MIP-based electrochemical sensor for direct detection of hepatitis C virus via E2 envelope protein.

Hepatitis C is the most common liver disease caused by Hepatitis C virus (HCV), and can evolve into serious health problems e.g. cirrhosis and hepatocellular carcinoma. Nowadays, the initial stage of the disease cannot be practically diagnosed, representing thus an extremely important problem of modern public health care. This study is aimed at the development of a sensor for direct detection of HCV. The sensor utilizes a synthetic recognition element prepared by the technology of molecular imprinting and representing a molecularly imprinted polymer (MIP) having molecular recognition sites of HCV envelope protein E2 (E2-MIP). E2-MIP integrated into an electrochemical sensor platform allows quantitative evaluation of binding of free E2 protein as well as HCV-mimetic particles (HCV-MPs) in human plasma with LOD value of 4.6 × 10-4 ng/mL (for HCV-MPs). The developed electrochemical HCV sensor represents a simple, fast and inexpensive alternative for the existing methods of HCV detection and paves the way for the point-of care diagnostics of Hepatitis C.

Polymer Particles Bearing Recombinant LEL CD81 as Trapping Systems for Hepatitis C Virus.

Hepatitis C is one of the most common social diseases in the world. The improvements in both the early diagnostics of the hepatitis C and the treatment of acute viremia caused by hepatitis C virus are undoubtedly an urgent task. In present work, we offered the micro- and nanotraps for the capturing of HCV. As a capturing moiety, we designed and synthesized in E. coli a fusion protein consisting of large extracellular loop of CD81 receptor and streptavidin as spacing part. The obtained protein has been immobilized on the surface of PLA-based micro- and nanoparticles. The developed trapping systems were characterized in terms of their physico-chemical properties. In order to illustrate the ability of developed micro- and nanotraps to bind HCV, E2 core protein of HCV was synthesized as a fusion protein with GFP. Interaction of E2 protein and hepatitis C virus-mimicking particles with the developed trapping systems were testified by several methods.

Macroporous Polymer Monoliths in Thin Layer Format.

Nowadays, macroporous polymer monoliths represent widely used stationary phases for a number of dynamic interphase mass exchange processes such as high-performance liquid chromatography, gas chromatography, electrochromatography, solid-phase extraction, and flow-through solid-state biocatalysis. This review represents the first summary in the field of current achievements on the preparation of macroporous polymer monolithic layers, as well as their application as solid phases for thin-layer chromatography and different kinds of microarray.

An electrochemical biosensor for direct detection of hepatitis C virus.

This paper is aimed at the development of a biosensor for direct detection of Hepatitis C virus (HCV) surface antigen: envelope protein (E2). A recombinant LEL fragment of biological cell receptor CD81 and two short synthetic peptides imitating the fragment of LEL sequence of CD81 (linear and loop-like peptides) capable of specific binding to E2 were tested as molecular recognition elements of the biosensor. For this purpose the selected ligands were immobilized to the surface of a screen-printed electrode utilized as an electrochemical sensor platform. The immobilization parameters such as the ligand concentration and the immobilization time were carefully optimized for each ligand. Differential pulse voltammetry used to evaluate quantitatively binding of E2 to the ligands revealed their similar binding affinity towards E2. Thus, the linear peptide was selected as a less expensive and easily prepared ligand for the HCV biosensor preparation. The resulting HCV biosensor demonstrated selectivity towards E2 in the presence of interfering protein, conalbumin. Moreover, it was found that the prepared biosensor effectively detected E2 bound to hepatitis C virus-mimetic particles (HC VMPs) at LOD value of 2.1∙10-5 mg/mL both in 0.01 M PBS solution (pH 7.4) and in simulated blood plasma.

Towards the Development of a 3-D Biochip for the Detection of Hepatitis C Virus.

The early diagnostics of hepatitis C virus (HCV) infections is currently one of the most highly demanded medical tasks. This study is devoted to the development of biochips (microarrays) that can be applied for the detection of HCV. The analytical platforms of suggested devices were based on macroporous poly(glycidyl methacrylate-co-di(ethylene glycol) dimethacrylate) monolithic material. The biochips were obtained by the covalent immobilization of specific probes spotted onto the surface of macroporous monolithic platforms. Using the developed biochips, different variants of bioassay were investigated. This study was carried out using hepatitis C virus-mimetic particles (VMPs) representing polymer nanoparticles with a size close to HCV and bearing surface virus antigen (E2 protein). At the first step, the main parameters of bioassay were optimized. Additionally, the dissociation constants were calculated for the pairs "ligand-receptor" and "antigen-antibody" formed at the surface of biochips. As a result of this study, the analysis of VMPs in model buffer solution and human blood plasma was carried out in a format of direct and "sandwich" approaches. It was found that bioassay efficacy appeared to be similar for both the model medium and real biological fluid. Finally, limit of detection (LOD), limit of quantification (LOQ), spot-to-spot and biochip-to-biochip reproducibility for the developed systems were evaluated.

Novel Formulations of C-Peptide with Long-Acting Therapeutic Potential for Treatment of Diabetic Complications.

The development and application of novel nanospheres based on cationic and anionic random amphiphilic polypeptides with prolonged stability were proposed. The random copolymers, e.g., poly(l-lysine-co-d-phenylalanine) (P(Lys-co-dPhe)) and poly(l-glutamic acid-co-d-phenylalanine) (P(Glu-co-dPhe)), with different amount of hydrophilic and hydrophobic monomers were synthesized. The polypeptides obtained were able to self-assemble into nanospheres. Such characteristics as size, PDI and ζ-potential of the nanospheres were determined, as well as their dependence on pH was also studied. Additionally, the investigation of their biodegradability and cytotoxicity was performed. The prolonged stability of nanospheres was achieved via introduction of d-amino acids into the polypeptide structure. The cytotoxicity of nanospheres obtained was tested using HEK-293 cells. It was proved that no cytotoxicity up to the concentration of 500 µg/mL was observed. C-peptide delivery systems were realized in two ways: (1) peptide immobilization on the surface of P(Glu-co-dPhe) nanospheres; and (2) peptide encapsulation into P(Lys-co-dPhe) systems. The immobilization capacity and the dependence of C-peptide encapsulation efficiency, as well as maximal loading capacity, on initial drug concentration was studied. The kinetic of drug release was studied at model physiological conditions. Novel formulations of a long-acting C-peptide exhibited their effect ex vivo by increasing activity of erythrocyte Na⁺/K⁺-adenosine triphosphatase.