Covalent bound sensing layers on surface acoustic wave (SAW) biosensors

This paper reports on the development of immunosensors based on commercially available surface acoustic wave (SAW) devices working at 380 MHz. Approaches for coating the sensor surface with a sensing layer of receptive biomolecules are presented and discussed. It was found that the sensitivity strongly relates to the immobilization method. Additionally, the sensitivity can be influenced by the density of accessible biomolecules on the active sensing area. Usually, by most of the standard immobilization procedures, two-dimensional layers of receptive biomolecules are obtained. We present a three-dimensional layer, which provides a higher absolute amount of recognition molecules. A dextran layer is photoimmobilized to the sensor surface and the recognition molecules are covalently embedded into the dextran matrix. The feasibility of specific immunosensing is investigated using SAW sensors connected to a fluid handling system.     

Plasma modified surfaces for covalent immobilization of functional biomolecules in the absence of chemical linkers: towards better biosensors and a new generation of medical implants

Plasma modification and plasma polymer deposition are valuable technologies for the preparation of surfaces for the covalent binding of biomolecules for applications such as biosensors, medical prostheses, and diagnostic devices as well as surfaces for enzyme-mediated reactions. Covalency is conveniently tested by the ability of the surface to retain the attached molecules after vigorous washing with sodium dodecyl sulphate (SDS). Covalency is indicated if the fraction of protein retained lies above the curve characteristic of physisorption. Confidence in covalency is strengthened when the washing protocol is aggressive enough to remove all adsorbed protein from a control significantly more hydrophobic than the test surface. The use of linker chemistry to space the molecules from the surface is in some cases beneficial. However, the use of linker chemistry is not necessary to retain molecular function for long periods when the polymer surface is modified by energetic bombardment. The energetic bombardment retains hydrophilicity of the surface by crosslinking the subsurface, and this appears to facilitate retention of protein function. Energetic bombardment also increases the functional life of molecules immobilized and then freeze dried on plasma-modified surfaces. Analysis of the surfaces shows that the covalent binding mechanism is related to the presence of free radicals on the surface and in the subsurface regions. The unpaired electrons associated with the radicals appear to be mobile within the modified region and can diffuse to the surface to take part in binding interactions. Proactive implantable devices can make use of these principles of covalent attachment by seeding the surface of an implant with a biomolecule that elicits the desired interaction with cells and prevents undesirable responses.     

Arraying prostate specific antigen PSA and Fab anti-PSA using light-assisted molecular immobilization technology

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti‐PSA biosensor arrays using UV light‐assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti‐PSA in solution and immobilized Fab anti‐PSA cross‐reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.     

Nanomechanics: Opening new frontiers in bio analyses and diagnostics

Micro-fabricated silicon cantilevers arrays offer a novel label-free approach where ligand-receptor binding interactions occurring on the sensor generate nanomechanical signals like bending or a change in mass that is optically detected in-situ. We report the detection of multiple unlabelled biomolecules simultaneously down to picomolar concentrations within minutes. Differential measurements including reference cantilevers on an array of eight sensors enables sequence-specifically detection of unlabelled DNA and is suitable to detect specific gene fragments within a complete genome (gene fishing). Expression of detection of inducible genes as well as the ultimate challenge: the detection of total RNA fragments in a unspecific back ground will be shown. Ligand-receptor binding interactions, such as antigen recognition will be presented. Antibody activated cantilevers with sFv (single chain fragments) which bind to the indicator proteins show a significant improved sensitivity which is comparable with SPR (Surface Plasmon Resonance). In addition this technology offers a brought variety of receptor molecules application such as e.g. membrane protein recognition, micro-organism detection, enantiomeric separation. New coating procedures, enlargement of the active surface area by dendritic molecules as well as improvement of the receptor-cantilever chemical bond will be presented. This new findings may lead to a novel individual diagnostic assay in a combined label-free GENOMIC and PROTEOMIC biomarker sensor (COMBIOSENS).     

Use of thiazolidine-mediated ligation for site specific biotinylation of mouse EGF for biosensor immobilisation

Summary Biosensors provide a sophisticated and discriminating means of probing biomolecular interactions. Specific ligands such as peptides and proteins can be immobilized onto sensor surfaces by a number of means including covalent attachment via amine, thiol or aldehyde chemistry, capture via biotin-avidin interaction or the use of specific tags. We have devised a simple chemoselective ligation method to selectively conjugate an anchoring functionality onto N-terminal serine or threonine residues of peptides and proteins allowing them to be immobilised onto the sensor surface in a defined orientation. It is based on the specific reaction of the 1,2-aminothiol of cysteine with an aldehyde under acidic conditions to form a stable thiazolidine product. The carbonyl precursors are derived from the 1,2-aminoalcohols of Ser or Thr that can be selectively and rapidly converted to the aldehyde form by periodate oxidation. Biotinylation of the aldehyde is then achieved via simple conjugation with a novel water-soluble dipeptide that contains a lysine residue bearing an Nε-cysteine-derived 1,2-aminothiol and an Nα-biotin moiety. Use of this method allowed selective biotinylation of a native form of murine EGF (mEGF_2-53) that has an N-terminal serine residue. This derivative was then immobilised onto a streptavidin biosensor surface, and the resultant surface activity compared with those obtained by immobilising recombinant human EGF or the soluble extracellular domain of the EGF receptor (sEGFR_1-621) using amine coupling (NHS/EDC) chemistry. The surface recognised the recombinant sEGFR with a similar K _D to that of human EGF immobilised using NHS/EDC chemistry, or if the receptor was immobilised and murine EGF injected.     

Label-free detection of single nucleotide polymorphisms utilizing the differential transfer function of field-effect transistors

We present a label-free method for the detection of DNA hybridization, which is monitored by non-metallized silicon field-effect transistors (FET) in a microarray approach. The described method enables a fast and fully electronic readout of ex situ binding assays. The label-free detection utilizing the field-effect is based on the intrinsic charge of the DNA molecules and/or on changes of the solid–liquid interface impedance, when biomolecules bind to the sensor surface. With our sensor system, usually a time-resolved, dc readout is used. In general, this FET signal suffers from sensor drift, temperature drift, changes in electrolyte composition or pH value, influence of the reference electrode, etc. In this article, we present a differential ac readout concept for FET microarrays, which enables a stable operation of the sensor against many of these side-parameters, reliable readout and a possibility for a quick screening of large sensor arrays. We present the detection of point mutations in short DNA samples with this method in an ex situ binding assay.     

Use of thiazolidine-mediated ligation for site specific biotinylation of mouse EGF for biosensor immobilisation

Biosensors provide a sophisticated and discriminating means of probing biomolecular interactions. Specific ligands such as peptides and proteins can be immobilized onto sensor surfaces by a number of means including covalent attachment via amine, thiol or aldehyde chemistry, capture via biotin-avidin interaction or the use of specific tags. We have devised a simple chemoselective ligation method to selectively conjugate an anchoring functionality onto N-terminal serine or threonine residues of peptides and proteins allowing them to be immobilised onto the sensor surface in a defined orientation. It is based on the specific reaction of the 1,2-aminothiol of cysteine with an aldehyde under acidic conditions to form a stable thiazolidine product. The carbonyl precursors are derived from the 1,2-aminoalcohols of Ser or Thr that can be selectively and rapidly converted to the aldehyde form by periodate oxidation. Biotinylation of the aldehyde is then achieved via simple conjugation with a novel water-soluble dipeptide that contains a lysine residue bearing an N-cysteine-derived 1,2-aminothiol and an N-biotin moiety. Use of this method allowed selective biotinylation of a native form of murine EGF (mEGF_2-53) that has an N-terminal serine residue. This derivative was then immobilised onto a streptavidin biosensor surface, and the resultant surface activity compared with those obtained by immobilising recombinant human EGF or the soluble extracellular domain of the EGF receptor (sEGFR_1-621) using amine coupling (NHS/EDC) chemistry.The surface recognised the recombinant sEGFR with a similar K_D to that of human EGF immobilised using NHS/EDC chemistry, or if the receptor was immobilised and murine EGF injected.     

Critical assessment of the Quartz Crystal Microbalance with Dissipation as an analytical tool for biosensor development and fundamental studies: Metallophthalocyanine-glucose oxidase biocomposite sensors

One of the challenges in electrochemical biosensor design is gaining a fundamental knowledge of the processes underlying immobilisation of the molecules onto the electrode surface. This is of particular importance in biocomposite sensors where concerns have arisen as to the nature of the interaction between the biological and synthetic molecules immobilised. We examined the use of the Quartz Crystal Microbalance with Dissipation (QCM-D) as a tool for fundamental analyses of a model sensor constructed by the immobilisation of cobalt(II) phthalocyanine (TCACoPc) and glucose oxidase (GOx) onto a gold-quartz electrode (electrode surface) for the enhanced detection of glucose. The model sensor was constructed in aqueous phase and covalently linked the gold surface to the TCACoPc, and the TCACoPc to the GOx, using the QCM-D. The aqueous metallophthalocyanine (MPc) formed a multi-layer over the surface of the electrode, which could be removed to leave a monolayer with a mass loading that compared favourably to the theoretical value expected. Analysis of frequency and dissipation plots indicated covalent attachment of glucose oxidase onto the metallophthalocyanine layer. The amount of GOx bound using the model system compared favourably to calculations derived from the maximal amperometric functioning of the electrochemical sensor (examined in previously-published literature, Mashazi, P.N., Ozoemena, K.I., Nyokong, T., 2006. Electrochim. Acta 52, 177-186), but not to theoretical values derived from dimensions of GOx as established by crystallography. The strength of the binding of the GOx film with the TCACoPc layer was tested by using 2% SDS as a denaturant/surfactant, and the GOx film was not found to be significantly affected by exposure to this. This paper thus showed that QCM-D can be used in order to model essential processes and interactions that dictate the functional parameters of a biosensor.     

Biological UV dosimetry using the DLR-biofilm.

Changes of environmental UV radiation as part of global atmospheric changes will influence the biosphere substantially. The determination of the biological effects of these changes requires accurate and reliable UV monitoring systems that weight the spectral irradiance according to the biological responses under consideration. Biological UV dosimeters, which directly weight the incident UV components of sunlight in relation to the effectiveness of the different wavelengths and the potential interactions between them, can complement weighted physical UV measurements. Up to now several UV-dependent endpoints in biomolecules (e.g. uracil, DNA, provitamin D3), bacteriophages (e.g. T7), bacteria (e.g.E. coli, B. subtilis) and cultured eukaryotic cells have been suggested as sensing elements in biological UV dosimeters. One example is the DLR-biofilm consisting of immobilised spores of the bacterium B. subtilis as a UV sensor. It weights per se the incident UV radiation according to its DNA-damaging effectiveness. In several examples the applicability of the DLR-biofilm technique for personal UV dosimetry as well as for the measurement of the biologically weighted irradiance of the sun and of artificial UV sources is demonstrated.     

A fluidised bed vessel for the culture of immobilised plant cells and its application for the continuous production of fine cell suspensions

With the expansion of immobilised plant cell technology the need has arisen for a suitable vessel in which systems can be efficiently manipulated.Described is a vessel which incorporates many of the features of a fluidised bed, together with some of those from airlift technology to enable immobilised plant cells to be cultured at high biomass concentrations while maintaining a high mass transfer and controlled aeration under continuous flow conditions. In the case described, the vessel has been used for the continuous production of fine plant cell suspensions, although it is easily adaptable for use in cell mediated biotransformation or de novo synthesis studies.Abbreviations 2,4D 2.4 dichlorophenoxyacetic acid     

In Situ Hydration of Enzymes in Non-Polar Organic Media Can Increase the Catalytic Rate

The nature of the buffer species used in the drying process is important when lyophilized enzyme preparations are suspended in organic media. The activity of subtilisin Carlsberg in a transesterification reaction was found to vary depending on the nature of the buffer used. It was postulated that the large excess of salt present in the dried powder could be affecting enzymatic activity by alterations to the microscopic structure of the powder. To establish if this were true, microscopic changes were eliminated by covalently immobilising the enzyme onto a macroporous polymer support so that the counter-ions could be exchanged by washing with dilute salt solutions. It was found that in the immobilised samples no significant effects of salt ions were noted. This was the case even when salt ions were in considerable excess of that needed to balance protein charges. Hence the activity variations noted in freeze-dried powders are probably due to changes to the microscopic structure, rather than to molecular scale interactions. Similarly the previously observed activating effect of crown ether solutions on freeze-dried powders is not repeated on an immobilised preparation suggesting that this too may be due to a microscopic effect on the powder.     

Electrochemical arrays coupled with magnetic separators for immunochemistry

Electrochemical methods are increasingly applied to immunoassays, because they overcome problems associated with other modes of detection. In particular, with respect to conventional immunoassays, electrochemical immunosensors show versatility, reliability, and fast analysis time. In immunosensor strategy, the antigen or antibody can be immobilized directly onto the surface of the electrochemical transducer that will finally be used to reveal the amount of the affinity reaction. However, the use of the electrode surface as a solid phase as well as an electrochemical transducer presents some problems: a shielding of the surface by biospecifically bound antibody molecules can cause hindrance in the electron transfer, resulting in a reduced voltammetric signal. Thus, as an alternative solid phase, magnetic beads because of their low toxicity and high biocompatibility have gained much attention in chemistry, associated with various analytical techniques, due to their suitability for immobilization of biomolecules. Magnetic micro- or nanobeads can be separated easily and quickly by magnetic forces and will be used together with bioaffine ligands, e.g., antibodies or proteins with a high affinity to the target. The special advantages of magnetic separation techniques are the fast and simple handling of a sample vial and the opportunity to deal with large sample volumes without the need for time-consuming centrifugation steps. This also makes biomagnetic separation ideal for automated assay/analysis systems which will play a very important role in the near future. This review presents some examples of immunochemical assay developed using magnetic beads as a solid phase coupled with electrochemical detection techniques, in particular, using electrochemical arrays as transducers. Applications related to static measurements, together with in-flow detection systems are presented.     

Determination of inhibitors’ potency (IC50) by a direct high-performance liquid chromatographic method on an immobilised acetylcholinesterase column

An immobilised acetylcholinesterase (AChE) stationary phase was prepared by using an in situ AChE immobilisation procedure. A stainless steel column packed with epoxide silica was connected to the HPLC system and the enzyme solution at pH 5.8 was recycled through the column at a flow-rate of 0.5 ml/min for 24 h. The activity of the immobilised AChE was determined by injecting the substrate acetylthiocholine, using as mobile phase 0.1 M phosphate buffer (pH 7.4) containing Ellman’s reagent [5,5′-dithio-bis(2-nitrobenzoic acid)] and measuring the area of the obtained peak with UV detection at 412 nm. The effect of AChE inhibitors tacrine, edrophonium and donepezil were evaluated by the simultaneous injection of each inhibitor with the substrate. The resulting decrease in the AChE activity, as expressed by the decrease of the peak area detected at 412 nm, was related to the concentration and potency of the solutes. The obtained IC₅₀ values were compared with those derived by the conventional spectrophotometric method. This immobilized enzyme reactor, included in a chromatographic system, can be used for the rapid screening for new inhibitors allowing for the on-line determination of a compound’s inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analysed continuously.     

The use of regenerable, affinity ligand-based surfaces for immunosensor applications.

The regeneration of antibody-binding surfaces is of major importance for re-usable sensor formats such as required for direct 'real-time' biosensing technologies and is often difficult to achieve. Antibodies commonly bind the antigen with high avidity and may themselves be sensitive to regeneration conditions. The interaction of polyclonal anti-chlorpyriphos antibody with an immobilised chlorpyriphos-ovalbumin (chlor-oval) conjugate and the interaction of soluble recombinant CD4 with covalently immobilised anti-CD4 IgG are presented in order to highlight these difficulties. Affinity-capture is suggested as an alternative format as it facilitates surface regeneration, directed immobilisation and the attainment of interaction progress curves that conform to the ideal pseudo-first-order kinetic interaction model. Protein A, protein G and polyclonal anti-mouse Fe-coated surfaces were used to observe the interaction of captured anti-GST monoclonal antibody with glutathione-s-transferase (GST). It was shown that a protein A affinity-capture surface produced ideal interaction progress curves while both protein G and polyclonal anti-mouse Fe resulted in systemic deviations.     

Emerging tools for real-time label-free detection of interactions on functional protein microarrays

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.     

The 96-well twin-layer system: a novel approach in the cultivation of microalgae

A novel system for the growth and maintenance of microalgae has been developed that allows the cultivation of a large number of strains with little manual effort. The system is based on a 96-well microtiter plate in which a membrane filter constitutes the bottom of each well. Algal strains are immobilised on the membranes and provided with culture medium through contact with layers of glass fibre located beneath the membranes in a special cultivation chamber. The configuration effectively separates culture medium from algal cells which allows the simultaneous exchange of the culture medium for 96 strains within a few minutes without the need to transfer the algae. If necessary, algal strains can be transferred using multi-channel pipettes. We demonstrate that a large variety of microalgal strains including delicate flagellates can be reliably grown in the system under axenic conditions and without cross-contamination. As an array system, the 96-well twin-layer system using immobilised algae is also amenable to high-throughput and massively parallel applications increasingly sought after in algal bio- and environmental technology.     

THE SEARCH FOR INTERSTELLAR GLYCINE

Millimeter arrays can be used to identify hot young molecular cores which contain large, highly saturated interstellar molecules, including biomolecules. These cores are prime locations for searching for interstellar glycine. The current status of the interstellar glycine search is discussed.     

Understanding enzyme action on immobilised substrates

With increasing interest in automated synthesis and screening protocols, solid supported chemistry and biochemistry are attractive technologies. Studies with surface-immobilised substrates have been carried out to analyse enzyme accessibility, kinetics and thermodynamics. Several interesting new methods have been developed to monitor enzyme action on substrates attached to a solid phase such as polymer beads glass or gold surfaces. These include fluorescence measurements, MALDI-TOF mass spectrometry, and the use of quartz crystal microbalances to measure weight changes of immobilised molecules directly on the surface. Approaches that allow spatial resolution in single beads have also been reported. The ability of enzymes to reach the inside of beads is becoming better characterised and new supports have been developed that allow improved accessibility. The equilibrium position of reactions on the solid surface can be substantially shifted compared with reactions in solution, and this can be usefully exploited using hydrolases in reverse. Research is also starting to tackle the way in which kinetics are modified when the substrates are surface immobilised.     

Sensing with electro-switchable biosurfaces

The conformation of charged molecules tethered to conducting substrates can be controlled efficiently through the application of external voltages. Biomolecules like DNA or oligopeptides can be forced to stretch away from??or fold onto??surfaces biased at moderate potentials of merely hundreds of millivolts. These externally controlled conformation changes can be used to switch the biological function of molecular monolayers on and off, by revealing or concealing molecular recognition sites at will. Moreover, the electrical actuation of biomolecular surface probes bears great potential as a novel, label-free, yet highly sensitive measurement modality for the analysis of molecular interactions. The binding of target molecules to an oscillating probe layer significantly alters the layer??s switching behavior in terms of the conformation switching amplitude and, most remarkably, with respect to the molecular switching dynamics. Analyzing the switching response of target?Cprobe complexes from the low- to the high-frequency regime reveals a wealth of previously inaccessible information. Besides ??classical?? interaction parameters like binding affinities and kinetic rate constants, information on the size, shape, bending flexibility, and elasticity of the target molecule may be obtained in a single assay. This review describes the advent of electrically switchable biosurfaces, focusing on DNA monolayers. The preparation of self-assembled switchable oligonucleotide monolayers and their electrical interactions with charged substrates are highlighted. Special attention is paid to the merits of evaluating the dynamic response of charged biolayers which are operated at high driving frequencies. Several applications of biosensors based on electrically manipulated molecules are exemplified. It is emphasized that the electrical actuation of biomolecules bears many advantages over passive sensor surfaces.     

Carbon nanotube array: A new MIP platform

Here we demonstrate that a free-standing carbon nanotube (CNT) array can be used as a large surface area and high porosity 3D platform for molecular imprinted polymer (MIP), especially for surface imprinting. The thickness of polymer grafted around each CNT can be fine-tuned to imprint different sizes of target molecules, and yet it can be thin enough to expose every imprint site to the target molecules in solution without sacrificing the capacity of binding sites. The performance of this new CNT–MIP architecture was first assessed with a caffeine-imprinted polypyrrole (PPy) coating on two types of CNT arrays: sparse and dense CNTs. Real-time pulsed amperometric detection was used to study the rebinding of the caffeine molecules onto these CNT-MIPPy sensors. The dense CNT-MIPPy sensor presented the highest sensitivity, about 15 times better when compared to the conventional thin film, whereas an improvement of 3.6 times was recorded on the sparse CNT. However, due to the small tube-to-tube spacing in the dense CNT array, electrode fouling was observed during the detection of concentrated caffeine in phosphate buffer solution. A new I–V characterization method using pulsed amperometry was introduced to investigate the electrical characterization of these new devices. The resistance value derived from the I–V plot provides insight into the electrical conductivity of the CNT transducer and also the effective surface area for caffeine imprinting.