TY - JOUR
T1 - Electrochemically Induced Nanoscale Stirring Boosts Functional Immobilization of Flavocytochrome P450 BM3 on Nanoporous Gold Electrodes
AU - Hengge, Elisabeth
AU - Steyskal, Eva-Maria
AU - Dennig, Alexander
AU - Nachtnebel, Manfred
AU - Fitzek, Harald Matthias
AU - Würschum, Roland
AU - Nidetzky, Bernd
PY - 2024
Y1 - 2024
N2 - Enzyme-modified electrodes are core components of electrochemical biosensors for diagnostic and environmental analytics and have promising applications in bioelectrocatalysis. Despite huge research efforts spanning decades, design of enzyme electrodes for superior performance remains challenging. Nanoporous gold (npAu) represents advanced electrode material due to high surface-to-volume ratio, tunable porosity, and intrinsic redox activity, yet its coupling with enzyme catalysis is complex. Here, the study reports a flexible-modular approach to modify npAu with functional enzymes by combined material and protein engineering and use a tailored assortment of surface and in-solution methodologies for characterization. Self-assembled monolayer (SAM) of mercaptoethanesulfonic acid primes the npAu surface for electrostatic adsorption of the target enzyme (flavocytochrome P450 BM3; CYT102A1) that is specially equipped with a cationic protein module for directed binding to anionic surfaces. Modulation of the SAM surface charge is achieved by electrochemistry. The electrode-adsorbed enzyme retains well the activity (33%) and selectivity (complete) from in-solution. Electrochemically triggered nanoscale stirring in the internal porous network of npAu-SAM enhances speed (2.5-fold) and yield (3.0-fold) of the enzyme immobilization. Biocatalytic reaction is fueled from the electrode via regeneration of its reduced coenzyme (NADPH). Collectively, the study presents a modular design of npAu-based enzyme electrode that can support flexible bioelectrochemistry applications.
AB - Enzyme-modified electrodes are core components of electrochemical biosensors for diagnostic and environmental analytics and have promising applications in bioelectrocatalysis. Despite huge research efforts spanning decades, design of enzyme electrodes for superior performance remains challenging. Nanoporous gold (npAu) represents advanced electrode material due to high surface-to-volume ratio, tunable porosity, and intrinsic redox activity, yet its coupling with enzyme catalysis is complex. Here, the study reports a flexible-modular approach to modify npAu with functional enzymes by combined material and protein engineering and use a tailored assortment of surface and in-solution methodologies for characterization. Self-assembled monolayer (SAM) of mercaptoethanesulfonic acid primes the npAu surface for electrostatic adsorption of the target enzyme (flavocytochrome P450 BM3; CYT102A1) that is specially equipped with a cationic protein module for directed binding to anionic surfaces. Modulation of the SAM surface charge is achieved by electrochemistry. The electrode-adsorbed enzyme retains well the activity (33%) and selectivity (complete) from in-solution. Electrochemically triggered nanoscale stirring in the internal porous network of npAu-SAM enhances speed (2.5-fold) and yield (3.0-fold) of the enzyme immobilization. Biocatalytic reaction is fueled from the electrode via regeneration of its reduced coenzyme (NADPH). Collectively, the study presents a modular design of npAu-based enzyme electrode that can support flexible bioelectrochemistry applications.
KW - bioelectrochemistry and biosensing
KW - biointerface engineering
KW - nanoporous gold
KW - self-assembled monolayer
KW - enzyme electrode
KW - flavocytochrome P450
KW - charge-directed functional immobilization
UR - http://www.scopus.com/inward/record.url?scp=85204280923&partnerID=8YFLogxK
U2 - 10.1002/smtd.202400844
DO - 10.1002/smtd.202400844
M3 - Article
SN - 2366-9608
JO - Small Methods
JF - Small Methods
M1 - 2400844
ER -