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Analytical and Bioanalytical Chemistry (v.394, #2)

Nanofluidics by Jan Eijkel (pp. 383-384).

is Associate Professor in microfluidics and nanofluidics in the BIOS/Lab on a Chip group (MESA⁺ Institute for Nanotechnology, University of Twente, The Netherlands). He studied pharmacy (University of Amsterdam) and theology (University of Utrecht) and obtained a Ph.D. in biosensor research at the University of Twente. His research interests include physical and colloid chemistry, electrochemistry, microseparation methods, and microplasma physics and applications.

Chemistry in nanochannel confinement by Han J. G. E. Gardeniers (pp. 385-397).

This review addresses the questions of whether it makes sense to use lithographically defined nanochannels for chemistry in liquids, and what it is possible to learn from experiments on that topic. The behavior of liquids in different classes of pores (categorized according to their size) is reviewed, with a focus on chemical reactions and protein dynamics. A number of interesting phenomena are discussed for nanochannels with feature sizes that are manufacturable with modern photolithography-based fabrication technology. The use of spectroscopic methods to investigate chemistry in nanochannels, where both spectroscopic method and nanochannels are integrated into a single device, will be evaluated.

Keywords: Water; Spectroscopy; Instrumentation; NMR; ESR; Nanoparticles; Nanotechnology; Microfluidics; Microfabrication

Estimation of surface desorption times in hydrophobically coated nanochannels and their effect on shear-driven and pressure-driven chromatography by Frederik Detobel; Veronika Fekete; Wim De Malsche; Selm De Bruyne; Han Gardeniers; Gert Desmet (pp. 399-411).

The present paper provides a detailed analysis of the analyte-wall adsorption effects in nanochannels, including a random walk study of the analyte-wall collision frequency, and uses these insights to estimate wall desorption times from chromatographic experiments in nanochannels. Using coumarin dye analytes and using a methanol/water mixture buffered at pH 3 in 120-nm deep channels, the surface desorption times on naked fused-silica glass were found to be maximally of the order of 60 to 150 μs, while they were found to be on the order of 100 to 500 μs on a hydrophobically coated wall. These nonzero adsorption and desorption times lead to an additional band broadening when conducting chromatographic separations. Shear-driven flows, requiring a noncoated moving wall and a stationary coated wall, intrinsically turn out to be more prone to this effect than pressure-driven or electro-driven flows for example. The present study also shows that, interestingly, the number of analyte-wall collisions increases with the inverse of the channel depth and not with its second power, as would be expected from the Einstein–Smoluchowski relationship for molecular diffusion.

Keywords: Shear-driven chromatography; Analyte-wall collisions; Band broadening; Random walk simulations

Control of ionic transport through gated single conical nanopores by Eric B. Kalman; Olivier Sudre; Ivan Vlassiouk; Zuzanna S. Siwy (pp. 413-419).

Control of ionic transport through nanoporous systems is a topic of scientific interest for the ability to create new devices that are applicable for ions and molecules in water solutions. We show the preparation of an ionic transistor based on single conical nanopores in polymer films with an insulated gold thin film “gate.” By changing the electric potential applied to the “gate,” the current through the device can be changed from the rectifying behavior of a typical conical nanopore to the almost linear behavior seen in cylindrical nanopores. The mechanism for this change in transport behavior is thought to be the enhancement of concentration polarization induced by the gate. Figure

Keywords: Nanotechnology; Electroanalytical methods; Microfluidics/microfabrication

Detecting proteins complex formation using steady-state diffusion in a nanochannel by Nicolas F. Y. Durand; Elli Saveriades; Philippe Renaud (pp. 421-425).

In this work, we present theoretical and experimental studies of nanofluidic channels as a potential biosensor for measuring rapid protein complex formation. Using the specific properties offered by nanofluidics, such as the decrease of effective diffusion of biomolecules in confined spaces, we are able to monitor the binding affinity of two proteins. We propose a theoretical model describing the concentration profile of proteins in a nanoslit and show that a complex composed by two bound biomolecules induces a wider diffusion profile than a single protein when driven through a nanochannel. To validate this model experimentally, we measured the increase of the fluorescent diffusion profile when specific biotinylated dextran was added to fluorescent streptavidin. We report here a direct and relatively simple technique to measure the affinity between proteins. Figure We present theoretical and experimental studies of nanofluidic channels as potential biosensors for rapidly measuring protein complex formation. Our system is based on steady-state diffusion effects which are observed inside a nanoslit.

Keywords: Bioassay; Nanofluidics; Nanochannel; Fluorescence; Proteomics; Microfabrication

Transport of biomolecules in asymmetric nanofilter arrays by Zi Rui Li; G. R. Liu; Jongyoon Han; Yu Zong Chen; Jian-Sheng Wang; Nicolas G. Hadjiconstantinou (pp. 427-435).

We propose a theoretical model for describing the electric-field-driven migration of rod-like biomolecules in nanofilters comprising a periodic array of shallow passages connecting deep wells. The electrophoretic migration of the biomolecules is modeled as transport of point-sized Brownian particles, with the orientational degree of freedom captured by an entropy term. Using appropriate projections, the formulation dimensionality is reduced to one physical dimension, requiring minimal computation and making it ideal for device design and optimization. Our formulation is used to assess the effect of slanted well walls on the energy landscape and resulting molecule mobility. Using this approach, we show that asymmetry in the well shape, such as a well with one slanted and one vertical wall, may be used for separation using low-frequency alternating-current fields because the mobility of a biomolecule is different in the two directions of travel. Our results show that, compared to methods using direct-current fields, the proposed method remains effective at higher field strengths and can achieve comparable separation using a significantly shorter device.

Keywords: Nanofluidics; Asymmetric nanofilter; Entropy barrier; DNA separation; Brownian ratchet; Molecular transport

Investigating the translocation of λ-DNA molecules through PDMS nanopores by Yi-Heng Sen; Rohit Karnik (pp. 437-446).

We investigate the translocation of λ-DNA molecules through resistive-pulse polydimethylsiloxane (PDMS) nanopore sensors. Single molecules of λ-DNA were detected as a transient current increase due to the effect of DNA charge on ionic current through the pore. DNA translocation was found to deviate from a Poisson process when the interval between translocations was comparable to the duration of translocation events, suggesting that translocation was impeded during the presence of another translocating molecule in the nanopore. Characterization of translocation at different voltage biases revealed that a critical voltage was necessary to drive DNA molecules through the nanopore. Above this critical voltage, frequency of translocation events was directly proportional to DNA concentration and voltage bias, suggesting that transport of DNA from the solution to the nanopore was the rate limiting step. These observations are consistent with experimental results on transport of DNA through nanopores and nanoslits and the theory of hydrodynamically driven polymer flow in pores.

Keywords: Nanopore; DNA; Resistive-pulse; Biosensing; PDMS; Translocation

Redox cycling in nanofluidic channels using interdigitated electrodes by Edgar D. Goluch; Bernhard Wolfrum; Pradyumna S. Singh; Marcel A. G. Zevenbergen; Serge G. Lemay (pp. 447-456).

Amperometric detection is ideally suited for integration into micro- and nanofluidic systems as it directly yields an electrical signal and does not necessitate optical components. However, the range of systems to which it can be applied is constrained by the limited sensitivity and specificity of the method. These limitations can be partially alleviated through the use of redox cycling, in which multiple electrodes are employed to repeatedly reduce and oxidize analyte molecules and thereby amplify the detected signal. We have developed an interdigitated electrode device that is encased in a nanofluidic channel to provide a hundred-fold amplification of the amperometric signal from paracetamol. Due to the nanochannel design, the sensor is resistant to interference from molecules undergoing irreversible redox reactions. We demonstrate this selectivity by detecting paracetamol in the presence of excess ascorbic acid. Figure

Keywords: Nanotechnology; Nanofluidic; Electrochemistry; Amperometric detection; Redox cycling; Interdigitated electrodes (IDEs); Paracetamol; Ascorbic acid; Sensor

Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number by Somanath Bhat; Jan Herrmann; Paul Armishaw; Philippe Corbisier; Kerry R. Emslie (pp. 457-467).

Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. PCR solution is distributed throughout numerous partitions, and following amplification, target DNA copy number is estimated based on the proportion of partitions containing amplified DNA. Here, we identify approaches for obtaining reliable digital PCR data. Single molecule amplification efficiency was significantly improved following fragmentation of total DNA and bias in copy number estimates reduced by analysis of short intact target DNA fragments. Random and independent distribution of target DNA molecules throughout partitions, which is critical to accurate digital PCR measurement, was demonstrated by spatial distribution analysis. The estimated relative uncertainty for target DNA concentration was under 6% when analyzing five digital panels comprising 765 partitions each, provided the panels contained an average of 212 to 3,365 template molecules. Partition volume was a major component of this uncertainty estimate. These findings can be applied to other digital PCR studies to improve confidence in such measurements. Figure Digital PCR amplification plot (left) and panel read out (right) of HindIII-digested pIRMM69. pIRMM69 contains one HindIII restriction enzyme site outside the hmg and transgene fragments used as targets in PCR. Red boxes with white shade denote positive hits containing one or more target DNA molecules, and white boxes with grey shade refer to no target being amplified.

Keywords: Bioanalytical methods; Biochips/high throughput screening; Nucleic acids (DNA|RNA); PCR; Clinical/biomedical analysis

Bioconjugation techniques for microfluidic biosensors by Julie M. Goddard; David Erickson (pp. 469-479).

We have evaluated five bioconjugation chemistries for immobilizing DNA onto silicon substrates for microfluidic biosensing applications. Conjugation by organosilanes is compared with linkage by carbonyldiimidazole (CDI) activation of silanol groups and utilization of dendrimers. Chemistries were compared in terms of immobilization and hybridization density, stability under microfluidic flow-induced shear stress, and stability after extended storage in aqueous solutions. Conjugation by dendrimer tether provided the greatest hybridization efficiency; however, conjugation by aminosilane treated with glutaraldehyde yielded the greatest immobilization and hybridization densities, as well as enhanced stability to both shear stress and extended storage in an aqueous environment. Direct linkage by CDI activation provided sufficient immobilization and hybridization density and represents a novel DNA bioconjugation strategy. Although these chemistries were evaluated for use in microfluidic biosensors, the results provide meaningful insight to a number of nanobiotechnology applications for which microfluidic devices require surface biofunctionalization, for example vascular prostheses and implanted devices.

Keywords: Microfluidics/microfabrication; Biosensors; DNA immobilization; Surface functionalization; Bioconjugation; Silane stability

Monitoring surface-assisted biomolecular assembly by means of evanescent-field-coupled waveguide-mode nanobiosensors by Subash C. B. Gopinath; Koichi Awazu; Makoto Fujimaki; Katsuaki Sugimoto; Yoshimichi Ohki; Tetsuro Komatsubara; Junji Tominaga; Penmetcha K. R. Kumar (pp. 481-488).

Biological self-assembly is a natural process that involves various biomolecules, and finding the missing partner in these interactions is crucial for a specific biological function. Previously, we showed that evanescent-field-coupled waveguide-mode sensor in conjunction with a SiO₂ waveguide, the surfaces which contain cylindrical nanometric holes produced by atomic bombardment, allowed us to detect efficiently the biomolecular interactions. In the present studies, we showed that the assembly of biomolecules can be monitored using the evanescent-field-coupled waveguide-mode biosensor and thus provide a methodology in monitoring assembly process in macromolecular machines while they are assembling. Evanescent-field-coupled waveguide-mode sensor

Keywords: Biomolecular assembly; Nanobiosensor; Evanescent-field-coupled waveguide; SiO₂ ; Aptamer

Single-molecule immunosorbent assay as a tool for human immunodeficiency virus-1 antigen detection by Jiangwei Li; Wenjun Xie; Ning Fang; Edward S. Yeung (pp. 489-497).

Ultrasensitive detection and quantification of viral antigen with a novel single-molecule immunosorbent assay (SMISA) was achieved. Antigen from human immunodeficiency virus type 1 (HIV-1), the major etiological agent of acquired immune deficiency syndrome, served as the screening target in this study. The target molecule was sandwiched between a polyclonal capture antibody and a monoclonal detector antibody. The capture antibody was covalently immobilized on (3-glycidoxypropyl) trimethoxy silane-modified glass slides. The detector antibody was conjugated with fluorescent Alexa Fluor 532 labeled secondary antibody prior to being used as a probe for the antigen. Imaging was performed with a total internal reflection fluorescence single-molecule detection system. This technique is demonstrated for detecting HIV-1 p24 antigen down to 0.1 pg/mL with a dynamic range of over four orders of magnitude. A Langmuir isotherm fits the molecule count dependence on the target concentration. The target antigen was further tested in 20% human serum, and the results showed that neither sensitivity nor dynamic range was affected by the biological matrix. SMISA is therefore a promising approach for the early diagnosis of viral induced diseases.

Keywords: Single-molecule immunosorbent assay (SMISA); Early diagnosis; HIV-1; Fluorescence detection

Miniaturized 96-well ELISA chips for staphylococcal enterotoxin B detection using portable colorimetric detector by Kim E. Sapsford; Jesse Francis; Steven Sun; Yordan Kostov; Avraham Rasooly (pp. 499-505).

A previously developed fluorescence sensing platform, combining spatial illumination using electroluminescence (EL) semiconductor strips with charge coupled device (CCD)-based detection (EL-CCD), was adapted to a new 96-well chip for colorimetric immunological assays, enhancing the capabilities of the EL-CCD platform. The modified system was demonstrated using a colorimetric-based enzyme linked immunosorbent assay (ELISA) for detection of staphylococcal enterotoxin B (SEB). Limits of detection (LODs) of 3.9 ng/mL (±2.4 ng/mL) SEB were determined with the ELISA chip measured using the EL-CCD platform, following a standard 4-h ELISA protocol. The LODs were comparable to those obtained using standard 96-well ELISA plates measured using a standard laboratory 96-well plate reader. The miniature 96-well ELISA chip however required as little as 5-µL samples, representing a tenfold reduction in sample volume compared to a standard 96-well ELISA plates. The ELISA chip also demonstrated detection of SEB spiked into various food matrices (milk, mushrooms, and mayonnaise) using limited-to-no sample preparation, with LODs ranging from 3.9 to 18.5 ng/mL depending on the matrix. The EL-CCD platform is versatile, capable of multi-mode detection (e.g., fluorescent and colorimetric along with solution and solid phase assays), and could readily be applied to other field portable or point-of-care applications. Figure Detection of SEB using miniature ELISA chips coupled with a portable electroluminiscent-charge couple device (EL-CCD) detection system.

Keywords: Electroluminescent (EL) illumination; CCD; Miniature ELISA; Staphylococcal enterotoxin B (SEB); Food; Colorimetric measurement; Biosensor

Homogeneous immunoassay based on gold nanoparticles and visible absorption detection by Laura Anfossi; Claudio Baggiani; Cristina Giovannoli; Gianfranco Giraudi (pp. 507-512).

A sensitive homogeneous immunoassay, using human serum albumin (HSA) as a model analyte coupled with simple visible absorption detection, has been developed. The new assay is based on the use of gold nanoparticles functionalized with the target protein, which compete with the analyte for the binding of a specific polyclonal antibody. The binding of antibodies to the functionalized nanoparticles determines a shift of the visible absorption maximum of the gold colloid, and quantification of the analyte could be obtained as the competitive inhibition of the binding of antibodies to the nanoparticles. The proposed immunoassay has been optimized and successfully applied to measuring HSA in human urine samples, in which results agreed well with those obtained by a nephelometric reference method.

Keywords: Gold nanoparticles; Homogeneous immunoassay; Human Serum Albumin in urine; Visible absorption detection

Simultaneous quantification of buprenorphine, norbuprenorphine, buprenorphine glucuronide, and norbuprenorphine glucuronide in human placenta by liquid chromatography mass spectrometry by Marta Concheiro-Guisan; Diaa M. Shakleya; Marilyn A. Huestis (pp. 513-522).

A LCMS method was developed and validated for the determination of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc), and norbuprenorphine glucuronide (NBUP-Gluc) in placenta. Quantification was achieved by selected ion monitoring of m/z 468.4 (BUP), 414.3 (NBUP), 644.4 (BUP-Gluc), and 590 (NBUP-Gluc). BUP and NBUP were identified monitoring MS² fragments m/z 396, 414 and 426 for BUP, and 340, 364 and 382 for NBUP, and glucuronide conjugates monitoring MS³ fragments m/z 396 and 414 for BUP-Gluc, and 340 and 382 for NBUP-Gluc. Linearity was 1–50 ng/g. Intra-day, inter-day and total assay imprecision (% RSD) were <13.4%, and analytical recoveries were 96.2–113.1%. Extraction efficiencies ranged from 40.7–68%, process efficiencies 38.8–70.5%, and matrix effect 1.3–15.4%. Limits of detection were 0.8 ng/g for all compounds. An authentic placenta from an opioid-dependent pregnant woman receiving BUP pharmacotherapy was analyzed. BUP was not detected but metabolite concentrations were NBUP-Gluc 46.6, NBUP 15.7 and BUP-Gluc 3.2 ng/g.

Keywords: Placenta; Buprenorphine; LCMS; Ion trap

Pollen-imprinted polyurethanes for QCM allergen sensors by Michael Jenik; Alexandra Seifner; Peter Lieberzeit; Franz L. Dickert (pp. 523-528).

Molecularly imprinted polymers for detecting plant pollen were designed as artificial recognition materials for quartz crystal microbalances in the gaseous phase. Imprints of birch (diameter, 25 μm) and nettle (diameter, 15 μm) pollen can be generated by polydimethylsiloxane stamping technique as proven by atomic force microscopy. If pollen grains are able to access the cavities and thus are incorporated, the resulting sensors display Sauerbrey-like negative frequency shifts. Non-Sauerbrey behaviour can be observed as soon as pollen is prevented from entering the selective hollows: this results in grain mobility on the electrode surface leading to frequency increases. Access to the cavities is determined by the diameter ratio between pollen grains and imprints as can be revealed during cross-selectivity measurements of nettle and birch pollen imprinted layers. When the amount of pollen grains on the electrode surface exceeds the number of available imprints, the excess particles move freely, resulting in positive, non-Sauerbrey frequency shifts.

Keywords: Molecular imprinting; Plant pollen; QCM; Non-Sauerbrey behaviour

A novel approach for the detection of DNA using immobilized peptide nucleic acid (PNA) probes and signal enhancement by real-time immuno-polymerase chain reaction (RT-iPCR) by Lillian Roth; Jutta Zagon; Anke Ehlers; Lothar W. Kroh; Hermann Broll (pp. 529-537).

A new approach for the detection of DNA target molecules is described, using capture probes and subsequent signal enhancement by a uniform polymerase chain reaction (PCR). Peptide nucleic acid probes were immobilized in real-time PCR-compatible microtiter plates. After hybridization of biotinylated DNA targets, detection was performed by real-time immuno-PCR, a method formerly used for protein detection. We demonstrate the feasibility of this strategy for the qualitative detection of DNA oligonucleotides with a detection limit (LOD) of 6 attomol. Furthermore, the method was applied to PCR-amplified samples from genetically modified maize DNA (Mon810). A 483-bp DNA fragment was detected in mixture with 99.9% of noncomplementary DNA with a sensitivity down to the level of attomole. Figure

Keywords: DNA detection; PNA probes; Real-time immuno-PCR; GMO; Method; Signal amplification

Effectiveness of natural and synthetic blocking reagents and their application for detecting food allergens in enzyme-linked immunosorbent assays by Denise Huber; Judith Rudolf; Parisa Ansari; Brigitte Galler; Manuela Führer; Christoph Hasenhindl; Sabine Baumgartner (pp. 539-548).

Blocking is an important step before an enzyme-linked immunosorbent assay (ELISA) can be performed. It reduces non-specific binding to the microtiter plate to a minimum. For detecting food allergens by means of ELISA, the problem with protein blocking solutions is obvious. The blocker might interfere with the antibodies of the assay and leads to false positive results. Therefore, other blocking solutions are greatly needed. There are some alternatives like synthetic blockers or carbohydrates. Comparisons of these different blocking agents, namely proteins, carbohydrates, and synthetic blockers, were made at different reaction conditions. The incubation periods and temperatures were varied, as well as the pH. The best combinations were evaluated and compared, in respect of their blocking efficiency. The two best non-proteinaceous blockers, i.e. polyvinylalcohol and Ficoll, were subsequently applied to ELISA tests for the determination of α-casein and peanut. The study showed that Ficoll and PVA did as well as BSA in buffer solution. Therefore, they can be considered as alternative blocking reagents for ELISA, especially for the detection of food allergens.

Keywords: ELISA; Blocking; PVA; Ficoll; Food allergens; α-casein

Combining silica-based adsorbents and SPME fibers in the extraction of the volatiles of beer: an exploratory study by César Luis Biazon; Rodrigo Brambilla; Arnaud Rigacci; Tânia M. Pizzolato; João H. Z. dos Santos (pp. 549-556).

A series of silica-based materials were employed as sorbents within solid-phase microextraction vials. The aim of the study was to evaluate the effect of an additional phase on the distribution of the volatile and less volatile analytes. The adsorption of six probe molecules, namely isoamyl acetate, ethyl hexanoate (ethyl caproate), phenylethyl alcohol, ethyl octanoate (ethyl caprilate), 2-phenylethyl acetate, and ethyl decanoate, was monitored by detecting the desorbed amount on a DVD–CAR–PDMS fiber from Pilsen beer. The microextraction process involved the presence of different silica-based phases produced via different methods: xerogel produced by hydrolytic and non-hydrolytic routes, aerogel, pyrogenic, and precipitated silica. The resulting data are discussed in correlation with sorbent texture properties (specific area and pore diameter). The modification of silica with alkyl groups also affects the preconcentrated amount of the target molecules in the headspace. The presence of sorbents was shown to affect the analyte signal more than the addition of NaCl or the use of ultrasound. Analyte’s equilibrium between fiber and sorbent

Keywords: Beer; SPME; Silica; Sorbent; Xerogel

On-line determination of aliphatic amines in water using in-tube solid-phase microextraction-assisted derivatisation in in-valve mode for processing large sample volumes in LC by P. Campíns-Falcó; R. Herráez-Hernández; J. Verdú-Andrés; C. Cháfer-Pericás (pp. 557-565).

This paper describes a cost-effective procedure for the analysis of short-chain aliphatic amines in water samples using a solid-phase microextraction device. Analyte preconcentration and derivatisation were effected into a capillary column coated with 95% polydimethylsiloxane–5% polydiphenylsiloxane, which was used as the injection loop of a Rheodyne injection valve. The coating was previously loaded with the derivatisation reagent, 9-fluorenylmethyl chloroformate. A volume of 1 mL of samples was then drawn into the capillary column, and the extracted analytes were left to react on the capillary coating for 5 min. Next, the capillary column was cleaned by passing water. Finally, the injection valve was rotated, and the derivatives formed were dynamically desorbed and transferred to the analytical column into the mobile phase. Methylamine, ethylamine, propylamine, n-butylamine and n-pentylamine were selected as model compounds. Excellent sensitivity was achieved, being the limits of detection of 15–200 µg/L when using UV detection and of 0.1–0.4 µg/L by fluorescence.

Keywords: Aliphatic amines; In-tube solid-phase microextraction; Derivatisation; Liquid chromatography; Water analysis

Environmental monitoring of phenolic pollutants in water by cloud point extraction prior to micellar electrokinetic chromatography by Patricia W. Stege; Lorena L. Sombra; Germán A. Messina; Luis D. Martinez; María F. Silva (pp. 567-573).

Many aromatic compounds can be found in the environment as a result of anthropogenic activities and some of them are highly toxic. The need to determine low concentrations of pollutants requires analytical methods with high sensitivity, selectivity, and resolution for application to soil, sediment, water, and other environmental samples. Complex sample preparation involving analyte isolation and enrichment is generally necessary before the final analysis. The present paper outlines a novel, simple, low-cost, and environmentally friendly method for the simultaneous determination of p-nitrophenol (PNP), p-aminophenol (PAP), and hydroquinone (HQ) by micellar electrokinetic capillary chromatography after preconcentration by cloud point extraction. Enrichment factors of 180 to 200 were achieved. The limits of detection of the analytes for the preconcentration of 50-ml sample volume were 0.10 μg L⁻¹ for PNP, 0.20 μg L⁻¹ for PAP, and 0.16 μg L⁻¹ for HQ. The optimized procedure was applied to the determination of phenolic pollutants in natural waters from San Luis, Argentina. Figure Schematic representation of the cloud point extraction process.

Keywords: Micellar electrokinetic chromatography; Cloud point extraction; p-nitrophenol; p-aminophenol; Hydroquinone.

Use of polyclonal antibodies to ochratoxin A with a quartz–crystal microbalance for developing real-time mycotoxin piezoelectric immunosensors by J. C. Vidal; P. Duato; L. Bonel; J. R. Castillo (pp. 575-582).

A piezoelectric immunosensor was tested for ochratoxin A (OTA) mycotoxin detection through the immobilization of OTA–bovine serum albumin (OTA–BSA) conjugate on gold-coated quartz crystals (AT-cut/5 MHz). Immunoassays were performed in a flow-injection system through frequency decreases in a quartz–crystal microbalance (QCM) because of a mass increasing during immunoreaction with anti-OTA antibodies. Three immobilization procedures for OTA–BSA (direct adsorption and covalent attachment to two alkane thiol self-assembled monolayers) were characterized with QCM in real time. Covalent attachment of the OTA–BSA conjugates through gold nanoparticles was also tested for amplifying the signal. Binding of the excess of antibodies to the immobilized OTA in an indirect competitive analysis decreased linearly the resonant frequency in the range of the OTA concentration from 10 to 128 ng/mL, with a detection limit of 8 ng/mL (signal/noise ratio of 3). A pepsin 2 mg/mL (pH = 2.1) solution was used to release antigen–antibody complexes, regenerating the biorecognition surface.

Keywords: Immunosensor; Quartz crystal microbalance; Piezoelectric immunosensor; Ochratoxin A; Gold nanoparticles; Voltammetry

Preparation of reference materials for the determination of RoHS-relevant flame retardants in styrenic polymers by Manfred Pöhlein; Raquel Urpi Bertran; Marion Wolf; Rudi van Eldik (pp. 583-595).

Reference materials for the analysis of polybrominated diphenyl ethers, polybrominated biphenyls and other common brominated flame retardants (FR) in styrenic polymers were prepared to suit the demands of actual restriction of the use of certain hazardous substances in electrical and electronic equipment analytics. Three methods of preparation were employed, viz. pellet forming, dissolution/vaporisation and extrusion, whereby extrusion proved to be the most suitable method. For extrusion, three procedures of pre-mixing were investigated: the polymers were either mixed with FR powder, FR solutions or FR concentrates that were taken from waste industrial polymers. The latter procedure proved to be most appropriate in terms of analyte concentration, predictability and recovery. The homogeneity of the samples, as well as the chemical and thermal long-term stabilities, was investigated. The result was an optimised method to prepare a suitable reference material for laboratory use. Figure

Keywords: Reference material; PBDE; PBB; RoHS; WEEE; Styrenic polymers

Enantioseparation of underivatised amino acids by ligand exchange capillary electrophoresis in a counter-electroosmotic mode by Abderrahim Aït Adoubel; Christophe J. Morin; Nadine Mofaddel; Georges Dupas; Paul-Louis Desbène (pp. 597-608).

Enantiomer separations of underivatised amino acids were carried out by using ligand exchange capillary electrophoresis (LECE). Chiral discrimination is based on the formation of ternary complexes between copper(II), a chiral selector (L-proline or trans-4-hydroxy-L-proline) and an amino acid. All amino acids containing aromatic moieties or not were detected at 214 nm because of their interactions with copper(II). In order to reduce copper(II) adsorption onto capillary walls, we used hexadimethrine bromide to reverse the electroosmotic flow. Using this original strategy, the studied enantiomers migrated in the opposite direction of the anodic electroosmosis. After optimising the analytical conditions taking into account the chiral resolution and the detection sensitivity, we performed very satisfactory enantioseparations not only of aromatic amino acids (tryptophan, tyrosine, phenylalanine and histidine) but also of aliphatic amino acids (threonine, serine, isoleucine and valine). These enantioseparations were performed in a short analysis time at 35 °C. In order to rationalise the obtained results, we evaluated the complexation constants corresponding to the formed ternary complexes by capillary electrophoresis and we used molecular mechanics modelling.

Keywords: Ligand exchange capillary electrophoresis; Enantioseparation; Underivatised amino acid; Complexation constant

Comparison of off- and in-line solid-phase extraction for enhancing sensitivity in capillary electrophoresis using ochratoxin as a model compound by S. Almeda; L. Arce; F. Benavente; V. Sanz-Nebot; J. Barbosa; M. Valcárcel (pp. 609-615).

This paper proposes and compares two approaches based on off- and in-line solid-phase extraction (SPE), intended to enhance sensitivity in capillary electrophoresis with ultraviolet detection (CE-UV) using as a model the determination of ochratoxin A (OA) in river water samples. In the off-line SPE mode, the reversed-phase sorbent (octadecilsylane, C₁₈) selectively retains the target analyte (OA) and allows large volumes of the sample (70 mL) to be introduced and subsequently eluted in a small volume (0.1 mL) of an appropriate solution. In the in-line SPE mode, a custom-made microcartridge is inserted near the inlet of the capillary, which is filled with the same C₁₈ sorbent. This solid phase selectively retains OA present in a sample volume as low as approximately 640 µL for subsequent elution with ca. 135 nL of an appropriate eluent. The limit of detection (LOD) obtained with the in-line SPE method was 1 ng L^-1, which is 3 orders of magnitude lower than that obtained with CE-UV and roughly 1 order lower than that provided by the off-line SPE-CE-UV method.

Keywords: Capillary electrophoresis; In-line preconcentration; Ochratoxin A; Solid-phase extraction

Fast preparation of polystyrene-based monolith using microwave irradiation for micro-column separation by Yu-Ping Zhang; Wei Li; Xue-Jin Wang; Ling-Bo Qu; Gong-Ling Zhao; Yi-Xin Zhang (pp. 617-623).

The conventional analytical HPLC was successfully developed for micro-column separation by using a simple eluate splitting system, a self-preparation of monolithic column and an on-capillary column detector in our laboratory. A typical polystyrene-based monolith was quickly prepared inside the fused-silica capillary, which in situ polymerization was carried out in 10 min by microwave irradiation. The reactant solution consisted of styrene (ST) as a functional monomer, divinylbenzene (DVB) as a cross-linking agent, toluene and isooctane as porogenic solvents, and azobisisobutyronitrile (AIBN) as an initiator. The monolith was proved to form in the center of the capillary and adhered to the column inner wall by the scanning electron micrograph. Its chromatographic behaviors were evaluated in detail by varying the flow rate and percentage of mobile phases, and under the optimal condition, baseline separation of the model analytes including thiourea, benzene, toluene, ethylbenzene was obtained with a highest theoretical plate number near 11,290 N/m by the developed capillary HPLC. Furthermore, the stability and porosity of the prepared monolith were systematically investigated by a simple flow method. Figure A polystyrene-based monolith was rapidly prepared inside the fused-silica capillary, which in situ polymerization was carried out about 10 min by microwave irradiation.

Keywords: HPLC; Polymers; Microwave irradiation/monolith

Alternating iterative regression method for dead time estimation from experimental designs by S. Pous-Torres; J. R. Torres-Lapasió; J. J. Baeza-Baeza; M. C. García-Álvarez-Coque (pp. 625-636).

An indirect method for dead time (t ₀) estimation in reversed-phase liquid chromatography, based on a relationship between retention time and organic solvent content, is proposed. The method processes the retention data obtained in experimental designs. In order to get more general validity and enhance the accuracy, the information from several compounds is used altogether in an alternating regression fashion. The method was applied to nitrosamines, alkylbenzenes, phenols, benzene derivatives, polycyclic aromatic hydrocarbons and β-blockers, among other compounds, chromatographed in a cyano and several C18 columns. A comprehensive validation was carried out by comparing the results with those provided by the injection of markers, the observation of the solvent front and the homologous series method. It was also found that different groups of compounds yielded the same t ₀ value with the same column, which was verified in different solvent composition windows. The method allows improved models useful for optimisation or for other purposes, since t ₀ can be estimated with the retention data of the target solutes.

Keywords: Dead time; Retention model; Reversed-phase liquid chromatography; Experimental design; Alternating regression

Using the Tsallis distribution and the fractional differentiation to resolve the overlapping bands by Yuanlu Li (pp. 637-645).

Using the Tsallis distribution, which facilitates the generalization of well-known distributions such as Gaussian and Lorentzian by varying a non-extensivity parameter q as a model of the individual band to correctly assign overlapping bands and the fractional differentiation as mathematical tool to help to determine the spectral parameters of the individual band, a new resolution method for the overlapping bands is presented. According to variation of the maximum and the zero-crossing of the Tsallis distribution at different differential order, two types of parameter estimators are obtained, which are utilized to calculate the parameters of position, height, and width of Tsallis distribution. To verify the suggested method, separation of several kinds of overlapping bands simulated by computer and the experimental infrared spectrum of 1,2-bromofluoroethane have been performed and discussed. Figure α-Order differentiation of the overlapping band

Keywords: Fractional differentiation; Overlapping bands; Tsallis distribution; Spectral parameters

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Analytical and Bioanalytical Chemistry (v.394, #2)

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Analytical and Bioanalytical Chemistry (v.394, #2)