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

The optimal conditions for the formation of a stable mixed urease/octadecylamine (ODA) film at the air-water interface are determined. This film is efficiently transferred onto the hydrophobized surface of a pH-ISFET, and the features of the urea enzymatic field-effect transistor (ENFET) are determined: detection limit 0.2 mM, response time 15 s, and dynamic range 0-20 mM.

Mixed urease/amphiphile LB films and their application for biosensor development.

Langmuir-Blodgett (LB) technique appears to be quite suitable for generating biospecific surfaces and it has potential application for fabricating biosensors. In this work, mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile have been transferred onto hydrophobic silver surface previously modified by 1-octadecanethiol (ODT) SAMs. In order to obtain stable LB films, the influences of different parameters - type of amphiphile, surface pressure and pH - on the properties of mixed IgG/amphiphile monolayer, were investigated. Electrochemical properties of the engineered immunosensor have been measured by impedimetric spectroscopy. The immunosensor obtained exhibits a high sensitivity and a good specificity in a linear dynamic range from 200 to 1000 ng ml(-1).

Study of mixed Langmuir-Blodgett films of immunoglobulin G/amphiphile and their application for immunosensor engineering.

To make ultrathin films for the fabrication of artificial olfactory systems, odorant biosensors, we have investigated mixed Langmuir and Langmuir-Blodgett films of odorant-binding protein/amphiphile. Under optimized experimental conditions (phosphate buffer solution, pH 7.5, OBP-1F concentration of 4 mg L(-1), target pressure 35 mN m(-1)), the mixed monolayer at the air/water interface is very stable and has been efficiently transferred onto gold supports, which were previously functionalized by self-assembled monolayers (SAMs) with 1-octadecanethiol (ODT). Atomic force microscopy and electrochemical impedance spectroscopy were used to characterize mixed Langmuir-Blodgett (LB) films before and after contact with a specific odorant molecule, isoamyl acetate. AFM phase images show a higher contrast after contact with the odorant molecule due to the new structure of the OBP-1F/ODA LB film. Non-Faradaic electrochemical spectroscopy (EIS) is used to quantify the effect of the odorant based on the electrical properties of the OBP-1F/ODA LB film, as its resistance strongly decreases from 1.18 MOmega (before contact) to 25 kOmega (after contact).

Study of Langmuir and Langmuir-Blodgett films of odorant-binding protein/amphiphile for odorant biosensors.

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.

Immobilization of native membrane-bound rhodopsin on biosensor surfaces.

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.

Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy.

In this study, we report a dose-dependent detection of odorant molecules in solution by rat olfactory receptor I7 (OR I7) in its membrane fraction. The OR I7 is immobilized on a gold electrode by multilayer bioengineering based on a mixed self-assembled monolayer and biotin/avidin system, which allows for a well-controlled immobilization of the bioreceptor within its lipid environment. The odorant detection is electronically performed in a quantitative manner by electrochemical impedance spectroscopy (EIS) measurements on samples and controls.

A novel detection strategy for odorant molecules based on controlled bioengineering of rat olfactory receptor I7.

Dyschromatosis symmetrica hereditaria (OMIM127400) is a rare autosomal dominant pigmentary genodermatosis caused by mutations in the RNA-specific adenosine deaminase (ADAR) gene. This study investigated 5 families and 3 sporadic patients with dyschromatosis symmetrica hereditaria in the Chinese Han population from Anhui province, China. By direct sequencing, 5 novel ADAR gene mutations (c.982C>T, c.1491insA, c.2568_2571delTAAC, c.2969C>G and c.3040G>T) and 3 mutations described previously (c.3203-2A>G, c.3247C>T and c.3286C>T) were identified, all of which were heterozygous. We reviewed a total of 48 mutations in the ADAR gene in patients with dyschromatosis symmetrica hereditaria by previous reports and speculated that the mutation hotspots on the ADAR gene might be located in exons 9-15. The tRNA-specific and double-stranded RNA adenosine deaminase domain is essential for the deaminase activity of the ADAR encoded protein.

Five novel mutations of RNA-specific adenosine deaminase gene with dyschromatosis symmetrica hereditaria.

There is substantial interest in engineering solid supports to achieve functional immobilization of membrane receptors both for investigation of their biological function and for the development of novel biosensors. Three simple and practical strategies for immobilization of a human olfactory receptor carried by nanosomes are presented. The basis of the functionalization of solid gold surfaces is a self-assembled monolayer (SAM) containing biotinyl groups. Biotinyl groups are subsequently used to attach neutravidin and then biotinylated monoclonal antibody directed against the receptor to allow its specific grafting. Surface plasmon resonance technique is implemented for real-time monitoring of step-by-step surface functionalization and, in addition, for testing the functional response of immobilized olfactory receptors. We show that OR1740 is functional when immobilized via a tag attached to its C-terminus, but not via its N-terminus. Finally, we demonstrate that gold surfaces can be patterned by the SAMs tested using microcontact printing. AFM images of immobilized nanosomes onto a patterned surface suggest that small nanosomes flatten and fuse into larger vesicles but do not merge into a continuous layer. The whole study emphasizes the outstanding performances of the BAT/PEGAT SAM, which could be useful for developing on-a-chip sensor formats for membrane receptor investigations and use.

Gold surface functionalization and patterning for specific immobilization of olfactory receptors carried by nanosomes.

An ion channel biosensor is described for label-free detection of inhibitors which bind to the coiled coil domain of human immunodeficiency virus type 1 gp41. gp41 is the viral transmembrane glycoprotein responsible for fusion between HIV-1 and host cells. The N-terminal coiled coil domain binds three antiparallel C-heptad repeat peptides in the six helix bundle structure of trimeric gp41 that forms during fusion. Compounds able to prevent six-helix bundle formation by binding to the gp41 coiled coil could inhibit fusion and have important therapeutic potential. We have immobilized on gold a positively charged metallopeptide mimic of a section of the gp41 coiled coil containing a hydrophobic pocket suitable for small molecule binding. We demonstrate that the resulting sensor is able to transmit a current in the presence of negatively charged redox marker ions, therefore acting as an artificial ion channel. The electrochemical signal, measured by cyclic voltammetry, was modulated by specific analyte binding to the coiled coil. Nanomolar quantities of peptides and small molecules that bind in the hydrophobic pocket could be selectively detected, providing a method for label-free detection of binding to gp41.

Artificial ion channel biosensor in human immunodeficiency virus gp41 drug sensing.

Bismuth film electrode (BiFE) was shown to be an attractive alternative to common mercury film electrode (MFE) for anodic stripping voltammetric measurements. In this study, bismuth film, that was in situ deposited onto glassy carbon electrode, was used to detect zinc content of milkvetch, used in traditional Chinese medicine. Variables affecting the response have been evaluated and optimized. Experimental results showed a high response, with a good linearity (between 0.5x10(-6)molL(-1) and 3x10(-6)molL(-1)) a good precision (R.S.D.=3.58%) and a low detection limit (9.6x10(-9)molL(-1) with a 120s anodic). The anodic stripping performance makes the bismuth film electrode very desirable for measurements of trace nutritive element zinc in milkvetch and should impart possible restrictions on the use of mercury electrode.

Quantitative determination of zinc in milkvetch by anodic stripping voltammetry with bismuth film electrodes.

T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8(+) T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8(+) T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP(89-97) and NP(198-206), respectively. The results indicate that peptides NP(89-97) (PKKTGGPIY) and NP(198-206) (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken.

Prediction and identification of T cell epitopes in the H5N1 influenza virus nucleoprotein in chicken.

Continuous evolution profiles for electronic-tongue-based 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.

Yanxia Hou

Structure et Propriétés d'Architectures Moléculaires

Institut Nanosciences et Cryogénie, CEA-Grenoble

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

Yanxia Hou

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Structure et Propriétés d'Architectures Moléculaires

Institut Nanosciences et Cryogénie, CEA-Grenoble

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

Structure et Propriétés d'Architectures Moléculaires