@article{BeissenhirtzSchellerLisdat2004,
  author    = {Beissenhirtz, Moritz Karl and Scheller, Frieder W. and Lisdat, Fred},
  title     = {A superoxide sensor based on a multilayer cytochrome c electrode},
  issn      = {0003-2700},
  year      = {2004},
  abstract  = {A novel multilayer cytochrome c electrode for the quantification of superoxide radical concentrations is introduced. The electrode consists of alternating layers of cytochrome c and poly(aniline(sulfonic acid)) on a gold wire electrode. The formation of multilayer structures was proven by SPR experiments. Assemblies with 2-15 protein layers showed electrochemical communication with the gold electrode. For every additional layer, a substantial increase in electrochemically active cytochrome c (cyt. c) was found. For electrodes of more than 10 layers, the increase was more than 1 order of magnitude as compared to monolayer electrode systems. Thermodynamic and kinetic parameters of the electrodes were characterized. The mechanism of electron transfer within the multilayer assembly was studied, with results suggesting a protein-protein electron-transfer model. Electrodes of 2-15 layers were applied to the in vitro quantification of enzymatically generated superoxide, showing superior sensitivity as compared to a monolayer-based sensor. An electrode with 6 cyt. c/PASA layers showed the highest sensitivity of the systems studied, giving an increase in sensitivity of half an order of magnitude versus the that of the monolayer electrode. The stability of the system was optimized using thermal treatment, resulting in no loss in sensor signal or protein loading after 10 successive measurements or 2 days of storage},
  language  = {en}
}
@article{BeissenhirtzKwanKoetal.2004,
  author    = {Beissenhirtz, Moritz Karl and Kwan, R. C. H. and Ko, K. M. and Renneberg, Reinhard and Scheller, Frieder W. and Lisdat, Fred},
  title     = {Comparing in vitro electrochemical measurement of superoxide scavenging activity with an in vivo assessment of antioxidant potential in Chinese tonifying herbs},
  year      = {2004},
  abstract  = {The in vitro superoxide scavenging activity (as determined by electrochemical measurement) and the in vivo antioxidant potential (as determined by a mouse model of carbon tetrachloride (CCl4) hepatotoxicity) of methanolic extracts prepared from 10 Chinese tonifying herbs were compared. Electrochemical measurement using a cytochrome c (Cyt. c) sensor showed that all of the tested herbal extracts exhibited a medium superoxide scavenging activity of different potency, as indicated by their IC50 values. The in vivo measurement demonstrated that 80\% of the herbal extracts displayed in vivo antioxidant potential, as assessed by the percentage of protection of the activity of plasma alanine aminotransferases and the hepatic glutathione regeneration capacity under CCl4-intoxicated condition. Although the in vitro antioxidant activity did not correlate quantitatively with the in vivo antioxidant potential, for 8 out of 10 samples a similar tendency was found. The rapid amperometric assessment of antioxidant potential by Cyt. c sensor may offer a convenient and direct method for screening as well as the quality control of herbal products. Copyright (C) 2004 John Wiley Sons, Ltd},
  language  = {en}
}
@article{BeissenhirtzSchellerStoeckleinetal.2004,
  author    = {Beissenhirtz, Moritz Karl and Scheller, Frieder W. and St{\"o}cklein, Walter F. M. and Kurth, D. and M{\"o}hwald, Helmuth and Lisdat, Fred},
  title     = {Electroactive cytochrome c multilayers within a polyelectrolyte assembly},
  year      = {2004},
  language  = {en}
}
@article{KappBeissenhirtzGeyeretal.2006,
  author    = {Kapp, A. and Beissenhirtz, Moritz Karl and Geyer, F. and Scheller, F. and Viezzoli, Maria Silvia and Lisdat, Fred},
  title     = {Electrochemical and sensorial behavior of SOD mutants immobilized on gold electrodes in aqueous/organic solvent mixtures},
  series = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis},
  volume    = {18},
  journal   = {Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis},
  publisher = {Wiley},
  address   = {Weinheim},
  issn      = {1040-0397},
  doi       = {10.1002/elan.200603620},
  pages     = {1909 -- 1915},
  year      = {2006},
  abstract  = {A cysteine mutant of a monomeric human Cu, Zn-SOD (Glycine 61, Serine 142) has been immobilized directly on gold electrodes using the thiol groups introduced. The electrochemical behavior of the surface confined protein was studied in mixtures of aqueous buffer and DMSO up to an organic solvent content of 60\%. The formal potential was found to be rather independent of the DMSO content. However, half peak width increased and the redoxactive amount clearly decreased with raising DMSO content. In addition, the kinetics of the heterogeneous electron transfer became slower; but still a quasireversible electrochemical conversion of the mutant SOD was feasible. Thus, the electrodes were applied for sensorial superoxide detection. At a potential of +220 mV vs. Ag/AgCl advantage was taken of the partial oxidation reaction of the enzyme. A defined superoxide signal was obtained in solutions up to 40\% DMSO. The sensitivity of the mutant electrodes decreased linearly with the organic solvent content in solution but was still higher compared to conventional cyt.c based sensors. At DMSO concentrations higher than 40\% no sensor response was detected.},
  language  = {en}
}
@article{KappBeissenhirtzGeyeretal.2006,
  author    = {Kapp, Andreas and Beissenhirtz, Moritz Karl and Geyer, F. and Scheller, Frieder W. and Viezzoli, Maria Silvia and Lisdat, Fred},
  title     = {Electrochemical and sensorial behaviour of SOD mutants immobilized on gold electrodes in aqueous / organic solvent mixtures},
  issn      = {1040-0397},
  doi       = {10.1002/elan.200603620},
  year      = {2006},
  language  = {en}
}
@article{Beissenhirtz2005,
  author    = {Beissenhirtz, Moritz Karl},
  title     = {Electrochemical quartz crystal microbalance studies on cytochrome c/polyelectrode multilayer assemblies on gold electrodes},
  year      = {2005},
  abstract  = {Polyelectrolyte multilayer assemblies containing proteins are of interest for applications such as sensors, bioreactors, and bioelectronics. A multilayer electrode was built up by the layer-by-layer strategy consisting of alternating layers of cytochrome c and poly(aniline sulfonic acid). The electrode showed a linear increase of redox active protein with the number of deposited layers. The principle of electrode preparation was transferred from needle electrodes to planar surfaces in order to further the understanding of electron transfer through the layer assembly by means of electrochemical quartz crystal microbalance studies. The deposition process was followed on-line by detection of the frequency shift of the crystals and was found to be rather fast (minutes). The total mass deposited was found to correlate well with the electrochemical response of the immobilized cyt.c. Furthermore, the influence of the polyelectrolyte was investigated by addition of PSS to the PASA solution. The strong interaction of the former polyelectrolyte seemed to hinder the electron transfer although a multilayer formation was proved. Dilution of the protein solution with redox inactive apo-cyt.c led to a strong decrease of the voltammetric signal, well beyond the percentage of apo-cyt.c inside the assembly. Thus, arguments for an electron transfer via protein-protein interaction were found},
  language  = {en}
}
@article{BeissenhirtzSchellerViezzolietal.2006,
  author    = {Beissenhirtz, Moritz Karl and Scheller, Frieder W. and Viezzoli, Maria Silvia and Lisdat, Fred},
  title     = {Engineered superoxide dismutase monomers for superoxide biosensor applications},
  issn      = {0003-2700},
  doi       = {10.1021/Ac051465g},
  year      = {2006},
  abstract  = {Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasireversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by similar to 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold},
  language  = {en}
}
@article{BuettemeyerPhilippSchlenzkaetal.2003,
  author    = {B{\"u}ttemeyer, R. and Philipp, A. W and Schlenzka, L. and Mall, J. W. and Beissenhirtz, Moritz Karl and Lisdat, Fred},
  title     = {Epigallocatechin gallate can significantly decrease free oxygen radicals in the reperfusion insury in vivo},
  year      = {2003},
  language  = {en}
}
@article{BeissenhirtzSchellerLisdat2003,
  author    = {Beissenhirtz, Moritz Karl and Scheller, Frieder W. and Lisdat, Fred},
  title     = {Immobilized cytochrome c sensor in organic / aqueous media for the characterization of hydrophilic and hydrophobic antioxidants},
  year      = {2003},
  language  = {en}
}
@phdthesis{Beissenhirtz2005,
  author    = {Beissenhirtz, Moritz Karl},
  title     = {Proteinmultischichten und Proteinmutanten f{\"u}r neuartige empfindliche Superoxidbiosensoren},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5661},
  school      = {Universit{\"a}t Potsdam},
  year      = {2005},
  abstract  = {Das Superoxidradikal kann mit fast allen Bestandteilen von Zellen reagieren und diese sch{\"a}digen. Die medizinische Forschung stellte eine Beteiligung des Radikals an Krebs, Herzinfarkten und neuraler Degeneration fest. Ein empfindlicher Superoxidnachweis ist daher zum besseren Verst{\"a}ndnis von Krankheitsverl{\"a}ufen wichtig. Dabei stellen die geringen typischen Konzentrationen und seine kurze Lebensdauer große Anforderungen. Ziel dieser Arbeit war es zum einen, zwei neuartige Proteinarchitekturen auf Metallelektroden zu entwickeln und deren elektrochemisches Ansprechverhalten zu charakterisieren. Zum anderen waren diese Elektroden zur empfindlichen quantitativen Superoxiddetektion einzusetzen. Im ersten Teil der Arbeit wurde eine Protein-Multischichtelektrode aus Cytochrom c und dem Polyelektrolyten Poly(anilinsulfons{\"a}ure) nach dem Layer-by-layer-Verfahren aufgebaut. F{\"u}r zwei bis 15 Schichten an Protein wurde eine deutliche Zunahme an elektrodenaktivem Cytochrom c mit jedem zus{\"a}tzlichen Aufbringungsschritt nachgewiesen. Die Zunahme verlief linear und ergab bei 15 Schichten eine Zunahme der redoxaktiven Proteinmenge um deutlich mehr als eine Gr{\"o}ßenordnung. W{\"a}hrend das formale Potential im Multischichtsystem sich im Vergleich zur Monoschichtelektrode nicht ver{\"a}nderte, wurde f{\"u}r die Kinetik eine Abh{\"a}ngigkeit der Geschwindigkeit des Elektronentransfers von der Zahl der Proteinschichten beobachtet. Mit zunehmender Scangeschwindigkeit trat ein reversibler Kontaktverlust zu den {\"a}ußeren Schichten auf. Die lineare Zunahme an elektroaktivem Protein mit steigender Zahl an Depositionsschritten unterscheidet sich deutlich von in der Literatur beschriebenen Protein/Polyelektrolyt-Multischichtelektroden, bei denen ab etwa 6-8 Schichten keine Zunahme an elektroaktivem Protein mehr festgestelltwurde. Auch ist bei diesen die Zunahme an kontaktierbaren Proteinmolek{\"u}len auf das Zwei- bis F{\"u}nffache limitiert. Diese Unterschiede des neu vorgestellten Systems zu bisherigen Multischichtassemblaten erkl{\"a}rt sich aus einem in dieser Arbeit f{\"u}r derartige Systeme erstmals beschriebenen Elektronentransfermechanismus. Der Transport von Elektronen zwischen der Elektrodenoberfl{\"a}che und den Proteinmolek{\"u}len in den Schichten verl{\"a}uft {\"u}ber einen Protein-Protein-Elektronenaustausch. Dieser Mechanismus beruht auf dem schnellen Selbstaustausch von Cytochrom c-Molek{\"u}len und einer verbleibenden Rotationsflexibilit{\"a}t des Proteins im Multischichtsystem. Die Reduzierung des Proteins durch das Superoxidradikal und eine anschließende Reoxidation durch die Elektrode konnten nachgewiesen werden. In einem amperometrischen Messansatz wurde das durch Superoxidradikale hervorgerufene elektrochemische Signal in Abh{\"a}ngigkeit von der Zahl an Proteinschichten gemessen. Ein maximales Ansprechverhalten auf das Radikal wurde mit 6-Schichtelektroden erzielt. Die Empfindlichkeit der 6-Schichtelektroden wurde im Vergleich zum Literaturwert der Monoschichtelektrode um Faktor 14, also mehr als eine Gr{\"o}ßenordnung, verbessert. Somit konnte eine Elektrode mit 6 Schichten aus Cytochrom c und Poly(anilinsulfons{\"a}ure) als neuartiger Superoxidsensor mit einer 14-fachen Verbesserung der Empfindlichkeit im Vergleich zum bislang benutzten System entwickelt werden. Der zweite Teil dieser Arbeit beschreibt die Auswahl, Gewinnung und Charakterisierung von Mutanten des Proteins Cu,Zn-Superoxiddismutase zur elektrochemischen Quantifizierung von Superoxidradikalen. Monomere Mutanten des humanen dimeren Enzyms wurden entworfen, die durch Austausch von Aminos{\"a}uren ein oder zwei zus{\"a}tzliche Cysteinreste besaßen, mit welchem sie direkt auf der Goldelektrodenoberfl{\"a}che chemisorbieren sollten. 6 derartige Mutanten konnten in ausreichender Menge und Reinheit in aktiver Form gewonnen werden. Die Bindung der Superoxiddismutase-Mutanten an Goldoberfl{\"a}chen konnte durch Oberfl{\"a}chen-plasmonresonanz und Impedanzspektroskopie nachgewiesen werden. Alle Mutanten wiesen einen quasi-reversiblen Elektronentransfer zwischen SOD und Elektrode auf. Durch Untersuchung von kupferfreien SOD-Mutanten sowie des Wildtyps konnte nachgewiesen werden, das die Mutanten {\"u}ber die eingef{\"u}gten Cysteinreste auf der Elektrode chemisorptiv gebunden wurden und der Elektronentransfer zwischen der Elektrode und dem Kupfer im aktiven Zentrum der SOD erfolgte. Die Superoxiddismutase katalysiert die Zersetzung von Superoxidmolek{\"u}len durch Oxidation und durch Reduktion der Radikale. Somit sind beide Teilreaktionen von analytischem Interesse. Zyklovoltammetrisch konnte sowohl die Oxidation als auch die Reduktion des Radikals durch die immobilisierten Superoxiddismutase-Mutanten nachgewiesen werden. In amperometrischen Messanordnungen konnten beide Teilreaktionen zur analytischen Quantifizierung von Superoxidradikalen genutzt werden. Im positiven Potentialfenster wurde die Empfindlichkeit um einen Faktor von etwa 10 gegen{\"u}ber der Cytochrom c-Monoschichtelektrode verbessert.},
  subject      = {Biosensor},
  language  = {de}
}
@article{KrylovBeissenhirtzAdamzigetal.2004,
  author    = {Krylov, Andrey V. and Beissenhirtz, Moritz Karl and Adamzig, Holger and Scheller, Frieder W. and Lisdat, Fred},
  title     = {Thick-film electrodes for measurement of superoxide and hydrogen peroxide based on direct protein-electrode contacts},
  year      = {2004},
  abstract  = {Cytochrome c was immobilized on screen-printed thick-film gold electrodes by a self-assembly approach using mixed monolayers of mercaptoundecanoic acid and mercaptoundecanol. Cyclic voltammetry revealed quasi-reversible electrochemical behavior of the covalently fixed protein with a formal potential of +10 mV vs. Ag/AgCl. Polarized at +150 mV vs. Ag/AgCl the electrode was found to be sensitive to superoxide radicals in the range 300-1200 nmol L-1. Compared with metal needle electrodes sensitivity and reproducibility could be improved and combined with the easiness of preparation. This allows the fabrication of disposable sensors for nanomolar superoxide concentrations. By changing the electrode potential the sensor can be switched from response to superoxide radicals to hydrogen peroxide-another reactive oxygen species. H2O2 sensitivity can be provided in the range 10-1000 mumol L-1 which makes the electrode suitable for oxidative stress studies},
  language  = {en}
}