Till T. Bachmann

Improved multianalyte detection of organophosphates and carbamates with disposable multielectrode biosensors using recombinant mutants of Drosophila acetylcholinesterase and artificial neural networks

Engineered variants of Drosophila melanogaster acetylcholinesterase (AChE) were used as biological receptors of AChE-multisensors for the simultaneous detection and discrimination of binary mixtures of cholinesterase-inhibiting insecticides. The system was based on a combination of amperometric multielectrode biosensors with chemometric data analysis of sensor outputs using artificial neural networks (ANN). The multisensors were fully manufactured by screen-printing, including enzyme immobilisation. Two types of multisensors were produced that consisted of four AChE variants each. The AChE mutants were selected in order to obtain high resolution, enhanced sensitivity and minimal assay time. This task was successfully achieved using multisensor I equipped with wild-type Drosophila AChE and mutants Y408F, F368L, and F368H. Each of the AChE variants was selected on the basis of displaying an individual sensitivity pattern towards the target analytes. For multisensor II, the inclusion of F368W, which had an extremely diminished paraoxon sensitivity, increased the sensors capacity even further. Multisensors I and II were both used for inhibition analysis of binary paraoxon and carbofuran mixtures in a concentration range 0-5 microg/l, followed by data analysis using feed-forward ANN. The two analytes were determined with prediction errors of 0.4 microg/l for paraoxon and 0.5 microg/l for carbofuran. A complete biosensor assay and subsequent ANN evaluation was completed within 40 min. In addition, multisensor II was also investigated for analyte discrimination in real water samples. Finally, the properties of the multisensors were confirmed by simultaneous detection of binary organophosphate mixtures. Malaoxon and paraoxon in composite solutions of 0-5 microg/l were discriminated with predication errors of 0.9 and 1.6 microg/l, respectively.

Design of acetylcholinesterases for biosensor applications

In recent years, the use of acetylcholinesterases (AChEs) in biosensor technology has gained enormous attention, in particular with respect to insecticide detection. The principle of biosensors using AChE as a biological recognition element is based on the inhibition of the enzymes natural catalytic activity by the agent that is to be detected. The advanced understanding of the structure-function-relationship of AChEs serves as the basis for developing enzyme variants, which, compared to the wild type, show an increased inhibition efficiency at low insecticide concentrations and thus a higher sensitivity. This review describes different expression systems that have been used for the production of recombinant AChE. In addition, approaches to purify recombinant AChEs to a degree that is suitable for analytical applications will be elucidated as well as the various attempts that have been undertaken to increase the sensitivity of AChE to specified organophosphates and carbamates using side-directed mutagenesis and employing the enzyme in different assay formats.

Development, validation, and application of an acetylcholinesterase-biosensor test for the direct detection of insecticide residues in infant food

A highly sensitive and rapid food-screening test based on disposable screen-printed biosensors was developed, which is suitable for monitoring infant food. The exposure of infants and children to neurotoxic organophosphates and carbamates is of particular concern because of their higher susceptibility to adverse effects. The European Union has, therefore, set a very low limit for pesticides in infant food, which must not contain concentrations exceeding 10 microg/kg for any given pesticide. The maximum residue limit (MRL) has been set to be near the determination threshold that is typically achieved for pesticides with traditional analytical methods. The biosensor method could detect levels lower than 5 microg/kg and thus clearly fulfills the demands of the EU. To substantiate these measurements, recovery rates were determined and amounted on average to 104% in food. Matrix effects were eliminated by the introduction of a special electrode treatment. The test was compared with two traditional pesticide multiresidue analysis methods (GC-MS, LC-MS) using 26 fruit and vegetable samples from local markets and 23 samples of processed infant food from Germany, Spain, Poland and USA. Three infant food samples exceeded the MRL of 10 microg/kg when analyzed by either biosensor test or multiresidue methods.

Expression level of heterologous proteins in Pichia pastoris is influenced by flask design

Abstract. The yeast Pichia pastoris is a convenient production system that enables expression of heterologous proteins in high amounts. As a fermentation method, shaking flasks are very popular because of their simplicity of handling and their low cost. We compared the expression level of the enzyme acetylcholinesterase in a transformed strain of P. pastoris grown in different flasks, presenting various designs but all with the same volume. A several-thousand-fold difference appeared in the expression levels; and the results could not be explained by differences between the flasks in the oxygenation of the medium. The data show that flask design is an important factor to consider for optimising fermentation processes.

How many genes encode cholinesterase in arthropods

Carbamate and organophosphate pesticides act by inhibiting the key enzyme acetylcholinesterase in arthropods. Conversely to the case in vertebrates, in which two genes encode two different cholinesterases (ChE) a single gene was found in the fruit fly Drosophila melanogaster. In this species, several mutations decreasing the enzyme sensitivity to insecticides and thus responsible for resistance have been identified. After the sequencing of ChE-like genes in other species, repeated attempts to identify resistance mutations failed. These intriguing results raise the question of the existence of several ChE-encoding genes in a Arthropoda.

Transforming cyanobacteria into bioreporters of biological relevance

Microbial bioreporters play an important role in environmental monitoring and ecotoxicology. Microorganisms that are genetically modified with reporter genes can be used in various formats to determine the bioavailability of chemicals and their effect on living organisms. Cyanobacteria are abundant in the photosynthetic biosphere and have considerable potential with regards to broadening bioreporter applications. Two recent studies described novel cyanobacterial reporters for the detection of environmental toxicants and iron availability.

A microbial sensor based on Pseudomonas putida for phenol, benzoic acid and their monochlorinated derivatives which can be used in water and n-hexane

Abstract A microbial sensor for the detection of phenol, benzoic acid and their monochlorinated derivatives was developed. Detection was based on oxygen consumption in relation to analyte oxidation. To this end, induced cells of Pseudomonas putida DSM 548 were immobilized on Clark-type oxygen electrodes. The sensors were linear in their current signal up to a phenol concentration of 50 μM and had a detection limit of 0.1μM. Monochlorophenols could be detected at a concentration of 8 μM with a strong dependency on the growth substrate used for cell cultivation. Further variations in cell induction protocols resulted in microbial sensors with different selectivities. Using sensors equipped with cells grown on benzoic acid, this substance was detectable in the range of 2–16 μM, whereas 3- and 4-chlorobenzoic acid were detected at concentrations of 7 μM. The sensors were found to be stable under storage at 4°C for over 21 days. The use of n-hexane as a solvent increased the sensitivity for phenol five fold, t...

Detection of 4-chlorobenzoate using immobilized recombinant Escherichia coli reporter strains

Abstract A test for the detection of 4-chlorobenzoic acid (4-CBA) based on reporter microorganisms is developed. A 1.7-kb DNA fragment, upstream of the dehalogenase operon from Arthrobacter sp. strain SU, is fused to the promoterless luciferase operon of Vibrio fischeri . This reporter construct is introduced into two different Escherichia coli strains (UTL2 and RFM443), which specifically respond to the presence of 4-CBA with light emission. The development and optimization of an alginate-based immobilization procedure of the microorganisms in microtiter plates is described. In this format, 4-CBA can be detected in the concentration range of 113 μM–3.6 mM. The membrane leaky mutant UTL2 of E. coli transformed with the reporter construct improves the detection limit to 28 μM when 150 mM KNO 3 is added to the medium.

Disposable sensor for measuring the biochemical oxygen demand for nitrification and inhibition of nitrification in wastewater.

Abstract A disposable-type microbial sensor was developed for the determination of both the biochemical oxygen demand for nitrification (N-BOD) and inhibiting effects on nitrifying bacteria. The sensor was based on the respiratory activity of nitrifying bacteria immobilized on a miniature oxygen electrode. Typical response times for measuring N-BOD of ammonium standard solutions as well as of wastewater samples were in the range of 6–12 min. A dynamic evaluation of the signals after a measuring time of 120 s also resulted in good reproducibility and sensitivity. A daily profile of a municipal sewage plant was recorded, comparing the biosensor data with two standard methods. For the measurement of nitrification-inhibiting effects a 120-s dynamic signal evaluation was preferred to a steady-state method because of the long recovery times resulting from extended exposure to inhibitors. However, steady-state measurement techniques allowed allylthiourea detection with a ten times higher sensitivity. Because of the advantages of this miniaturized electrode, e.g. short response time, simple measuring procedure and low costs of production, this sensor system is considered to be suitable for commercial application in environmental analysis.

Multiparametric determination of genes and their point mutations for identification of beta-lactamases.

More than half of all currently used antibiotics belong to the beta-lactam group, but their clinical effectiveness is severely limited by antibiotic resistance of microorganisms that are the causative agents of infectious diseases. Several mechanisms for the resistance of Enterobacteriaceae have been established, but the main one is the enzymatic hydrolysis of the antibiotic by specific enzymes called beta-lactamases. Beta-lactamases represent a large group of genetically and function-ally different enzymes of which extended-spectrum beta-lactamases (ESBLs) pose the greatest threat. Due to the plasmid localization of the encoded genes, the distribution of these enzymes among the pathogens increases every year. Among ESBLs the most widespread and clinically relevant are class A ESBLs of TEM, SHV, and CTX-M types. TEM and SHV type ESBLs are derived from penicillinases TEM-1, TEM-2, and SHV-1 and are characterized by several single amino acid substitutions. The extended spectrum of substrate specificity for CTX-M beta-lactamases is also associated with the emergence of single mutations in the coding genes. The present review describes various molecular-biological methods used to identify determinants of antibiotic resistance. Particular attention is given to the method of hybridization analysis on microarrays, which allows simultaneous multiparametric determination of many genes and point mutations in them. A separate chapter deals with the use of hybridization analysis on microarrays for genotyping of the major clinically significant ESBLs. Specificity of mutation detection by means of hybridization analysis with different detection techniques is compared.