Peptide-protein microarrays and surface plasmon resonance detection: biosensors for versatile biomolecular interaction analysis.

International audienceBiosensors in microarray format provide promising tools for high-throughput analyses of complex samples. Although they are able to detect, quantify and characterize a multitude of compounds, most of the available devices are specialized in the analysis of one type of interaction, limiting their application to a define area. The aim of our work was to develop and characterize versatile protein (or peptide) microarrays suitable for the simultaneous analysis of a large panel of biological interactions. Our system involved a simple procedure to immobilized proteins or peptides, based on pyrrole electropolymerization, and ligand binding was detected by imaging the surface plasmon resonance. We demonstrated its suitability in three different contexts, i.e. humoral response characterization, ion binding analysis and cell detection. This work evidences the potentiality of this approach which allows multiparametric, high-throughput and label-free analysis of biological samples suitable for the detection of compounds as various as proteins, ions or cells and the characterization of their interaction with peptides or proteins

Relationship between humoral response against hepatitis C virus and disease overcome

International audienceConclusionHumoral response against hepatitis C virus linear epitopes is partly modified according to the disease state. This study highlights the importance of considering relative quantities of antibodies with different specificities rather than the amount of each antibody.Hepatitis C virus infection leads to liver disease whose severity can range from mild to serious lifelong illness. However the parameters involved in the evolution of the disease are still unknown. Among other factors, the virus-elicited antibody profile is suspected to play a role in the outcome of the disease. Analysis of the relationship between anti-virus antibodies and disease state requires the analysis of a large number of serums from patients (hepatitis C virus+) and of epitopes from the viral proteins. Such a study would benefit from microarray-based screening systems that are appropriate for high-throughput assays.We used a method combining peptide chips and surface plasmon resonance imaging previously shown to be suitable for analyzing complex mediums and detecting peptide-protein interactions. 56 peptides covering the entire viral proteome were grafted on chips and their interaction with antibodies present in the 68 injected serums from infected and non-infected donors was measured. Statistical analyses were conducted to determine a possible relationship between antibodies (specificity and amount) and disease states.A good discrimination between infected and non-infected donors validated our approach, and several correlations between antibodies profiles and clinical parameters have been identified. In particular, we demonstrated that ratios between particular antibodies levels allow for accurate discrimination of patients according to their pathologic states

Fabrication of Bio-Functionalised Polypyrrole Nanoarrays for Bio-Molecular Recognition

International audienceThe present study demonstrates that nanosphere lithography and electro-polymerization can be successfully combined to produce bioactive protein nanoarrays. In particular, we describe a method to produce well-defined nanoarrays of polypyrrole functionalized with biomolecules. The nanoarrayed surfaces were fabricated on gold coated surface plasmon resonance prisms by first creating silicon oxide or polyethylene oxide nanotemplate using nanosphere lithography. The nanotemplate was subsequently used to grow bio-functionalized polypyrrole nanoarrays by electrocopolymerization. Atomic force microscopy analysis showed that the fabricated surfaces have a well-organized 2D hexagonal geometry with nanoscale dimensions. The biological activity of the bio-functionalized polypyrrole was assessed by surface plasmon resonance detection. The results showed that the immobilized biomolecules within the nanoarrayed polypyrrole films had the necessary bioactivity for successful molecular recognition. Moreover the detection signals normalized to the bioactive area were increased by a factor 5 as compared to non-structured bio-functionalized polypyrrole in the nanoarrayed surfaces using polyethylene oxide

Protein/peptide arrays to detect anti-delta genotype 1, 6, 8 specific antibodies among hepatitis D virus infectedpatients

International audienceLiver diseases linked to Hepatitis B-Hepatitis Delta virus co- or superinfections are more severe than during HBV mono-infection. Diagnosis of HDV infection therefore remains crucial in monitoring patients, but is often overlooked. To integrate delta markers into high throughput viral hepatitis diagnostic, we studied the binding of anti-delta antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides.Full-length and truncated S-HDAg recombinant proteins of HDV1 and 11 HDV peptides of HDV1, 6 and 8, representing various portion of the delta antigen were grafted onto biochips, allowing SPRi measurements. Sixteen to seventeen sera from patients infected with different HDV-genotypes were injected onto protein or peptide chips, respectively.In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94,12%), although the amplitude of the SPR signal varied. Amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant as 9 patient's samples led to a specific SPR signal on peptide 65#1, independently of the infecting genotype.In this pilot study, we confirmed that HDV-infection screening based on patient's Abs reactivity against carefully chosen HDV peptides and/or proteins can be included in a syndrome–based viral hepatitis diagnostic assay. Preliminary results indicated that SPRi studying direct physical HDAg/Anti-Delta Ab interactions was more convenient using linear peptide epitopes than full length S-HDAg protein, due to regeneration process and may represent an innovative approach for a hepatitis syndrome-viral etiology-exploring array