55 research outputs found
Cystein-Mutanten der Cu,Zn-Superoxiddismutase und ihre Anwendung in Proteinelektroden für die Detektion von freien Sauerstoffradikalen
Das Enzym Superoxiddismutase (SOD) bietet wegen seiner hohen Reaktionsrate und seiner extrem hohen Substratspezifi tät große Vorteile für eine Anwendung als Superoxidbiosensor. In dieser Arbeit wurden durch molekularbiologische Methoden Mutanten der humanen Cu,Zn-SOD gewonnen, welche ein oder zwei zusätzliche Cystein-Reste enthielten, die eine einfache Immobilisierung des Proteins durch Bindung des Cystein-Schwefels auf Goldelektroden ermöglichten. Sechs solcher Mutanten wurden entworfen, exprimiert, aufgereinigt und elektrochemisch charakterisiert. Alle Mutanten konnten durch einen einfachen Inkubationsschritt auf Goldelektroden gebunden werden und zeigten ein quasi-reversibles elektrochemisches Ansprechen. Für eine Mutante wurde die Anwendung als Superoxidsensor genauer untersucht und für beide Teilreaktionen der Dismutation ein Ansprechen des Sensors auf das Radikal gefunden. Bei Verwendung einer Teilreaktion konnte die Empfindlichkeit herkömmlicher Monoschichtsensoren um etwa eine Größenordnung übertroffen werden
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
The First Electrochemical MIP Sensor for Tamoxifen
We present an electrochemical MIP sensor for tamoxifen (TAM)—a nonsteroidal anti-estrogen—which is based on the electropolymerisation of an O-phenylenediamine‒resorcinol mixture directly on the electrode surface in the presence of the template molecule. Up to now only “bulk” MIPs for TAM have been described in literature, which are applied for separation in chromatography columns. Electro-polymerisation of the monomers in the presence of TAM generated a film which completely suppressed the reduction of ferricyanide. Removal of the template gave a markedly increased ferricyanide signal, which was again suppressed after rebinding as expected for filling of the cavities by target binding. The decrease of the ferricyanide peak of the MIP electrode depended linearly on the TAM concentration between 1 and 100 nM. The TAM-imprinted electrode showed a 2.3 times higher recognition of the template molecule itself as compared to its metabolite 4-hydroxytamoxifen and no cross-reactivity with the anticancer drug doxorubucin was found. Measurements at +1.1 V caused a fouling of the electrode surface, whilst pretreatment of TAM with peroxide in presence of HRP generated an oxidation product which was reducible at 0 mV, thus circumventing the polymer formation and electrochemical interferences
- …