Roberto Calemczuk

Point Mutation Detection by Surface Plasmon Resonance Imaging Coupled with a Temperature Scan Method in a Model System

The detection of point mutations in genes presents clear biological and medical interest. Various methods have been considered. In this paper, we take advantage of surface plasmon resonance imaging, a technique allowing detection of unlabeled DNA hybridization. Coupled with a temperature scan, this approach allows us to determine the presence of single-point mutations in oligonucleotide samples from the analysis of DNAs melting curves in either the homozygous or heterozygous case. Moreover, these experimental data are confirmed in good agreement with numerical calculations.

Effects of formamide on the thermal stability of DNA duplexes on biochips

In molecular biology, formamide (FA) is a commonly used denaturing agent for DNA. Although its influence on DNA duplex stability in solution is well established, little is known about immobilized DNA on microarrays. We measured thermal denaturation curves for oligonucleotides immobilized by two standard protocols: thiol self-assembling and pyrrole electrospotting. A decrease of the DNA denaturation temperature with increasing FA fraction of the solvent was observed on sequences with mutations for both surface chemistries. The average dissociation temperature decrease was found to be -0.58+/-0.05 degrees C/% FA (v/v) independently of grafting chemistry and probe sequence.

Continuous Evolution Profiles for Electronic‐Tongue‐Based Analysis

Conventional biosensors and biochips rely on a “lock-andkey” recognition principle, for which specific ligands, such as aptamers, antibodies, or mimetics are needed. In contrast, electronic nose/tongue devices (eN/eT) use the differential binding of analytes, either alone or in mixtures, to an array of cross-reactive receptors (CRRs) whose combined responses create a characteristic pattern for each component. As a result, none of the CRRs need to be highly specific for any given analyte and the time-consuming design and generation of specific sensors may be circumvented. Based on this principle, CRR arrays associated with appropriate detection have been developed. For example, synthetic tetraphenylporphyrin derivatives, whose fluorescence quenching upon protein binding was used for detection, or a receptor library, integrating a peptide cavity coupled with indicator-uptake colorimetric detection, were developed for protein sensing above concentrations of ten micromolar. A sensor array composed of gold nanoparticles, grafted with different amino-functionalized thiols and conjugated with an anionic fluorescent polymer (poly(p-phenyleneethynylene, PPE), allowed detection of proteins in the low nanomolar range, as well as detection of bacteria or discrimination of normal cells from their cancerous counterparts. Furthermore, replacing PPE with green fluorescent protein led to a concentration-sensitive CRR array able to discriminate different proteins in human serum at physiologically relevant concentrations. However, though simplified with regard to conventional biosensors, the preparation of eN/eT sensing arrays still requires the design and synthesis 5–29 CRRs and conjugation to a ligand-independent detection system. The development of new eN/eTapplications would thus benefit from simplified production of the sensing elements. In this regard, eN applications take advantage of the differential diffusion of volatile biomarkers through films of a single sensing material in varying morphology and surface coverage. For an eT, we anticipated that the design and synthetic efforts could also be drastically reduced if the CRRs were prepared by selfassembly of different combinations of building blocks (BBs), that is easily accessible molecules displaying different physicochemical properties. In this way, simply mixing different BBs in varying and controlled proportions and allowing them to assemble, either alone or on a template, should lead to a collection of combinatorial cross-reactive receptors (CoCRRs) with evolutionary properties. Interestingly, a high diversity of CoCRRs can be produced from a restricted number of BBs: 11 with only two BBs mixed in concentrations varying from 0 to 100% in 10% increments and 66 by simply adding a third BB. Further generalization to n BBs and i% concentration increment leads to [(100/i)+ n 1]!/[(n 1)!(100/i)!] potential different CoCRRs. To assure 1) reproducible assembly; 2) close correlation between the CoCRRs topology and the precursor combination of BBs; 3) spatial encoding of the CoCRRs compositions, we reasoned that the BBs would best be combined through the formation of self-assembled monolayers (SAMs). Interestingly, if the gold surface of a surface plasmon resonance imaging (SPRi) prism was used for the formation of SAMs through thiol or disulfide bonds, a label-free, synchronous, parallel, and real-time observation of binding events on the CoCRRs array could also be performed (Figure 1). To investigate this hypothesis, we designed a model array inspired by the way cell-surface heparan sulfates (HS) recognize HS binding proteins (HSbps), an important group of extracellular mediators involved in many physiological and pathological processes. HS are negatively charged polysaccharides displaying a high degree of molecular diversity, which arises from the regulated generation of various epimerization and sulfation patterns during biosynthesis. According to cell type and activation state, HS chains with different negatively charged topologies are expressed, presumably to promote selective interactions with HSbps. [*] Dr. Y. Hou, M. Genua, Dr. D. Tada Batista, Dr. R. Calemczuk, Dr. A. Buhot, Dr. T. Livache SPrAM, UMR 5819 (CEA-CNRS-UJF-Grenoble 1), INAC/CEA-Grenoble 38054 Grenoble cedex 9 (France) E-mail: yanxia.hou-broutin@cea.fr thierry.livache@cea.fr

Temperature scans/cycles for the detection of low abundant DNA point mutations on microarrays.

The possibility to detect low abundant DNA point mutations is essential for early cancer diagnosis and/or prognosis. Furthermore, in order to be less invasive, the somatic mutations are not only sought in tumor extract samples but also from body fluids or stools rendering their content even more diluted compared to the wild type sequences. In this short communication, we propose two protocols based on temperature scans or cycles for the enrichment of the mutation strands hybridized on microarrays. We predict numerically and confirm experimentally a 10-fold increase in the fraction of mutated DNA hybridized on the microarray compared to the sample content. Coupled to more standard solution phase enrichment techniques, it would be possible to lower by one order of magnitude the current detection limit with the advantage of multiple mutation detections offered by the microarray technology.

Relationship between humoral response against hepatitis C virus and disease overcome.

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.ConclusionHumoral 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.

SPR imaging based electronic tongue via landscape images for complex mixture analysis.

Electronic noses/tongues (eN/eT) have emerged as promising alternatives for analysis of complex mixtures in the domain of food and beverage quality control. We have recently developed an electronic tongue by combining surface plasmon resonance imaging (SPRi) with an array of non-specific and cross-reactive receptors prepared by simply mixing two small molecules in varying and controlled proportions and allowing the mixtures to self-assemble on the SPRi prism surface. The obtained eT generated novel and unique 2D continuous evolution profiles (CEPs) and 3D continuous evolution landscapes (CELs) based on which the differentiation of complex mixtures such as red wine, beer and milk were successful. The preliminary experiments performed for monitoring the deterioration of UHT milk demonstrated its potential for quality control applications. Furthermore, the eT exhibited good repeatability and stability, capable of operating after a minimum storage period of 5 months.

Fabrication of nanotweezers and their remote actuation by magnetic fields

A new kind of nanodevice that acts like tweezers through remote actuation by an external magnetic field is designed. Such device is meant to mechanically grab micrometric objects. The nanotweezers are built by using a top-down approach and are made of two parallelepipedic microelements, at least one of them being magnetic, bound by a flexible nanohinge. The presence of an external magnetic field induces a torque on the magnetic elements that competes with the elastic torque provided by the nanohinge. A model is established in order to evaluate the values of the balanced torques as a function of the tweezers opening angles. The results of the calculations are confronted to the expected values and validate the overall working principle of the magnetic nanotweezers.

SPR-imaging based assays on an oligonucleotide-array to analyze DNA lesions recognition and excision by repair proteins

An original oligonucleotide-array, coupled with SPR-imaging detection, has been developed to study biological interactions between DNA base lesions and DNA repair enzymes. This bioanalytical tool constitutes an efficient screening platform to quantify DNA repair activities and to search for new DNA repair inhibitors.