Electrochemical evaluation of dsDNA—Liposomes interactions

The aim of the present work was to evaluate the interaction between double-stranded DNA (dsDNA) and liposomes by voltammetric methods. The experimental results were analyzed considering the initial studies regarding the oxidation mechanism of dsDNA purine bases by cyclic and differential pulse voltammetry at the glassy carbon electrode (GCE). The interaction between dsDNA and 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was studied in a suspension containing both dsDNA and DMPC liposomes, prepared in pH = 7.0, 0.1 M phosphate buffer and using different incubation time periods. The formation of dsDNA-liposome complex was put in evidence by the decrease of the dsDNA oxidation peaks, dependent upon the incubation time. This behavior was explained considering the electroactive centers of dsDNA, guanosine monophosphate and adenosine monophosphate residues, part of them hidden inside the dsDNA-liposome complex structure and thus being unable to reach the GC electrode and preventing their oxidation. The electrochemical results are relevant for a better physicochemical characterisation of the dsDNA and dsDNA-liposome complex, which can be important for the development of gene therapy vectors

Development and characterization of layer-by-layer biosensors based on PEI(+)/GOx(-) layers using label-free methods

Biosensors, as analytical devices, demonstrate unique efficiency in translating biochemical events into easily measurable electrical signals by using biological recognition elements, especially enzymes. Of the possible enzyme immobilisation methods, the layer-by-layer (LBL) technique, based on electrostatic interactions between layers, has the advantages of low cost, using small amount of materials, and leads to the formation of highly ordered and reproducible biosensor architectures. In this study, LbL biosensor construction has been evaluated. The substrates used were Au surfaces and mediated carbon-ink screen-printed electrodes. The gold electrodes were first functionalized with amino moieties by covalent linkage of cysteamine (Cys) through Au-S bonds. These allowed the linking of polyethyleneimine (PEI) through hydrogen-bonding to the gold surface and increased the stability of subsequent multilayers. PEI was directly adsorbed on the SPE surface. PEI is a short chain polymer and thence an efficient electron carrier, and being positively charged, it allows the formation of LBL structures with negatively charged enzymes. The multilayer formation of PEI(+)/GOx(-) was monitored by cyclic voltammetry, electrochemical impedance spectroscopy, and gravimetry. The influence of each enzymatic layer on the performance of the developed biosensor was analysed by fixed potential amperometric measurements

New strategies for early diagnosis of heart allograft rejection.

BACKGROUND: Allograft rejection is mediated by T cells that recognize allogeneic major histocompatibility complex (MHC) molecules via the direct and indirect pathway. The direct pathway involves T cells that react against MHC/peptide complexes expressed on the surface of donor antigen-presenting cells (APCs). In contrast, T cells involved in the indirect pathway recognize peptides derived from processing and presentation of allogeneic MHC molecules by self (recipient) APCs. To explore the relative contribution of these two pathways to rejection, we have evaluated the response of peripheral blood T cells from 50 heart transplant recipients against donor APCs (direct recognition) and against self APCs pulsed with synthetic peptides corresponding to the hypervariable region of the mismatched HLA-DR antigens of the donor (indirect recognition). METHODS: T cell reactivity against donor APCs was quantitated by measuring the expression of CD69 on allostimulated CD3+ LDA1+ cells. Reactivity to synthetic allopeptides was determined in limited dilution assays. RESULTS: Serial studies of the kinetics of direct and indirect recognition showed that both pathways contribute to early acute rejection episodes. Primary rejection was accompanied invariably by indirect recognition of a dominant allopeptide. Intermolecular spreading of T cell epitopes was observed during recurrent rejections. Enhanced recognition of donor alloantigens via the direct pathway was found predominantly during early rejection episodes. A single form of allorecognition was shown to occur in some rejection episodes. CONCLUSIONS: Monitoring of the direct and indirect pathway of allorecognition provides a reliable method for prediction and differential diagnosis of acute rejection of heart allografts