Nanobioelectrocatalysis Using Human Liver Microsomes and Cytochrome P450 Bactosomes: Pyrenyl-Nanocarbon Electrodes.

Human liver microsomes containing various drug-metabolizing cytochrome P450 (P450) enzymes, along with their NADPH-reductase bound to phospholipid membranes, were absorbed onto 1-pyrene butylamine pi-pi stacked with amine-functionalized multiwalled carbon nanotube-modified graphite electrodes. The interfaced microsomal biofilm demonstrated direct electrochemical communication with the underlying electrode surface and enhanced oxygen reduction electrocatalytic activity typical of heme enzymes such as P450s over the unmodified electrodes and nonenzymatic currents. Similar enhancements in currents were observed when the bioelectrodes were constructed with recombinant P450 2C9 (single isoform) expressed bactosomes. The designed liver microsomal and 2C9 bactosomal bioelectrodes successfully facilitated the electrocatalytic conversion of diclofenac, a drug candidate, into 4'-hydroxydiclofenac. The enzymatic electrocatalytic metabolite yield was several-fold greater on the modified electrodes than on the unmodified bulk graphite electrodes adsorbed with a microsomal or bactosomal film. The nonenzymatic metabolite production was less than the enzymatically catalyzed metabolite yield in the designed microsomal and bactosomal biofilm electrodes. To test the throughput potential of the designed biofilms, eight-electrode array configurations were tested with the microsomal and bactosomal biofilms toward electrochemical 4'-hydroxydiclofenac metabolite production from diclofenac. The stability of the designed microsomal bioelectrode was assessed using nonfaradaic impedance spectroscopy over 40 h, which indicated good stability.

Buckypaper-Bilirubin Oxidase Biointerface for Electrocatalytic Applications: Buckypaper Thickness.

Electrode materials play an important role on the electrocatalytic properties of immobilized biocatalysts. In this regard, achieving direct electronic communication between the electrode and redox sites of biocatalysts eliminates the need for additional electron transfer mediators for biocatalytic applications in fuel cells and other electrochemical energy devices. In order to increase electrocatalytic currents and power in fuel cells and metal-air batteries, conductive carbon-nanostructure-modified large surface area electrodes are quite useful. Among various electrode materials, freestanding buckypapers made from carbon nanotubes have gained significance as they do not require a solid support material and thus facilitate miniaturization. In this article, we present the effect of buckypaper (BP) thickness on the electrocatalytic properties of a bilirubin oxidase (BOD) enzyme. In this study, we prepared BPs of varying thicknesses ranging from 87 µm, the minimum thickness for suitable handling with a good stability in aqueous experiments, to 380 µm. BOD was adsorbed overnight onto the BPs, mostly via hydrophobic and π-π interactions since the nanotubes used were not chemically functionalized. Furthermore, intercalation of the BOD molecules onto the nanotubes' multicylindrical network is feasible. We determined that the lower range BP thickness (<220 µm) exhibited better sigmoidal shaped electrocatalytic currents than the higher BP-thickness-based BOD biofilms with larger capacitive currents. An oxygen reduction current density of up to 3 mA cm-2 is achieved without the use of any redox mediators or tedious electrode modifications. Using the 87 µm thick BP as the representative case, we were able to obtain distinguishable peaks for all Cu sites of BOD and assign their types, T1, T2, and T3, based on the peak-width at half-maximum in anaerobic cyclic voltammograms. Our peak assignment is further supported by the appearance of dual electrocatalytic oxygen reduction waves at a higher scan rate region (>10 mV s-1) in oxygen-saturated buffer, which is identified to be driven by an ∼3.5 times faster electron transfer rate from the buckypaper to the T2/T3 center than the T1 Cu site. Findings from this study are significant for designing enzyme electrocatalytic systems and biosensors in general and fuel cells and aerobic energy storage devices in particular, where the cathodic oxygen reduction current is often inadequate.

Electrochemical and Surface-Plasmon Correlation of a Serum-Autoantibody Immunoassay with Binding Insights: Graphenyl Surface versus Mercapto-Monolayer Surface.

We present here the correlation of picomolar affinities between surface-plasmon and electrochemical immunoassays for the binding of serum glutamic acid decarboxylase 65 autoantibody (GADA), a biomarker of type 1 diabetes (T1D), to its antigen GAD-65. Carboxylated (∼5.0%)-graphene-modified immunoassembly on a gold surface-plasmon chip or on an electrochemical array provided significantly larger binding affinity, higher sensitivity, and lower detection limits than a self-assembled monolayer surface of mercaptopropionic acid (MPA). Estimation of the relative surface -COOH groups by covalent tagging of an electroactive aminoferrocene showed that the graphenyl surface displayed a greater number of -COOH groups (9-fold) than the MPA surface. X-ray-photoelectron-spectroscopy analysis showed more C-O and C═O functionalities on the graphene-COOH surface than on the MPA surface. The graphene-COOH coating on gold exhibited ∼5.5-fold enhancement of plasmon signals compared with a similar coating on a plain glass surface. In summary, this article provides a quantitative comparison of carboxylated graphene with a mercapto-monolayer immunoassembly. Additionally, we propose that the binding-constant value can be useful as a quality-control checkpoint for reproducible and reliable production of large-scale biosensors for clinical bioassays.

Pyrenyl-carbon nanostructures for scalable enzyme electrocatalysis and biological fuel cells.

The objective of this article is to demonstrate the electrode geometric area-based scalability of pyrenyl-carbon nanostructure modification for enzyme electrocatalysis and fuel cell power output using hydrogenase anode and bilirubin oxidase cathode as the model system.

Combined covalent and noncovalent carboxylation of carbon nanotubes for sensitivity enhancement of clinical immunosensors.

We report here for the first time with quantitative details that the combination of pi-pi stacking of pyrenecarboxylic acid with chemically carboxylated multiwalled carbon nanotubes (MWNT-COOH) offers superior sensitivity compared to MWNT-COOH alone for serum insulin measurements and that this combination is broadly applicable for biosensors, drug delivery, and catalytic systems.