A layer-by-layer structure of enzyme multilayers composed of glucose oxidase (GOx) or lactate oxidase (LOx) and ascorbate oxidase (AOx) was prepared on the surface of a platinum electrode. The amperometric response to glucose or lactate was studied in the presence of ascorbic acid as a possible interference. An alternating and repeated deposition of avidin and the biotin-labeled enzymes resulted in the layer-by-layer structure of GOx/AOx and LOx/AOx multilayers. Optical and gravimetric measurements based on an ultraviolet-visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of monomolecular layers of the proteins. The GOx/AOx and LOx/AOx enzyme multilayers were useful to eliminate ascorbic acid interference in the glucose and lactate biosensors, because ascorbic acid can be converted to an electrochemically inert form, dehydroascorbic acid, before being oxidized directly on the Pt electrode. Thus, the GOx/AOx or LOx/AOx multilayer-modified biosensors can be used to determine the normal blood level of glucose (5 mM) and lactate (1 mM) in the presence of a physiological level of ascorbic acid (0.1 mM). The effects of the number of the AOx layers and geometry of the enzyme layers in the multilayer on the performance characteristics of the biosensors are discussed.

A platinum electrode was coated with polyelectrolyte multilayer (PEM) films to prepare an amperometric hydrogen peroxide sensor which can be used in the presence of possible interferences such as ascorbic acid, uric acid, and acetaminophen. The PEM films were prepared on the surface of a Pt disk electrode by an alternate deposition of polycation and polyanion from the aqueous solutions through electrostatic force of attraction. The Pt electrodes coated with a poly(allylamine)/poly(vinyl sulfate) or poly(allylamine)/poly(styrenesulfonate) film were used successfully for detecting H2O2 selectively in the presence of the possible interfering agents. It was suggested that H2O2 can diffuse into the PEM film smoothly while the ascorbic acid, uric acid, and acetaminophen cannot penetrate the film by a size exclusion mechanism. On the other hand, the electrodes coated with PEM films containing poly(ethyleneimine) or poly(diallyldimethylammonium chloride) were not useful for the selective determination of H2O2. The results were rationalized based on the different permeability of the films due to the different molecular density or packing in the PEM films. The PEM film-coated electrode was useful for constructing glucose biosensors by coupling with glucose oxidase.

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroidresistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

The surface of a gold (Au) electrode was modified with 4-mercaptophenylboronic acid (MPBA) and dithiobis(4-butyrylamino-m-phenylboronic acid) (DTBA-PBA) to prepare sugar-sensitive electrodes. MPBA and DTBA-PBA formed well-packed monomolecular layers on the Au electrode through a sulfur-Au bond. The MPBA- and DTBA-PBA-modified electrodes exhibited a nearly reversible cyclic voltammogram (CV) for the Fe(CN)(6)(3-)(/4-) ion in acidic solution, while the CVs were significantly attenuated in alkaline media as a result of addition of OH(-) ion to the boron atom to generate the negatively charged surface. In other words, the negatively charged monolayers blocked the surface of the Au electrode from the access of the Fe(CN)(6)(3-)(/4-) ion. The pK(a) values of the addition equilibrium of the OH(-) ion to the MPBA and DTBA-PBA monolayers were estimated to be 9.2 +/- 0.1 and 8.0 +/- 0.2, respectively, on the basis of the pH-dependent peak current (i(p)) in the CV of the Fe(CN)(6)(3-)(/4-) ion. On the other hand, in the presence of sugars, the addition of the OH(-) ion was accelerated by forming the phenylboronate esters of sugars on the surface of the monolayers. The pK(a) values for the MPBA and DTBA-PBA monolayers were 8.3 +/- 0.1 and 7.2 +/- 0.1, respectively, in the presence of 50 mM D-fructose. The MPBA- and DTBA-PBA-modified electrodes can be used for determining sugars on the basis of the change in i(p) of the Fe(CN)(6)(3-)(/4-) ion in the presence of sugars. The calibration curves useful for determining 1-100 mM D-glucose, D-mannose, and D-fructose were obtained.