Michael Keusgen

Biosensors: new approaches in drug discovery.

Abstract. The development of biosensors for analytical purposes has attracted a great deal of attention in recent years. A biosensor is defined as an analytical device consisting of a biological component (e.g., enzyme, antibody, entire cell, DNA) and a physical transducer (e.g., electrode, optical device). Biosensors are mostly designed for routine analysis, such as clinical diagnosis, quality control of food, in-process control of fermentations, and in environmental analysis. Many of these sensors are also suitable for screening purposes in order to find new drugs. Such systems should yield information either about compounds with known bioactivity or about the bioactivity of samples with known or unknown chemical composition. Biosensors intended for the latter purpose are essentially based on whole cells carrying receptors and ion channels at their surfaces. Miniaturization of structures, primarily based on silicon, allows integration of many sensors into arrays, which may be suitable for the screening of natural and chemical products as well as combinatorial libraries. Until now, no commercially available sensors of this kind exist but they are expected in the near future. Different biosensors, based on enzymes, antibodies, cells, artificial membranes and entire animal tissues, which can be used in drug discovery and may lead to efficient screening systems in the future, are described in this review.

Cysteine Sulfoxides and Alliinase Activity of Some Allium Species

The flavor precursors of 17 species belonging to the Alliaceae family were analyzed by HPLC, and results were evaluated with respect to the classification of species into their genus, subgenus, and section. Identification and quantification of these precursors were carried out by synthetic and natural reference materials. In addition, nine of these species were investigated in terms of their alliinase activity. Alliinase (EC 4.4.1.4) catalyzes the conversion of odorless (+)-S-alk(en)yl-L-cysteine sulfoxides into volatile thiosulfinates. Cysteine sulfoxides as well as alliinase activity were found in all investigated samples, and (+)-S-methyl-L-cysteine sulfoxide was most abundant. (+)-S-Propyl-L-cysteine sulfoxide was detected in only a few, not closely related, species. Analysis of the crude protein extract of nine species gave evidence that alliinase activities of samples were similar in terms of pH and temperature optimum, K(M) value, and substrate specificity. For all investigated protein extracts, the highest specific alliinase activity was found for (+)-S-(2-propenyl)-L-cysteine sulfoxide (alliin). The substrate specificity of these enzymes was not related to relative abundance of the cysteine sulfoxides. However, SDS-PAGE yielded some significant differences among species in terms of their total protein compositions. Species belonging to different subgenera exhibited a specific protein pattern with molecular masses between 13 and 35 kDa.

A haem cofactor is required for redox and light signalling by the AppA protein of Rhodobacter sphaeroides.

The AppA protein of Rhodobacter sphaeroides is unique in its ability to sense and transmit redox signals as well as light signals. By functioning as antagonist to the PpsR transcriptional repressor, it regulates the expression of photosynthesis genes in response to these environmental stimuli. Here we show binding of the cofactor haem to a domain in the C‐terminal part of AppA and redox activity of bound haem. This is supported by the findings that: (i) the C‐terminal domain of AppA (AppAΔN) binds to haemin agarose, (ii) AppAΔN isolated from Escherichia coli shows absorbance at 411 nm and absorbances at 424 nm and 556 nm after reduction with dithionite and (iii) AppAΔN can be reconstituted with haem in vitro. Expression of AppA variants in R. sphaeroides reveals that the haem binding domain is important for normal expression levels of photosynthesis genes and for normal light regulation in the presence of oxygen. The haem cofactor affects the interaction of the C‐terminal part of AppA to PpsR but also its interaction to the N‐terminal light sensing AppA‐BLUF domain. Based on this we present a model for the transmission of light and redox signals by AppA.

Surface plasmon resonance (SPR) as a rapid tool for serotyping of Salmonella.

An SPR-based sandwich immunoassay for serotyping of Salmonella is demonstrated. The Salmonella are captured on an SPR chip using polyclonal capture antibody. SPR sensorgrams are obtained for the immunoreactions of the somatic (O) and flagellar (H) surface antigens, of the captured bacteria, to their respective antibodies. The sensorgram data are compiled to determine the antigenic formula in accordance with the Kauffmann-White scheme. Salmonella Enteritidis was completely serotyped using this SPR-based method. In addition, Salmonella belonging to serogroups B, C and D were successfully assigned to their respective serogroups. Before serotyping the bacteria are grown to a concentration of 1x10(10) mL(-1). This SPR-based serotyping provides quantitative data, and thus, eliminates the possibility of false detections as encountered in the conventional slide agglutination test (SAT). This method was also proved to work with rough strains.

Immobilization of alliinase on porous aluminum oxide

Membrane filters prepared from porous aluminum oxide (Anopore) were investigated for their potential use as a durable support for enzymes. Alliinase (EC 4.4.1.4) was chosen as a model enzyme for immobilization experiments. To allow for smooth fixation, the enzyme was immobilized indirectly by sugar-lectin binding. Monomolecular layers of the lectin concanavalin A and alliinase were applied by self-assembling processes. As an anchor for these layers, the sugar, mannan, was covalently coupled to the membrane surface. This procedure exhibits several advantages: (i) enzyme immobilization can be carried out under smooth conditions; (ii) immobilization needs little time; and (iii) protein layers may be renewed.

Phlorotannins from the brown algae Cystophora torulosa and Sargassum spinuligerum

Phlorotannins often have various toxic effects against a large number of organisms. From the ethyl acetate fraction of the ethanolic extract of the brown alga Cystophora torulosa 33 phlorotannins were obtained. Twenty of them are described in this report: phlorethols and fuhalols, and fucophlorethols and hydroxyfucophlorethols. Seven of them were isolated for the first time. New phlorotannins bearing additional hydroxy groups belong to the hydroxyfucophlorethols. NMR- and MS-data were used for structural elucidation. Several of the substances described for C. torulosa occur in Sargassum spinuligerum as well.

A rapid serological assay for prediction of Salmonella infection status in slaughter pigs using surface plasmon resonance

We present a rapid surface plasmon resonance-based serological assay for the detection of Salmonella Typhimurium infection in pigs using the Plasmonic((R)) SPR device. Lipopolysaccharide (LPS, 10 microg mL(-1)) from Salmonella Typhimurium was immobilised by self-assembly on a hydrophobic SPR chip. Using this LPS-coated chip, it was possible to bind and detect the anti-Salmonella Typhimurium antibodies in serum of pigs infected with the bacteria. The developed SPR assay is able to differentiate between sera obtained from pigs having low, medium, and high levels of Salmonella infection. A commercial ELISA kit was used to classify the sera for levels of Salmonella infection on the basis of optical density (OD%). A strong positive correlation was observed between the SPR-based assay and the ELISA (n=38, r=0.90, p<0.01). The sensitivity and specificity of the assay are 0.93 and 0.87, respectively. The SPR-based assay is label-free and does not require any sample preparation or dilution steps. The total analysis time is 45 min for each serum sample. The assay was found to be specific for Salmonella Typhimurium and shows no cross-reactivity to Salmonella Choleraesuis or Escherichia coli antibodies. As no sample preparation is required the developed assay has the potential to be used as a reliable tool for Salmonella monitoring programmes in pork production.

Development of a biosensor specific for cysteine sulfoxides

S-Alk(en)yl cysteine sulfoxides have been observed in several plants, mainly belonging to the onion family (Alliaceae), which are of high commercial interest (e.g. garlic, Allium sativum). The quality of most garlic containing herbal remedies produced from garlic powder is determined by their content of the cysteine sulfoxide alliin. Therefore, a comprehensive method for the documentation of alliin amounts present in the fresh plant material through to the final remedy is desirable. The newly developed biosensoric method described in this paper was designed in order to fulfil these demands. In contrast to conventional HPLC-methods, neither a pre-column derivatization nor a chromatographic separation are required allowing a high throughput of samples. This technique is based on immobilized alliinase (EC 4.4.1.4), which was combined with an ammonia-gas electrode. The enzyme was either placed in a small cartridge or was immobilized in direct contact of the electrode surface giving detection limits of 3.7 x 10(-7) and 5.9 x 10(-6) M. Founded on these experiments, a pH-sensitive electrolyte/insulator/semiconductor (EIS) layer structure made of Al/p-Si/SiO(2)/Si(3)N(4) was also combined with immobilized alliinase. Measurements could be performed in a range between 1 x 10(-5) and 1 x 10(-3) M alliin. All sensors were operated in the flow-through modus. A high specificity for alliin could be demonstrated for the electrode and a number of garlic samples were analyzed. Results gained with the new method showed a good correlation with those obtained with conventional HPLC-methods. In addition, onion and a variety of wild Allium species were analyzed in order to determine the amount of isoalliin or total cysteine sulfoxides present, respectively.

Immobilization of enzymes on PTFE surfaces

Membranes and powders prepared from PTFE (polytetrafluorethylene) were investigated for their potential use as multifunctional supports for enzymes. The obtained bioactive materials are valuable for the construction of biosensors and enzyme reactors. To allow covalent coupling of enzymes to PTFE, the surface of the material was treated with elementary sodium followed by oxidation with ozone or hydrogen peroxide.%Derivatization steps were optimized in order to achieve highest enzyme loading and short reaction times. Alliinase (EC 4.4.1.4) and L-lactic dehydrogenase (EC 1.1.1.27) were chosen as model enzymes and were either immobilized by covalent coupling or fixed indirectly by a sugar-lectin binding. For the latter method, the sugar mannan was bound to the membrane surface as an anchor for layers of the lectin concanavalin A and the alliinase. Highest alliinase loading was achieved at 0.2 microg x cm(-2). Immobilization of alliinase via the lectin concanavalin A and a bifunctional epoxide gave the best long-term stability.%L-Lactic dehydrogenase was most sufficiently immobilized by using benzoquinone as spacer. These procedures show several advantages: 1) enzymes can be immobilized under physiological conditions, 2) an enzyme-multilayer can be achieved, and 3) protein layers are renewable.

DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

Miniaturized setup, compatibility with advanced micro- and nanotechnologies, and ability to detect biomolecules by their intrinsic molecular charge favor the semiconductor field-effect platform as one of the most attractive approaches for the development of label-free DNA chips. In this work, a capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor covered with a layer-by-layer prepared, positively charged weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was used for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization. The negatively charged probe single-stranded DNA (ssDNA) molecules were electrostatically adsorbed onto the positively charged PAH layer, resulting in a preferentially flat orientation of the ssDNA molecules within the Debye length, thus yielding a reduced charge-screening effect and a higher sensor signal. Each sensor-surface modification step (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), reducing an unspecific adsorption by a blocking agent, incubation with noncomplementary DNA (ncDNA) solution) was monitored by means of capacitance-voltage and constant-capacitance measurements. In addition, the surface morphology of the PAH layer was studied by atomic force microscopy and contact-angle measurements. High hybridization signals of 34 and 43 mV were recorded in low-ionic strength solutions of 10 and 1 mM, respectively. In contrast, a small signal of 4 mV was recorded in the case of unspecific adsorption of fully mismatched ncDNA. The density of probe ssDNA and dsDNA molecules as well as the hybridization efficiency was estimated using the experimentally measured DNA immobilization and hybridization signals and a simplified double-layer capacitor model. The results of field-effect experiments were supported by fluorescence measurements, verifying the DNA-immobilization and hybridization event.