TY - JOUR A1 - Ida, Jeunice A1 - Chan, Soo Khim A1 - Glökler, Jörn A1 - Lim, Yee Ying A1 - Choong, Yee Siew A1 - Lim, Theam Soon T1 - G-Quadruplexes as An Alternative Recognition Element in Disease-Related Target Sensing JF - Molecules N2 - G-quadruplexes are made up of guanine-rich RNA and DNA sequences capable of forming noncanonical nucleic acid secondary structures. The base-specific sterical configuration of G-quadruplexes allows the stacked G-tetrads to bind certain planar molecules like hemin (iron (III)-protoporphyrin IX) to regulate enzymatic-like functions such as peroxidase-mimicking activity, hence the use of the term DNAzyme/RNAzyme. This ability has been widely touted as a suitable substitute to conventional enzymatic reporter systems in diagnostics. This review will provide a brief overview of the G-quadruplex architecture as well as the many forms of reporter systems ranging from absorbance to luminescence readouts in various platforms. Furthermore, some challenges and improvements that have been introduced to improve the application of G-quadruplex in diagnostics will be highlighted. As the field of diagnostics has evolved to apply different detection systems, the need for alternative reporter systems such as G-quadruplexes is also paramount. KW - G-quadruplex KW - DNAzyme KW - colorimetric KW - fluorescence KW - luminescence KW - diagnostics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-11438 SN - 1420-3049 VL - 24 IS - 6 ER - TY - JOUR A1 - Morlock, Sascha A1 - Subramanian, Senthil Kumar A1 - Zouni, Athina A1 - Lisdat, Fred T1 - Closing the green gap of photosystem I with synthetic fluorophores for enhanced photocurrent generation in photobiocathodes JF - Chemical Science N2 - One restriction for biohybrid photovoltaics is the limited conversion of green light by most natural photoactive components. The present study aims to fill the green gap of photosystem I (PSI) with covalently linked fluorophores, ATTO 590 and ATTO 532. Photobiocathodes are prepared by combining a 20 μm thick 3D indium tin oxide (ITO) structure with these constructs to enhance the photocurrent density compared to setups based on native PSI. To this end, two electron transfer mechanisms, with and without a mediator, are studied to evaluate differences in the behavior of the constructs. Wavelength-dependent measurements confirm the influence of the additional fluorophores on the photocurrent. The performance is significantly increased for all modifications compared to native PSI when cytochrome c is present as a redox-mediator. The photocurrent almost doubles from −32.5 to up to −60.9 μA cm−2. For mediator-less photobiocathodes, interestingly, drastic differences appear between the constructs made with various dyes. While the turnover frequency (TOF) is doubled to 10 e−/PSI/s for PSI-ATTO590 on the 3D ITO compared to the reference specimen, the photocurrents are slightly smaller since the PSI-ATTO590 coverage is low. In contrast, the PSI-ATTO532 construct performs exceptionally well. The TOF increases to 31 e−/PSI/s, and a photocurrent of −47.0 μA cm−2 is obtained. This current is a factor of 6 better than the reference made with native PSI in direct electron transfer mode and sets a new record for mediator-free photobioelectrodes combining 3D electrode structures and light-converting biocomponents. Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-17022 VL - 14 SP - 1696 EP - 1708 PB - Royal Society of Chemistry (RSC) ER - TY - JOUR A1 - Morlock, Sascha A1 - Subramanian, Senthil Kumar A1 - Zouni, Athina A1 - Lisdat, Fred T1 - Bio-inorganic hybrid structures for direct electron transfer to photosystem I in photobioelectrodes JF - Biosensors and Bioelectronics N2 - Synthetic materials can be combined with biological components in many ways. One example that provides scientists with multiple challenges is a photobioelectrode that converts sunlight into electrons in a biohybrid approach. In the present study several key parameters are evaluated concerning their influence on the direct electron transfer from a 3D indium tin oxide (ITO) electrode material to photosystem I (PSI) as a light-harvesting biomolecule. In contrast to previous investigations, no mediating molecule is added to shuttle the electrons to the luminal side of PSI. Thus, this setup is less complex than foregoing ones. The solution composition drastically influences the interaction of PSI with the ITO surface. Here, the application of higher buffer concentrations and the addition of salts are advantageous, whereas the nature of the buffer ions plays a minor role. The artificial electrode material's thickness is adjustable since a spin-coating procedure is used for preparation. With a 30 μm thick structure and immobilized PSI cathodic photocurrents up to 10.1 μA cm−2 are obtained at 100 mW cm−2 illumination intensity and an applied potential of −0.1V vs. Ag/AgCl. Over a period of three days the photobioelectrodes are illuminated for a total of 90 min and stored between the measurements at ambient temperature. The stability of the setup is noteworthy as still about 90% of the photocurrent is retained. The photocathode described here offers many positive features, including a high onset potential for the photocurrent starting sligthly above the redox potentail of P700, and applicability in a wide pH range from pH 5 to 8. Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-16770 SN - 1873-4235 VL - 204 PB - Elsevier ER - TY - JOUR A1 - Glökler, Jörn A1 - Lim, Theam Soon A1 - Ida, Jeunice A1 - Frohme, Marcus T1 - Isothermal amplifications – a comprehensive review on current methods JF - Critical Reviews in Biochemistry and Molecular Biology N2 - AbstractThe introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole. Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-15154 SP - 1 EP - 44 PB - Taylor & Francis ER - TY - JOUR A1 - Kallabis, Conrad A1 - Beyerlein, Peter A1 - Lisdat, Fred T1 - Quantitative determination of dopamine in the presence of interfering substances supported by machine learning tools JF - Bioelectrochemistry N2 - In the field of neuroscience as well as in the clinical setting, the neurotransmitter dopamine (DA) is an analyte which is important for research as well as medical purposes. There are plenty of methods available to measure dopamine quantitatively, with voltammetric ones such as differential pulse voltammetry (DPV) being among the most convenient and simple ones. However, dopamine often occurs, either naturally or because of the requirements of involved enzymatic systems, alongside substances that can influence the signal it produces upon electrochemical conversion. An example for such substances is the magnesium ion, which itself is not electrochemically active in the potential range needed for DA oxidation, but influences the dopamine signal. We have characterized the properties of DPV signals subject to the interaction between DA and Mg2+ and show that, although these properties are changing in a nonlinear fashion when both concentrations are varying, relatively simple linear mathematical models can be used to determine dopamine concentrations quantitatively in the presence of magnesium ions. The focus of this study is thus, the mathematical treatment of experimental data in order to overcome an analytical problem and not the investigation of the chemical background of DA-Mg2+ interaction. KW - dopamine KW - voltammetric detection KW - interference KW - magnesium ion KW - multiple linear regression KW - Bayesian linear regression KW - multivariate calibration models Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-19277 SN - 1567-5394 VL - 157 IS - 108667 ER - TY - JOUR A1 - Göbel, Gero A1 - Müller, Florian A1 - Talke, Anja A1 - Ahnert, Uwe A1 - Lisdat, Fred T1 - Qualitative and quantitative protease activity tests based on protein degradation in three-dimensional structures JF - Bioelectrochemistry N2 - The pattern of the activity of proteases is related to distinct physiological states of living organisms. Often activity changes of a certain protease can be assigned to a specific disease. Hence, they are useful biomarkers and a simple and fast determination method of their activity could be a valuable tool for the efficient monitoring of numerous diseases. Here, two different methods for the qualitative and quantitative determination of protease activity are demonstrated using the model system of proteinase K. The first test system is based on a protein-modified and colored 3D silica structure that changes color when exposed to the enzyme. This method has also been used for the detection of matrix metallo-protease 2 (MMP2) with gelatine as protease substrate on the plates. The second detection system uses the decrease in the voltammetric signal of a cytochrome c/DNA multilayer electrode after incubation with a protease to quantitatively determine its proteolytic activity. While activities down to 0.15 U/ml can be detected with the first method, the second one provides detection limits of about 0.03 U/ml (for proteinase K.) The functionality of both systems can be demonstrated and ways for further enhancement of sensitivity have been elucidated. KW - multilayer electrode KW - protease activity KW - proteinase K KW - silica gel Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-19294 SN - 1567-5394 VL - 160 ER - TY - JOUR A1 - Suzuki, Juliana Yumi A1 - Herkenhoff, Marcos Edgar A1 - Brödel, Oliver A1 - Cucick, Ana Clara Candelaria A1 - Frohme, Marcus A1 - Saad, Susana Marta Isay T1 - Exploring the potential of red pitaya pulp (Hylocererus sp.) as a plant-based matrix for probiotic delivery and effects on betacyanin content and flavoromics JF - Food Research International N2 - This study evaluated the potential of red pitaya pulp fermented with Lacticaseibacillus paracasei subsp. paracasei F-19 (F-19) as a base for probiotic products. Physicochemical parameters, sugar, betacyanin, and phenolic contents, and antioxidant activity were analyzed over 28 days at 4 °C and compared to a non-fermented pulp, and to a pulp fermented with Bifidobacterium animalis subsp. lactis BB-12 (BB-12). Volatile compounds were identified using HS-SPME/GC–MS. Probiotic viability during storage and survival through in vitro-simulated gastrointestinal tract (GIT) stress were assessed. Red pitaya pulp, rich in moisture (85.83 g/100 g), carbohydrates (11.65 g/100 g), and fibers (2.49 g/100 g), supported fermentation by both strains. F-19 and BB-12 lowered pH, with F-19 showing stronger acidification, and maintained high viability (8.85–8.90 log CFU/mL). Fermentation altered sugar profiles and produced unique volatile compounds, enhancing aroma and sensory attributes. F-19 generated 2-phenylethanol, a unique flavor compound, absent in BB-12. Phenolic content initially increased but antioxidant activity decreased during storage. Betacyanin remained stable for up to 14 days. Red pitaya improved F-19 viability through the simulated GIT, while BB-12 populations significantly decreased (p < 0.05). These results suggest red pitaya pulp is a promising plant-based matrix for F-19, offering protection during digestion and highlighting its potential as a functional food with enhanced bioactive compound bioavailability and sensory attributes. KW - Pitaya KW - probiotic KW - PMA-qPCR KW - betacyanin KW - prebiotic KW - metabolome Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-19843 VL - 192 PB - Elsevier ER - TY - JOUR A1 - Morlock, Sascha A1 - Subramanian, Senthil Kumar A1 - Zouni, Athina A1 - Lisdat, Fred T1 - Scalable Three-Dimensional Photobioelectrodes Made of Reduced Graphene Oxide Combined with Photosystem I JF - ACS Applied Materials & Interfaces N2 - Photobioelectrodes represent one of the examples where artificial materials are combined with biological entities to undertake semi-artificial photosynthesis. Here, an approach is described that uses reduced graphene oxide (rGO) as an electrode material. This classical 2D material is used to construct a three-dimensional structure by a template-based approach combined with a simple spin-coating process during preparation. Inspired by this novel material and photosystem I (PSI), a biophotovoltaic electrode is being designed and investigated. Both direct electron transfer to PSI and mediated electron transfer via cytochrome c from horse heart as redox protein can be confirmed. Electrode preparation and protein immobilization have been optimized. The performance can be upscaled by adjusting the thickness of the 3D electrode using different numbers of spin-coating steps during preparation. Thus, photocurrents up to ∼14 μA/cm2 are measured for 12 spin-coated layers of rGO corresponding to a turnover frequency of 30 e– PSI–1 s–1 and external quantum efficiency (EQE) of 0.07% at a thickness of about 15 μm. Operational stability has been analyzed for several days. Particularly, the performance at low illumination intensities is very promising (1.39 μA/cm2 at 0.1 mW/cm2 and −0.15 V vs Ag/AgCl; EQE 6.8%). KW - biophotovoltaics KW - reduced graphene oxide (rGO) KW - scalable template process KW - spin-coating KW - 3D electrode structure KW - carbon material Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-16760 VL - 13 IS - 9 SP - 11237 EP - 11246 PB - American Chemical Society (ACS) ER - TY - GEN A1 - Keil, Karolin A1 - Radke, Lars A1 - Tillich, Ulrich M. A1 - Frohme, Marcus T1 - Automatisierung des Bio-Plex Pro Analyseverfahrens T2 - Wissenschaftliche Beiträge 2015 N2 - Für den simultanen Nachweis mehrerer Analyten innerhalb einer Probe ist die Bead-basierte Multiplexanalytik ein häufig verwendetes Verfahren und wird beispielsweise zur Quantifizierung von Proteinen genutzt. Die aufwendige Durchführung der Assays soll durch Automatisierung einerseits dem Anwender abgenommen werden, während andererseits die Genauigkeit und Reproduzierbarkeit der Analyse gesteigert wird. Die Automatisierung des Bio-Plex Pro™ Assays ist mit dem Pipettierroboter Tecan Freedom EVO 200 umgesetzt worden. Es ist ein Skript mit der Software Freedom EVOware® entwickelt worden, welches die Probenvorbereitung des Assays vollständig übernimmt. Für einen Vergleich der manuellen und automatisierten Methode sind die humanen Zytokine IL-2, IL-4, IL-10, GM-CSF, IFN-γ und TNF-α in einer achtstufigen Standardverdünnungsreihe und in unterschiedlich konzentrierten Proben gemessen worden. Die Berechnung der Streuungen (Standardabweichung und Variationskoeffizient) der einzelnen Standardverdünnungsreihen sowie der Vergleich von gemessenen und erwarteten Konzentrationen der automatisierten und manuellen Methode zeigen, dass die Automatisierung neben der zeitlichen Optimierung auch die Genauigkeit und Reproduzierbarkeit der Analyse verbessert. N2 - Bead-based multpiplex analysis is frequently used for the simultaneous detection of multiple analytes within a sample. Such assays are commonly used to quantify proteins. The automation of the process relieves the user from the complex assay conductance and on the other hand increases the accuracy and reproducibility of the analysis. The automation of the Bio-Plex Pro™ assay has been successfully implemented with the pipetting-robot Tecan Freedom EVO 200. A script has been developed, using the Freedom EVOware® which has the ability to perform the complete assay procedure. For a comparison between manual and automated methods, the human cytokines IL-2, IL-4, IL-10, GM-CSF, IFN-γ and TNF-α were analysed by an eight-point standard dilution series and with samples of different concentrations. The calculation of the variances (standard deviation and coefficient of variation) of the single standard dilution series as well as the comparison of the observed and expected concentrations of the manual or automated method show the higher precision and reproducibility of the automated process in addition to its time-saving nature. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-3524 SN - 0949-8214 VL - 19 SP - 15 EP - 19 ER - TY - GEN A1 - Bütow, Sandra M. A1 - Pänke, Oliver A1 - Kafka, Jan A1 - Lisdat, Fred T1 - Goldchipelektroden zur elektrochemischen DNA-Detektion T2 - Wissenschaftliche Beiträge 2008 N2 - Im folgenden Artikel werden einfache DNA-Sensoren vorgestellt, mit deren Hilfe es durch voltammetrische und impedimetrische Messmethoden möglich ist, schnell, sensitiv und kostengünstig Einzelstrang-DNA (ssDNA) nachzuweisen. Beide Messprinzipien lassen neben der spezifischen Detektion auch die Quantifizierung von DNA-Sequenzen sowie den Nachweis von einzelnen Basenfehlpaarungen innerhalb dieser Sequenzen zu. Fänger- DNA wurde zu diesem Zweck mit dem 5’-Ende auf einer Goldoberfläche immobilisiert. Die Hybridisierung mit einem Methylenblau (MB) markierten oder unmarkierten Probenstrang konnte dann mit Hilfe der Differenzpulsvoltammetrie DPV oder der elektrochemischen Impedanzspektroskopie nachgewiesen werden. Die voltammetrische Quantifizierung erfolgte in einem direkten und kompetitiven Ansatz, mit einem Detektionslimit von 30 nM bzw. 3 nM (bei Einsatz von 0,1 μM Kompetitor- DNA). Das Detektionslimit beim impedimetrischen Nachweis lag bei 100 nM DNA. Die hier vorgestellten Sensoren sind zum einen regenerierbar und können zum anderen über einen Zeitraum von zwei Monaten gelagert werden. N2 - This paper describes simplistic electrochemical DNA sensors for the sensitive, more rapid and cost effective detection of single-stranded DNA (ssDNA). The used methodes are the voltammetric detection and the detection by impedance spectroscopy. Beside the specific detection of ssDNA both techniques allow quantification of DNA and verification of single base pair mismatches within the sequences. Therefore a single-stranded 18mer oligonucleotide (DNA) was immobilised via a thiol-linker on gold film electrodes and served as probe DNA. Hybridisation was detected by means of the electroactive redox-marker methylene blue (MB), which was covalently bound to the 5’-end of the target DNA, by differential pulse voltammetry (DPV) or by measuring the differences in the charge transfer resistance (RCT) by electrochemical impedance spectroscopy (EIS) using non-labelled ssDNA targets. MB-labelled target DNA was verified down to 30 nM DNA. By application of a competitive binding assay non-labelled DNA was detected down to 3 nM DNA. The detection limit for impedimetric DNA sensors was 100 nM ssDNA. The sensors were found to be reusable and could be stored for more than two month at 4 °C without significant loss in their activity. Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus-653 SN - 0949-8214 VL - 13 SP - 25 EP - 34 ER - TY - GEN A1 - Radke, Lars A1 - López-Hemmerling, Diana A1 - Lubitz, Annika A1 - Giese, Christoph A1 - Wildenauer, Franz-Xaver A1 - Frohme, Marcus T1 - Etablierung verschiedener Bead-basierter Multiplexmethoden mit einem Suspensions Array-System für molekulardiagnostische Zwecke T2 - Wissenschaftliche Beiträge 2009/2010 N2 - Die simultane Bestimmung mehrerer Analyten und die Erstellung komplexer Parameterprofile erlangt immer größere Bedeutung in der heutigen Labordiagnostik. Die Bead-basierte Multiplexanalytik bietet hier eine flexible, schnelle und einfache Methode zur Erstellung individueller Analysen. Aufgrund der Vielzahl von Anwendungsmöglichkeiten, die diese moderne Nachweismethode im Bereich molekularbiologischer Fragestellungen bietet, ist die Etablierung der Beadbasierten Multiplexanalytik im Labor für Molekularbiologie und funktionelle Genomik der Technischen Hochschule Wildau von großem Nutzen. Zur Einarbeitung in das Testsystem wurden die Konzentrationen der Zytokine IL-2, IL-4, IFN-γ und TNF-α in Zellkulturüberständen mit kommerziellen Fertigsystemen gemessen und mit mRNA-Expressionsraten der gleichen Proben verglichen. Des Weiteren wurde ein Testsystem zum Nachweis von humanen Antikörpern der Klassen IgG und IgM sowie deren antigen-spezifischer Anteil in Zellkulturüberständen entwickelt. Außerdem konnte durch die erfolgreiche Detektion von DNA-gekoppelten Beads mittels markierter Oligonukleotidsequenz die Kopplung und die Anwendbarkeit der Methode auf Bindungsexperimente mit Nukleotidsequenzen gezeigt werden. N2 - The simultaneous determination of multiple analytes and the generation of complex parameter profiles gains increasing importance in today’s laboratory diagnostics. The bead-based multiplex assay is offering a flexible, rapid and easy to handle method for the creation of individual analyses. Because of the multitude of applications this modern detection system offers, the establishment of the bead-based multiplex technique is of great benefit for the Laboratory for Molecular Biology and Functional Genomics of the Technical University of Applied Sciences Wildau. To familiarize with the testing system the concentrations of the cytokines IL-2, IL-4, IFN-γ and TNF-α in cell culture supernatant were analysed with commercially available assays and compared with mRNA expression ratios of the same samples. Furthermore a custom testing system was developed for the detection of human IgG and IgM antibodies and also antigen specific antibodies in cell culture supernatants. The assignability of the method to binding experiments with nucleotide sequences could be shown by the successful detection of DNA-modified beads by conjugated oligonucleotides. Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus-802 SN - 0949-8214 VL - 14 SP - 6 EP - 12 ER - TY - GEN A1 - Fischer, Susanne T1 - Experimentelle Therapieentwicklung für die akute lymphatische Leukämie T2 - Wissenschaftliche Beiträge 2013 N2 - Bei Leukämie handelt es sich um eine Entartung von Vorläuferzellen der weißen Blutzellen (Leukozyten). Die hier betrachtete Form dieser Erkrankung ist die akute lymphatische Leukämie(ALL ), bei der vor allem Rückfallpatienten eine schlechte Diagnose haben. Demnach wird nach neuen Therapien gesucht, die neue Angriffspunkte für die Behandlung der ALL bieten. Die getestete Substanz 5-Azacytidin hat zwei therapeutische Wirkmechanismen. Zum einen das Hervorrufen des selbstinduzierte Zelltod (Apoptose) und zum anderen einen epigenetischen Effekt. Der epigenetische Effekt bewirkt eine Veränderung der Genregulation (Veränderung der Genaktivität) ohne die DNA - Sequenz zu beeinflussen. Dies kann mithilfe eines Screenings (Micoarraytechnologie) in behandelten und unbehandelten Proben verglichen werden. Da das Screening sehr global ist, erfolgt für eine Auswahl therapeutisch relevanter Gene eine Verifizierung mittels einer molekularbiologischen Methode (qRT -PCR ). Dabei wurden Gene mit erhöhter Aktivität bestätigt, MA EL; TM EM56; IL 1B; BCL 6; MS4A1 und FLT 3. Für jedes dieser Gene konnte ein positiver Nutzen für die Therapie einer ALL diskutiert werden. Für FLT 3 erfolgte sogar eine positive Kombination mit einer anderen therapeutischen Substanz. Der therapeutische Einsatz des 5-Azacytidins kann zukünftig eine wichtige Therapieoption für Patienten mit ALL darstellen. Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-3146 SN - 0949-8214 VL - 17 SP - 13 EP - 16 ER - TY - GEN A1 - Tanne, Christoph K. A1 - Göbel, Gero A1 - Lisdat, Fred T1 - Entwicklung einer Glucosedehydrogenase-basierten Anode und deren Anwendung in einer Glucose/O2-Biobrennstoffzelle T2 - Wissenschaftliche Beiträge 2011 N2 - Unter Verwendung von mehrwandigen Kohlenstoffnanoröhren wurde in dieser Studie eine neuartige Anode zum Einsatz in Biobrennstoffzellen entwickelt. Dazu wurde das rekombinante Enzym Pyrrolochinolinchinon(PQQ)- abhängige Glucosedehydrogenase kovalent an eine aus PQQ bestehenden Zwischenschicht gekoppelt, welche zuvor an die Kohlenstoffnanoröhren adsorbiert war. Die Nanoröhren wurden aufgrund ihrer Thiolmodifikation chemisorptiv auf einer Goldelektrode gebunden. In glucosehaltiger Lösung konnte der Start eines katalytischen Stroms bei einem Potential von -80 mV vs. Ag/AgCl (1 MKCl) beobachtet werden. Unter Substratsättigung wurden Stromdichten im Bereich von 170 bis 200 μA/cm2 gemessen. Dieses System basiert auf einem mediatorvermittelten Elektronentransfer. Die entwickelte (PQQ)-GDH-MWCNT-Elektrode wurde mit einer MWCNT-modifizierten Elektrode kombiniert, bei der Bilirubinoxidase (BOD) als Biokatalysator fungiert. Daraus resultierte eine membranfreie Biobrennstoffzelle mit einem leichgewichtspotential von 600 mV und Leistungsdichten im Bereich von 20-25 μW/cm2. N2 - In this study a biofuel cell anode is developed on the basis of multi-walled carbon nanotubes (MWCNTs). Recombinant pyrroloquinoline quinone (PQQ) dependent glucose dehydrogenase is covalently coupled to a PQQ-layer which is adsorbed onto thiolmodified MWCNTs. The MWCNTs are chemisorbed to a gold electrode. In the presence of glucose a catalytic current starts at a potential of -80 mV vs. Ag/AgCl, 1 M KCl. Under substrate saturation current densities of 170 to 200 μA/cm2 can be achieved. The operation is based on mediated electron transfer of the enzyme. This (PQQ)-GDH-MWCNT-electrode is combined with a MWCNT-modifi ed electrode to which bilirubin oxidase (BOD) is covalently coupled. The resulting membrane-free biofuel cell has an open cell potential of 600 mV and can achieve power densities in the range of 20-25 μW/cm2. Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus-1039 SN - 0949-8214 VL - 15 SP - 13 EP - 21 ER - TY - JOUR A1 - Fischbach, Jens A1 - Loh, Qiuting A1 - Bier, Frank Fabian A1 - Lim, Theam Soon A1 - Frohme, Marcus A1 - Glökler, Jörn T1 - Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method JF - Scientific Reports N2 - We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate. KW - biochemical assay KW - fluorescent dye KW - screening Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9248 SN - 2045-2322 VL - 7 ER - TY - GEN A1 - Riedel, Marc A1 - Kartchemnik, Julia A1 - Schöning, Michael J. A1 - Lisdat, Fred T1 - Impedimetrischer DNA Nachweis - Schritte in Richtung sensorischer Anwendung T2 - Wissenschaftliche Beiträge 2015 N2 - Diese Studie beschreibt einen labelfreien impedimetrischen Sensor auf der Grundlage von kurzen einzelsträngigen DNA-Erkennungselementen für den Nachweis von Hybridisierungsereignissen. Der Fokus der Arbeit liegt auf der Aufklärung des Einflusses der Ziel-DNA-Länge und der Erkennungssequenzposition auf die sensorische Leistungsfähigkeit. Die impedimetrischen Messungen werden in Anwesenheit des Redoxsystems Kaliumhexacyanoferrat (II/III) durchgeführt und zeigen einen Anstieg des Durchtrittswiderstandes nach der Hybridisierung mit komplementärer Ziel-DNA mit einer Nachweisgrenze im unteren nanomolaren Bereich. Nach der Hybridisierung kann die Regeneration des Sensors mit deionisiertem Wasser durch die Einstellung effektiver Konvektionsbedingungen erreicht werden und ermöglicht somit eine Wiederverwendbarkeit des Sensors. Untersuchungen zu längeren Ziel-DNA-Strängen mit einem zur Lösung exponierten Überhang demonstrieren die Anwendbarkeit des impedimetrischen Nachweises für längere Sequenzen. Allerdings resultiert eine zunehmende Überhanglänge in einer verringerten Durchtrittswiderstandsänderung. Um die Impedanzänderung für längere Ziel-DNA zu erhöhen, wird die Erkennungssequenzposition verändert, sodass ein kleiner Überhang zur Elektrode ausgerichtet ist. Die Ergebnisse legen nahe, dass DNA in direkter Nähe zur Elektrode einen größeren Einfluss auf das impedimetrische Signal besitzt als weiter entfernte DNA. N2 - This study describes a label-free impedimetric sensor based on short ssDNA recognition elements for the detection of hybridisation events. We concentrate on the elucidation of the influence of target length and recognition sequence position on the sensorial performance. The impedimetric measurements are performed in the presence of the redox system ferri-/ferrocyanide and show an increase in charge transfer resistance upon hybridisation of complementary ssDNA in the nanomolar range. After hybridisation, a sensor regeneration can be achieved with deionised water by adjustment of effective convection conditions, ensuring sensor reusability. By investigation of longer targets with overhangs exposed to the solution, we can demonstrate applicability of the impedimetric detection for longer ssDNA. However, a decreasing charge transfer resistance change (ΔRct) is found by extending the overhang. As a strategy to increase the impedance change for longer target strands, the position of the recognition sequence can be designed in a way that a small overhang is exposed to the electrode surface. These results suggest that DNA near the electrode possesses a larger impact on the impedimetric signal than DNA further away. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-3538 SN - 0949-8214 VL - 19 SP - 21 EP - 28 ER - TY - JOUR A1 - Kagel, Heike A1 - Frohme, Marcus A1 - Glökler, Jörn T1 - Photoacids in biochemical applications JF - Journal of Cellular Biotechnology N2 - BACKGROUND: After excitation with light photoacids can change the pH in a solution by release of a proton. They have been used mostly for excited state proton transfer studies. In this review the general functionality and mechanisms and the subdivision of photoacids is explained. STATE OF THE ART: Different uses of photoacids are described, covering a wide range of various biochemical topics, focusing on biochemical applications. Examples for the introduced subdivisions are covered. CONCLUSIONS AND OUTLOOK: The areas in which photoacids can be employed are diverse. Photoacids have a promising future in biotechnology and biochemistry and should be considered for upcoming applications, especially in non-invasive control of biochemical reactions. Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-16123 SN - 2352-3697 VL - 4 IS - 1-2 SP - 23 EP - 30 PB - IOS Press ER - TY - GEN A1 - Standfuß, Christoph A1 - Klein, Andreas A1 - Pospisil, Heike T1 - Einfluss von Kopienzahlvariationen auf die Tumorentwicklung T2 - Wissenschaftliche Beiträge 2013 N2 - Tumorentstehung ist ein Prozess, bei dem die Abläufe innerhalb der Zelle schrittweise verändert werden. Die vielfältigen Interaktionen bei der Tumorentstehung sind jedoch bislang nicht vollständig erforscht. Bisher wurden vorwiegend Genexpressionsanalysen genutzt, die jedoch nur eine Zeitaufnahme aller Genexpressionen innerhalb der Zelle darstellen und somit allein nicht ausreichend zur Charakterisierung eines Tumors. Wir haben mithilfe von Affymetrix Mouse Diversity Genotyping Microarrays Mausbrustdrüsengewebe entsprechend unserem Dreistufen-Mausmodell analysiert und die Kopienzahländerungen berechnet. Wir fanden eine zunehmende stufenweise Änderung von den transgenen zu den Tumorproben. Die Berechnung von chromosomalen Segmenten mit gleicher Kopienzahl zeigte deutliche Fragmentmuster. Unsere Analysen zeigen, dass die Tumorentstehung ein schrittweiser Prozess ist, der sowohl durch Amplifikationen als auch Deletionen chromosomaler Abschnitte definiert ist. Wir fanden charakteristisch konservierte Fragmentierungsmuster und individuelle Unterschiede welche zur Tumorgenese beitragen. Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-3435 SN - 0949-8214 VL - 17 SP - 27 EP - 30 ER - TY - GEN A1 - Gladisch, Johannes A1 - Sarauli, David A1 - Schäfer, Daniel A1 - Dietzel, Birgit A1 - Schulz, Burkhard A1 - Lisdat, Fred T1 - Elektrogesponnene Polymerfasern als neuartiges Material für die Bioelektrokatalyse des Enzyms Pyrrolochinolinchinon-abhängige Glucosedehydrogenase T2 - Wissenschaftliche Beiträge 2016 N2 - Es wurde ein dreidimensionales Polymerfasernetzwerk aufgebaut, charakterisiert und anschließend daran das Enzym Pyrrolochinolinchinon-abhängige Glukosedehydrogenase (PQQ)GDH gebunden. Das Polymerfasernetzwerk wurde durch Elektrospinnen einer Mischung des Polymers Polyacrylnitril und verschiedener leitfähiger Polymere der Polyanilin-Familie auf Indium-Zinn-Oxid-Elektroden aufgebracht. Die so hergestellten Fasermatten erwiesen sich bei mikroskopischen Untersuchungen gleichförmig präpariert und die Faserdurchmesser bewegten sich im Bereich weniger hundert Nanometer. Das Redoxpaar Kaliumhexacyanoferrat (II/III) zeigte an diesen Polymer-Elektrodenstrukturen eine quasi-reversible Elektrochemie. Bei weitergehenden Untersuchungen an den enzymmodifizierten Fasern ((PQQ)GDH) konnten unter Substratzugabe (Glukose) bioelektrokatalytische Ströme nachgewiesen werden. Das Fasernetzwerk fungiert hier nicht nur als Immobilisierungsmatrix, sondern als auch als Teil des Signalwandlers. N2 - A three-dimensional polymeric electrode structure was developed, characterized and subsequently coupled with the enzyme pyrroloquinoline quinone-dependent Glucosedehydrogenase (PQQ)GDH. The polymeric fiber network is produced by means of electrospinning from mixtures of polyacrylonitrile (PAN) and three different sulfonated poylanilines on top of ITO electrodes. The mats are uniform in their overall appearance; average diameters of the fibers produced are in the range of a few hundred nanometers. These polymeric structures can be shown to allow electrochemical conversions as verified with the ferri-/ferrocyanide redox couple. In addition, application in bioelectrocatalysis can be demonstrated. For two of three selected blends of PAN with sulfonated polyanillines, a well-defined bioelectrochemical response is obtained upon covalent fixation of PQQ-GDH to the fiber network and subsequent addition of substrate glucose. The electrospun matrix does not only act here as an immobilization support, but at the same time as a transducing element. Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-5293 SN - 0949-8214 VL - 20 SP - 15 EP - 21 ER - TY - GEN A1 - Seifert, Felix A1 - Pospisil, Heike T1 - Reannotation des Maize oligonucleotide arrays T2 - Wissenschaftliche Beiträge 2014 N2 - Die Microarray-Technologie hat sich zu einem etablierten Ansatz der Hochdurchsatz-Genexpressionsanalyse entwickelt. Das „maize oligonucleotide array“ (maizearray) ist eine der wenigen Microarray-Plattformen, welche für die genomweite Genexpressionsanalyse von Mais (Zea mays L.) erzeugt wurden. Die Sonden wurden basierend auf ESTs (expressed sequence tags) generiert. Mittlerweile ist die Genomsequenz von Mais verfügbar und ermöglicht eine genauere Annotation dieser Sonden. In dieser Arbeit wurden die Genompositionen aller Sonden und basierend darauf die zugrunde liegenden Gene sowie deren funktionelle Annotation bestimmt. Durch die Analyse konnten Redundanzen und nicht eindeutig bindende Sonden aufgedeckt und gleichzeitig die Zahl der Gene mit funktioneller Annotation verdoppelt werden. Unsere Reannotation wird funktionelle Analysen bereits existierender und zukünftiger Datensätze stark verbessern. Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-3315 SN - 0949-8214 VL - 18 SP - 33 EP - 35 ER - TY - GEN A1 - Stieger, Kai Ralf A1 - Feifel, Sven Christian A1 - Lokstein, Heiko A1 - Hejazi, Mahdi A1 - Zouni, Athina A1 - Lisdat, Fred T1 - Biohybride Architekturen für eine effiziente Umwandlung von Licht in elektrische Energie durch Integration von Photosystem I in skalierbare mesoporöse 3D Elektroden T2 - Wissenschaftliche Beiträge 2018 N2 - Die Kombination von fortschrittlichen Materialien und kontrolliertem Oberflächendesign mit komplexen Proteinen aus der natürlichen Photosynthese ist derzeit eines der Hauptthemen bei der Entwicklung von Biohybridsystemen und Biophotovoltaik. In dieser Studie werden transparente makroporöse Indium-Zinn-Oxid-(μITO-) Elektroden mit dem trimeren Superkomplex Photosystem I (PSI) aus dem Cyanobakterium Thermosynechococcus elongatus sowie dem kleinen Redoxprotein Cytochrom c (Cyt c) kombiniert, um neuartige und effiziente biohybride Photokathoden herzustellen. Mit diesen bis zu 40 μm hohen 3D-Strukturen können beide Proteine in einer annähernden Monolage abgeschieden werden und die elektrische Kommunikation mit der Elektrode kann erzielt werden. Der generierte Photostrom folgt dabei linear der kontrollierbaren Schichtdicke der μITO-Elektrode, wobei Stromdichten von bis zu 150 μA cm –2 erhalten werden. Eine effiziente elektrische Kopplung der Proteine kann durch die hohe interne Quanteneffizienz von 30 % gezeigt werden. N2 - The combination of advanced materials and a controlled surface design with complex proteins from natural photosynthesis is currently one of the major topics in the development of biohybrid systems and biophotovoltaic devices. In this study, transparent macroporous indium tin oxide (μITO) electrodes are combined with the trimeric supercomplex, Photosystem I (PSI) from the cyanobacterium Thermosynechococcus elongatus, and the small redox protein cytochrome c (cyt c) to create novel and efficient biohybrid photocathodes. With these 3D structures up to 40 μm in size, both proteins can be deposited in a monolayer fashion and electrical communication with the electrode can be established. The generated photocurrent linearly follows the controllable layer thickness of the μITO electrode up to 40 μm, whereby current densities of up to 150 μA cm –2 are obtained. An efficient electrical coupling of the proteins can be demonstrated by the high internal quantum efficiency of 30 %. Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-10220 SN - 0949-8214 VL - 22 SP - 17 EP - 24 ER - TY - CHAP A1 - Riedel, Marc A1 - Göbel, Gero A1 - Parak, Wolfgang J. A1 - Lisdat, Fred T1 - Light-addressable amperometric electrodes for enzyme sensors based on direct quantum dot-electrode contacts N2 - Quantum dots allow the generation of charge carriers upon illumination. When these particles are attached to an electrode a photocurrent can be generated. This allows their use as a light-switchable layer on the surface. The QDs can not only exchange electronics with the electrode, but can also interact with donor or acceptor compounds in solution providing access to the construction of signal chains starting from an analytic molecule. The magnitude and the direction of the photocurrent depend on several factors such as electrode polarization, solution pH and composition. These defined dependencies have been evaluated with respect to the combination of QD-electrodes with enzyme reactions for sensorial purpose. CdSe/ZnS-QD-modified electrodes can be used to follow enzymatic reactions in solution based on the oxygen sensitivity. In order to develop a photoelectrochemical biosensor, e.g. glucose oxidase is immobilized on the CdSe/ZnS-electrode. One immobilization strategy applies the layer-by-layer-technique of GOD and a polyelectrolyte. Photocurrent measurements of such a sensor show a clear concentration dependent behavior. The principle of combing QD oxidase. The sensitivity of quantum dot electrodes can be influenced by additional nanoparticles, but also by multiple layers of the QDs. In another direction of research it can be influenced by additional nanoparticles, but also by multiple layers of the QDs. In another direction of research it can be demonstrated that direct electron transfer from excited quantum dots can be achieved with the redox protein cytochrome c. This allows the detection of the protein, but also interaction partners such as a enzymes or superoxide. KW - quantum dot electrode KW - enzyme sensor KW - signal chain KW - photobioelectrochemistry KW - light adressability Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-15266 SP - 195 EP - 200 PB - Society of Photo-Optical Instrumentation Engineers (SPIE) ER - TY - JOUR A1 - Kölsch, Adrian A1 - Hejazi, Mahdi A1 - Stieger, Kai Ralf A1 - Feifel, Sven Christian A1 - Kern, Jan F. A1 - Müh, Frank A1 - Lisdat, Fred A1 - Lokstein, Heiko A1 - Zouni, Athina T1 - Insights into the binding behavior of native and non-native cytochromes to photosystem I from Thermosynechococcus elongatus JF - Journal of Biological Chemistry N2 - The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c6 and cyt c from horse heart (cyt cHH) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Although PS I has a higher affinity for cyt cHH than for cyt c6, the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations revealed the existence of unspecific binding sites for cyt cHH besides one specific binding site close to P700. Binding to PS I was found to be the same for reduced and oxidized cyt cHH. Based on this information, suitable conditions for cocrystallization of cyt cHH with PS I were found, resulting in crystals with a PS I:cyt cHH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt cHH cannot be identified in the electron density map because of unspecific binding sites and/or high flexibility at the specific binding site. Modeling the binding of cyt c6 to PS I revealed a specific binding site where the distance and orientation of cyt c6 relative to P700 are comparable with cyt c2 from purple bacteria relative to P870. This work provides new insights into the binding modes of different cytochromes to PS I, thus facilitating steps toward solving the PS I–cyt c costructure and a more detailed understanding of natural electron transport processes. Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-12780 SN - 1083-351X VL - 293 IS - 23 SP - 9090 EP - 9100 ER - TY - GEN A1 - Wettstein, Christoph A1 - Kano, Kenji A1 - Schäfer, Daniel A1 - Wollenberger, Ulla A1 - Lisdat, Fred T1 - Die Flavin-abhängige Fruktosedehydrogenase und Cytochrom c: Elektronentransfer und Sensorstrategien T2 - Wissenschaftliche Beiträge 2017 N2 - Die hier durchgeführten Untersuchungen ermöglichen ein besseres Verständnis der Elektronentransferprozesse zwischen der Flavin-abhängigen Fruktosedehydrogenase (FDH) und dem Redoxprotein Cytochrom c (Cyt c). Dies liefert im Hinblick auf sensorische Anwendungen wichtige Erkenntnisse für vorteilhafte Sensorarchitekturen und deren Messbedingungen. Es wurden zwei unterschiedliche pH-Optima für die Redoxreaktion der beiden Proteine untereinander entdeckt. Die Reaktion wurde im Weiteren mit Elektroden kombiniert und so eine Fruktose-abhängige Stromantwort detektiert. Darüber hinaus konnten definierte dreidimensionale Sensorarchitekturen der beiden Proteine, mit Hilfe von DNA als zusätzlichen biologischen Baustein erzeugt und für die Sensorik genutzt werden. N2 - With regard to sensor application, flavin-dependent fructose dehydrogenase (FDH) and the redox protein cytochrome c (cyt c) were examined for a better understanding of electron transfer (ET) pathways. First, two different pH optima could be determined for the reaction of both proteins. A fructose-dependent signal can be achieved on a cyt c electrode with adsorbed FDH as well as with the enzyme in solution. Furthermore, fructose sensors could be produced using a defined arrangement of cyt c and FDH by the layer-by-layer technique with DNA as a second building block. Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9379 SN - 0949-8214 VL - 21 SP - 13 EP - 21 ER -