title: DNA-directed immobilization of horseradish peroxidase-DNA conjugates on microelectrode arrays: towards electrochemical screening of enzyme libraries.
abstract: This work is aimed towards the generation of enzyme arrays on electrochemically active surfaces by taking advantage of the DNA-directed immobilization (DDI) technique. To this end, two different types of horseradish peroxidase (HRP)-DNA conjugates were prepared, either by covalent coupling with a bifunctional cross-linker or by the reconstitution of apo-HRP, that is, HRP lacking its prosthetic heme (protoporphyrin IX) group, with a covalently DNA-modified heme cofactor. Both conjugates were characterized in bulk and also subsequent to their immobilization on gold electrodes through specific DNA hybridization. Electrochemical measurements by using the phenolic mediator ortho-phenylendiamine indicated that, due to the high degree of conformational orientation, the apparent Michaelis-Menten constants of the reconstituted HRP conjugate were lower than those of the covalent conjugate. Due to the reversible nature of DDI, both conjugates could be readily removed from the electrode surface by simple washing and, subsequently, the electrodes could be reloaded with fresh enzymes, thereby restoring the initial amperometric-response activity. Moreover, the specific DNA hybridization allowed us to direct the two conjugates to distinct sites on a microelectrode array. Therefore, the self-assembly and regeneration capabilities of this approach should open the door to the generation of arrays of redox-enzyme devices for the screening of enzymes and their effectors.
authors: Universität Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Strasse 6, 44227 Dortmund, Germany.
authors: Ljiljana
authors: L
authors: Fruk
authors: Joachim
authors: J
authors: Müller
authors: Günther
authors: G
authors: Weber
authors: Arántzazu
authors: A
authors: Narváez
authors: Elena
authors: E
authors: Domínguez
authors: Christof M
authors: CM
authors: Niemeyer
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002853:Chromatography, Liquid; D004796:Clinical Enzyme Tests; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes; D004563:Electrochemistry; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006046:Gold / Q000737:chemistry; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008839:Microelectrodes; D010084:Oxidation-Reduction; D010636:Phenols / Q000737:chemistry; D010655:Phenylenediamines / Q000737:chemistry; D011524:Protoporphyrins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D015342:DNA Probes; D004800:Enzymes, Immobilized; D010636:Phenols; D010655:Phenylenediamines; D011524:Protoporphyrins; D006418:Heme; D006046:Gold; C034193:1,2-diaminobenzene; D004247:DNA; C028025:protoporphyrin IX; D006735:Horseradish Peroxidase