Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.

A study of the peculiarities and a comparative analysis of the technologies used for the fabrication of elements of novel hybrid microfluidic biochips for express biomedical analysis have been carried out. The biochips were designed with an incorporated microfluidic system, which enabled an accumulation of the target compounds in a biological fluid to be achieved, thus increasing the biochip system’s sensitivity and even implementing a label-free design of the detection unit. The multilevel process of manufacturing a microfluidic system of a given topology for label-free fluorometric detection of protein structures is presented. The technological process included the chemical modification of the working surface of glass substrates by silanization using (3-aminopropyl) trimethoxysilane (APTMS), formation of the microchannels, for which SU-8 technologies and a last generation dry film photoresist were studied and compared. The solid-state phosphor layers were deposited using three methods: drop application; airbrushing; and mechanical spraying onto the adhesive surface. The processes of sealing the system, installing input ports, and packaging using micro-assembly technologies are described. The technological process has been optimized and the biochip was implemented and tested. The presented system can be used to design novel high-performance diagnostic tools that implement the function of express detection of protein markers of diseases and create low-power multimodal, highly intelligent portable analytical decision-making systems in medicine.

The problems of chronic or noncommunicable diseases (NCD) that now kill around 40 million people each year require multiparametric combinatorial diagnostics for the selection of effective treatment tactics. This could be implemented using the biosensor principle based on peptide aptamers for spatial recognition of corresponding protein markers of diseases in biological fluids. In this paper, a low-cost label-free principle of biomarker detection using a biosensor system based on fluorometric registration of the target proteins bound to peptide aptamers was investigated. The main detection principle considered includes the re-emission of the natural fluorescence of selectively bound protein markers into a longer-wavelength radiation easily detectable by common charge-coupled devices (CCD) using a specific luminophore. Implementation of this type of detection system demands the reduction of all types of stray light and background fluorescence of construction materials and aptamers. The latter was achieved by careful selection of materials and design of peptide aptamers with substituted aromatic amino acid residues and considering troponin T, troponin I, and bovine serum albumin as an example. The peptide aptamers for troponin T were designed in silico using the «Protein 3D» (SPB ETU, St. Petersburg, Russia) software. The luminophore was selected from the line of ZnS-based solid-state compounds. The test microfluidic system was arranged as a flow through a massive of four working chambers for immobilization of peptide aptamers, coupled with the optical detection system, based on thick film technology. The planar optical setup of the biosensor registration system was arranged as an excitation-emission cascade including 280 nm ultraviolet (UV) light-emitting diode (LED), polypropylene (PP) UV transparent film, proteins layer, glass filter, luminophore layer, and CCD sensor. A laboratory sample has been created.

Impedimetric biosensors are used for detecting a wide range of analytes. The detection principle is a perspective for the development of new types of analytical devices for biomolecular diagnosis of diseases. Of particular interest are biosensors with very high sensitivities, capable of detecting trace amounts of biomarkers or drugs in biological fluids. Impedimetric biosensors possess a potential for increased sensitivity, since their electrodes can be modified with nanostructured materials, in particular zinc oxide. In this work, a miniature biosensor with an array of zinc oxide nanorods synthesized by the hydrothermal method has been created. Protein A was immobilized on the resulting structure, which was previously tested for binding to omalizumab by capillary electrophoresis. Using impedance spectroscopy, it was possible to detect the binding of omalizumab at concentrations down to 5 pg/mL. The resulting structures are suitable for creating reusable biosensor systems, since ZnO-coated electrodes are easily cleaned by photocatalytic decomposition of the bound molecules. The biosensor is promising for use in Point-of-Care systems designed for fast, multimodal detection of molecular markers of a wide range of diseases.

The in silico modelling of peptides complementary to lactoferrin was carried out using the Protein 3D software package and replication of the natural bonding site between pneumococcal surface protein (PSP) and lactoferrin (LF). The modeling was based on analysis of the conjugated ion–hydrogen bond systems between these proteins (CIHBS). The oligopeptide EEVAPQAQAKIAELENQVHRLE was proposed via computer modelling and synthesized using the solid phase synthesis technique, purified, and analyzed with MS and HPLC methods to confirm >95% purity. The peptide was then studied by capillary electrophoresis (CE). The CE experiments demonstrated the split of peptide zone in the presence of LF, due to complex formation and subsequent mobility change of the system peptide-protein. The reference experiments with homomyeloperoxidase and myoglobin did not show binding with LETI-11.