electronic-tongue-generating-continuous-recognition-patterns-for

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Institut Nanosciences et Cryogénie, CEA-Grenoble

Rigorous bounds to retarded learning.

We study the out-of-equilibrium fluctuation-dissipation (FD) relations in the low-temperature, finite-time, physical-aging regime of two simple models with strong glass behavior, the Fredrickson-Andersen model and the square-plaquette interaction model. We explicitly show the existence of unique, waiting-time independent dynamical FD relations. While in the Fredrickson-Andersen model the FD theorem is obeyed at all times, the plaquette model displays piecewise-linear FD relations, similar to what is found in disordered mean-field models and in simulations of supercooled liquids, and despite the fact that its static properties are trivial. We discuss the wider implications of these results.

Fluctuation-dissipation relations in the activated regime of simple strong-glass models.

Diagnostic DNA arrays for detection of point mutations as markers for cancer usually function in the presence of a large excess of wild-type DNA. This excess can give rise to false positives as a result of competitive hybridization of the wild-type target at the mutation spot. Analysis of the DNA array data is typically qualitative, aimed at establishing the presence or absence of a particular point mutation. Our theoretical approach yields methods for quantifying the analysis to obtain the ratio of concentrations of mutated and wild-type DNA.

Hybridization isotherms of DNA microarrays and the quantification of mutation studies.

In this paper, we present a cluster algorithm for the numerical simulations of nonadditive hard-core mixtures. This algorithm allows one to simulate and equilibrate systems with a number of particles two orders of magnitude larger than previous simulations. The phase separation for symmetric binary mixtures is studied for different nonadditivities as well as for the Widom-Rowlinson model [B. Widom and J. S. Rowlinson, J. Chem. Phys. 52, 1670 (1970)] in two and three dimensions. The critical densities are determined from finite size scaling. The critical exponents for all the nonadditivities are consistent with the Ising universality class.

Cluster algorithm for nonadditive hard-core mixtures.

We have found strong supporting evidence for the helical structures of single-stranded nucleic acids by stretching individual molecules of polyadenylic acid [poly(A)] and polycytidylic acid [poly(C)]. Analyzing the force versus extension data using a two-state elastic model in which random-coil domains alternate with rigid helical domains allows one to extract the thermodynamic and structural properties. In addition, it also yields moderate to low cooperativity of the helix-coil transition for poly(A) and poly(C), respectively.

Stretching of homopolymeric RNA reveals single-stranded helices and base-stacking.

Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine and (5'S)-5',8-cyclopurine-2'-deoxynucleoside, were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5'S)-5',8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins.

SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes.

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.

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

The secretions of molecules by cells are of tremendous interest for both fundamental insights studies and medical purposes. In this study, we propose a new biochip-based approach for the instantaneous monitoring of protein secretions, using antibody production by B lymphocytes cultured in vitro. This was possible thanks to the Surface Plasmon Resonance imaging (SPRi) of a protein biochip where antigen proteins (Hen Egg Lysozyme, HEL) were micro-arrayed along with series of control proteins. B cell hybridomas were cultured on the chip and the secretion of immunoglobulins (antibody) specific to HEL was monitored in real-time and detected within only few minutes rather than after a 30-60 min incubation with standard ELISA experiments. This fast and sensitive detection was possible thanks to the sedimentation of the cells on the biochip sensitive surface, where local antibody concentrations are much higher before dilution in the bulk medium. An other interesting feature of this approach for the secretion monitoring was the independence of the SPR response--after normalization--regarding to the density of the surface-immobilized probes. Such biosensor might thus pave the way to new tools capable of both qualitative and semi-quantitative analysis of proteins secreted by other immune cells.

On chip real time monitoring of B-cells hybridoma secretion of immunoglobulin.

Platinating agents are commonly prescribed anticancer drugs damaging DNA. Induced lesions are recognized by a wide range of proteins. These are involved in cellular mechanisms such as DNA repair, mediation of cytotoxicity or chromatin remodeling. They therefore constitute crucial actors to understand pharmacology of these drugs. To expand our knowledge about this subproteome, we developed a ligand fishing trap coupled to high throughput proteomic tools. This trap is made of damaged plasmids attached to magnetic beads, and was exposed to cell nuclear extracts. Retained proteins were identified by nanoHPLC coupled to tandem mass spectrometry. This approach allowed us to establish a list of 38 proteins interacting with DNA adducts generated by cisplatin, oxaliplatin and satraplatin. Some of them were already known interactome members like high mobility group protein 1 (HMGB1) or the human upstream binding factor (hUBF), but we also succeeded in identifying unexpected proteins such as TOX HMG box family member 4 (TOX4), phosphatase 1 nuclear targeting subunit (PNUTS), and WD repeat-containing protein 82 (WDR82), members of a recently discovered complex. Interaction between TOX4 and platinated DNA was subsequently validated by surface plasmon resonance imaging (SPRi). These interactions highlight new cellular responses to DNA damage induced by chemotherapeutic agents.

TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs.

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.

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

The use of biological-probe-modified solid-state pores in biosensing is currently hindered by difficulties in pore-wall functionalization. The surface to be functionalized is small and difficult to target and is usually chemically similar to the bulk membrane. Herein, we demonstrate the contactless electrofunctionalization (CLEF) approach and its mechanism. This technique enables the one-step local functionalization of the single pore wall fabricated in a silica-covered silicon membrane. CLEF is induced by polarization of the pore membrane in an electric field and requires a sandwich-like composition and a conducting or semiconducting core for the pore membrane. The defects in the silica layer of the micropore wall enable the creation of an electric pathway through the silica layer, which allows electrochemical reactions to take place locally on the pore wall. The pore diameter is not a limiting factor for local wall modification using CLEF. Nanopores with a diameter of 200 nm fabricated in a silicon membrane and covered with native silica layer have been successfully functionalized with this method, and localized pore-wall modification was obtained. Furthermore, through proof-of-concept experiments using ODN-modified nanopores, we show that functionalized nanopores are suitable for translocation-based biosensing.

Polarization-induced local pore-wall functionalization for biosensing: from micropore to nanopore.

Aptamers are raising an increasing interest for biosensor applications as replacements for antibodies due to their high stability and low cost. Thrombin, a key enzyme in the coagulation cascade, is an archetypical target against which two different aptamers, binding to two different exosites, have been selected. Recent studies dedicated to thrombin monitoring applications of biosensors have taken advantage of a potential sandwich-like structure between thrombin and these two aptamers for amplification purposes. However, in most cases, only end-point analysis was observed as a result of labeling requirements, thus preventing access to the kinetics of the complex formation. By using Surface Plasmon Resonance (SPR) imaging of aptamer-functionalized biosensors, we followed the binding of thrombin on the sensor and its interaction with a second reporter aptamer in real-time and in a label-free manner. Surprisingly, we showed that the injection of a second unlabeled-aptamer following the previous thrombin injection destabilized the thrombin-aptamer complex formed on the sensor surface, thus limiting any further amplification. However, the direct co-injection of thrombin, pre-complexed with a biotinylated aptamer bound to streptavidin efficiently increased the SPR signal by comparison to single thrombin detection. The various injection sequences performed may be rationalized considering a poor selectivity of one of the aptamers towards its exosite and a further negative allosteric effect upon sandwich complexation of the thrombin with its aptamers.

Real time monitoring of thrombin interactions with its aptamers: insights into the sandwich complex formation.

Continuous evolution profiles for electronic-tongue-based analysis.

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments.

Aptamers are selected DNA ligands that target biomolecules such as proteins. In recent years, they are showing an increasing interest as potential therapeutic agents or recognition elements in biosensor applications. In both cases, the need for characterizing the mating between the target and the aptamer either in solution or immobilized on a surface, is pressing. In this context, we have developed a kinetic biosensor made of micro-arrayed anti-thrombin aptamers to assess the kinetic parameters of this interaction. The binding of label-free thrombin on the biosensor was monitored in real-time by Surface Plasmon Resonance imaging. Remarkable performances were obtained for the quantification of thrombin without amplification (sub-nanomolar limit of detection and linear range of quantification to two orders of magnitude). The independent determinations of both the solution- and surface-phase affinities, respectively KD(Sol) and KD(Surf), revealed distinct values illustrating the importance of probes, targets or surface interactions in biosensors. Interestingly, KD(Surf) values depend on the aptamer grafting density and linearly extrapolate towards KD(Sol) for highly diluted probes. This suggests a lesser impact of the surface compared to the probe or target cooperativity interactions since the latter decrease with a reduced grafting density.

Solution-phase vs surface-phase aptamer-protein affinity from a label-free kinetic biosensor.

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.

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

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.

Article	Year
Rigorous bounds to retarded learning. Physical review letters\| PubMed ID: 11864063	2002
Fluctuation-dissipation relations in the activated regime of simple strong-glass models. Physical review letters\| PubMed ID: 12059430	2002
Hybridization isotherms of DNA microarrays and the quantification of mutation studies. Clinical chemistry\| PubMed ID: 15388635	2004
Cluster algorithm for nonadditive hard-core mixtures. The Journal of chemical physics\| PubMed ID: 15638570	2005
Stretching of homopolymeric RNA reveals single-stranded helices and base-stacking. Physical review letters\| PubMed ID: 17501388	2007
SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes. The Analyst\| PubMed ID: 18645645	2008
Effects of formamide on the thermal stability of DNA duplexes on biochips. Analytical biochemistry\| PubMed ID: 19796625	2010
On chip real time monitoring of B-cells hybridoma secretion of immunoglobulin. Biosensors & bioelectronics\| PubMed ID: 20961746	2011
TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs. Archives of biochemistry and biophysics\| PubMed ID: 21184731	2011
Temperature scans/cycles for the detection of low abundant DNA point mutations on microarrays. Biosensors & bioelectronics\| PubMed ID: 22047974	2012
Polarization-induced local pore-wall functionalization for biosensing: from micropore to nanopore. Analytical chemistry\| PubMed ID: 22364436	2012
Real time monitoring of thrombin interactions with its aptamers: insights into the sandwich complex formation. Biosensors & bioelectronics\| PubMed ID: 22863116	2013
Continuous evolution profiles for electronic-tongue-based analysis. Angewandte Chemie (International ed. in English)\| PubMed ID: 22968809	2012
Physico-chemical foundations underpinning microarray and next-generation sequencing experiments. Nucleic acids research\| PubMed ID: 23307556	2013
Solution-phase vs surface-phase aptamer-protein affinity from a label-free kinetic biosensor. PloS one\| PubMed ID: 24069412	2013
Relationship between humoral response against hepatitis C virus and disease overcome. SpringerPlus\| PubMed ID: 24516785	2014
SPR imaging based electronic tongue via landscape images for complex mixture analysis. Talanta\| PubMed ID: 25159378	2014

Arnaud Buhot

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