Biologische Erkennungselemente in elektrochemischen Biosensoren - eine Übersicht

Bereits Anfang der sechziger Jahre wurde erstmals das Prinzip von Enzymelektroden als Anordnung von Enzymen direkt auf einen Messfühler patentiert. Leland C. Clark Jr. beschrieb eine Enzymelektrode zur Bestimmung von Blutzucker, bei der Glucoseoxidase mit einer semipermeablen Membran vor einer Sauerstoffelektrode fixiert wurde. Seit dieser Zeit ist eine große Zahl von Enzymen verwendet worden. Aber auch andere Biokomponenten, wie Organellen, intakte Zellen, Antikörper und Rezeptoren werden als Erkennungselemente eingesetzt. Diese traditionell verwendeten Erkennungssysteme werden in neuerer Zeit durch Nukleinsäuren, synthetische und semisynthetische (biomimetische) Erkennungssubstanzen erweitert. Hier sind besonders DNA , RNA, PNA, Synzyme und ‚Molecular Imprints’ zu nennen. Durch Nutzung des Protein-Engineering werden gezielt veränderte Proteine, Antikörper und Enzyme erhalten. Die Erzeugung von optimalen Bindungsmolekülen auf Aminosäure-und Nukleinsäurebasis wird durch Kombination von Synthese großer kombinatorischer Peptid- und RNA-Bibliotheken, Selektion nach Bindungsstärke zum Analyten und enzymatischer Verstärkung realisiert. Der Nachweis der biospezifischen Erkennung erfolgt mit vorwiegend mit optischen und elektrochemischen Transduktoren. Im vorliegenden Beitrag werden Biomoleküle in ihrer Kombination mit amperometrischen Elektroden beschrieben

Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer

An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV–Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V (vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 µM sulfite with a sensitivity of 2.19 mA M−1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample

Molecularly imprinted polymer-based electrochemical sensors for biopolymers

Electrochemical synthesis and signal generation dominate among the almost 1200 articles published annually on protein-imprinted polymers. Such polymers can be easily prepared directly on the electrode surface, and the polymer thickness can be precisely adjusted to the size of the target to enable its free exchange. In this architecture, the molecularly imprinted polymer (MIP) layer represents only one ‘separation plate’; thus, the selectivity does not reach the values of ‘bulk’ measurements. The binding of target proteins can be detected straightforwardly by their modulating effect on the diffusional permeability of a redox marker through the thin MIP films. However, this generates an ‘overall apparent’ signal, which may include nonspecific interactions in the polymer layer and at the electrode surface. Certain targets, such as enzymes or redox active proteins, enables a more specific direct quantification of their binding to MIPs by in situ determination of the enzyme activity or direct electron transfer, respectively

Kapillar-Immuno-Assay mit elektrochemischer Detektion

Alkylphenolethoxylate werden als Bestandteile von Waschmitteln und als Weichmacher eingesetzt und gelangen so in großer Zahl ins Abwasser. Alkylphenole, als relativ hydrophobe Abbauprodukte, gelten aufgrund ihres hohen östrogenen Potential als Umwelthormone (endocrine disruptors). Deshalb werden verschiedene klassische und biochemische Analysenmethoden für die Substanzklasse entwickelt. Das hohe Probenaufkommen verlangt nach schnellen Nachweisverfahren, die mit Hilfe von automatisierbaren Fließ-Systemen realisiert werden kann. In dieser Arbeit präsentieren wir einen elektrochemischen Fließ-Immunoassay (EFIA), der eine Kombination eines empfindlichen Biosensors mit einem Kapillar-Immuno-Reaktor darstellt. Im vorgestellten Ansatz werden Antikörper gegen die Zielsubstanzen Nonyl-,Oktylphenol, und Nonylphenolethoxylat in Mikro-Kapillaren immobilisiert, in denen Kompetition stattfindet. Dazu wird die Probenlösung mit Tracer versetzt und gemeinsam in den Kapillaren inkubiert. Nach einem anschließenden Spülschritt wird die gebundene Menge markierte Analyt quantifiziert, indem das aminophenylierte Substrat des Tracers injiziert wird. Nach zeitlich kontrollierter Hydrolyse wird das gebildete p-Aminophenol am Biosensor vorbei geführt und dabei vermessen. Als Markierungssubstanz für den Tracer wird ß-D-Galactosidase verwendet, welche Amino- und Nitrophenylgalactopyranosid hydrolysiert und so Amino- bzw. Nitrophenol freisetzt. Die Tracer werden durch chemische Kopplung der zur Immunisierung verwendeten Haptene mit dem Enzym (ßGal) dargestellt

Electrosynthesized MIPs for Transferrin: Plastibodies or Nano-Filters?

Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered

Prediction of wastewater quality using amperometric bioelectronic tongues

Wastewater samples from a Swedish chemi-thermo-mechanical pulp (CTMP) mill collected at different purification stages in a wastewater treatment plant (WWTP) were analyzed with an amperometric enzyme-based biosensor array in a flow-injection system. In order to resolve the complex composition of the wastewater, the array consists of several sensing elements which yield a multidimensional response. We used principal component analysis (PCA) to decompose the array's responses, and found that wastewater with different degrees of pollution can be differentiated. With the help of partial least squares regression (PLS-R), we could link the sensor responses to the Microtox (R) toxicity parameter, as well as to global organic pollution parameters (COD, BOD, and TOC). From investigating the influences of individual sensors in the array, it was found that the best models were in most cases obtained when all sensors in the array were included in the PLS-R model. We find that fast simultaneous determination of several global environmental parameters characterizing wastewaters is possible with this kind of biosensor array, in particular because of the link between the sensor responses and the biological effect onto the ecosystem into which the wastewater would be released. In conjunction with multivariate data analysis tools, there is strong potential to reduce the total time until a result is yielded from days to a few minutes. (C) 2015 Elsevier B.V. All rights reserved

Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes

International audienceSulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a " closed " conformation, suitable for internal electron transfer, and an " open " conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization lead to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modelled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the 1 electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization

A glucose dehydrogenase biosensor as an additional signal amplification step in an enzyme-flow immunoassay.

Both the antibody affinity and the detectability of the label are essential in deciding the final characteristics of a heterogeneous immunoassay. This paper describes an approach to obtain a supplementary enhancement of the signal generated by using an enzyme label, e.g., by including the product of the enzymatic reaction in an additional amplification cycle during the detection step performed with an amperometric biosensor based on glucose dehydrogenase (GDH). An immunoassay format with a labelled analyte derivative that competes with the analyte present in the sample for a limited amount of antibody binding sites was employed. The beta-galactosidase label hydrolyses the substrate aminophenyl-beta-galactopyranoside, and the generated aminophenol enters then into a bioelectrocatalytic amplification cycle at the GDH biosensor. The principle was applied for determination of 4-nitrophenol, with the best minimal concentration of 1.5 microM and a midpoint of the calibration of 24 microM. The potentials and limitations of such a system are discussed