Analytical and Bioanalytical Chemistry (v.398, #6)
Focus on bioanalysis by Antje J. Baeumner; Günter Gauglitz; Frieder W. Scheller (2337-2339).
is Professor Biological Engineering and Director of Graduate Studies in the Department of Biological and Environmental Engineering at Cornell University in Ithaca, NY, USA. She is on the Board of Directors of the Society of Electroanalytical Chemistry, a member of the Extended Executive Committee of the International Association of Environmental Analytical Chemistry, was the 2010 Chair of the Gordon Research Conference on Bioanalytical Sensors, and has received numerous honors in recent years including being Finalist for the Blavatnik Award of the NY Academy of Sciences, and receiving a Humboldt Research Fellowship and a German National Science Foundation Mercator Guest professorship. Her research is focused on the development of microTotal Analysis Systems and smart lateral flow assays for the detection of pathogens and toxins in food, in the environment, and for medical diagnostics. is Professor at the Eberhard-Karls-University of Tübingen working 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 10 years his main scientific interests have centered on research and development in chemical and biochemical sensors with special focus on the characterization of interfaces of polymers and biomembrane surfaces, spectroscopic techniques, use of spectral interferometry to monitor changes in optical thickness of thin layers, and effects of Fresnel reflectivity at interfaces. He has been Editor of Analytical and Bioanalytical Chemistry (ABC) since 2002. is visiting Professor at the University of Potsdam and consultant at the Fraunhofer Institute for Biomedical Engineering. 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. He has been Professor of Analytical Biochemistry at the University of Potsdam since 1993. In 2001 he was President of the German Society of Biochemistry and Molecular Biology. His current research interests include biomimetic sensors based on molecularly imprinted polymers and protein electrochemistry.
Electrochemical impedance spectroscopy in label-free biosensor applications: multivariate data analysis for an objective interpretation by Britta Lindholm-Sethson; Josefina Nyström; Martin Malmsten; Lovisa Ringstad; Andrew Nelson; Paul Geladi (2341-2349).
Electrochemical impedance spectroscopy plays an important role in biosensor science thanks to the possibility of finding specific information from processes with different kinetics at a chosen electrode potential in one experiment. In this paper we briefly discuss label-free impedimetric biosensors described in the literature. A novel method for neutral interpretation of impedance data is presented that includes complex number chemometrics. Three examples are given based on impedance measurements on synthetic biomembranes, in this case a lipid monolayer deposited on a mercury electrode. The interaction of various compounds with the monomolecular lipid layer is illustrated with the following: (1) different concentrations of magainin (Geladi et al. in Proc. Int. Fed. Med. Biomed. Eng. 9:219–220, 2005); (2) different derivatives of gramicidin A (Lindholm-Sethson et al. in Langmuir 24:5029–5032, 2007), and (3) an antimicrobial peptide (Ringstad et al. in Langmuir 24:208–216, 2008).
Keywords: Impedance; Biosensor; Complex number chemometrics
Trends in solid-state NMR spectroscopy and their relevance for bioanalytics by Silvia Paasch; Eike Brunner (2351-2362).
Based on continuous methodical advances and developments, solid-state NMR spectroscopy has become a powerful tool for the investigation of various materials, including polymers, glasses, zeolites, fullerenes, and many others. During the past decade, solid-state NMR spectroscopy also found increasing interest for the study of biomolecules. For example, membrane proteins reconstituted into lipid environments such as bilayers or vesicles, protein aggregates such as amyloid fibrils, as well as carbohydrates can now be studied by solid-state NMR spectroscopy. This review briefly introduces the principles of solid-state NMR spectroscopy and highlights novel methodical trends. Selected applications demonstrate the possibilities of solid-state NMR spectroscopy as a valuable bioanalytical tool. Figure Trends in solid-state NMR spectroscopy
Keywords: Bioanalytics; Nuclear magnetic resonance; Solid state; Biomolecules
Direct optical detection in bioanalysis: an update by Günter Gauglitz (2363-2372).
In biomolecular interaction analysis, direct optical detection is attracting increasing interest in academia and industry. Therefore, a previous review has been updated. Optical principles are given in brief, focussing especially on modern and frequently used techniques. Commercialized methods are listed with some specific applications. In addition, some of the many applications found in the literature are listed; others which have been reviewed elsewhere are cited. Overall, the growing interest in direct optical monitoring of biomolecular interaction is demonstrated and future trends are outlined. Because optical methods is a very wide field, the paper concentrates on the currently most common methods, microrefractometry and microreflectivity.
Keywords: Optical biosensor; Refractometry; Reflectometry; Transduction; Optical monitoring
Utilization of microparticles in next-generation assays for microflow cytometers by Jason S. Kim; Frances S. Ligler (2373-2382).
Micron-sized particles have primarily been used in microfabricated flow cytometers for calibration purposes and proof-of-concept experiments. With increasing frequency, microparticles are serving as a platform for assays measured in these small analytical devices. Light scattering has been used to measure the agglomeration of antibody-coated particles in the presence of an antigen. Impedance detection is another technology being integrated into microflow cytometers for microparticle-based assays. Fluorescence is the most popular detection method in flow cytometry, enabling highly sensitive multiplexed assays. Finally, magnetic particles have also been used to measure antigen levels using a magnetophoretic micro-device. We review the progress of microparticle-based assays in microflow cytometry in terms of the advantages and limitations of each approach. Figure Multiplexed assays using coded microspheres provide sensitive assays for on-site microflow cytometers
Keywords: Microparticle; Flow cytometry; Microfluidics; Microflow cytometer; Biosensors
Progress and possible applications of miniaturised separation techniques and elemental mass spectrometry for quantitative, heteroatom-tagged proteomics by Daniel Pröfrock (2383-2401).
The application of miniaturised separation techniques such as capillary LC, nano LC or capillary electrophoresis offers a number of advantages in terms of analytical performance, solvent consumption and the ability to analyse very small sample amounts. These features make them attractive for various bioanalytical tasks, in particular those related to the analysis of proteins and peptides. The skillful combination of such techniques with inductively coupled plasma mass spectrometry (ICP-MS) has recently permitted the design of combined analytical approaches utilising either elemental or molecule-specific detection techniques such as electrospray ionisation (ESI) or matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry in a highly complementary manner for, as an example, proteomics-orientated research (heteroatom-tagged proteomics). Such hybrid approaches are, in particular, providing promising new options for the fast screening of complex samples for specific metal-containing or—more generally speaking—heteroatom-containing biomolecules, as well as the accurate absolute quantification of biomolecules, which is still an unsolved problem in bioanalysis. Here, progress in as well as the potential and the special requirements of hyphenating miniaturised separation techniques with ICP-MS are reviewed and critically discussed. In addition, selected applications are highlighted to indicate current and possible future trends within this emerging area of research. Figure Application of miniaturized separation techniques and ICP-MS for heteroatom-tagged proteomics
Keywords: Miniaturisation; ICP-MS; Capillary LC; Nano LC; Capillary electrophoresis; Mass spectrometry; Proteomics; Bioanalysis
Biosensors with label-free detection designed for diagnostic applications by Bastian E. Rapp; Friederike J. Gruhl; Kerstin Länge (2403-2412).
Since the first biosensor was introduced in 1962 by Clark and Lyons, there has been increasing demand for such analytical devices in diagnostic applications. Research initially focussed mainly on detector principles and recognition elements, whereas the packaging of the biosensors and the microfluidic integration has been discussed only more recently. However, to obtain a user-friendly and well-performing analytical device, those components have to be considered all together. This review outlines the requirements and the solutions suggested for the integration of suitable biosensors in packaging and the integration of those encapsulated biosensors into a microfluidic surrounding resulting in a complete and efficient analytical device for diagnostic applications. The components required for a complete biosensor instrument are described and the latest developments which meet the requirements for diagnostic applications, such as single-use components and arrays for multiparameter detection, are discussed. The current state and the future of biosensors in the field of clinical diagnostics are outlined, particularly on the basis of label-free assay formats and the detection of prominent biomarkers for cancer and autoimmune disorders. Figure Caption Components to be considered in an efficient biosensor system
Keywords: Biosensors; Label-free; Single-use; Packaging; Microfluidics/microfabrication; Diagnostics
Emerging food contaminants: a review by Lina Kantiani; Marta Llorca; Josep Sanchís; Marinella Farré; Damià Barceló (2413-2427).
Governments throughout the world are intensifying their efforts to improve food safety. These efforts come as a response to an increasing number of food-safety problems and increasing consumer concerns. In addition, the variety of toxic residues in food is continuously increasing as a consequence of industrial development, new agricultural practices, environmental pollution, and climate change. This paper reviews a selection of emerging contaminants in food, including the industrial organic pollutants perfluorinated compounds (PFCs), polybrominated diphenyl ethers (PBDEs), and nanomaterials; the pharmaceutical residues antibiotics and coccidiostats; and emerging groups of marine biotoxins. The main analytical approaches for their detection and quantification in food will be presented and discussed with special emphasis on biological techniques, when these are feasible. In the last section, a summary of recent publications reporting the concentrations of these compounds in food will be presented and discussed.
Keywords: Emerging food contaminants; PFCs; PBDEs; Nanomaterials; Antibiotics; Biotoxins
In-situ imaging sensors for bioprocess monitoring: state of the art by Arne Bluma; Tim Höpfner; Patrick Lindner; Christoph Rehbock; Sascha Beutel; Daniel Riechers; Bernd Hitzmann; Thomas Scheper (2429-2438).
Over the last two decades, more and more applications of sophisticated sensor technology have been described in the literature on upstreaming and downstreaming for biotechnological processes (Middendorf et al. J Biotechnol 31:395–403, 1993; Lausch et al. J Chromatogr A 654:190–195, 1993; Scheper et al. Ann NY Acad Sci 506:431–445, 1987), in order to improve the quality and stability of these processes. Generally, biotechnological processes consist of complex three-phase systems—the cells (solid phase) are suspended in medium (liquid phase) and will be streamed by a gas phase. The chemical analysis of such processes has to observe all three phases. Furthermore, the bioanalytical processes used must monitor physical process values (e.g. temperature, shear force), chemical process values (e.g. pH), and biological process values (metabolic state of cell, morphology). In particular, for monitoring and estimation of relevant biological process variables, image-based inline sensors are used increasingly. Of special interest are sensors which can be installed in a bioreactor as sensor probes (e.g. pH probe). The cultivation medium is directly monitored in the process without any need for withdrawal of samples or bypassing. Important variables for the control of such processes are cell count, cell-size distribution (CSD), and the morphology of cells (Höpfner et al. Bioprocess Biosyst Eng 33:247–256, 2010). A major impetus for the development of these image-based techniques is the process analytical technology (PAT) initiative of the US Food and Drug Administration (FDA) (Scheper et al. Anal Chim Acta 163:111–118, 1984; Reardon and Scheper 1995; Schügerl et al. Trends Biotechnol 4:11–15, 1986). This contribution gives an overview of non-invasive, image-based, in-situ systems and their applications. The main focus is directed at the wide application area of in-situ microscopes. These inline image analysis systems enable the determination of indirect and direct cell variables in real time without sampling, but also have application potential in crystallization, material analysis, polymer research, and the petrochemical industry. Figure Photo of an In-situ microscope manufactured by Sartorius Stedim Biotech (Göttingen, Germany)
Keywords: In-situ Microscopy; In-situ monitoring; Image-based sensors; Cell-size distribution; Inline estimation
New opportunities in multiplexed optical bioanalyses using quantum dots and donor–acceptor interactions by W. Russ Algar; Ulrich J. Krull (2439-2449).
This review highlights recent trends in the development of multiplexed bioanalyses using quantum dot bioconjugates and donor–acceptor interactions. In these methods, multiple optical signals are generated in response to biorecognition through modulation of the photoluminescence of populations of quantum dots with different emission colors. The donor–acceptor interactions that have been used include fluorescence resonance energy transfer, bioluminescence resonance energy transfer, charge transfer quenching, and quenching via proximal gold nanoparticles. Assays for the simultaneous detection of between two and eight target analytes have been developed, where spectral deconvolution is an important tool. Target analytes have included small molecules, nucleic acid sequences, and proteases. The unique optical properties of quantum dots offer several potential advantages in multiplexed detection, and a large degree of versatility, for example, one pot multiplexing at the ensemble level, where only wavelength discrimination is required to differentiate between detection channels. These methods are not being developed to compete with array-based technologies in terms of overall multiplexing capacity, but rather to enable new formats for multiplexed bioanalyses. In particular, quantum dot bioprobes based on donor–acceptor interactions are anticipated to provide future opportunities for multiplexed biosensing within living cells. Figure Multicolor optical bioanalyses using donor-acceptor interactions between quantum dots and gold nanoparticles, fluorescent dyes, or redox active complexes.
Keywords: Quantum dots; Multiplexing; Fluorescence resonance energy transfer; Bioluminescence resonance energy transfer; Gold nanoparticles; Charge transfer quenching
Recent advances in analytical and bioanalysis applications of noble metal nanorods by Ilaria Mannelli; M.-Pilar Marco (2451-2469).
In the last decade the use of anisotropic nanoparticles in analytical and bioanalytical applications has increased substantially. In particular, noble metal nanorods have unique optical properties that have attracted the interest of many research groups. The localized surface plasmon resonance (LSPR) generated by interaction of light at a specific wavelength with noble metal nanoparticles was found to depend on particle size and shape and on the constituting material and the surrounding dielectric solution. Because of their anisotropic shape, nanorods are characterized by two LSPR peaks: the transverse, fixed at approximately 530 nm, and the longitudinal, which is in the visible–near infra-red region of the spectrum and varies with nanorod aspect ratio. The intense surface plasmon band enables nanorods to absorb and scatter light in the visible and near infra-red regions, and fluorescence and two-photon induced luminescence are also observed. These optical properties, with the reactivity towards binding events that induce changes in the refractive index of the surrounding solution, make nanorods a useful tool for tracking binding events in different applications, for example assembly, biosensing, in-vivo targeting and imaging, and single-molecule detection by surface-enhanced Raman spectroscopy. This review presents the promising strategies proposed for functionalizing gold nanorods and their successful use in a variety of analytical and biomedical applications.
Keywords: Gold nanorod; Nanorod functionalization; (Bio)analytical applications
Aptamer-based molecular recognition for biosensor development by Jing Zhou; Mark R. Battig; Yong Wang (2471-2480).
Nucleic acid aptamers are an emerging class of synthetic ligands and have recently attracted significant attention in numerous fields. One is in biosensor development. In principle, nucleic acid aptamers can be discovered to recognize any molecule of interest with high affinity and specificity. In addition, unlike most ligands evolved in nature, synthetic nucleic acid aptamers are usually tolerant of harsh chemical, physical, and biological conditions. These distinguished characteristics make aptamers attractive molecular recognition ligands for biosensing applications. This review first concisely introduces methods for aptamer discovery including upstream selection and downstream truncation, then discusses aptamer-based biosensor development from the viewpoint of signal production. Figa Aptamer-based molecular recognition for analyte detection.
Keywords: Molecular recognition; Biosensor; Ultrasensitive detection; Nucleic acid aptamer; Upstream aptamer selection; Downstream aptamer truncation
Molecularly imprinted polymers: synthetic receptors in bioanalysis by Bernadette Tse Sum Bui; Karsten Haupt (2481-2492).
Molecularly imprinted polymers (MIPs) are tailor-made synthetic materials possessing specific cavities designed for a target molecule. Since they recognise their target analyte with affinities and selectivities comparable to those of antibody–antigen, enzyme–substrate and ligand–receptor interactions, they are often referred to as synthetic receptors or plastic antibodies. In this review, we describe the great potential and recent developments of MIPs in affinity separations, with emphasis on their application to the solid-phase extraction (SPE) of analytes from complex matrices. Research efforts made in this field to obtain water-compatible polymers for their applicability in aqueous environments are described. We particularly discuss problems encountered in the use of MIPs in SPE and the attempts carried out to improve their efficiency. Figure
Keywords: Molecular imprinting; Molecularly imprinted polymers; Bioanalysis; Solid-phase extraction; Affinity separation
Analytical techniques for single-cell metabolomics: state of the art and trends by Andrea Amantonico; Pawel L. Urban; Renato Zenobi (2493-2504).
Single-cell metabolomics is an emerging field that addresses fundamental biological questions and allows one to observe metabolic phenomena in heterogeneous populations of single cells. In this review, we assess the suitability of different detection techniques and present considerations on sample preparation for single-cell metabolomics. Although targeted analysis of single cells can readily be conducted using fluorescent probes and optical instruments (microscopes, fluorescence detectors), a comprehensive metabolomic approach requires a powerful label-free method, such as mass spectrometry (MS). Mass-spectrometric techniques applied to study small molecules in single cells include electrospray MS, matrix-assisted laser desorption/ionization MS, and secondary ion MS. Sample preparation is an important aspect to be taken into account during further development of methods for single-cell metabolomics.
Keywords: Cell individuality; Cell-to-cell variability; Single-cell analysis; Small-molecule analysis; Ultrasensitive analysis
Advanced dynamic monitoring of cellular status using label-free and non-invasive cell-based sensing technology for the prediction of anticancer drug efficacy by Toshihiro Ona; Junko Shibata (2505-2533).
Quantitative evaluation of anticancer drug efficacy using in vitro cell-based assays is useful for cancer patients, particularly those who show unconventional cancer development. Nevertheless, conventional chemosensitivity testing often requires widely used labeling agents and time-consuming laboratory procedures that provide low reliability. Label-free non-invasive cell-based assays are desired for dynamic monitoring of cellular status. This critical review first describes conventional chemosensitivity testing and then advanced label-free cell-based technology used to screen anticancer drugs through dynamic monitoring of cellular status, focusing on dosage and the use of drug-resistant cancer cells. Results from label-free cell-based approaches are compared with those of conventional chemosensitivity testing. The cellular statuses, addressed in terms of respective mechanisms and disadvantages, are extracellular fluxes of proton (H+), O2, and anticancer drugs, cell morphology changes, cell–environment interaction, and mitochondrial membrane potential. Finally, a cell-based systems outlook is presented. This paper represents a step toward efficient and accurate initial screening of anticancer drugs and development of compounds and their combined use to achieve pharmacodynamic and pharmacokinetic interactions, and chemotherapy evaluation of particular anticancer drugs for individual patients.
Keywords: Bioanalysis; Cell-based assay; Label-free; Anticancer drug; Screening; Biosensor
Sizing up the future of microRNA analysis by Abraham J. Qavi; Jared T. Kindt; Ryan C. Bailey (2535-2549).
In less than 20 years, our appreciation for micro-RNA molecules (miRNAs) has grown from an original, curious observation in worms to their current status as incredibly important global regulators of gene expression that play key roles in many transformative biological processes. As our understanding of these small, non-coding transcripts continues to evolve, new approaches for their analysis are emerging. In this critical review we describe recent improvements to classical methods of detection as well as innovative new technologies that are poised to help shape the future landscape of miRNA analysis. Figure Driven by the ever increasing appreciation of the critical biological roles played by microRNAs, new technologies are continually reshaping the landscape of microRNA analysis. This review highlights existing and emerging technologies for the detection of microRNAs
Keywords: Bioanalytical methods; Bioassays; Biosensors; Nucleic acids (DNA | RNA)
Analysis of DNA–protein interactions: from nitrocellulose filter binding assays to microarray studies by Reham Helwa; Jörg D. Hoheisel (2551-2561).
Determination of the sequence of the human genome and knowledge of the genetic code have allowed rapid progress in the identification of mammalian proteins. However, far less is known about the molecular mechanisms that control expression of human genes and about the variations in gene expression that underlie many pathological states, including cancer. This is caused in part by lack of information about the binding specificities of DNA-binding proteins and particularly regulative important molecules such as transcription factors. It is consequently crucial to develop new technologies or improve existing ones for the analysis of DNA–protein interaction in order to identify and characterise DNA response elements and the related transcription factors or other DNA-binding proteins. The techniques that are currently available vary with respect to the type of result that can be expected from the assay: a mere qualitative demonstration of binding; the identification of response element sequences at high throughput; or a quantitative characterisation of affinities. This article gives an overview of early and recent methodologies applied to such ends.
Keywords: DNA structure; DNA-binding proteins; Transcription factors; Regulation
Analytical methods in DNA and protein adduct analysis by Pertti Koivisto; Kimmo Peltonen (2563-2572).
DNA or protein adducts are reaction products of endogenous or exogenous chemicals and cellular macromolecules. Adducts are useful in toxicological studies and/or human biomonitoring exercises. In particular, DNA damage provides invaluable information for risk analysis. Second, metabolites or conjugates can be regarded as markers of phase II reactions though they may not give accurate information about the levels of reactive and damage-provoking reactive compounds or intermediates. Electrophiles are often short-lived molecules and therefore difficult to monitor. In contrast, adducts are often chemically stable, though their levels in biological samples are low, which makes their detection challenging. The assay of adducts is similar to the analysis of any other trace organic molecule, i.e. problems with the matrix and small amounts of analytes in samples. The 32P-postlabelling assay is a specific method for DNA adducts but immunochemical and fluorescence-based methods have been developed which can detect adducts linked to both DNA and protein. Tandem mass spectrometry, particularly if combined with ultrahigh-performance liquid chromatography, is currently the recommended detection technique; however investigators are striving to develop novel ways to achieve greater sensitivity. Standards are a prerequisite in adduct analysis, but unfortunately they are seldom commercially available.
Keywords: DNA; Protein; Adduct; Postlabelling; HPLC; Mass spectrometry
Therapeutic drug monitoring by LC–MS–MS with special focus on anti-infective drugs by Daniel M. Müller; Katharina M. Rentsch (2573-2594).
Liquid chromatography coupled to mass spectrometry nowadays plays an important role in the field of therapeutic drug monitoring (TDM), especially of new compounds for which no immunoassays are available. This paper reviews LC–MS(–MS) methods published recently for anti-infective drugs: antiretroviral drugs, other antiviral drugs, antibacterial drugs, antihelmintic drugs, antimalarial drugs, and other antiprotozoal drugs. An overview of the different methods is given, with special focus on selection of the internal standard and validation procedures.
Keywords: Therapeutic drug monitoring; LC–MS(–MS); Antiviral drugs; Antibacterial drugs; Antihelmintic drugs; Antimalarial drugs; Antiprotozoal drugs
Microtable arrays for culture and isolation of cell colonies by Jeng-Hao Pai; Wei Xu; Christopher E. Sims; Nancy L. Allbritton (2595-2604).
Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new three-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10-μJ pulses to each of the four support structures. In contrast, microstructures with a 150 × 150-μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie–Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array. Figure Three-dimensional structures were microfabricated by a novel two-step photolithography process to create a unique array platform for cell biology applications
Keywords: Microtable; Micropallet; Microfabrication; 1002F; SU-8; Cell separation; Cell array
Low-temperature co-fired ceramic microchannels with individually addressable screen-printed gold electrodes on four walls for self-contained electrochemical immunoassays by Eyitayo S. Fakunle; Ingrid Fritsch (2605-2615).
Microchannel devices were constructed from low-temperature co-fired ceramic (LTCC) materials with screen-printed gold (SPG) electrodes in three dimensions—on all four walls—for self-contained enzyme-linked immunosorbant assays with electrochemical detection. The microchannel confines the solution to a small volume, allowing concentration of electroactive enzymatically generated product and nearby electrodes provide high-speed and high-sensitivity detection: it also facilitates future integration with microfluidics. LTCC materials allow easy construction of three-dimensional structures compared with more traditional materials such as glass and polymer materials. Parallel processing of LTCC layers is more amenable to mass production and fast prototyping, compared with sequential processing for integrating multiple features into a single device. LTCC and SPG have not been reported previously as the basis for microchannel immunoassays, nor with integrated, individually addressable electrodes in three dimensions. A demonstration assay for mouse IgG at 5.0 ng/mL (3.3 × 10-11 M) with electrochemical detection was achieved within a 1.8 cm long × 290 μm high × 130 μm wide microchannel (approximately 680 nL). Two of four SPG electrodes span the top and bottom walls and serve as the auxiliary electrode and the assay site, respectively. The other two (0.7 cm long × 97 μm wide) are centered lengthwise on the sidewalls of the channel. One serves as the working and the other as the pseudoreference electrode. The immunoassay components were immobilized at the bottom SPG region. Enzymatically generated p-aminophenol was detected at the internal working electrode within 15 s of introducing the enzyme substrate p-aminophenyl phosphate. A series of buffer rinses avoided nonspecific adsorption and false-positive signals. Figure Microchannel constructed from low-temperature co-fired ceramic layers containing screen-printed gold electrodes and immunoassay site that converts p-aminophenylphosphate (PAPP) to p-aminophenol (PAPR) for subsequent electrochemical detection.
Keywords: Low-temperature co-fired ceramic; Electrochemical immunoassay; Microchannel; Self-contained electrochemistry; Individually addressable microelectrodes
A microelectrochemical sensing system for the determination of Epstein–Barr virus antibodies by Michael Bandilla; Andreas Zimdars; Sebastian Neugebauer; Manfred Motz; Wolfgang Schuhmann; Gerhard Hartwich (2617-2623).
An electrochemical method for the detection of Epstein–Barr virus (EBV) infections is described. The method relies on an immunoassay with electrochemical read-outs based on recombinant antigens. The antigens are immobilised on an Au electrode surface and used to complementarily bind antibodies from serum samples found during different stages of infection with EBV. Thiol chemistry under formation of self-assembled monolayers functions as a means to immobilise the antigens at the Au electrodes. A reporter system consisting of a secondary antibody labelled with alkaline phosphatase is used for electrochemical detection. The feasibility of the assay design is demonstrated and the assay performance is tested against the current gold standard in EBV detection. Close correlation is obtained for the results found for the developed electrochemical immunoassay and a standard line assay. Moreover, the electrochemical immunoassay is combined with a nanoporous electrode system allowing signal amplification by means of redox recycling. An amplification factor of 24 could be achieved. Figure Amplified immunoassay for the determination of anti-EBV antibodies in serum based on enzyme amplification coupled with electrochemical amplification by redox cycling in nanopore electrodes
Keywords: Immunoassay; Recombinant antigen; Biosensor; Redox cycling; Amplification; Epstein–Barr virus; Nanopore electrode
An SPR biosensor for the detection of microcystins in drinking water by Sonia Herranz; Markéta Bocková; María Dolores Marazuela; Jiří Homola; María Cruz Moreno-Bondi (2625-2634).
A surface plasmon resonance (SPR) biosensor for the detection of microcystins (MCs) in drinking water has been developed. Several assay formats have been evaluated. The selected format is based on a competitive inhibition assay, in which microcystin-LR (MCLR) has been covalently immobilized onto the surface of an SPR chip functionalized with a self-assembled monolayer. The influence of several factors affecting sensor performance, such as the nature and concentration of the antibody, the composition of the carrier buffer, and the blocking and regeneration solutions, has been evaluated. The optimized SPR biosensor provides an IC50 0.67 ± 0.09 µg L−1, a detection limit of 73 ± 8 ng L−1, and a dynamic range from 0.2 to 2.0 µg L−1 for MCLR. Cross-reactivity to other related MCs, such as microcystin-RR (88%) and microcystin-YR (94%), has also been measured. The SPR biosensor can perform four simultaneous determinations in 60 min, and each SPR chip can be reused for at least 40 assay–regeneration cycles without significant binding capacity loss. The biosensor has been successfully applied to the direct analysis of MCLR in drinking water samples, below the provisional guideline value of 1 µg L−1 established by the World Health Organization for drinking water.
Keywords: Microcystin-LR; Label-free biosensor; Self-assembled monolayer; Surface plasmon resonance
Aptamer sandwich assays: label-free and fluorescence investigations of heterogeneous binding events by Katie A. Edwards; Antje J. Baeumner (2635-2644).
We studied aptamer binding events in a heterogeneous format using label-free and fluorescence measurements for the purpose of developing an aptamer-based sandwich assay on a standard microtiter plate platform. The approach allowed visualization of the underlying aptamer immobilization and target binding events rather than relying on only an endpoint determination for method optimization. This allowed for a better understanding of these multi-step assays and optimal conditions specific to aptamers. α-thrombin was chosen as a prototypical analyte as two well-studied aptamers (15 and 29-mer) binding distinct epitopes are available. The Corning Epic® system, which utilizes a resonance waveguide diffraction grating in a 384-well microtiter plate format, was employed to measure relative immobilization and binding levels for various modified aptamers. Parameters investigated included the effects of aptamer orientation, label orientation, spacer length, spacer type, immobilization concentration, and binding buffer. Most notably, the 15-mer aptamer was preferable for capture over the 29-mer aptamer and aptamers with increasing poly(dT) spacer length between the biotin modification and the aptamer yielded decreased immobilization levels. This decreased immobilization resulted in increased α-thrombin binding ability for 15-mer aptamers with the poly(dT) spacer. Fluorescence measurements of fluorescein-labeled 29-mer aptamers with varying spacers were used to visualize sandwich complex formation. Using both label-free and traditional fluorescence measurements, an in-depth understanding of the overall assay was obtained, thus the inclusion of label-free measurements is recommended for future method development.
Keywords: Label-free; Aptamer; Fluorescence; Epic®; Sandwich assay; Immobilization
Aptamer sandwich assays: human α-thrombin detection using liposome enhancement by Katie A. Edwards; Yang Wang; Antje J. Baeumner (2645-2654).
Fluorescent dye-encapsulating liposomes tagged with aptamers were developed and used as reporting signals in an aptamer-based sandwich assay. α-Thrombin was utilized as a prototypical analyte as two well-studied aptamers binding distinct epitopes are available to form a sandwich complex. Cholesteryl–TEG-modified aptamers were embedded into the liposomal lipid bilayer while the interior cavity of the liposomes encapsulated fluorescent sulforhodamine B dye. Such liposomes successfully formed a sandwich complex with α-thrombin and a microtiter plate immobilized aptamer, proving that aptamers retain their ability to fold when anchored to the liposome surface. Parameters studied included liposomal aptamer coverage, sandwich aptamer orientation, aptamer label orientation, aptamer spacer length and type, incubation buffer, and aptamer concentration. The optimized conditions found here in the fluorescence assay led to a limit of detection of 64 pM or 2.35 ng/mL, corresponding to 6.4 fmol or 235 pg, respectively, in a 100 μL volume. This is an order of magnitude lower than previous sandwich aptamer assays using the same sequences with lowest reported limits of detection of 0.45 nM. In addition, the assay was applied successfully to the detection of α-thrombin in human plasma. The success of this method in a standard microtiter plate format and the relatively facile functionalization of liposomes with aptamers suggest that this approach provides a versatile option for routine analytical applications.
Keywords: Aptamer; Liposome; Fluorescence; Sandwich assay; Bioanalytical methods; Nucleic acids/DNA
Development and characterization of rat monoclonal antibodies for N-acylated homoserine lactones by Xiao Chen; Elisabeth Kremmer; Marie-Françoise Gouzy; Ernst Clausen; Mandy Starke; Karin Wöllner; Gerd Pfister; Anton Hartmann; Petra M. Krämer (2655-2667).
Quorum sensing (QS) is a communication mechanism between bacteria using diffusible chemical signaling molecules, which are called autoinducers (AI). By detecting the concentration of quorum sensing molecules through binding to a specific receptor protein, bacteria regulate their gene expressions when the concentration of autoinducers and thus the cell density reaches a threshold level. Many Gram-negative bacteria use acylated homoserine lactones (HSLs) as autoinducers. Because of the broad biological functions of HSLs, interest in detection and analysis of HSLs is increasing with a view to their medical, biotechnological, and agricultural applications. In this study, an anti-HSL antibody-based immunochemical detection method has been developed. Four structurally distinct HSL haptens, named HSL1, HSL2, HSL3, and HSL4, have been designed for antibody and assay development. New rat anti-HSL monoclonal antibodies (mAbs) have been produced in-house and characterized with enzyme-linked immunosorbent assays (ELISA), both in the coating antigen and in the enzyme tracer format. Eight mAbs (HSL1-1A5, HSL1-8E1, HSL1/2-2C10, HSL1/2-4H5, HSL4-4C9, HSL4-5E12, HSL4-5H3, and HSL4-6D3) will be presented in this paper. We demonstrate that the anti-HSL mAbs have distinguished sensitivity and selectivity toward HSLs depending upon their chemical structures. The optimized assays are capable of detecting HSLs in the microgram per liter (low micromolar to nanomolar) range. The best IC50 (test midpoint) was 134 ± 30 μg L−1 (n = 54) for N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) using mAb HSL1/2-2C10 and HSL1–HRP in the enzyme tracer format. In the coating antigen format, the most selective mAb for N-octanoyl-l-homoserine lactone (C8-HSL) was mAb HSL4-4C9. Additionally, anti-HSL mAbs showed higher sensitivity against hydrolyzed HSLs, namely homoserines. These compounds might also occur under certain biological conditions. This study marks the beginning of new ways for quick and cost-effective HSL detection, requiring small sample amounts (less than 1 mL) and little to no sample preparation.
Keywords: Quorum sensing; Quorum quenching; N-Acyl homoserine lactone; N-Acyl homoserine; Enzyme-linked immunosorbent assay (ELISA); Monoclonal antibodies
Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays by Xiao Chen; Katharina Buddrus-Schiemann; Michael Rothballer; Petra M. Krämer; Anton Hartmann (2669-2676).
The Burkholderia cepacia complex (Bcc) employs a quorum sensing (QS) mechanism which is a cell density-dependent bacterial communication system to regulate certain gene expressions. As with many other Gram-negative bacteria, Burkholderia cepacia species use (N-acyl-)homoserine lactones (AHLs or HSLs) as signalling molecules. Because of the essential role of QS in bacterial behavior, the aim of this study was to demonstrate the applicability of our in-house-developed enzyme-linked immunosorbent assays (ELISAs) for the detection of bacterial activities via HSLs in B. cepacia strain LA3 culture supernatants. For this purpose the previously developed monoclonal antibodies (mAbs) HSL1/2-2C10 and HSL1/2-4H5 were exploited. N-3-Oxo-decanoyl-L-homoserine lactone (3-oxo-C10-HSL) was used as main analyte throughout all experiments. With the bacterial culture medium (named ABC medium) a matrix effect in both ELISAs was visible (slight increase in optical density, shift in test midpoints (IC50) and working ranges). For example, ELISA with mAb HSL1/2-2C10 and enzyme tracer HSL3-HRP (HSL derivative conjugated to horseradish peroxidase) had an IC50 of 120 μg L−1 for 3-oxo-C10-HSL in phosphate-buffered saline versus 372 μg L−1 in ABC medium. A significant increase of HSLs in B. cepacia strain LA3 culture supernatants after 12 h to 48 h of growth was observed. Although the analytical result of these immunoassays cannot distinguish HSLs from homoserines (HSs), the appearance of these compounds can be easily followed. Hydrolysis and spiking experiments were carried out with these biological samples. According to our knowledge, these are the first immunoassays for the detection of quorum sensing molecules in biological culture supernatants. This study provides a cost-effective, fast, and sensitive analytical method for detection of HSLs/HSs in biological samples without complex sample preparation and will offer a quick idea about B. cepacia activities. The low sample amount requirement (less than 1 mL) constitutes a tremendous advantage for many analytical questions with biological samples.
Keywords: Quorum sensing; (N-acyl-)homoserine lactone (AHL or HSL); (N-acyl-)homoserine; Burkholderia cepacia ; Enzyme-linked immunosorbent assay (ELISA); Monoclonal antibodies; Biological samples
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for detection and identification of albumin phosphylation by organophosphorus pesticides and G- and V-type nerve agents by Harald John; Felicitas Breyer; Jörg Oliver Thumfart; Hans Höchstetter; Horst Thiermann (2677-2691).
Toxic organophosphorus compounds (OPC), e.g., pesticides and nerve agents (NA), are known to phosphylate distinct endogenous proteins in vivo and in vitro. OPC adducts of butyrylcholinesterase and albumin are considered to be valuable biomarkers for retrospective verification of OPC exposure. Therefore, we have detected and identified novel adducts of human serum albumin (HSA) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Pure albumin and plasma were incubated with numerous pesticides and NA of the V- and G-type in different molar ratios. Samples were prepared either by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by in-gel enzymatic cleavage using endoproteinase Glu-C (Glu-C) or by combining highly albumin-selective affinity extraction with ultrafiltration followed by reduction, carbamidomethylation, and enzymatic cleavage (Glu-C) prior to MALDI-TOF MS analysis. Characteristic mass shifts for phosphylation revealed tyrosine adducts at Y411 (Y401KFQNALLVRY411TKKVPQVSTPTLVE425), Y148 and Y150 (I142ARRHPY148FY150APE153, single and double labeled), and Y161 (L154LFFAKRY161KAAFTE167) produced by original NA (tabun, sarin, soman, cyclosarin, VX, Chinese VX, and Russian VX) as well as by chlorpyrifos-oxon, diisopropyl fluorophosphate (DFP), paraoxon-ethyl (POE), and profenofos. MALDI-MS/MS of the single-labeled I142–E153 peptide demonstrated that Y150 was phosphylated with preference to Y148. Aged albumin adducts were not detected. The procedure described was reproducible and feasible for detection of adducts at the most reactive Y411-residue (S/N ≥ 3) when at least 1% of total albumin was labeled. This was achieved by incubating plasma with molar HSA/OPC ratios ranging from approximately 1:0.03 (all G-type NA, DFP, and POE) to 1:3 (V-type NA, profenofos). Relative signal intensity of the Y411 adduct correlated well with the spotted relative molar amount underlining the usefulness for quantitative adduct determination. In conclusion, the current analytical design exhibits potential as a verification tool for high-dose exposure. Human albumin adducts with organophosphorus
Keywords: Albumin adducts; MALDI-TOF MS; Nerve agents; Organophosphorus compounds; Verification
Microfluidic immunosensor with integrated liquid core waveguides for sensitive Mie scattering detection of avian influenza antigens in a real biological matrix by Brian C. Heinze; Jessica R. Gamboa; Keesung Kim; Jae-Young Song; Jeong-Yeol Yoon (2693-2700).
This work presents the use of integrated, liquid core, optical waveguides for measuring immunoagglutination-induced light scattering in a microfluidic device, towards rapid and sensitive detection of avian influenza (AI) viral antigens in a real biological matrix (chicken feces). Mie scattering simulations were performed and tested to optimize the scattering efficiency of the device through proper scatter angle waveguide geometry. The detection limit is demonstrated to be 1 pg mL−1 in both clean buffer and real biological matrix. This low detection limit is made possible through on-chip diffusional mixing of AI target antigens and high acid content microparticle assay reagents, coupled with real-time monitoring of immunoagglutination-induced forward Mie scattering via high refractive index liquid core optical waveguides in close proximity (100 μm) to the sample chamber. The detection time for the assay is <2 min. This device could easily be modified to detect trace levels of any biological molecules that antibodies are available for, moving towards a robust platform for point-of-care disease diagnostics.
Keywords: Lab-on-a-chip; Optofluidic; Immunoassay; Bird flu; Latex agglutination; Influenza A
Quantification of drugs of abuse in municipal wastewater via SPE and direct injection liquid chromatography mass spectrometry by Kevin J. Bisceglia; A. Lynn Roberts; Michele M. Schantz; Katrice A. Lippa (2701-2712).
We present an isotopic-dilution direct injection reversed-phase liquid chromatography–tandem mass spectrometry method for the simultaneous determination of 23 drugs of abuse, drug metabolites, and human-use markers in municipal wastewater. The method places particular emphasis on cocaine; it includes 11 of its metabolites to facilitate assessment of routes of administration and to enhance the accuracy of estimates of cocaine consumption. Four opioids (6-acetylmorphine, morphine, hydrocodone, and oxycodone) are also included, along with five phenylamine drugs (amphetamine, methamphetamine, 3,4-methylenedioxy-methamphetamine, methylbenzodioxolyl-butanamine, and 3,4-methylenedioxy-N-ethylamphetamine) and two human-use markers (cotinine and creatinine). The method is sufficiently sensitive to directly quantify (without preconcentration) 18 analytes in wastewater at concentrations less than 50 ng/L. We also present a modified version of this method that incorporates solid-phase extraction to further enhance sensitivity. The method includes a confirmatory LC separation (selected by evaluating 13 unique chromatographic phases) that has been evaluated using National Institute of Standards and Technology Standard Reference Material 1511 Multi-Drugs of Abuse in Freeze-Dried Urine. Seven analytes (ecgonine methyl ester, ecgonine ethyl ester, anhydroecgonine methyl ester, m-hydroxybenzoylecgonine, p-hydroxybenzoyl-ecgonine, ecgonine, and anhydroecgonine) were detected for the first time in a wastewater sample.
Keywords: Wastewater; Illicit drugs; Urinary metabolites; LC/MS/MS; Direct injection; SPE
Differentiation of walnut wood species and steam treatment using ATR-FTIR and partial least squares discriminant analysis (PLS-DA) by Alison J. Hobro; Julia Kuligowski; Markus Döll; Bernhard Lendl (2713-2722).
Wood is a ubiquitous material used in everyday life. Accurate identification of species can be of importance in a historical context enabling appropriate conservation treatment and adequate choice of material to be applied to historic wooden objects, and in a more modern context, in the identification of forgeries. Wood is also often treated to improve certain physical characteristics, often strength and durability. However, determination of whether or not a piece of wood has been treated can be very difficult. Infrared spectroscopy has previously been applied to differentiate between different wood species or between treated and untreated wood, often in conjunction with chemometric analysis techniques. Here, we report the use of mid-IR spectroscopy, coupled with partial least squares discriminant analysis for the discrimination between two walnut wood species and to differentiate between steam-treated and untreated samples of each of these wood species. We show that the discrimination between species and between steam-treated and non-steam-treated wood from Juglans nigra is very clear and, while analysis of the quality of the discrimination between steam-treated and non-steam-treated J. regia samples is not as good, it is, nevertheless, sufficient for discrimination between the two groups with a statistical significance of P < 0.0001. Figure ATR-IR spectra of walnut wood from J. nigra and J. regia.
Keywords: Walnut wood; Steam treatment; Species differentiation; Infrared spectroscopy; PLS-DA
Analysis of hydrophilic and lipophilic choline compounds in radioresistant and radiosensitive glioblastoma cell lines by HILIC-ESI-MS/MS by D. Desoubzdanne; C. Claparols; N. Martins-Froment; C. Zedde; S. Balayssac; V. Gilard; F. Tercé; R. Martino; M. Malet-Martino (2723-2730).
A new method based on hydrophilic interaction chromatography-electrospray ionisation–tandem mass spectrometry (HILIC-ESI-MS/MS) coupled to the use of a stable isotope labelled substrate was developed to study the metabolism of choline (Cho) compounds in two human glioblastoma multiform (GBM) cell lines with different responses to ionising radiation. Analysis was performed in the positive ion mode using multiple reaction monitoring. This fast, sensitive and selective method enabled the profiling of both hydrophilic and lipophilic Cho-containing compounds, to analyse specifically different phosphatidylcholine (PtdCho) molecular species, and to measure simultaneously native and labelled Cho metabolites. Radioresistant (SF763) and radiosensitive (SF767) cells were incubated for 8 h with d 9-Cho. Higher native Cho and phosphocholine (PCho) concentrations and higher uptake of d 9-Cho and formation of d 9-PCho were found in the radioresistant cell line. The similar low concentrations of native cytidine 5′-diphosphocholine (CDP-Cho) and d 9-CDP-Cho in both cell lines show that CDP-Cho is the limiting metabolite in the two models. The turnovers (percentage of each d 9-Cho compound in its respective pool, i.e. native + labelled) were lower in radioresistant cells for all Cho compounds, suggesting a global PtdCho metabolism more active in radiosensitive cells that could be related to their higher proliferation rate. Figure HILIC-ESI-MS/MS applied to the analysis of hydrophilic and lipophilic choline (Cho) compounds in radioresistant (RR) and radiosensitive (RS) glioblastoma cell lines incubated for 8h with d9-Cho showed (i) higher uptake of d9-Cho and formation of d9-phosphocholine (d9-PCho) in the RR cell line; (ii) very low and similar amounts of d9-cytidine 5’-diphosphocholine (d9-CDP-Cho) in both cell lines suggesting that this compound is the limiting metabolite; and (iii) lower formation of d9-phosphatidylcholines (d9-PtdCho) in the RR cell line.
Keywords: HILIC-ESI-MS/MS; GBM cell lines; d 9-Choline; Hydrophilic and lipophilic choline compounds; Native and labelled choline compounds; Kinetics
Retention prediction and hydrophobicity estimation of weak acidic compounds by reversed-phase liquid chromatography using acetic and perchloric acids as ion suppressors by Shu-ying Han; Xin Ming; Zheng-chun Qi; Dong Sheng; Hong-zhen Lian (2731-2743).
Simple acids are usually applied to suppress the ionization of weakly ionizable acidic analytes in reversed-phase liquid chromatography. The purpose of this study is to investigate the retention behavior of various weak acidic compounds (monoprotic, diprotic, triprotic, and tetraprotic acids) using acetic or perchloric acid as ion suppressor in a binary hydroorganic mobile phase. The apparent n-octanol–water partition coefficient (K ow ″) was proposed to calibrate the n-octanol–water partition coefficient (K ow) of weak acidic compound. LogK ow ″ was found to have a better linear correlation with logk w, the logarithm of the retention factor obtained by extrapolating to neat aqueous fraction of the mobile phase, for all weakly ionizable acidic compounds. This straightforward relationship offers a potential medium for direct measurement of K ow data of weak acidic analytes and can be used to predict retention behavior of these compounds in the ion suppression reversed-phase liquid chromatographic mode. Figure The linear correlation of logK ow vs logk w (a and c) and logK ow″ vs logk w (b and d) of all 28 model weak acidic compounds eluted by methanol–aqueous acetic (a and b) and perchloric (c and d) acid solutions, respectively, at pH 3.00. The approaching of the outliers marked by circles and squares from the line of logK ow vs logk w to that of logK ow″ vs logk w evidently testified that K ow″ is more suitable to be adopted than K ow to describe the correlation between hydrophobicity and RPLC retention of ionizable compounds
Keywords: Reversed-phase liquid chromatography; Retention behavior; Ion suppressor; n-Octanol–water partition coefficient (K ow); Apparent n-octanol–water partition coefficient (K ow ″); Weakly ionizable acidic compound
DNA microarrays for visual detection of human pathogenic microorganisms based on tyramine signal amplification coupled with gold label silver stain by Hong-Juan Qi; Su-Hong Chen; Min-Li Zhang; Hua Shi; Sheng-Qi Wang (2745-2750).
The utility of DNA microarrays is severely limited by their restricted sensitivity. Tyramine signal amplification (TSA) coupled with gold label silver stain (GLSS) was introduced in DNA microarrays for visual detection of human pathogenic microorganisms. First, a TSA system was introduced to the microarrays after the microarrays were prepared and hybridized with biotinylated targets. This procedure leads to large amounts of biotin-conjugated tyramine depositing at the site of enzyme reaction under HRP catalysis. Second, streptavidin–nanogold was introduced and accumulated by specific binding of biotin and streptavidin. Finally, silver staining was performed. The images of the spots were scanned with a visible light scanner and quantified with ArrayVision 7.0 software. Detection conditions were systematically optimized. Then the sensitivity among TSA coupled with GLSS, GLSS, and TSA coupled with Cy3 was compared. The optimized conditions were: streptavidin–HRP (1 mg mL−1) dilution 1:1500, biotin–tyramine dilution 1:200 (+0.5% H2O2), streptavidin–nanogold dilution 1:100 (all diluted in 1 × PBS + 1% BSA) and silver stain time of 10 min. The sensitivity of TSA coupled with GLSS was 100-fold higher than that of GLSS, and was identical with that of TSA coupled with Cy3. Meanwhile, the specificity of the microarrays were not affected. This implied that TSA coupled with GLSS was a sensitive visual detection method and would be an ideal alternative to fluorescence-based detection for DNA microarrays. Online abstract figure Scanned images and quantification of the microarrays in comparison of the sensitivity between TSA–GLSS and GLSS. The result showed that the sensitivity of this method was 100-fold higher than that of GLSS
Keywords: DNA microarray; Sensitivity; Visual detection; Gold label silver stain; Tyramine signal amplification