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.

Bioelectroanalysis with nanoelectrode ensembles and arrays

“…15. NEEs functionalized with the DNA probe are then hybridized with the target ssDNA labelled with glucose oxidase (GOx) [89]. The occurrence of the hybridization event is detected by adding, to the supporting electrolyte, excess glucose as the substrate and the ferrocenyltrimethylammonium cation as suitable redox mediator.…”

Tetra(p‐tolyl)borate‐Functionalized Solvent Polymeric Membrane: A Facile and Sensitive Sensing Platform for Peroxidase and Peroxidase Mimetics

“…[19] By using the visible absorbance, [20] fluorescence, [21] chemiluminescence, [22] and redox behavior [23] of DAP, this reaction has been transduced by various optical and electrochemical techniques, and corresponding peroxidase detection protocols have been developed. According to the pK a of DAP (5.1), [24] it exists mainly in the electrically neutral form at the optimal pH of HRP (6.0 to 6.5).…”

Functionalized ensembles of nanoelectrodes as affinity biosensors for DNA hybridization detection

“…For the synthesis of ssDNA conjugated with GO x , a procedure previously described (Fruk et al, 2007) was used. Experimental details are reported in Supplementary information.…”

“…To enable the detection of the DNA hybridization event, thiolated complementary DNA (SHD1) was modified with glucose oxidase (GO x ) using sulfoSMCC methodology (Fruk et al, 2007) and subsequently hybridized to the capture acD1 immobilized on the PC (Scheme 1b). Glucose oxidase was chosen as the enzymatic label since it is readily available; moreover, the modification with single-stranded DNA is simple and it enables the amplification of the redox probe signal (Cass et al, 1984).…”