Ioanis Katakis

Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors

The developments in the techniques of NADH catalytic oxidation relevant for incorporation in amperometric biosensors with dehydrogenase enzymes are reviewed with special emphasis in the years following 1990. The review stresses the direct electro-catalytic methods of NAD+ recycling as opposed to enzymatic regeneration of the coenzyme. These developments are viewed and evaluated from a mechanistic perspective of recycling of NADH to enzymatically active NAD+, and from the point of view of development of technologically useful reagentless dehydrogenase biosensors. An effort is made to propose a method for the standardization of evaluation of new mediating and direct coenzyme recycling schemes. A perspective is given for the requirements that have to be met for successful biosensor development incorporating dehydrogenase enzymes that open the analytical possibilities to a number of new analytes. The intrinsic limitations of the system are finally discussed and a view of the future of the field is presented.

“Wiring” of glucose oxidase within a hydrogel made with polyvinyl imidazole complexed with [(Os-4,4′-dimethoxy-2,2′-bipyridine)Cl]+/2+1

Abstract Glucose electrodes based on hydrogels made by crosslinking glucose oxidase and the redox polymer formed upon complexing polyvinyl imidazole (PVI) with [Os(dmo-bpy) 2 Cl] +/2+ (dmo-bpy= 4,4′-dimethoxy-2,2′-bipyridine) on vitreous carbon electrode surfaces were investigated. The redox potential of the hydrogels was −69 mV (SCE) and their glucose electrooxidation current reached a plateau at +50 mV (SCE). Urate and acetaminophen were not electrooxidized at this potential at rates that would interfere with the glucose assays. At 32 mM glucose concentration switching of the atmosphere from argon to O 2 reduced the current only by 5%.

‘Wiring’ of glucose oxidase and lactate oxidase within a hydrogel made with poly(vinyl pyridine) complexed with [Os(4,4′-dimethoxy-2,2′-bipyridine)2Cl]+/2+

Glucose and lactate electrodes based on hydrogels made by crosslinking glucose oxidase and the redox polymer formed upon complexing poly(vinyl pyridine)(PVP) with [Os(dmo-bpy)2Cl]+/2+(dmo-bpy = 4,4′-dimethoxy-2,2′-bipyridine) on vitreous carbon electrode surfaces have been investigated. The redox potential of the hydrogels was +35 mV vs. SCE and their glucose electrooxidation current reached a plateau at +150 mV vs. SCE. Urate and acetaminophen were not electrooxidized at this potential at rates that would interfere with the glucose and lactate assays. At a glucose concentration of 1 mM, the addition of 0.1 mM ascorbate increased the current by 17%. At 5 mM glucose, switching the atmosphere from argon to oxygen reduced the currents by 11%.

Reagentless biosensors based on self-deposited redox polyelectrolyte-oxidoreductases architectures

Reagentless fructose and alcohol biosensors have been produced with a versatile enzyme immobilisation technique which mimics natural interactions and flexibility of living systems. The electrode architecture is built up on electrostatic interactions by the sequential adsorption of redox polyelectrolytes and redox enzymes giving rise to the efficient transformation of substrate fluxes into electrocatalytic currents. All investigated multilayer structures were self-deposited on 3-mercapto-1-propanesulfonic acid monolayers self-assembled on gold electrodes. Fructose dehydrogenase, horseradish peroxidase (HRP) and the couple HRP-alcohol oxidase were electrochemically connected with a cationic poly[(vinylpyridine)Os(bpy)2Cl] redox polymer (RP) interface in a layer-by-layer self-deposited architecture. The dependence of the distance on the electrochemical response of this interface was also studied showing a clear decrease in the Faradaic current when the distance to the electrode surface was increased. The sensitivities obtained for each biosensor were 19.3, 58.1 and 10.6 mA M(-1) cm(-1) for fructose, H2O2 and methanol, respectively. The sensitivity values can be easily controlled by a rational deposition and manipulation of the charge in the catalytic layers. The electrostatic assembly of the electrochemical interface and the catalytic layers resulted in integrated biochemical systems in which mass transfer diffusion and heterogeneous catalytic and electron transfer steps are efficiently coupled and can be easily manipulated.

Improved mediated tyrosinase amperometric enzyme electrodes

Abstract Tyrosinase amperometric enzyme electrodes in a carbon paste configuration based on the electroreduction of quinoid enzymatic products have been constructed. The electroreduction was mediated by the inclusion in the carbon paste material of an osmium (4,4′-dimethyl 2,2′-bipyridine) 2 (1,10-phenanthroline-5,6-dione) mediator. The mediated electrodes showed a three-fold increase in current density, almost two orders of magnitude decrease in detection limit, and a more than one order of magnitude increase in lifetime of the sensor under FIA conditions compared with the unmediated electrodes. Furthermore, they showed a clear mass transport limited response, changing the response limiting step compared with unmediated electrodes. These findings suggest that the use of suitable mediators and electrode configurations can improve the characteristics of tyrosinase electrodes.

Amperometric mediated carbon paste biosensor based on D-fructose dehydrogenase for the determination of fructose in food analysis

A new mediated amperometric biosensor for fructose is described. The sensor is based on a commercially available D-fructose dehydrogenase. The enzyme is incorporated in a carbon paste matrix containing Os(bpy)2Cl2 as redox mediator that achieves electron transfer at 0·1 V (versus Ag/AgCl) with maximum apparent current densities of 1·2 mA/cm2. The dependence of the steady-state current on the loading of the mediator and the enzyme, other electrode construction parameters, the operating potential, the pH and the temperature was studied. In the steady-state mode the response current was directly proportional to D-fructose concentration from 0·2 to 20mM with a detection limit of 35 μM (signal-to-noise ratio, S/N, 3). In the flow injection analysis mode the response current was directly proportional to D-fructose concentration from 0·5 to 15 M with a detection limit of 115 μM (S/N 3). The sensor was used for the determination of fructose in food samples in a flow injection system and validated with a commercial enzyme kit.

Electrocatalytic oxidation of NADH at graphite electrodes modified with osmium phenanthrolinedione

We report here a detailed study concerning the electrochemical behavior of Os(4,4′-dimethyl, 2,2′-bipyridine)2(1,10-phenanthroline 5,6-dione) complex, adsorbed on spectrographic graphite, and about its electrocatalytic activity for NADH oxidation. Cyclic voltammetric measurements, performed in aqueous phosphate buffer solutions, at different scan rates and pH values, allowed us: (i) to relate the redox response of the o-quinone ligand (phendione) to that of the Os(II) central ion; (ii) to confirm that, in aqueous solutions, the phendione based redox process globally involves two electrons and two protons; (iii) to estimate the rate constant for the heterogeneous electron transfer corresponding to the phendione redox couple (ks≈20.1 s−1). The second order rate constant for electrocatalytic oxidation of NADH (k1,[NADH]=0=1.9×103 M−1 s−1, at pH 6.1) as well as its pH dependence (from pH 5.5 to 8.1) were evaluated from RDE experiments, using both Koutecky–Levich and Lineweaver–Burk data interpretations.

Microperoxidase-11-mediated reduction of hemoproteins: electrocatalyzed reduction of cytochrome c, myoglobin and hemoglobin and electrocatalytic reduction of nitrate in the presence of cytochrome-dependent nitrate reductase

Microperoxidase-11 (MP-11) assembled onto an Au electrode catalyzes the electroreduction of a series of hemoproteins: cytochrome c (Cyt. c) myoglobin (Mb) and hemoglobin (Hb). The electrical contact between MP-11 and the hemoproteins originates from intermolecular complexes between MP-11 and the different hemoproteins. The MP-11-functionalized electrode catalyzes the reduction of cytochrome-dependent nitrate reductase and mediates the bioelectrocatalyzed reduction of nitrate to nitrite. The systems provide assemblies mimicking inter-protein electron transfer in hemoproteins.

Design and testing of a packaged microfluidic cell for the multiplexed electrochemical detection of cancer markers

We present the rapid prototyping of electrochemical sensor arrays integrated to microfluidics towards the fabrication of integrated microsystems prototypes for point‐of‐care diagnostics. Rapid prototyping of microfluidics was realised by high‐precision milling of polycarbonate sheets, which offers flexibility and rapid turnover of the desired designs. On the other hand, the electrochemical sensor arrays were fabricated using standard photolithographic and metal (gold and silver) deposition technology in order to realise three‐electrode cells comprising gold counter and working electrodes as well as silver reference electrode. The integration of fluidic chips and electrode arrays was realised via a laser‐machined double‐sided adhesive gasket that allowed creating the microchannels necessary for sample and reagent delivery. We focused our attention on the reproducibility of the electrode array preparation for the multiplexed detection of tumour markers such as carcinoembryonic antigen and prostate‐specific antigen as well as genetic breast cancer markers such as estrogen receptor‐α, plasminogen activator urokinase receptor, epidermal growth factor receptor and erythroblastic leukemia viral oncogene homolog 2. We showed that by carefully controlling the electrode surface pre‐treatment and derivatisation via thiolated antibodies or short DNA probes that the detection of several key health parameters on a single chip was achievable with excellent reproducibility and high sensitivity.

Reagentless amperometric glucose dehydrogenase biosensor based on electrocatalytic oxidation of NADH by osmium phenanthrolinedione mediator

The mediator Os(4,4′-dimethyl, 2,2′-bipyridine)2(1,10-phenanthroline-5,6-dione) was used for the catalytic oxidation and recycling of NADH. The mediator, with a redox potential of almost 0 V versus Ag/AgCl, shows clear electrocatalysis of NADH at 0.1 V versus Ag/AgCl at pH 6.0. Carbon paste electrodes modified with the mediator show a clear electrocatalytic wave reaching limiting current densities at 0.15 V versus Ag/AgCl of the order of 140 µA cm–2 l mmol–1 NADH. Reagentless dehydrogenase carbon paste amperometric electrodes for glucose were developed, mixing the mediator, glucose dehydrogenase and NAD+ in the paste. These electrodes were optimized with respect to amounts of enzyme, mediator and NAD+ and were studied with a variety of electrochemical techniques. The results suggest that the response is limited by the enzymic step of the reduction of NAD+ or the oxidation of the substrate. The glucose electrodes show maximum current densities of >0.5 mA cm–2 and very good operational stability in continuous operation; under dry storage conditions their lifetime exceeded 1 month.