariate analysis of blood serum ¹H-NMR spectra by Tim De Meyer; Davy Sinnaeve; Bjorn Van Gasse; Ernst-R Rietzschel; Marc L. De Buyzere; Michel R. Langlois; Sofie Bekaert; José C. Martins; Wim Van Criekinge (pp. 1781-1790).

Proton nuclear magnetic resonance (¹H-NMR)-based metabolomics enables the high-resolution and high-throughput assessment of a broad spectrum of metabolites in biofluids. Despite the straightforward character of the experimental methodology, the analysis of spectral profiles is rather complex, particularly due to the requirement of numerous data preprocessing steps. Here, we evaluate how several of the most common preprocessing procedures affect the subsequent univariate analyses of blood serum spectra, with a particular focus on how the standard methods perform compared to more advanced examples. Carr–Purcell–Meiboom–Gill 1D ¹H spectra were obtained for 240 serum samples from healthy subjects of the Asklepios study. We studied the impact of different preprocessing steps—integral (standard method) and probabilistic quotient normalization; no, equidistant (standard), and adaptive-intelligent binning; mean (standard) and maximum bin intensity data summation—on the resonance intensities of three different types of metabolites: triglycerides, glucose, and creatinine. The effects were evaluated by correlating the differently preprocessed NMR data with the independently measured metabolite concentrations. The analyses revealed that the standard methods performed inferiorly and that a combination of probabilistic quotient normalization after adaptive-intelligent binning and maximum intensity variable definition yielded the best overall results (triglycerides, R = 0.98; glucose, R = 0.76; creatinine, R = 0.70). Therefore, at least in the case of serum metabolomics, these or equivalent methods should be preferred above the standard preprocessing methods, particularly for univariate analyses. Additional optimization of the normalization procedure might further improve the analyses.

Keywords: NMR metabolomics preprocessing; Serum; Normalization; Binning; Bucketing

Development and validation of a liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of tamoxifen, anastrozole, and letrozole in human plasma and its application to a clinical study by Beate Beer; Birthe Schubert; Anne Oberguggenberger; Verena Meraner; Michael Hubalek; Herbert Oberacher (pp. 1791-1800).

There is substantial evidence that circulating estrogens promote the proliferation of breast cancer. Consequently, adjuvant hormonal treatment strategies targeting estrogen action have been established. Such hormonal therapies include selective estrogen receptor modulators, such as tamoxifen, which interfere at the estrogen receptors directly, or non-steroidal aromatase inhibitors, such as anastrozole and letrozole, which inhibit estrogen synthesis through blocking the aromatase, a key enzyme of estrogen production. Despite considerable therapeutic success, in several cases, the use of these drugs is limited by side effects that have been described to significantly impair the adherence of patients to endocrine treatment. However, objective data concerning patient adherence and its clinical relevance are limited. One promising approach to check patient-reported adherence is drug monitoring in human plasma. Therefore, a liquid chromatography–tandem mass spectrometry method to determine the plasma concentrations of tamoxifen, anastrozole, and letrozole has been developed and fully validated according to guidelines for clinical and forensic toxicology. The validation criteria evaluated were selectivity, linearity, accuracy and precision, limit of quantification, recovery and matrix effects, sample stability, and carryover. The six-point calibration curves showed linearity over the range of concentrations from 25 to 500 ng/ml for tamoxifen, 5 to 200 ng/ml for anastrozole, and 10 to 300 ng/ml for letrozole. The intra- and inter-day precision and accuracies were always better than 15%. The validated procedure was successfully applied to a clinical study (Patient-Reported Outcomes in Breast Cancer Patients undergoing Endocrine Therapy, PRO-BETh). A major aim of PRO-BETh study is the comprehensive evaluation of adherence to treatment in pre- and post-menopausal women with breast cancer. Plasma samples of 310 breast cancer patients undergoing anti-estrogen therapy were analyzed. Eight samples did not contain a quantifiable amount of drug, strongly indicating non-adherence of the corresponding patients to adjuvant breast cancer treatment. Furthermore, plasma concentrations at the lower end of the observed plasma level distribution might represent a hint but not a confirmation for non-adherence in terms of non-daily and irregular intake of the prescribed drug.

Keywords: LC/MS/MS; Drug monitoring; Adherence; Tamoxifen; Anastrozole; Letrozole

A rapid and sensitive assay for determining human brain levels of farnesyl-(FPP) and geranylgeranylpyrophosphate (GGPP) and transferase activities using UHPLC–MS/MS by Gero P. Hooff; Nina Patel; W. Gibson Wood; Walter E. Müller; Gunter P. Eckert; Dietrich A. Volmer (pp. 1801-1808).

The isoprenoids farnesyl-(FPP) and geranylgeranylpyrophosphate (FPP and GGPP) are two major lipid intermediates in the mevalonate pathway. They participate in post-translational modification of members of the superfamily of small guanosine triphosphatases (GTPases; Ras, Rab, Rac, etc.) via prenylation reactions. Due to the important role of these proteins in a number of cell processes, in particular cell growth, division, and differentiation, investigation of the involvement of isoprenoids in these processes is of great interest. In a previously published report, we described a fully validated assay for the quantitation of the two isoprenoids using a high-performance liquid chromatography (HPLC)–fluorescence detection (FLD) method. The current work expands on the previous method and enhances it greatly by using a much faster state-of-the-art ultrahigh-performance liquid chromatography (UHPLC) technique coupled to tandem mass spectrometry (MS/MS). The method exhibited a linear concentration range of 5–250 ng/mL for FPP and GGPP in human brain tissue; it was shown to be unaffected by ion suppression and provided results almost six times faster than the HPLC–FLD assay. Comparison of UHPLC–MS/MS and HPLC–FLD yielded excellent comparability of the two assays for both isoprenoids. Based on the UHPLC–MS/MS assay, a novel in vitro test system was implemented to study enzyme specificity for distinct amino acid CAAX motifs, which is potentially useful for investigating target interactions of new therapeutics for diseases involving pathological regulation of isoprenoids and/or small GTPases. Figure Abbreviated mevalonate/isoprenoid/cholesterol pathway. 3-HMG-CoA reductase activity leads to mevalonate (MVA), which is a precursor of farnesyl pyrophosphate (FPP) formed by FPP synthase. FPP is a branching point and serves as a precursor of geranylgeranyl pyrophosphate (GGPP) by activation of GGPP synthase. Both FPP and GGPP are involved in post-translational modification of proteins (protein prenylation). FPP is also the precursor of cholesterol

Keywords: Prenylation kinetics; Post-translational modification (PTM); Farnesyl pyrophosphate (FPP); Geranylgeranyl pyrophosphate (GGPP); Ultrahigh-performance liquid chromatography (UHPLC); Tandem mass spectrometry; Brain tissue

Coelenterazine-v ligated to Ca²⁺-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase by Galina A. Stepanyuk; James Unch; Natalia P. Malikova; Svetlana V. Markova; John Lee; Eugene S. Vysotski (pp. 1809-1817).

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some “yellow” Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca²⁺-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

Keywords: Bioluminescence; Coelenterazine; Calcium; Imaging

Multi-walled carbon nanotubes as solid-phase extraction adsorbent for the ultra-fast determination of chloramphenicol in egg, honey, and milk by fused-core C18-based high-performance liquid chromatography–tandem mass spectrometry by Yanbin Lu; Qing Shen; Zhiyuan Dai; Hong Zhang (pp. 1819-1826).

In the present work, a high-performance liquid chromatography–tandem mass spectrometry method has been developed for the residue analysis of chloramphenicol (CAP) in several food matrices. Following the addition of D₅-CAP as internal standard, egg, honey, and milk were extracted and cleaned by means of solid-phase extraction, utilizing multi-walled carbon nanotubes as sorbent. The extracts were separated on a Halo fused-core C18 column (50 mm × 2.1 mm, 2.7 μm) and quantified by a 4000 Q-trap mass spectrometer equipped with a TurboIonSpray™ interface using electrospray ionization and multiple-reaction monitoring mode. The method validation was performed according to the criteria of Commission Decision 2002/657/EC. The decision limit (CCα) and detection capability (CCβ) of CAP in milk were calculated for m/z 320.8 > 151.9. Due to the existence of slight signal suppression, quantification was performed by matrix-matched calibration curves, ranging from 0.1 to 100 ng mL⁻¹, with regression coefficients of 0.9993, 0.9998, and 0.9997 for egg, honey, and milk, respectively. Mean recoveries of the CAP ranged from 95.8% to 102.3%, with the corresponding intra- and inter-day variation (relative standard deviation) less than 7.13% and 8.89%, respectively. The limit of detection and limit of quantification of the method were also reported. This method successfully applied to several food matrixes (egg, honey, and milk) and can serve as a monitoring tool to avoid unacceptable levels of residues of CAP entering the food chain.

Keywords: Multi-walled carbon nanotubes; Solid-phase extraction; Fused-core technology; High-performance liquid chromatography–tandem mass spectrometry; Chloramphenicol

Quantitative analysis of thymine with surface-enhanced Raman spectroscopy and partial least squares (PLS) regression by Lei Zhang; Qingqing Li; Wei Tao; Bohao Yu; Yiping Du (pp. 1827-1832).

Silver sol surface-enhanced Raman spectroscopy (SERS) was considered as a technique in the quantitative analysis of low-concentration thymine. Because of the poor stability and reproducibility of SERS signal, a polymer of polyacrylic acid sodium was selected as a stable medium to add into silver sol in order to obtain a surface-enhanced Raman spectroscopy signal. Assignments of Raman shift for solid thymine, SERS of thymine, and SERS of thymine containing stable medium were given. The comparison of Raman peaks between them showed that the addition of stable medium had a little influence on the SERS of thymine and is suitable for the quantitative analysis of low-concentration thymine.

Keywords: SERS; Thymine; Stable medium; Polyacrylic acid sodium

Erratum to: Mechanical ion gate for electrospray-ionization ion-mobility spectrometry by Li Zhou; David C. Collins; Edgar D. Lee; Alan L. Rockwood; Milton L. Lee (pp. 1833-1833).

Erratum to: Measuring the intra-individual variability of the plasma proteome in the chicken model of spontaneous ovarian adenocarcinoma by Adam M. Hawkridge; Rebecca B. Wysocky; James N. Petitte; Kenneth E. Anderson; Paul E. Mozdziak; Oscar J. Fletcher; Jonathan M. Horowitz; David C. Muddiman (pp. 1835-1835).

Erratum to: Part I: characterization of the extracellular proteome of the extreme thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS² by Genna Andrews; Derrick Lewis; Jaspreet Notey; Robert Kelly; David Muddiman (pp. 1837-1837).

Erratum to: Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS² and spectral counting by Genna Andrews; Derrick Lewis; Jaspreet Notey; Robert Kelly; David Muddiman (pp. 1839-1839).

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Analytical and Bioanalytical Chemistry (v.398, #4)