Allosteric Indicator Displacement Enzyme Assay for a Cyanogenic Glycoside

Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme β‐glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. β‐Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for β‐glucosidase uses amygdalin as natural substrate and allows measuring Michaelis–Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples.     

DNA‐Modified Screen‐Printed Electrodes with Nanostructured Films of Multiwall Carbon Nanotubes,Hydroxyapatite and Montmorillonite

Nanostructured films were deposited at the surface of working electrode of the screen‐printed assembly and utilized for the surface modification with double‐stranded DNA. The basic electrochemical properties of the sensors were investigated using voltammetric methods. Modified electrodes were also characterized by scanning electron microscopy and electrochemical impedance measurements. It was found that the electrode modification with DNA and nanomodifier leads to an enhanced sensitivity of the DNA voltammetric detection. New potentialities of the utilization of the K₃[Fe(CN)₆] cyclic voltammetric signal and electrochemical impedance spectroscopy were found. The DNA‐based biosensors showed good repeability and necessary stability within several days.     

New Strategy for Dehydrogenase Amperometric Biosensors Using Surfactant to Enhance the Sensitivity of Diaphorase/Ferrocene Modified Carbon Paste Electrodes for Electrocatalytic Oxidation of NADH

A carbon paste electrode (CPE) modified with diaphorase (DAP) and ferrocene (FcH) has been developed for determination of NADH at low working potential. The sensitivity and operational stability, towards the detection of the reduced form of the nicotinamide adenine dinucleotide (NADH) in flow injection analysis (FIA), were greatly improved (5 times) upon adding Tween 20 into the electrode matrix. The magnitude of the amperometric signal was dependent on DAP, FcH and surfactant loading, into the modified carbon paste electrode. A rapid and repeatable response was observed to the variation of NADH concentration in the vicinity of the electrode surface. Such advantages of the DAP/FcH/Tween 20 modified carbon paste were successfully used in the construction of L ‐lactate dehydrogenase modified electrodes. The use of this new approach can be generalized to other dehydrogenases and represents a decisive step for a versatile preparation method of amperometric biosensors.     

Electrically Addressed Fabrication of Aptamer‐Based Array Electrodes

An aptamer immobilization method based electrically addressed fabrication has been developed for the preparation of aptamer‐modified arrayed electrodes, by which the human IgE aptamer was oriented and immobilized on the gold electrode surface. The optimization of the experimental conditions including the applied potential, time and scan rate of potential was investigated. The method was successfully used to immobilize the aptamer onto the desired electrodes, pixel by pixel, based on the electrically addressed approach. Compared to the control electrodes, the resulting aptamer‐modified electrodes showed their specific recognition for human IgE. The present method owns several advantages such as rapid and simple immobilization as well as its automatic addressed capability by the electric approach.     

Research activities in the field of enzyme engineering in the framework of the russian state scientific program “novel methods in bioengineering”

The article describes the research activities in the field of enzyme engineering in Russia. The discussion is focused on fundamental studies of biocatalytic processes that expand utilization of enzymes, biocatalytic synthesis of organic products from renewable raw mate rials, enzymes for hydrolysis of cellulose and lignocellulose materials, immobilized cells, new enzyme-based drugs, enzymes in fine organic synthesis, bioanalytic devices, biosensors, and biofuels.     

Detection Mechanism of Metallized Carbon Epoxy Oxidase Enzyme Based Sensors

The coenzyme FAD has been identified to play an important role in the detection mechanism of oxidase enzyme based biosensors. Incorporating FAD into the carbon composite improved sensitivity to H₂O₂ consequently increasing sensitivity to the respective analyte. The amount of active enzyme also increased thus enhancing the overall performance of the sensors. Polycarboxybetaine (PCB) has been used as a biocompatible membrane coating. The PCB coated sensors gave reproducible calibrations in protein solutions, which has been shown to be a valid protocol for testing biocompatibility. The importance of reporting selectivity in a manner which indicates the “fitness of purpose” of biosensors has been discussed.     

Simultaneous determination of creatine and creatinine using amperometric biosensors

In order to determine creatine and creatinine amperometric biosensors were proposed. A bienzymatic biosensor based on creatinase (CI) and sarcosine oxidase (SO) was used for the assay of creatine and a trienzymatic biosensor based on CI, SO and creatininase (CA) for the assay of creatinine. The linear concentration ranges are of pmol l⁻¹ to nmol l⁻¹ magnitude order, with very low limits of detection. The biosensors proved high reliability for determination of creatine and creatinine as raw material, and in the pharmaceutical formulation.