L. Boguslavsky

Applications of redox polymers in biosensors

Abstract Polymers containing covalently attached redox molecules can be highly effective electron transfer mediators for flavin adenine dinucleotide redox centers of many oxidases. Highly flexible siloxane and ethylene oxide polymers containing covalently attached ferrocene molecules are shown to be capable of mediating electron transfer between enzymes and an electrode. The construction and response of bienzyme cholesterol biosensor, acetylcholine and glucose biosensor are described and discussed. Our data showed that the flexibility, hydrophilicity of the polymer, the density of redox centers in the polymer matrices and the self-exchange reaction rate of the redox molecules control the efficiency of the electron transfer mediation.

Amperometric biosensors based on electrocatalytic regeneration of NAD+ at redox polymer-modified electrodes

Abstract Amperometric biosensors based on redox polymer-mediated electron transfer from NADH to carbon paste electrodes, regenerating the NAD + needed for the dehydrogenase-catalysed reaction, are described. These sensors, operating around 0 mV vs SCE, can drive an unfavourable equilibrium of a dehydrogenase-catalysed reaction to the product side, and are reagentless in that NAD + need not be added to the analyte solution. By covering the sensor with an additional polymer, protection of the electrode surface, inclusion of water soluble components, exclusion of interferents and a diffusion controlled linear response current can be obtained.

Amperometric thin film biosensors based on glucose dehydrogenase and Toluidine blue O as catalyst for NADH electrooxidation

Amperometric glucose sensors were constructed based on solid graphite electrodes, surface-modified with NAD+ dependent glucose dehydrogenase (GDH), Toluidine Blue O (TBO), and protective ionic polymers. The electrocatalytic oxidation of NADH was evaluated from cyclic voltammetry with TBO dissolved, adsorbed, and electrostatically or covalently bound to polymers. The NADH and glucose sensors constructed were investigated and operated at 0 mV vs. Ag/AgCl using single potential step chronoamperometry. The operational stability of the glucose sensors was limited by leakage of NAD+. A glucose sensitivity much higher than carbon paste electrode was found. A sensitivity as high as 25 microA cm-2 mM-1 was achieved.

Thin film bienzyme amperometric biosensors based on polymeric redox mediators with electrostatic bipolar protecting layer

Amperometric bienzyme sensors were constructed based on solid graphite electrodes modified with polymer redox mediator, horseradish peroxidase and a variety of oxidases and covered with a bipolar electrostatic protective layer. Glucose, ethanol, choline, glycero-3-phosphate and cholesterol sensors were designed and operated at 0 mV vs. AgAgCl using single potential step chronoamperometry. The protection of the sensor against interference from ascorbic acid and linearity of response were provided by a bipolar electrostatic layer. Sensitivities as high as 0.74 μA cm−2 mM−1 of glucose, 0.71 μA cm−2 mM−1 of ethanol 5.7 μA cm2 mM−1 of glycerol-1-phosphate, and 28.3 nA cm−2 per 10 μM of choline, were achieved.

Amperometric glyceride biosensor

Glycerol dehydrogenase (GDH) and lipase have been used for the amperometric determination of glycerol and triglycerides on modified carbon electrodes. Carbon electrodes were modified with adsorbed Meldola Blue, Nile Blue or Toluidine Blue O. Electrochemical oxidation of NADH was realized at 0V vs saturated Ag/AgCl electrode. NADH was produced by the catalytic oxidation of glycerol in the presence of glycerol dehydrogenase immobilized on the surface of an electrode. GDH was adsorbed on the electrode, entrapped in gelatin, immobilized in polylysine gel, or trapped in two types of organic salts. Sensitivity of the electrodes vary from 2 to 9 nA/mM glycerol with steady state achieved in a time of between 20 s and 8 min, depending on the method of immobilization. Triglycerides were determined after a 5 min pre-incubation period in a mixture of lipases with different specificity.

Amperometric biosensors based on dehydrogenase/NAD and heterocyclic quinones☆

The electrocatalytic oxidation of NADH was evaluated from cyclic voltammetry with heterocyclic quinones dissolved in a water solution. The process of oxidation is not sensitive to the nature of the electrode surface and takes place on platinum, glassy carbon or carbon electrodes. NADH and glucose sensors constructed were investigated and operated at 0 mV vs. Ag/AgCl (in saturated KCl) using single potential step chronoamperometry.

AMPEROMETRIC BIOSENSOR FOR FREE CHOLESTEROL BASED ON ELECTRICAL COMMUNICATION BETWEEN HORSERADISH PEROXIDASE AND NOVEL REDOX POLYMERS

Publisher Summary This chapter discusses the development of a bienzyme based cholesterol sensor involving several major improvements over previous designs. The peroxide formed in this process can be reacted with 4-aminoantipyrine and phenol, in the presence of a peroxidase, to form a quinoneimine dye that can be monitored spectrophotometrically. Alternatively, one could also electrochemically monitor the change in oxygen concentration that occurs during the reaction. Effective method to avoid interference with easy oxidizable species involves the use of the enzyme horseradish peroxidase that consumes the hydrogen peroxide produced in reaction. It is indicated that catalysts can be incorporated in polypyrrole by covalent attachment or a counter ion. This includes phthaloryanines, porphyries, ferrocene, and viologen. The results show that the sensor operations in a negative potential that avoids the interference with electroactive species in blood.

2.25 – An Amperometric Electrode Based on Carbon Paste, Chemically Modified with D-Lactate Dehydrogenase, NAD+ and Mediator Containing Polymer for D-Lactic Acid Analysis

This chapter presents results of a study examining an amperometric electrode based on carbon paste and chemically modified with d -lactate dehydrogenase, NAD+ and mediator containing polymer for d -lactic acid analysis. It is interesting to use the carbon paste as biosensor with dehydrogenase because the consumption of expensive cofactor can be significantly reducing by constraining the water soluble cofactor inside the paste. Furthermore, the influence of the unfavorable equilibrium of the dehydrogenase catalyzed reaction can be reduced by means of the mediator electron transfer driving force coupled with the oxidation of the NADH. In this study, a carbon paste electrode was designed for quantification of d -lactic acid. d -Lactate dehydrogenase from Leuconostoc mesenteroides ssp. cremoris purified by affinity precipitation with Eudragit-cibarcon blue and DEAE ion exchange chromatography, NAD + , a synthetic redox polymer, containing a covalently attached Toluidine Blue O moiety as mediator, and paraffin oil were used for the construction of the electrode. According to the preliminary data, good selectivity was obtained between d - and l -form lactic acid.

Amperometric Biosensors Based on Dehydrogenases in Carbon Paste

This chapter discusses amperometric biosensors based on dehydrogenases in carbon paste. The use of carbon paste for the construction of biosensors based on dehydrogenases is highly motivated, as it gives means to immobilize and constrain the water soluble nicotinamide cofactor in close proximity with the enzyme. A close coupling between all necessary components for a stable and selective dehydrogenase based sensor also counteracts the commonly unfavorable equilibrium of the dehydrogenase catalyzed reaction when the oxidation of NADH is coupled to a mediated electron transfer to the graphite, thereby driving the equilibrium to the product side. Investigations of sensors based on glucose dehydrogenase (GDH) indicated excellent activity and stability of GDH, when contained in carbon paste. The chapter also discusses the investigations of biosensors based on other dehydrogenases for example, l -lactate- and l -glutamate dehydrogenase. The properties of these sensors are presented, and aspects such as allosteric inhibition and the coupling of the dehydrogenase catalyzed reaction with other enzyme systems are considered.

Amperometric Biosensors Based on Dehydrogenase/NAD and Heterocyclic Quinones

This chapter describes the amperometric biosensors based on dehydrogenase/NAD and heterocyclic quinones. Amperometric biosensors based on dehydrogenase and nicotinamide adenine dinucleotide (NAD) require electrocatalytic oxidation of NADH. Because of the low redox potential of NADH, dehydrogenase based amperometric sensors provide the possibility of operating at applied potentials close to zero that offers the advantage of significantly reduced electrochemical interference from common existing electroactive species. Phenoxazine and phenothiazine have been widely studied as electron transfer mediators for the electrochemical oxidation of NADH. The catalytic activity of these phenoxazines is very low at platinum and glassy carbon surfaces. Dehydrogenase/NAD-based biosensors using a heterocyclic quinone as the electron transfer mediator have been studied. Because the electrochemical oxidation of NADH catalyzed by heterocyclic quinones occur under homogeneous conditions, no specific adsorption is required for the catalysis.