[Physico-chemical methods for studing ß-amyloid aggregation].

Alzheimer's disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, a key event of the Alzheimer's disease pathogenesis is a transition of the ß-amyloid peptide (Аß) from the monomeric form to the aggregated state. The mechanism of Ðß aggregation is intensively studied in vitro, by means of synthetic peptides and various physico-chemical methods allowing evaluation of size, molecular structure, and morphology of the formed aggregates. The paper reviews both the well-known and recently introduced physico-chemical methods for analysis of Ðß aggregation, including microscopу, optical and fluorescent methods, method of electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Merits and drawbacks of the methods are discussed. The unique possibility to simultaneously observe Ðß monomers as well oligomers and large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy is emphasized. The high detection sensitivity of the latter method, monitoring the aggregation process in Ðß solutions at low peptide concentrations is underlined. Among mass spectrometric methods, the ion mobility mass spectrometry is marked out as a method enabling to obtain information about both the spectrum of Ðß oligomers and their structure. It is pointed out that the use of several methods giving the complementary data about Ðß aggregates is the best experimental approach to studying the process of b-amyloid peptide aggregation in vitro.

[Electrochemical methods for biomedical investigations].

In the review, authors discussed recently published experimental data concerning highly sensitive electrochemical methods and technologies for biomedical investigations in the postgenomic era. Developments in electrochemical biosensors systems for the analysis of various bio objects are also considered: cytochrome P450s, cardiac markers, bacterial cells, the analysis of proteins based on electro oxidized amino acids as a tool for analysis of conformational events. The electroanalysis of catalytic activity of cytochromes P450 allowed developing system for screening of potential substrates, inhibitors or modulators of catalytic functions of this class of hemoproteins. The highly sensitive quartz crystal microbalance (QCM) immunosensor has been developed for analysis of bio affinity interactions of antibodies with troponin I in plasma. The QCM technique allowed real-time monitoring of the kinetic differences in specific interactions and nonspecific sorption, with out multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, calculated using experimental data. Based on the electroactivity of bacterial cells, the electrochemical system for determination of sensitivity of the microbial cells to antibiotics cefepime, ampicillin, amikacin, and erythromycin was proposed. It was shown that the minimally detectable cell number corresponds to 106 CFU per electrode. The electrochemical method allows estimating the degree of E.coli JM109 cells resistance to antibiotics within 2-5 h. Electrosynthesis of polymeric analogs of antibodies for myoglobin (molecularly imprinted polymer, MIP) on the surface of graphite screen-printed electrodes as sensor elements with o- phenylenediamine as the functional monomer was developed. Molecularly imprinted polymers demonstrate selective complementary binding of a template protein molecule (myoglobin) by the "key-lock" principle.

[Electrochemical measurement of intraprorein and interprotein electron transfer].

Intramolecular and intermolecular direct (unmediated) electron transfer was studied by means of electrochemical techniques in flavohemoprotein cytochrome P450 BM3 (CYP102A1 from Bacillius megaterium) and between cytochrome b5 and cytochrome c. Flavohemoprotein cytochrome P450 BM3 was immobilized on a screen printed graphite electrode, modified with a biocompatible nanocomposite material based on the didodecyldimethylammonium bromide DDAB and gold nanoparticles. Analytical characterictics of DDAB/Au/P450 BM3 electrodes were studied with cyclic voltammetry and square wave voltammetry. It was shown that intramolecular electron transfer was realized between diflavin (FAD/FMN) and heme domain of CYP102A1. An electron transport chain of flavohemoprotein P450 BM3 immobilized at nanostructued electrode is realized as: electrode --> FAD --> FMN --> heme. Electron transfer occurs inside the protein, and it is an evidence of functional interaction between diflavin and heme domains. The effect of a substrate (lauric acid) or inhibitors (metyrapone or imidazole) binding on the electrochemical parameters of flavohemoprotein P450 BM3 was also studied. Interprotein electron transfer was analyzed between cytochrome b5 and cytochrome c. Electrochemical analysis revealed that electron transfer takes place in protein-protein complexes with participants possessing different redox potentials.

[Electrochemical sensor systems based on one dimensional (1D) nanostructures for analysis of bioaffinity interactions].

It was shown that modification of screen printed graphite electrodes with gold nanoparticles (AuNPs) decorated Pb nanowires (PbNWs) demonstrates the enhancement of sensor's analytical characteristics such as effective surface area, electro catalytic properties and heterogeneous electron transfer kinetics. The reason for such improvement may be the synergistic effect ofAuNPs and PbNWs. Nanowires ensembles on electrode surface were employed for the detection of hemeproteins cytochrome P450 2B4, cytochrome c, and cardiac myoglobin in human plasma. Composite materials based on nanoparticles with different dimentions (3D three dimensional gold nanoparticles and 1D one dimensional Pb nanowires make it possible to construct biosensors with low detection limit of proteins.

[Electrochemical immunoanalysis of cardiac myoglobin].

Method targeting the direct monitoring of myoglobin based on analysis of electrochemical parameters of modified electrodes were proposed. Method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of hemeprotein. Myocardial infarction biomarker myoglobin was quantified at biological level using screen printed electrodes modified with gold nanoparticles stabilized with didodecyldimethylammonium bromide (DDAB) and antibodies. Proposed method did not require signal enhancement and amplification and also labeled secondary antibodies. Electro analysis has high specificity and sensitivity. Myoglobin -antibodies interaction was studied also with electrochemical impedance spectroscopy. Sensor has low detection limit and broad diapason of working concentrations (17.8 ng/ml-1780 ng/ml; 1 nM-10 nM). Method based on gold nanoparticles detection on the surface of electrodes was treated for myoglobin identification. AuNP worked as an electrochemical sensing platform: the oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. The difference of cathodic peak area and peak high of gold oxide reduction in the case of electrodes with antibodies and electrodes with antibodies-myoglobin complex, was registered.

[Sensor systems for medical application based on hemoproteins and nanocomposite materials].

Recent advances in nanotechnologies stimulate the development of sensor systems based on nanocomposite materials. This review discusses the prospects and challenges of sensors coupled with functionally important for medicine hemoproteins and nanoscale materials. Authors summarized their own experimental results and literature data on hemoprotein-based sensor systems. Mechanisms and the main function principles of electrochemical nanosensors are also discussed.

Bi-enzyme sensor based on thick-film carbon electrode modified with electropolymerized tyramine.

Bi-enzyme sensor based on thick-film epoxy-carbon electrode modified with polytyramine has been developed and examined for the determination of peroxidase substrates and cholinesterase inhibitors. Polytyramine was obtained on the electrode surface by repeated scanning of the potential from +600 to +1800 mV vs. Ag/AgCl in tyramine solution. The enzymes were immobilized in the polytyramine matrix by cross-linking with glutaraldehyde. The biosensor developed provides a reliable and inexpensive way for preliminary testing of common environmental pollutants with a single sensor in accordance with assumed toxic effect by the choice of appropriate substrate and measurement conditions. The bi-enzyme sensor makes it possible to determine substituted phenols and aromatic amines in the micromolar range of their concentrations and anticholinesterase pesticides with detection limits of 0.1 (Coumaphos) and 0.03 micromol l(-1) (Chloropyrifos-methyl).