Analytical and Bioanalytical Chemistry (v.391, #5)

Nascent hydrogen challenge by Juris Meija; Alessandro D’Ulivo (1475-1476).

Solution to quality assurance challenge 6 by Manfred Reichenbächer; Jürgen W. Einax (1477-1479).

Europt(r)ode IX: Ninth European conference on optical chemical sensors and biosensors by Stefanie Jaeger; Markus Ehni; Christiane Albrecht; Florian Proell (1481-1482).

Focus on bioanalysis by Günter Gauglitz; Frieder W. Scheller; Tuan Vo-Dinh (1483-1484).
is Professor at the Eberhard Karls University in Tübingen, where he works in analytical and physical chemistry. He is chairman of the GDCh Division of Analytical Chemistry and chaired the Europt(r)ode VIII meeting. For the last ten years, his main scientific interests have centered on research and development in the area of chemical and biochemical sensors, especially the characterization of interfaces of polymers and biomembranes by surface spectroscopic techniques, the application of spectral interferometry to monitor changes in the optical thicknesses of thin layers, and the effects of Fresnel reflectivity at interfaces. has been Professor of Analytical Biochemistry at the University of Potsdam since 1993. He received his diploma in chemistry from the Technical College of Merseburg and his Ph.D. from Humboldt University in Berlin. For many years he headed the Department of Bioelectrochemistry/Biosensors at the Central Institute of Molecular Biology of the German Academy of Sciences, Berlin–Buch. In 2001 and 2002 he was Vice President of the German Society of Biochemistry and Molecular Biology. Since January 2004 he has been Vice President of the University of Potsdam. His current research interests include biomimetic sensors based on aptamers and molecularly imprinted polymers, the enzyme electrochemistry of c-type cytochromes, cytochrome P-450 and hemoglobin, and piezoelectric affinity sensors using antibodies, aptamers and enzymes. is currently Director of the Fitzpatrick Institute for Photonics (FIP), R. Eugene and Susie E. Goodson Distinguished Professor of Biomedical Engineering, and Professor of Chemistry at Duke University. Before joining Duke University, Dr. Vo-Dinh was Group Leader of the Advanced Biomedical Science and Technology Group, Director of the Center for Advanced Biomedical Photonics, and a Corporate Fellow, one of the highest honors for distinguished scientists at the Oak Ridge National Laboratory (ORNL), Oak Ridge, TN, USA. He received his Ph.D. in biophysical chemistry in 1975 from the ETH (Swiss Federal Institute of Technology) in Zurich, Switzerland. His research interests focus on the development of advanced technologies for human health protection and environmental sensing. His research activities involve nanobiotechnology, medical diagnostics, molecular imaging, laser spectroscopy, biophotonics, nanophotonics, biosensors, nanosensors, and biochips. Dr. Vo-Dinh has authored over 350 publications in peer-reviewed scientific journals. He is the author and editor of seven books, Editor-in-Chief of the international journal NanoBiotechnology, and holds over 32 US patents, six of which have been licensed to private companies for commercial development. Dr. Vo-Dinh has received numerous awards, including seven RD-100 Awards for the Most Technologically Significant Advance in Research and Development for his development of various technologies, the Gold Medal Award from the Society for Applied Spectroscopy, the Languedoc-Roussillon Award (France), the Scientist of the Year Award from Oak Ridge National Laboratory, the Thomas Jefferson Award from the Martin Marietta Corporation, the Distinguished Inventors Award from the Battelle Memorial Institute, and the Exceptional Services Award for Contributing to a Healthy Citizenry from the US Department of Energy.

The good, the bad, and the tiny: a review of microflow cytometry by Daniel A. Ateya; Jeffrey S. Erickson; Peter B. Howell Jr; Lisa R. Hilliard; Joel P. Golden; Frances S. Ligler (1485-1498).
Recent developments in microflow cytometry have concentrated on advancing technology in four main areas: (1) focusing the particles to be analyzed in the microfluidic channel, (2) miniaturization of the fluid-handling components, (3) miniaturization of the optics, and (4) integration and applications development. Strategies for focusing particles in a narrow path as they pass through the detection region include the use of focusing fluids, nozzles, and dielectrophoresis. Strategies for optics range from the use of microscope objectives to polymer waveguides or optical fibers embedded on-chip. While most investigators use off-chip fluidic control, there are a few examples of integrated valves and pumps. To date, demonstrations of applications are primarily used to establish that the microflow systems provide data of the same quality as laboratory systems, but new capabilities—such as automated sample staining—are beginning to emerge. Each of these four areas is discussed in detail in terms of the progress of development, the continuing limitations, and potential future directions for microflow cytometers.
Keywords: Flow cytometry; Microfluidics; Fluid focusing; Integrated optics; Cell sorter

Fluorescence polarization immunoassay (FPIA) is a homogeneous (without separation) competitive immunoassay method based on the increase in fluorescence polarization (FP) of fluorescent-labeled small antigens when bound by specific antibody. The minimum detectable quantity of FPIAs with fluorescein label (about 0.1 ng analyte) is comparable with chromatography and ELISA methods, although this may be limited by sample matrix interference. Because of its simplicity and speed, FPIA is readily automated and therefore suitable for high-throughput screening (HTS) in a variety of application areas. Systems that involve binding of ligands to receptor proteins are also susceptible to analysis by analogous FP methods employing fluorescent-labeled ligand and HTS applications have been developed, notably for use in candidate drug screening.
Keywords: Fluorescence polarization immunoassay; Fluorescence polarization; High-throughput screening; Food safety monitoring; Environmental monitoring

Surface acoustic wave biosensors: a review by Kerstin Länge; Bastian E. Rapp; Michael Rapp (1509-1519).
This review presents an overview of 20 years of worldwide development in the field of biosensors based on special types of surface acoustic wave (SAW) devices that permit the highly sensitive detection of biorelevant molecules in liquid media (such as water or aqueous buffer solutions). 1987 saw the first approaches, which used either horizontally polarized shear waves (HPSW) in a delay line configuration on lithium tantalate (LiTaO3) substrates or SAW resonator structures on quartz or LiTaO3 with periodic mass gratings. The latter are termed “surface transverse waves” (STW), and they have comparatively low attenuation values when operated in liquids. Later Love wave devices were developed, which used a film resonance effect to significantly reduce attenuation. All of these sensor approaches were accompanied by the development of appropriate sensing films. First attempts used simple layers of adsorbed antibodies. Later approaches used various types of covalently bound layers, for example those utilizing intermediate hydrogel layers. Recent approaches involve SAW biosensor devices inserted into compact systems with integrated fluidics for sample handling. To achieve this, the SAW biosensors can be embedded into micromachined polymer housings. Combining these two features will extend the system to create versatile biosensor arrays for generic lab use or for diagnostic purposes. SAW based biosensor immersed in a sample flow. Analyte molecules binding to the immobilized antibodies on the sensor surface will influence the velocity of the SAW and hence the output signal generated by the driving electronics.
Keywords: Surface acoustic wave; Biosensors; Interaction analysis; Chip

Automated analytical microarrays: a critical review by Michael Seidel; Reinhard Niessner (1521-1544).
Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment. The specific affinity reaction of nucleic acids (hybridization) and antibodies towards antigens is the most common bioanalytical method for generating multiplexed quantitative results. Nucleic acid-based analysis is restricted to the detection of cells and viruses. Antibodies are more universal biomolecular receptors that selectively bind small molecules such as pesticides, small toxins, and pharmaceuticals and to biopolymers (e.g. toxins, allergens) and complex biological structures like bacterial cells and viruses. By producing an appropriate antibody, the corresponding antigenic analyte can be detected on a multiplexed immunoanalytical microarray. Food and water analysis along with clinical diagnostics constitute potential application fields for multiplexed analysis. Diverse fluorescence, chemiluminescence, electrochemical, and label-free microarray readout systems have been developed in the last decade. Some of them are constructed as flow-through microarrays by combination with a fluidic system. Microarrays have the potential to become widely accepted as a system for analytical applications, provided that robust and validated results on fully automated platforms are successfully generated. This review gives an overview of the current research on microarrays with the focus on automated systems and quantitative multiplexed applications. Figure MCR 3: A fully automated chemiluminescence microarray reader for analytical microarrays
Keywords: Analytical microarrays; Biosystems technology; Environmental analysis; Food analysis; Clinical diagnostics

The evaluation of the effects of drugs or chemicals on the functions of the immune system is an increasingly important task. Due to the accessibility of primary cells and cell lines, in vitro cellular functional tests are frequently being performed with cells representing the innate immune system, in particular those with phagocytotic activities, such as neutrophils and macrophages. Suitable functional parameters are the efficiency of phagocytosis, the efficiency with which viable pathogens are killed, the production of reactive oxygen and nitrogen species (ROS and RNS) and that of cytokines. Corresponding analytical procedures are available, but standardization is required, as varying the procedure may influence the outcomes of the assays.
Keywords: Bioanalytical methods; Bioassays; Cell systems/Single cell analysis; Fluorescence/Luminescence; Immunoassays/ELISA

This review introduces the basic concepts and terms associated with impedance and techniques of measuring impedance. The focus of this review is on the application of this transduction method for sensing purposes. Examples of its use in combination with enzymes, antibodies, DNA and with cells will be described. Important fields of application include immune and nucleic acid analysis. Special attention is devoted to the various electrode design and amplification schemes developed for sensitivity enhancement. Electrolyte insulator semiconductor (EIS) structures will be treated separately. Figure An alternating current which is forced to pass an interface is sensitive to surface changes and will detect impedance changes due to biomolecule immobilisation or formation of a recognition complex. This can be used for the construction of biosensor electrodes
Keywords: Impedance; Sensor; DNA; Antibodies; Cells; Enzymes

Cubic phases in biosensing systems by Ewa Nazaruk; Renata Bilewicz; Göran Lindblom; Britta Lindholm-Sethson (1569-1578).
Incorporation of membrane proteins with retained activity in artificial membranes for use in membrane-based sensors has attracted scientists for decades. This review briefly summarises general concepts on relevant cubic phases with and without incorporated proteins and provides some insight into the development of biosensors where bicontinuous cubic phases are used for incorporation of an enzyme. Some new data on impedance characterisation of a supported cubic phase are also shown. An efficient membrane-based electrochemical biosensor requires that the analyte has free access to the immobilised membrane protein and that regeneration of the catalysing enzyme is fast. Long-term stability of the system is also necessary for the biosensor to find applications outside the research laboratory. These basic concepts are discussed in the review along with presentation of those biosensing systems based on cubic phases that are reported in the literature.
Keywords: Biosensors; Cubic phase; Membrane-based sensors

Development and trends of biosurfactant analysis and purification using rhamnolipids as an example by M. Heyd; A. Kohnert; T.-H. Tan; M. Nusser; F. Kirschhöfer; G. Brenner-Weiss; M. Franzreb; S. Berensmeier (1579-1590).
During the last few decades, increasing interest in biological surfactants led to an intensification of research for the cost-efficient production of biosurfactants compared with traditional petrochemical surface-active components. The quest for alternative production strains also is associated with new demands on biosurfactant analysis. The present paper gives an overview of existing analytical methods, based on the example of rhamnolipids. The methods reviewed range from simple colorimetric testing to sophisticated chromatographic separation coupled with detection systems like mass spectrometry, by means of which detailed structural information is obtained. High-performance liquid chromatography (HPLC) coupled with mass spectrometry currently presents the most precise method for rhamnolipid identification and quantification. Suitable approaches to accelerate rhamnolipid quantification for better control of biosurfactant production are HPLC analysis directly from culture broth by adding an internal standard or Fourier transform infrared attenuated total reflectance spectroscopy measurements of culture broth as a possible quasi-online quantification method in the future. The search for alternative rhamnolipid-producing strains makes a structure analysis and constant adaptation of the existing quantification methods necessary. Therefore, simple colorimetric tests based on whole rhamnolipid content can be useful for strain and medium screening. Furthermore, rhamnolipid purification from a fermentation broth will be considered depending on the following application.
Keywords: Rhamnolipids; Glycolipids; Biosurfactants; Pseudomonas aeruginosa ; Analysis; Purification

Intracellular signaling can be monitored in vivo in living cells by genetically encoded intracellular fluorescent probes. In this review, three aspects of these probes are introduced: 1) the imaging dynamics of endogenous mitochondrial RNA; 2) nuclear receptor and coactivator/corepressor interactions, and; 3) the signal sequence in mitochondrial intermembrane space. These probes are generally applicable to fundamental biological studies as well as for assaying and screening possible pharmaceutical or toxic chemicals that facilitate or inhibit cellular signaling pathways.
Keywords: Fluorescent probe; Mitochondrial RNA; Nuclear receptor; Mitochondrial intermembrane space

Bioimaging of cells and tissues using accelerator-based sources by Cyril Petibois; Mariangela Cestelli Guidi (1599-1608).
A variety of techniques exist that provide chemical information in the form of a spatially resolved image: electron microprobe analysis, nuclear microprobe analysis, synchrotron radiation microprobe analysis, secondary ion mass spectrometry, and confocal fluorescence microscopy. Linear (LINAC) and circular (synchrotrons) particle accelerators have been constructed worldwide to provide to the scientific community unprecedented analytical performances. Now, these facilities match at least one of the three analytical features required for the biological field: (1) a sufficient spatial resolution for single cell (< 1 μm) or tissue (<1 mm) analyses, (2) a temporal resolution to follow molecular dynamics, and (3) a sensitivity in the micromolar to nanomolar range, thus allowing true investigations on biological dynamics. Third-generation synchrotrons now offer the opportunity of bioanalytical measurements at nanometer resolutions with incredible sensitivity. Linear accelerators are more specialized in their physical features but may exceed synchrotron performances. All these techniques have become irreplaceable tools for developing knowledge in biology. This review highlights the pros and cons of the most popular techniques that have been implemented on accelerator-based sources to address analytical issues on biological specimens.
Keywords: Bioanalytical methods; X-ray spectroscopy (XPS/XRF/EDX); Spectroscopy/Instrumentation; Laser spectroscopy; Speciation; IR spectroscopy/Raman spectroscopy

Quantum dots (QDs) have a number of unique optical properties that are advantageous in the development of bioanalyses based on fluorescence resonance energy transfer (FRET). Researchers have used QDs as energy donors in FRET schemes for the analysis of nucleic acids, proteins, proteases, haptens, and other small molecules. This paper reviews these applications of QDs. Existing FRET technologies can potentially be improved by using QDs as energy donors instead of conventional fluorophores. Superior brightness, resistance to photobleaching, greater optimization of FRET efficiency, and/or simplified multiplexing are possible with QD donors. The applicability of the Förster formalism to QDs and the feasibility of using QDs as energy acceptors are also reviewed. Figure A ligand capped core/shell quantum dot acting as energy donor in a FRET process with aconjugated Cy3 labeled oligonucleotide
Keywords: Quantum dots; Fluorescence resonance energy transfer; Nucleic acids; Maltose binding protein; Nanobiotechnology

Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples by Anjali Kumari; Patrizia Pasini; Sylvia Daunert (1619-1627).
Bacteria communicate among themselves using certain chemical signaling molecules. These signaling molecules generally are N-acyl homoserine lactones (AHLs) in Gram-negative bacteria and oligopeptides in Gram-positive bacteria. In addition, both Gram-positive and Gram-negative bacteria produce a family of signaling molecules known as autoinducer-2 that they employ for their communications. Bacteria coordinate their behavior by releasing and responding to the chemical signaling molecules present in proportion to their population density. This phenomenon is known as quorum sensing. The role of bacteria in the pathogenesis of several diseases, including gastrointestinal (GI) disorders, is well established. Moreover, rather recently bacterial quorum sensing has been implicated in the onset of bacterial pathogenicity. Thus, we hypothesized that the signaling molecules involved in bacterial communication may serve as potential biomarkers for the diagnosis and management of several bacteria-related diseases. For that, we previously developed a method based on genetically engineered whole-cell sensing systems for the rapid, sensitive, cost-effective and quantitative detection of AHLs in biological samples, such as saliva and stool, from both healthy and diseased individuals with GI disorders. Although various analytical methods, based on physical-chemical techniques and bacterial whole-cell biosensors, have been developed for the detection of AHLs in the supernatants of bacterial cultures, only a few of them have been applied to AHL monitoring in real samples. In this paper, we report work performed in our laboratory and review that from others that describes the detection of AHLs in biological, clinical samples, and report some of our recent experimental results.
Keywords: Bacteria; Quorum sensing; N-Acyl homoserine lactones; Pathogenicity; Physiological samples

The increasing importance of biological analytes in chemistry has triggered the development of a vast number of techniques for rapidly assessing them. Aside from microbiological test methods, a wide range of analytical sensor and detection methods are being developed. Within this article, we review the literature on this topic from the last five years, stressing two main aspects of method development. The first aspect is the design of novel analytical strategies and transducers to generate signals more sensitively, more rapidly and more efficiently. Most of the progress in this field has focused on electrochemical detection, although novel approaches to optical and mass-sensitive measurements have been reported. Second, we provide an overview of two main approaches to creating artificial interaction layers for sensors based on tailored interaction sites in polymeric or biomimetic systems. The most prominent of these approaches is (molecular) imprinting, where selectivity is achieved by directly templating a polymer material with the target analyte or a model compound, thus achieving biomimetic interaction sites within both thin films and particles. Figure Sensors as means for rapid analysis gain increasing interest and importance in bioanalyte sensing. This article reviews recent developments in the design of transducers and artificial recognition membranes for assessing different biological species.
Keywords: Chemical sensors; Bioanalyte detection; Artificial recognition materials

The objective of this contribution is to review the application of advanced multivariate data-analysis techniques in the field of mid-infrared (MIR) spectroscopic biomedical diagnosis. MIR spectroscopy is a powerful chemical analysis tool for detecting biomedically relevant constituents such as DNA/RNA, proteins, carbohydrates, lipids, etc., and even diseases or disease progression that may induce changes in the chemical composition or structure of biological systems including cells, tissues, and bio-fluids. However, MIR spectra of multiple constituents are usually characterized by strongly overlapping spectral features reflecting the complexity of biological samples. Consequently, MIR spectra of biological samples are frequently difficult to interpret by simple data-analysis techniques. Hence, with increasing complexity of the sample matrix more sophisticated mathematical and statistical data analysis routines are required for deconvoluting spectroscopic data and for providing useful results from information-rich spectroscopic signals. A large body of work relates to the combination of multivariate data-analysis techniques with MIR spectroscopy, and has been applied by a variety of research groups to biomedically relevant areas such as cancer detection and analysis, artery diseases, biomarkers, and other pathologies. The reported results indeed reveal a promising perspective for more widespread application of multivariate data analysis in assisting MIR spectroscopy as a screening or diagnostic tool in biomedical research and clinical studies. While the authors do not mean to ignore any relevant contributions to biomedical analysis across the entire electromagnetic spectrum, they confine the discussion in this contribution to the mid-infrared spectral range as a potentially very useful, yet underutilized frequency region. Selected representative examples without claiming completeness will demonstrate a range of biomedical diagnostic applications with particular emphasis on the advantageous interaction between multivariate data analysis and MIR spectroscopy.
Keywords: Multivariate data analysis; Mid-infrared spectroscopy; Biomedical diagnostics; Cell analysis; Tissue analysis; Bio-fluid analysis

Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores by Dimitra N. Stratis-Cullum; Guy D. Griffin; Joel Mobley; Tuan Vo-Dinh (1655-1660).
This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 × 105 Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use. Figure Schematic diagram of the miniature biochip detection system
Keywords: Biochips; Biosensors immunoassays; ELISA; Spectroscopy; Bacillus globigii ; Chemiluminescence; Spores; Intensifier; Bioluminescence

Genotypic resistance testing in HIV by arrayed primer extension by Michaela Schanné; Jochen Bodem; Aslihan Gerhold-Ay; Anette Jacob; Kurt Fellenberg; Hans-Georg Kräusslich; Jörg D. Hoheisel (1661-1669).
The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5′-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere.
Keywords: Arrayed Primer Extension (APEX); Oligonucleotide microarray; Human immunodeficiency virus (HIV); Genotypic drug resistance testing

Reverse transcription-polymerase chain reaction on a microarray: the integrating concept of “active arrays” by Markus von Nickisch-Rosenegk; Xenia Marschan; Dennie Andresen; Frank F. Bier (1671-1678).
In this report we describe the proof of principle of a reverse transcription polymerase chain reaction (RT-PCR) but on-chip, with immobilized specific primers using a transcriptome of mouse-muscle fibroblasts for detection of muscle-specific expression products of these cells. The isolated total mRNA was directly incubated on an array of immobilized and solubilized specific primers, which allow the amplification of certain muscle-specific RNAs via its immobilized cDNAs. In contrast to others, the immobilized cDNA-products were directly synthesized on the chip by applying covalently bound specific primers. The products were detected by the incorporated and fluorophore-modified specific primers of the subsequently synthezised second strand. In addition, this second-strand served as a further template (like the basically used mRNA) in the subsequent solid-phase-PCR to amplify first-strand cDNA copies at the remaining immobilized specific primer-probes. This is the intrinsic factor of the amplification of certain signals of this application. The specific cDNA templates of genes coding for subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm), and ATPase (Atp2a2) were amplified on a biochip by RT-PCR directly from freshly isolated mRNA. The resulting procedure allows the detection of mRNA sequences from less than 5 pg of total RNA preparations.
Keywords: Microarray; Biochip; Transcription analysis; Reverse transcription-PCR; Active array

A microarray chip for label-free detection of narcotics by Goran Klenkar; Bo Liedberg (1679-1688).
A protein array chip for label-free optical detection of low molecular weight compounds has been developed. As a proof of principle, the chip is proven capable of rapidly (approximately 1 min) determining hits from aqueous cocktails composed of four common narcotics, cocaine, ecstasy, heroin, and amphetamine, using imaging surface plasmon resonance (SPR) as the detection principle. The chip is produced by injecting a mixture of antibodies and letting them self-sort and bind to narcotic analog coupled proteins already present in a predefined pattern on the supporting substrate. An indirect detection method, where antibodies are displaced from the surface upon recognition of their corresponding narcotics, is used to obtain the optical contrast and thus a detectable SPR and/or ellipsometric signal. Two types of readouts are possible from the present setup: intensity SPR images and SPR/ellipsometric sensorgrams. Positive hits were routinely obtained for analyte concentrations of 50 pg/μL and the limit of detection, without any parameter optimizations, seems to fall in the range 0.5 pg/μL (1.4 nM) for heroin, 2.5 pg/μL (8.2 nM) for cocaine, and 5 pg/μL for the other two narcotics (26 nM for ecstasy and 37 nM for amphetamine). With improved readout possibilities (sampling frequency), signal evaluation algorithms, and antibody–antigen design strategies, we believe this limit can be further improved. The chip is shown to work for many measurement cycles with excellent reproducibility. Moreover, with a more advanced fluidic system, excess injected antibodies could be collected and reused for many cycles, which could make the running costs of the system very low. The chip is in no way limited to detection of narcotics. Other low molecular weight compounds could easily be detected on the same chip. For example, trinitrotoluene detection has already been demonstrated using our chip. Possible areas of application for the system are therefore envisaged in airport and underground transport security, customs, drug interdiction, forensics, and as warning alerts on military equipment and personnel. Figure Narcotics chip (left) composed of spots of piezodispensed analog-coupled proteins that are loaded with antibodies to form a patterned regions represented by the capital letter of the four different narcotics in focus. (Right) The same chip showing hits for ectasy and herion in the cocktail. Both images are obtained in imaging surface plasmon resonance mode
Keywords: Protein microarray; Imaging surface plasmon resonance; Imaging ellipsometry; Narcotics trace detection; Biosensors; Antibody displacement

Universal liposomes: preparation and usage for the detection of mRNA by Katie A. Edwards; Katherine L. Curtis; Jessica L. Sailor; Antje J. Baeumner (1689-1702).
Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L−1 and an assay range spanning four orders of magnitude (5 pmol L−1−50 nmol L−1) with a coefficient of variation ≤5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.
Keywords: Sandwich hybridization; Liposomes; Nucleic acids (DNA/RNA); Fluorescence; Microtiter plate

A multianalyte ELISA for immunochemical screening of sulfonamide, fluoroquinolone and ß-lactam antibiotics in milk samples using class-selective bioreceptors by Javier Adrian; Daniel G. Pinacho; Benoit Granier; Jean-Marc Diserens; Francisco Sánchez-Baeza; M.-Pilar Marco (1703-1712).
A multianalyte ELISA has been developed for the simultaneous determination of the most frequently used antibiotic families in the veterinary field following the typical planar microarray configuration, where the identity of the target analyte is encoded by its location in the detection platform (Master et al. in Drug Discovery Today 11:1007–1011, 2006). To accomplish this aim, two individual enzyme-linked immunosorbent assays for sulfonamide and fluoroquinolone antibiotics and an enzyme-linked receptor assay for ß-lactam antibiotics have been combined. The strategy uses microplates coated with the corresponding haptenized proteins in specific sections of the microplate. The samples are mixed with a cocktail containing the bioreagents, and distributed in the wells of the microplate. Identification of the antibiotic present in a particular sample is consequently accomplished by detecting a positive response on the corresponding microplate section. Since the bioreceptors used show a wide recognition of the congeners of each antibiotic family, the multianalyte method is able to detect more than 25 different antibiotics from the three most important antibiotic families. The detectability reached in full-fat milk samples is below the European maximum residue limits. The accuracy and reliability of this multiplexed bioanalytical method have been demonstrated by analyzing blind spiked samples. Figure Several milk samples can be screening for more than 25 antibiotic residues of β-lactam, fluoroquinolone or sulfonamide families in just two hours in a single microplate ELISA. Milk samples are mixed with a cocktail of bioreceptors and added to the microplate. The assay provides a specific response for each antibiotic family on a different microplate section.
Keywords: Multianalyte detection; enzyme-linked immunosorbent assay; enzyme-linked receptor assay; antibiotic residues; sulfonamides; fluoroquinolones; ß-Lactams

Off-target activity of TNF-α inhibitors characterized by protein biochips by Oliver Feyen; Angelika Lueking; Axel Kowald; Christian Stephan; Helmut E. Meyer; Ulrich Göbel; Tim Niehues (1713-1720).
Tumor necrosis factor-alpha inhibitors are widely and successfully used to treat rheumatic diseases. However, significant side effects have been reported. To detect the potential off-target activities of such inhibitors we characterized two therapeutic antibodies (adalimumab, infliximab) and one receptor fusion protein (etanercept) on protein biochips (UNIchip® AV-400) containing a printed serial dilution of tumor necrosis factor-alpha and about 384 different human proteins. Etanercept binds to ten proteins (affinity: 20–33% of tumor necrosis factor-alpha recognition), and six of these proteins are related to ribosomal proteins. Interestingly, adalimumab binds to the same six proteins related to ribosomal proteins (affinity: 12–18%) as well as to four proteins crucially involved in ribosomal protein synthesis. Alignment of protein sequences indicates no significant sequence homology between these ten proteins bound by the biological drugs with the highest off-target activities. Taken together, our in vitro results demonstrate that a significant number of proteins are recognized by tumor necrosis factor-alpha inhibitors and are related to ribosome biogenesis.
Keywords: Specificity analysis; Off-target activity; Adalimumab; Etanercept; Infliximab; Protein biochip

Molecular biosensing system based on intrinsically disordered proteins by Kyle A. Cissell; Suresh Shrestha; Jennifer Purdie; Derrick Kroodsma; Sapna K. Deo (1721-1729).
Intrinsically disordered proteins (IDPs) that undergo structural transition upon binding their target molecules are becoming increasingly known. IDPs, because of their binding specificity and induced folding properties, can serve as biological recognition elements for sensing applications. In this paper, BRCA1, an IDP, was utilized as the biological recognition element to detect tumor suppressor protein p53 through the BRCA1/p53 binding interaction to serve as a proof-of-concept for the use of IDPs as recognition elements. The binding resulted in a disordered-to-ordered BRCA1 conformational change, as seen in our circular dichroism (CD) measurements. This conformational change in BRCA1 (residues 219–498) was utilized in the detection of p53 (residues 311–393) via both intrinsic and extrinsic fluorescent probes. Intrinsic tryptophan residues within the BRCA1 sequence detected p53 (311–393) with a detection limit of 0.559 nM (0.112 pmol). Two environmentally sensitive fluorophores, tetramethylrhodamine-5-maleimide (TMR) and 6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid, succinimidyl ester (dansyl-X, SE) were conjugated to BRCA1 (219–498). Dansyl-X, SE-conjugated BRCA1 (219–498) detected p53 (311–393) with a detection limit of 1.50 nM (0.300 pmol). The sensitivities for TMR and dansyl-X, SE-conjugated BRCA1 for the detection of p53 were nearly threefold and twofold higher, respectively, than the sensitivity reported using intrinsic BRCA1 tryptophan fluorescence. CD measurements did not reveal a disruption of p53/dye-conjugated BRCA1 binding, thus validating the applicability of environmentally sensitive fluorophores as transduction moieties to detect molecules which bind to IDPs and induce a structural change.
Keywords: Bioanalytical methods; Biosensors; Fluorescence; Intrinsically disordered protein (IDP); Breast cancer protein (BRCA1); p53

Sensing of oxygen in microtiter plates: a novel tool for screening drugs against pathogenic yeasts by Janine Wesolowski; Rabeay Y. A. Hassan; Stephan Hodde; Christian Bardroff; Ursula Bilitewski (1731-1737).
Most antibiotics were discovered via their inhibition of growth of target organisms. However, yeasts in particular have the capability to adapt metabolic pathways to the availability of nutrients e.g. yeasts can easily switch between respiratory and fermentative pathways in response to oxygen concentration, or can even use both simultaneously. Thus, we cultivated S. cerevisiae BY4741 and C. albicans 1386 in microtiter plates with integrated oxygen sensors to characterize the availability of oxygen for the organisms and to detect influences of fungicides on the oxygen consumption rates. The relevance of the respiratory pathway was indicated by the almost total consumption of oxygen during the first 1–3 h of the cultivation in the microtiter plates, when an increase in turbidity could hardly be seen. Moreover, the sensitivity of S. cerevisiae to inhibitors of the respiratory chain, such as myxothiazol, could be detected via a reduced oxygen consumption rate, whereas no inhibition of growth was observed. Thus, not only was the sensitivity of the test organism for the test compound detectable, but the affected pathway was also highlighted. Other compounds, such as pyrrolnitrin and ambruticin VS-3, inhibited growth of C. albicans 1386 and of S. cerevisiae (only pyrrolnitrin), which was additionally observed as reduced oxygen consumption rates. Thus, the determination of oxygen in microtiter plates via fluorescent dyes is a versatile supplement to standard growth inhibition tests.
Keywords: Myxothiazol; Ambruticin VS-3; Pyrrolnitrin; Candida albicans 1386; Saccharomyces cerevisiae ; Oxygen consumption; Fluorescent oxygen-sensitive dye

ADSA-TRIS: a new method to study interfacial phenomena at polymer–aqueous solution interfaces by Kerstin Nötzold; Stefanie Jäger; Stefan Michel; Senta Reichelt; Klaus-Jochen Eichhorn; Günter Gauglitz; Karina Grundke (1739-1749).
Axisymmetric drop shape analysis-profile (ADSA-P) was combined with total reflectometric interference spectroscopy (TRIS) in one experimental setup to study the interfacial phenomena at solid–liquid and liquid–vapor interfaces caused by adsorption/desorption (dissolution) of surface-active substances. Using sessile liquid droplets on polymer film/chromium-coated glass substrates that were optically matched with an immersion oil to a TRIS reflection prism, the optical thickness (product of physical thickness d and refractive index n) of the polymer film can be estimated by evaluating the wavelength-dependent intensity of reflected light. The sessile droplet is analyzed simultaneously by an ADSA setup arranged in a transverse direction to the path of the white-light beam of TRIS. From this analysis, the solid–vapor interfacial tension γ lv(t), contact angle θ(t), contact radius r(t), drop volume V(t), and solid–liquid interfacial tension γ sl(t) can be monitored as a function of time. The new method was applied to study polystyrene and poly(4-hydroxystyrene) surfaces in contact with aqueous buffer solutions and with protein solutions. The time-dependent changes in the optical film thickness caused by the adsorption of human serum albumin (HSA) and lysozyme (LSZ) were accompanied by changes in the solid–liquid interfacial tension. From the detailed study of both parameters, conclusions can be drawn with regard to the adsorption kinetics of the proteins on the hydrophobic polystyrene surfaces and to conformational changes occurring within the adsorbed protein layers. Figure Photo of the ADSA-TRIS setup
Keywords: Axisymmetric drop shape analysis-profile; Total reflectometric interference spectroscopy; Polystyrene; Poly(4-hydroxystyrene); Adsorption; Proteins

This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2′-bipyridine. In double-stranded DNA possessing free 3′-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 105 nucleotides in supercoiled plasmid DNA.
Keywords: DNA damage; Strand breaks; Apurinic sites; Electrochemistry; Carbon electrodes; Electroactive marker; Osmium

Electrically detected displacement assay (EDDA): a practical approach to nucleic acid testing in clinical or medical diagnosis by P. Liepold; T. Kratzmüller; N. Persike; M. Bandilla; M. Hinz; H. Wieder; H. Hillebrandt; E. Ferrer; G. Hartwich (1759-1772).
This paper introduces the electrically detected displacement assay (EDDA), a electrical biosensor detection principle for applications in medical and clinical diagnosis, and compares the method to currently available microarray technologies in this field. The sensor can be integrated into automated systems of routine diagnosis, but may also be used as a sensor that is directly applied to the polymerase chain reaction (PCR) reaction vessel to detect unlabeled target amplicons within a few minutes. Major aspects of sensor assembly like immobilization procedure, accessibility of the capture probes, and prevention from nonspecific target adsorption, that are a prerequisite for a robust and reliable performance of the sensor, are demonstrated. Additionally, exemplary results from a human papillomavirus assay are presented.
Keywords: Microarray; Electrical detection; Biosensor; HPV; Genotyping

SECM visualization of the spatial variability of enzyme-polymer spots. 3. Enzymatic feedback mode by Emad Mohamed Hussien; Thomas Erichsen; Wolfgang Schuhmann; Monika Maciejewska (1773-1782).
The responses of a PQQ-GDH entrapped in a polymer structure to mixtures of glucose and maltose were evaluated. Each compound was considered in the concentration range of 0–0.2 mM. Imaging was performed at constant height in the enzymatic feedback mode of scanning electrochemical microscopy (SECM). The enzyme-polymer spot was discretized into 15 × 15 μm2 substructures which were treated as independent individual microsensors. The response surfaces of the individual microsensors were approximated with a linear regression model. The coefficients in the derived equations represent contributions from topography, glucose concentration, maltose concentration, and the competition of glucose and maltose for the same active site of PQQ-GDH to the measured signal. The ratio of glucose and maltose contributions to the current at the SECM tip was constant for all microsensors and it was predominantly determined by the ratio of the turnover rates of both analytes in the PQQ-GDH catalyzed reaction. Using the difference between these coefficients, it was possible to select the microsensors within the overall enzyme-polymer spot that provided the best data for quantifying glucose and maltose by the artificial neural network used. The quantification of glucose and maltose was successful, except when the contributions from the components of the mixture were n g =k n units of glucose and simultaneously n m = 1.86(1−k)n units of maltose, for each constant n > 0 and k∈<0,1>.
Keywords: Scanning electrochemical microscopy; SECM; Amperometric biosensors; Discretization; Sensor array; Interference elimination

Characterisation of morphology of self-assembled PEG monolayers: a comparison of mixed and pure coatings optimised for biosensor applications by Jochen Mehne; Goran Markovic; Florian Pröll; Nina Schweizer; Stefan Zorn; Frank Schreiber; Günter Gauglitz (1783-1791).
For detection of low concentrations of analytes in complex biological matrices using optical biosensors, a high surface loading with capture molecules and a low nonspecific binding of nonrelevant matrix molecules are essential. To tailor biosensor surfaces in such a manner, poly(ethylene glycols) (PEG) in varying lengths were immobilised covalently onto glass-type surfaces in different mixing ratios and concentrations, and were subsequently modified with three different kinds of receptors. The nonspecific binding of a model protein (ovalbumin, OVA) and the maximum loading of the respective analytes to these prepared surfaces were monitored using label-free and time-resolved reflectometric interference spectroscopy (RIfS). The three different analytes used varied in size: 150 kDa for the anti-atrazine antibody, 60 kDa for streptavidin and 5 kDa for the 15-bp oligonucleotide. We investigated if the mixing of PEG in different lengths could increase the surface loadings of analyte mimicking a three-dimensional matrix as was found using dextrans as sensor coatings. In addition, the effect on the surface loading was investigated with regard to the size of the analyte molecule using such mixed PEGs on the sensor surface. For further characterisation of the surface coatings, polarisation modulation infrared reflection absorption spectroscopy, atomic force microscopy, and ellipsometry were applied.
Keywords: Surface chemistry; Poly(ethylene glycol); Reflectometric interference spectroscopy (RIfS); Ellipsometry; Biosensor; Atomic force microscopy (AFM); Polarisation modulation infrared reflection absorption spectroscopy (PM-IRRAS)

Biosensor-based on-site explosives detection using aptamers as recognition elements by Eva Ehrentreich-Förster; Dagmar Orgel; Andrea Krause-Griep; Birgit Cech; Volker A. Erdmann; Frank Bier; F. W. Scheller; Martina Rimmele (1793-1800).
Reliable observation, detection and characterisation of polluted soil are of major concern in regions with military activities in order to prepare efficient decontamination. Flexible on-site analysis may be facilitated by biosensor devices. With use of fibre-optic evanescent field techniques, it has been shown that immunoaffinity reactions can be used to determine explosives sensitively. Besides antibodies as molecular recognition elements, high-affinity nucleic acids (aptamers) can be employed. Aptamers are synthetically generated and highly efficient binding molecules that can be derived for any ligand, including small organic molecules like drugs, explosives or derivatives thereof. In this paper we describe the development of specific aptamers detecting the explosives molecule TNT. The aptamers are used as a sensitive capture molecule in a fibre-optic biosensor. In addition, through the biosensor measurements the aptamers could be characterised. The advantages of the aptamer biosensor include its robustness, its ability to discriminate between different explosives molecules while being insensitive to other chemical entities in natural soil and its potential to be incorporated into a portable device. Results can be obtained within minutes. The measurement is equally useful for soil that has been contaminated for a long time and for urgent hazardous spills.
Keywords: Aptamer; TNT; In-field measurement; Biosensor

Preparation of antibodies and development of a sensitive immunoassay with fluorescence detection for triazine herbicides by Sonia Herranz; Javier Ramón-Azcón; Elena Benito-Peña; María Dolores Marazuela; María Pilar Marco; María Cruz Moreno-Bondi (1801-1812).
Specific polyclonal antibodies against s-triazine herbicides were obtained by preparing immunogens coupling home-synthesized haptens derivatives of simazine (6-chloro-N-ethyl-N′-ethyl-1,3,5-triazine-2,4-diamine) to lysine groups of hemocyanin from keyhole limpets and bovine serum albumin carrier proteins. Three highly sensitive rabbit antisera were obtained and evaluated with a battery of six enzyme tracers derived from triazine structures in an optimized ELISA format. The antiserum As8 and the HRP-2f tracer, which yield the best assay sensitivity for simazine (detection limit 0.11 ± 0.02 μg L−1, IC50 0.88 ± 0.04 μg L−1), were applied to the development of a sensitive flow-through immunoassay for the analysis of this herbicide. The automated assay was based on a direct competitive immunosorbent assay and fluorescence detection. The optimized method presents an IC50 value of 0.35 ± 0.04 μg L−1 with a detection limit of 1.3 ± 0.9 ng L−1 and a dynamic range from 0.010 to 7.5 μg L−1 simazine. The generic nature of the antiserum was shown by good relative cross-reactivities with other triazines such as atrazine (420%) or propazine (130%) and a lower response to terbutylazine (6.4%) and desethyl-atrazine (2.2%). No cross-reactivity was obtained for nonrelated pesticides such as 2,4-dichlorophenoxyacetic acid or linuron and the assay could be applied as a screening method for triazine herbicides. The total analysis time was 30 min per determination and the immunosensor could be reused for more than 150 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of simazine in surface water samples at the nanogram per liter level. The results obtained by comparative analysis of the immunosensor with a chromatographic procedure for triazines showed a close correspondence.
Keywords: Triazines; Anti-simazine polyclonal antibodies; Flow-through immunoassay; Environmental analysis; Surface waters

Colloidal-based localized surface plasmon resonance (LSPR) biosensor for the quantitative determination of stanozolol by Mark P. Kreuzer; Romain Quidant; J.-Pablo Salvador; M.-Pilar Marco; Gonçal Badenes (1813-1820).
This work reports the systematic preparation of biosensors through the use of functionalized glass substrates, noble metal gold colloid, and measurement by localized surface plasmon resonance (LSPR). Glass substrate was modified through chemical silanization, and the density of gold colloid was carefully controlled by optimizing the conditions of silanization through the use of mixed silanes and selective mixing procedures. At this point, samples were exposed to bioreagents and changes in the shallow dielectric constant around the particles were observed by dark-field spectroscopy. Biological binding of high affinity systems (biotin/streptavidin and antigen/antibody) was subsequently investigated by optimizing coating layers, receptor concentration profiling, and finally quantitative determination of the analyte of interest, which in this case was a small organic molecule—the widely used, synthetic anabolic steroid called stanozolol. For this system, high specificity was achieved (>97%) through extensive nonspecific binding tests, with a sensitivity measurable to a level below the minimum required performance level (MRPL) as determined by standard chromatographic methods. Analytical best-fit parameters of Hillslope and regression coefficient are also commented on for the final LSPR biosensor. The LSPR biosensor showed good reproducibility (<5% RSD) and allowed for rapid preparation of calibration curves and determination of the analyte (measurement time of each sample ca. 2 min). As an alternative method for quantitative steroidal analysis, this approach significantly simplifies the detection setup while reducing the cost of analysis. In addition the system maintains comparable sensitivity to standard surface plasmon resonance methods and offers great potential for miniaturization and development of multiplexed devices. Figure Schematic of sensor configuration indicating both min and max controls and associatedexample localized resonance curves
Keywords: Localized surface plasmon resonance; Stanozolol; Colloid; Nanoparticles; Biosensor

The development and characterization of one rat monoclonal antibody (mAb) for 2,4-dinitroaniline and of two rat mAbs for 2,6-dinitroaniline are described. With the immunization of rats with 2,4,6-trinitrophenyl-glycylglycine–keyhole limpet hemocyanine (KLH) conjugate one mAb (PK 5H6) has been developed and formatted into a competitive enzyme-linked immunosorbent assay (ELISA). This assay no. 1 is very sensitive for 2,4-dinitroaniline with a test midpoint of 0.24 ± 0.06 μg L−1 (n = 19) in 40 mM phosphate-buffered saline (PBS). A second hapten, 3-(4-amino-2,6-dinitrophenyl)propionic acid, which was also conjugated to KLH and used for the immunization of rats, led to two sensitive ELISAs for 2,6-dinitroaniline in 40 mM PBS with test midpoints of 0.61 ± 0.08 μg L−1 (n = 15; mAb DNT4 3C6; assay no. 2) and 0.94 ± 0.29 μg L−1 (n = 17; mAb DNT4 1A7, assay no. 3). Selectivities of all mAbs were checked with more than 20 compounds, including nitroaromatic compounds, 2,6-dinitroaniline pesticides, and other substituted derivatives of aniline. As very noticeable cross-reactivities, all mAbs recognize 2-chloro-4,6-dinitroaniline, 4-chloro-2,6-dinitroaniline and 2-bromo-4,6-dinitroaniline, the last of these being a major metabolite of the azo dye Disperse Blue 79. As first demonstrations of applications, two ELISAs (assays no. 1 and 2) were used for the analysis of 2,4- or 2,6-dinitroaniline in spiked water and soil samples. Recovery data were determined and the majority of these data were in the range of 90–120%. These assays can contribute to a very cost-effective and environmentally friendly immunochemical surveillance monitoring of environmental samples for contaminations with these compounds. To the best of the authors’ knowledge, these are the first antibodies described for 2,4-dinitroaniline and for 2,6-dinitroaniline.
Keywords: 2-Bromo-4,6-dinitroaniline; 2-Chloro-4,6-dinitroaniline; 4-Chloro-2,6-dinitroaniline; Disperse Blue 79; Rat monoclonal antibodies; Water; Soil

A new optical platform for biosensing based on fluorescence anisotropy by F. Baldini; A. Carloni; A. Giannetti; G. Porro; C. Trono (1837-1844).
A novel fluorescence-based optical platform for the interrogation of an optical biochip was designed and developed. The optical biochip was made of poly(methyl methacrylate) (PMMA) formed by two pieces of PMMA appropriately shaped in order to obtain four microchannels that are 500-μm wide and 400-μm high. The lower part includes the microchannels and the inlet and outlet for the fluidics, while the sensing biolayer was immobilized on the upper part. The optical signal comprised the fluorescence emitted by the biolayer, which was anisotropically coupled to the PMMA cover and suitably guided by the PMMA chip. The potentiality of the optical chip as a biosensor was investigated by means of a direct IgG/anti-IgG interaction carried out inside the flow channels. The mouse-IgG was covalently immobilized on the internal wall of the PMMA cover, and the Cy5-labelled anti-mouse IgG was used for the specific interaction. Several chemical treatments of the PMMA surface were investigated, poly(L-lactic acid), Eudragit L100 and NaOH, in order to obtain the most effective distribution of carboxylic groups useful for the covalent immobilisation of the mouse-IgG. The treatment with Eudragit L100 was found to be the most successful. Limits of detection and quantification of 0.05 μg mL−1 and 0.2 μg mL−1, respectively, were obtained with the configuration described.
Keywords: Optical biosensor; Optical chip; PMMA; Fluorescence anisotropy; IgG

Two immunoassay formats for fully automated CRP detection in human serum by Christiane Albrecht; Nina Kaeppel; Guenter Gauglitz (1845-1852).
Immunoassays are a proven approach towards fast, sensitive, cost-effective and easy-to-use analytical systems which are able to measure a variety of interesting analytes, especially in medical diagnostics. Herein, we report two assay formats, binding inhibition and sandwich assay format, for detection of C-reactive protein (CRP) in human serum. Both assays were characterised and compared with respect to their suitability and adaption into a complete sensor system. An automated, optical biosensor system, based on evanescent field technology, was used to carry out a full threefold calibration in each case. Owing to the resulting working ranges, 0.044–2.9 mg L−1 and 0.13–22.9 mg L−1, respectively, the assays qualify for use in detecting high-sensitivity CRP (C-reactive protein).
Keywords: Optical immunosensor; River analyser (RIANA); C-reactive protein (CRP); High-sensitivity CRP (hsCRP); Healthcare; TIRF

Lectin-modified piezoelectric biosensors for bacteria recognition and quantification by B. Serra; M. Gamella; A. J. Reviejo; J. M. Pingarrón (1853-1860).
The use of lectins for microorganism biosensors fabrication is proposed. Lectins are immobilised onto a gold-plated quartz crystal for direct piezoelectric label-free transduction of the bacteria–lectin binding event using an electrochemical quartz crystal microbalance (EQCM). Concanavalin A (Con A) and Escherichia coli were used for the evaluation of the lectin immobilisation method and the biosensor performance. Adsorption on nonpolarised and polarised (−0.200 V) gold-coated quartz crystals and immobilisation through avidin–biotin binding were checked for Con A surface attachment. Lectin–bacteria binding was evaluated in all cases. With a crystal modified with Con A via avidin–biotin immobilisation we obtained a linear calibration plot between 5.0 × 106 and 2.0 × 107 cfu mL−1 by measuring frequency changes with E. coli concentration 1 h after bacteria addition. A remarkable increase in sensitivity was achieved when the analytical solution contained free biotinylated Con A, as a consequence of multiple lectin adhesion to Escherichia coli cell wall, which produced an accumulation of Con A–E. coli conjugates in the form of multilayers at the electrode surface. A detection limit of approximately 1.0 × 104 cfu mL−1 was achieved. Moreover nonspecific adsorptions were minimised. Using Con A and lectin from Arachis hypogaea, different response profiles were achieved for Escherichia coli, Staphylococcus aureus and Mycobacterium phlei, thus demonstrating the feasibility of bacteria discrimination. An approach involving filtering of free and lectin-bound bacteria and introduction of a filter in the measuring cell allowed a significant frequency change to be obtained for an E. coli concentration of 1.0 × 103 cfu mL−1 in order to further increase the sensitivity and discriminate between viable and nonviable cells; an approach using electrochemical measurements of bacterial catalase activity was also checked.
Keywords: Lectins; Quartz crystal microbalance; Microorganisms; Biosensors

Effect of the immobilisation of DNA aptamers on the detection of thrombin by means of surface plasmon resonance by Veronika Ostatná; Hana Vaisocherová; Jiří Homola; Tibor Hianik (1861-1869).
We report a multichannel surface plasmon resonance (SPR) sensor for detection of thrombin via DNA aptamers immobilized on the SPR sensor surface. A detailed investigation of the effect of the immobilisation method on the interaction between thrombin and DNA aptamers is presented. Three basic approaches to the immobilisation of aptamers on the surface of the SPR sensor are examined: (i) immobilisation based on chemisorption of aptamers modified with SH groups, (ii) immobilisation of biotin-tagged aptamers via previously immobilized avidin, neutravidin or streptavidin molecular linkers, and (iii) immobilisation employing dendrimers as a support layer for subsequent immobilisation of aptamers. A level of nonspecific binding of thrombin to immobilized human serum albumin (HSA) for each of the immobilisation methods is determined. Immobilisation of aptamers by means of the streptavidin–biotin system yields the best results both in terms of sensor specificity and sensitivity.
Keywords: DNA aptamer; Thrombin; Dendrimers; Surface plasmon resonance

Time-domain fluorescence lifetime imaging for intracellular pH sensing in living tissues by Carsten Hille; Maik Berg; Lena Bressel; Dorit Munzke; Philipp Primus; Hans-Gerd Löhmannsröben; Carsten Dosche (1871-1879).
pH sensing in living cells represents one of the most prominent topics in biochemistry and physiology. In this study we performed one-photon and two-photon time-domain fluorescence lifetime imaging with a laser-scanning microscope using the time-correlated single-photon counting technique for imaging intracellular pH levels. The suitability of different commercial fluorescence dyes for lifetime-based pH sensing is discussed on the basis of in vitro as well of in situ measurements. Although the tested dyes are suitable for intensity-based ratiometric measurements, for lifetime-based techniques in the time-domain so far only BCECF seems to meet the requirements of reliable intracellular pH recordings in living cells.
Keywords: Fluorescence lifetime imaging microscopy; Time domain; Two-photon excitation; Intracellular pH; pH sensors; BCECF; Salivary glands

Analytical nanoscience and nanotechnology today and tomorrow by M. Valcárcel; B. M. Simonet; S. Cárdenas (1881-1887).
This article discusses the mutual impact of nanotechnology and analytical science and illustrates how this technical trend can be expected to strengthen the role of analytical chemists. To this end, it defines the limits of actual nanotechnological approaches and uses selected examples to illustrate the three major relationships between nanotechnology and analytical science, namely: the design and use of nanodevices; the use of nanoparticles (and nanostructures) in analytical processes; and the extraction of accurate chemical information from the nanoworld. Finally, the future of analytical nanotechnology in the short and medium term is briefly addressed.
Keywords: Analytical nanotechnology; Analytical nanoscience; Nanoparticles

An in vitro, rapid, and quantitative cell-based assay is needed to predict the efficacy of cancer drugs in individual patients, because a cancer patient may have unconventional aspects of tumor development. Here we report a rapid and label-free quantitative method for verifying apoptosis in living cancer cells cultured on a sensor chip with a newly developed high-precision surface plasmon resonance (SPR) sensor. The time-course cell reaction was monitored as the SPR angle change rate for 5 min during a 35-min cell culture of pancreatic cancer lines with a drug. The time-course cell reaction was significantly related to cell viability counted after 48 h as assessed by caspase-3 activity assay of apoptosis. Furthermore, the detected SPR signal was derived from the decrease in inner mitochondrial membrane potential. The results obtained are universally valid for various cancer drugs mediating apoptosis through different cell-signaling pathways and even for combined use in various pancreatic cancer cell lines. This system can be applied in a clinical setting to evaluate the personal therapeutic potential of drugs including pharmacodynamic interactions.
Keywords: Cell-based assay; Surface plasmon resonance; Cancer drug; Personal therapeutic potential; Apoptosis; Inner mitochondrial membrane potential; Pharmacodynamic interaction

Towards chemical analysis of nanostructures in biofilms I: imaging of biological nanostructures by Thomas Schmid; Johannes Burkhard; Boon-Siang Yeo; Weihua Zhang; Renato Zenobi (1899-1905).
Due to their direct influence on the stability of bacterial biofilms, a better insight into the nanoscopic spatial arrangement of the different extracellular polymeric substances (EPS), e.g., polysaccharides and proteins, is important for the improvement of biocides and for process optimization in wastewater treatment and biofiltration. Here, the first application of a combination of confocal laser-scanning microscopy (CLSM) and atomic force microscopy (AFM) to the investigation of river-water biofilms and related biopolymers is presented. AFM images collected at selected areas of CLS micrographs dramatically demonstrate the heterogeneity of biofilms at the nanometer scale and the need for a chemical imaging method with nanoscale resolution. The nanostructures (e.g., pili, flagella, hydrocolloids, and EPS) found in the extracellular matrix are classified according to shape and size, which is typically 50–150 nm in width and 1–10 nm in thickness, and sets the demands regarding spatial resolution of a potential chemical imaging method. Additionally, thin layers of the polysaccharide alginate were investigated. We demonstrate that calcium alginate is a good model for the EPS architecture at the nanometer scale, because of its similar network-like structure. Figure CLSM-AFM allows imaging of nanometer-sized extracellular structures
Keywords: Biofilm; Extracellular polymeric substances (EPS); Alginate; Confocal laser-scanning microscopy (CLSM); Atomic force microscopy (AFM)

Towards chemical analysis of nanostructures in biofilms II: tip-enhanced Raman spectroscopy of alginates by Thomas Schmid; Andreas Messmer; Boon-Siang Yeo; Weihua Zhang; Renato Zenobi (1907-1916).
This study examines the feasibility of using tip-enhanced Raman spectroscopy (TERS) for label-free chemical characterization of nanostructures in biological systems. For this purpose, a well-defined model system consisting of calcium alginate fibers is studied. In a companion paper, calcium alginate fibers and their network structures were shown to be a good model for the extracellular polysaccharides of biofilms at the nanoscale. TERS analysis of biological macromolecules, such as alginates, is complicated by heterogeneity in their sequence, molecular weight, and conformations, their small Raman cross-section, and the large number of functional groups, which can chemically interact with the silver surface of the tip and cause significant band shifts. Due to these effects, Raman frequencies in TERS spectra of biopolymers do not necessarily resemble band positions in the normal Raman spectrum of the bulk material, as is the case for less complex samples (e.g., dye molecules) studied so far. Additionally, analyte decomposition due to laser heating can have a significant influence, and carbon contamination signals can sometimes even overwhelm the weak analyte signals. Based on the investigation of alginates, strategies for spectra correction, choice of appropriate reference samples, and data interpretation are presented. With this approach, characteristic frequency ranges and specific marker bands can be found for biological macromolecules that can be employed for their identification in complex environments. Figure TERS spectrum of a calcium alginate fiber bundle
Keywords: Alginate; Biological samples; Biofilm; Tip-enhanced Raman spectroscopy (TERS); Atomic force microscopy (AFM)

Metabolic profiling of major vitamin D metabolites using Diels–Alder derivatization and ultra-performance liquid chromatography–tandem mass spectrometry by Pavel A. Aronov; Laura M. Hall; Katja Dettmer; Charles B. Stephensen; Bruce D. Hammock (1917-1930).
Biologically active forms of vitamin D are important analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous metabolic profiling of entire biochemical pathways. The impediment to the metabolic profiling of vitamin D metabolites is the low level of 1α,25-dihydroxyvitamin D3 in human serum (15–60 pg/mL). Here, we demonstrate that liquid–liquid or solid-phase extraction of vitamin D metabolites in combination with Diels–Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)–electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1α,25-dihydroxyvitamin D3, 1α,25-dihydroxyvitamin D2, 24R,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6–4.8 % and 5–16 % for 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3, respectively, using solid-phase extraction.
Keywords: 1α,25-Dihydroxyvitamin D3 ; 25-Hydroxyvitamin D3 ; 24R,25-Dihydroxyvitamin D3 ; UPLC; LC-MS; Metabolic profiling; Derivatization

Quantification of atrazine and its metabolites in urine by on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry by Parinya Panuwet; Johnny V. Nguyen; Peter Kuklenyik; Simeon O. Udunka; Larry L. Needham; Dana B. Barr (1931-1939).
We have developed a method using on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry (SPE-HPLC-MS/MS) and isotope dilution quantification to measure atrazine and seven atrazine metabolites in urine. The metabolites measured were hydroxyatrazine, diaminochloroatrazine, desisopropylatrazine, desethylatrazine, desethylatrazine mercapturate, atrazine mercaturate and atrazine itself. Our method has good precision (relative standard deviations ranging from 4 to 20% at 5, 10 and 50 ng/mL), extraction efficiencies of 67 to 102% at 5 and 25 ng/mL, relative recoveries of 87 to 112% at 5, 25, 50 and 100 ng/mL limits of detection (LOD) ranging from 0.03 to 2.80 ng/mL. The linear range of our method spans from the analyte LOD to 100 ng/mL (40 ng/mL for atrazine and atrazine mercapturate) with R 2 values of greater than 0.999 and errors about the slope of less than 3%. Our method is rapid, cost-effective and suitable for large-scale sample analyses and is easily adaptable to other biological matrices. More importantly, this method will allow us to better assess human exposure to atrazine-related chemicals. Figure A schematic representation showing the elution of the analytes from the solid-phase extraction cartridge onto the analytical column for chromatographic separation prior to MS/MS analysis
Keywords: Atrazine; Metabolites; Urine; Mass spectrometry

The application of a new kind of technique involving solid-phase extraction coupled with thermal desorption (SPE-TD) to the qualitative analysis of water used in pharmaceutical products was evaluated. Comparative analyses performed by the purge and trap (PT) technique were also conducted. The application of this SPE-TD technique resulted in the isolation of a large number of compounds from the water sample. The SPE-TD technique is applied to less volatile compounds, whereas the PT technique is used for more volatile and nonpolar ones. These two techniques should be applied in order to achieve complete identification and quantitative determination. Additionally, an attempt to identify organic compounds in pharmaceutical products was also conducted. The compounds present in such products include aldehydes, ketones, hydrocarbons, alcohols, esters. The influence of storage on the quality of water was also investigated. For samples characterized by a longer storage time, qualitatively richer chromatograms were obtained, which confirmed that components were released from the packaging (especially polyethylene) which entered the stored product.
Keywords: Solid-phase extraction; Trace organic compounds; Pharmaceutical purpose for water

Three-dimensionally structured, silica based, organic–inorganic hybrid nanoparticles (NPs) were prepared by a simple and feasible water-in-oil (W/O) microemulsion method and a promising platform for bioelectrochemical analysis was obtained. The commonly used phenathiazine organic compound, toluidine blue (TB) was readily captured in the three-dimensional cage of the inorganic SiO2 network, which was considered to serve as a protective “shell” toward the embedded TB. A TEM image indicated the size of the thus prepared TB-doped SiO2 (TB@SiO2) NPs was 21 ± 3 nm. UV–visible and IR spectroscopy confirmed successful formation of the organic–inorganic composite and possible interaction between TB and SiO2, which favored enhanced stability of the hybrid. A sensitive amperometric sensor for hemoglobin (Hb) biomolecules based on TB@SiO2 NPs conjugated with a biopolymer chitosan (CHIT) membrane was then developed. The surface of the silica NPs was highly biocompatible and the TB captured inside maintained its high electron-transfer efficiency. Dye leakage of TB from the TB@SiO2 hybrid was proved to be minimal, owing to the inorganic SiO2 network and the force of interaction between TB and SiO2. The amperometric sensor had a detection limit of 2.5 × 10−9 mol L−1 (S/N = 3) with a linear range from 5.0 × 10−9 to 3.0 × 10−6 mol L−1 for Hb. When it was applied to determine the concentration of a clinical blood sample, satisfactory results were obtained which were in good agreement with those obtained by the standard method.
Keywords: Toluidine blue-doped silica nanoparticles; Water-in-oil microemulsion; Amperometric sensor; Hemoglobin

Laser induced breakdown spectroscopy as a tool for discrimination of glass for forensic applications by E. M. Rodriguez-Celis; I. B. Gornushkin; U. M. Heitmann; J. R. Almirall; B. W. Smith; J. D. Winefordner; N. Omenetto (1961-1968).
Materials analysis and characterization can provide important information as evidence in legal proceedings. The potential of laser induced breakdown spectroscopy (LIBS) for the discrimination of glass fragments for forensic applications is presented here. The proposed method is based on the fact that glass materials can be characterized by their unique spectral fingerprint. Taking advantage of the multielement detection capability and minimal to no sample preparation of LIBS, we compared glass spectra from car windows using linear and rank correlation methods. Linear correlation combined with the use of a spectral mask, which eliminates some high-intensity emission lines from the major elements present in glass, provides effective identification and discrimination at a 95% confidence level.
Keywords: Glass; Forensic; Laser induced breakdown spectroscopy; Correlation analysis; Material identification

A label-free method based on MALDI-TOF mass spectrometry for the absolute quantitation of troponin T in mouse cardiac tissue by Mariano Bizzarri; Chiara Cavaliere; Patrizia Foglia; Chiara Guarino; Roberto Samperi; Aldo Laganà (1969-1976).
A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed. This methodology was applied to mouse heart in order to quantify cardiac troponin T (cTnT), which is considered to be a sensitive marker of heart damage. The cTnT was extracted, isolated by reversed-phase high-performance liquid chromatography, digested, and analyzed by MALDI-TOF MS. The MS-based quantitation was performed using matrix-matched calibration curves (due to a matrix effect) of two synthetic peptides, one cTnT-specific peptide and one internal standard peptide, respectively. Recoveries at three spiking levels ranged from 87–96%, with relative standard deviations of below 10%. The method detection limit and the method quantitation limit, expressed as the amount of cTnT for the amount of total sarcomeric protein extract, were 0.03 mg g−1 and 0.15 mg g−1, respectively. This method appears to be accurate and generally suitable for improving absolute protein quantitation.
Keywords: Absolute quantitation; cTnT; Label-free; MALDI-TOF MS; Mouse cardiac troponin T; Quantitative proteomics

Polyurethane foam (PUF) has been suggested as a solid polymeric reagent for determination of nitrite. The determination is based on the diazotization of end toluidine groups of PUF with nitrite in acidic medium followed by coupling of polymeric diazonium cation with 3-hydroxy-7,8-benzo-1,2,3,4-tetrahydroquinoline. The intensely colored polymeric azodye formed in this reaction can be used as a convenient analytic form for the determination of nitrite by diffuse reflectance spectroscopy (c min = 0.7 ng mL−1). The possibility of using a desktop scanner, digital camera, and computer data processing for the numerical evaluation of the color intensity of the polymeric azodye has been investigated. A scanner and digital camera can be used for determination of nitrite with the same sensitivity and reproducibility as with diffuse reflectance spectroscopy. The approach developed was applied for determination of nitrite in river water and human exhaled breath condensate.
Keywords: Nitrite determination; Polyurethane foam; 3-Hydroxy-7,8-benzo-1,2,3,4-tetrahydroquinoline; Diffuse reflectance spectroscopy; Colorimetry