Emmanuelle Trévisiol

Preparation of Tethered-Lipid Bilayers on Gold Surfaces for the Incorporation of Integral Membrane Proteins Synthesized by Cell-Free Expression

There is an increasing interest to express and study membrane proteins in vitro. New techniques to produce and insert functional membrane proteins into planar lipid bilayers have to be developed. In this work, we produce a tethered lipid bilayer membrane (tBLM) to provide sufficient space for the incorporation of the integral membrane protein (IMP) Aquaporin Z (AqpZ) between the tBLM and the surface of the sensor. We use a gold (Au)-coated sensor surface compatible with mechanical sensing using a quartz crystal microbalance with dissipation monitoring (QCM-D) or optical sensing using the surface plasmon resonance (SPR) method. tBLM is produced by vesicle fusion onto a thin gold film, using phospholipid-polyethylene glycol (PEG) as a spacer. Lipid vesicles are composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethyleneglycol)-2000-N-[3-(2-pyridyldithio)propionate], so-called DSPE-PEG-PDP, at different molar ratios (respectively, 99.5/0.5, 97.5/2.5, and 95/5 mol %), and tBLM formation is characterized using QCM-D, SPR, and atomic force technology (AFM). We demonstrate that tBLM can be produced on the gold surface after rupture of the vesicles using an α helical (AH) peptide, derived from hepatitis C virus NS5A protein, to assist the fusion process. A cell-free expression system producing the E. coli integral membrane protein Aquaporin Z (AqpZ) is directly incubated onto the tBLMs for expression and insertion of the IMP at the upper side of tBLMs. The incorporation of AqpZ into bilayers is monitored by QCM-D and compared to a control experiment (without plasmid in the cell-free expression system). We demonstrate that an IMP such as AqpZ, produced by a cell-free expression system without any protein purification, can be incorporated into an engineered tBLM preassembled at the surface of a gold-coated sensor.

Dendrimer functionalization of gold surface improves the measurement of protein–DNA interactions by surface plasmon resonance imaging

Surface Plasmon Resonance imaging (SPRi) is a label free technique typically used to follow biomolecular interactions in real time. SPRi offers the possibility to simultaneously investigate numerous interactions and is dedicated to high throughput analysis. However, precise determination of binding constants between partners is not highly reliable. We report here a dendrimer functionalization of gold surface that significantly improves selectivity of the detection of protein-DNA interactions. We showed that amino-gold surface functionalization with phosphorus dendrimers of fourth generation (G4) allowed complete coverage of the gold surface and the increase of the surface roughness. We optimized the conditions for DNA probe deposition to allow accurate detection of a well-known protein-DNA interaction involved in bacterial chromosome segregation. Using this G4-functionalized surface, the specificity of the SPRi response was significantly improved allowing discrimination between protein and DNA interactions of different strengths. Kinetic constants similar to those obtained with other techniques currently used in molecular biology were only obtained with the G4 dendrimer functionalized surface. This study demonstrated the benefit of using dendrimeric surfaces for sensitive high throughput SPRi analysis.

Interaction of biomolecules sequentially deposited at the same location using a microcantilever-based spotter.

A microspotting tool, consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels is introduced. This spotter, called Bioplume, is able to address on active surfaces and in a time-contact controlled manner picoliter of liquid solutions, leading to arrays of 5 to 20-μm diameter spots. In this paper, this device is used for the successive addressing of liquid solutions at the same location. Prior to exploit this principle in a biological context, it is demonstrated that: (1) a simple wash in water of the microcantilevers is enough to reduce by >96% the cross-contamination between the successive spotted solutions, and (2) the spatial resolution of the Bioplume spotter is high enough to deposit biomolecules at the same location. The methodology is validated through the immobilization of a 35mer oligonucleotide probe on an activated glass slide, showing specific hybridization only with the complementary strand spotted on top of the probe using the same microcantilevers. Similarly, this methodology is also used for the interaction of a protein with its antibody. Finally, a specifically developed external microfluidics cartridge is utilized to allow parallel deposition of three different biomolecules in a single run.

Tailored SU-8 micropillars superhydrophobic surfaces enhance conformational changes in breast cancer biomarkers

Here we report the fabrication of lotus-leaves-like tailored SU8 micropillars and their application in the context of a multi-technique characterization protocol for the investigation of the structural properties of the two estrogen receptors (ERα66/ERα46). ER (α) expression is undoubtedly the most important biomarker in breast cancer, because it provides the index for sensitivity to endocrine treatment. Beside the well-characterized ERα66 isoform, a shorter one (ERα46) was reported to be expressed in breast cancer cell line. The superhydrophobic supports were developed by using a double step approach including an optical lithography process and a plasma reactive ion roughening one. Upon drying on the micropillars, the bio-samples resulted in stretched fibers of different diameters which were then characterized by synchrotron X-ray diffraction (XRD), Raman and FTIR spectroscopy. The evidence of both different spectroscopic vibrational responses and XRD signatures in the two estrogen receptors suggests the presence of conformational changes between the two biomarkers. The SU8 micropillar platform therefore represents a valid tool to enhance the discrimination sensitivity of structural features of this class of biocompunds by exploiting a multi-technique in-situ characterization approach.

Molecular analysis for medicine: a new technological platform based on nanopatterning and label-free optical detection

RésuméDans ce travail nous montrons que la structuration à l’échelle nanométrique de biomolécules sondes par lithographie douce permet de fabriquer des puces à protéines à un coût de production suffisamment réduit pour entrevoir leur utilisation dans le domaine de l’analysemoléculaire médicale. La combinaison d’un procédé d’impression moléculaire et d’une détection optique sans marquage fondée sur le principe de la diffraction de la lumière est mise en oeuvre afin de produire des supports d’analyse en verre comportant des motifs nanométriques et un scanner de diffraction qui permet la lecture d’un test biologique multiplexé.AbstractIn this article, we show that by biopatterning probe molecules at the nanoscale using soft lithography, protein biochips can be produced at a significantly lower cost for their use as a systematic method of molecular analysis for medical diagnosis purposes. The combination of multiplexed nanoscale microcontact printing and label-free optical detection using the principle of light diffraction is implemented for generating engineered glass slides for analysis, and a dedicated diffractive scanner for reading the multiplexed results of an assay.

Automated and Multiplexed Soft Lithography for the Production of Low-Density DNA Microarrays.

Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics in which DNA molecules are precisely addressed to the surface of a solid support. This study assesses the fabrication of low-density oligonucleotide arrays using an automated microcontact printing device, the InnoStamp 40®. This automate allows a multiplexed deposition of oligoprobes on a functionalized surface by the use of a MacroStampTM bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStampTM were shown to be fast and robust by a simple washing step in 96% ethanol. The low-density microarrays printed on either epoxysilane or dendrimer-functionalized slides (DendriSlides) showed excellent hybridization response with complementary sequences at unusual low probe and target concentrations, since the actual probe density immobilized by this technology was at least 10-fold lower than with the conventional mechanical spotting. In addition, we found a comparable hybridization response in terms of fluorescence intensity between spotted and printed oligoarrays with a 1 nM complementary target by using a 50-fold lower probe concentration to produce the oligoarrays by the microcontact printing method. Taken together, our results lend support to the potential development of this multiplexed microcontact printing technology employing soft lithography as an alternative, cost-competitive tool for fabrication of low-density DNA microarrays.

Direct patterning of probe proteins on an antifouling PLL-g-dextran coating for reducing the background signal of fluorescent immunoassays

The limit of detection of advanced immunoassays, biochips and micro/nano biodetection devices is impacted by the non-specific adsorption of target molecules at the sample surface. In this paper, we present a simple and versatile low cost method for generating active surfaces composed of antibodies arrays surrounded by an efficient anti-fouling layer, capable to decrease drastically the fluorescence background signal obtained after interaction with a solution to be analyzed. The technological process involves the direct micro-contact printing of the antibodies probe molecules on a pre-coated PLL-g-dextran thin layer obtained by contact printing using a flat PDMS stamp. Compared to other blocking strategies (ethanolamine blocking treatment, PLL-g-PEG incubation, PLL-g-dextran incubation, printing on a plasma-deposited PEO layer), our surface chemistry method is more efficient for reducing non-specific interactions responsible for a degraded signal/noise ratio.

Dynamic inking of large-scale stamps for multiplexed microcontact printing and fabrication of cell microarrays

Microcontact printing has become a versatile soft lithography technique used to produce molecular micro- and nano-patterns consisting of a large range of different biomolecules. Despite intensive research over the last decade and numerous applications in the fields of biosensors, microarrays and biomedical applications, the large-scale implementation of microcontact printing is still an issue. It is hindered by the stamp-inking step that is critical to ensure a reproducible and uniform transfer of inked molecules over large areas. This is particularly important when addressing application such as cell microarray manufacturing, which are currently used for a wide range of analytical and pharmaceutical applications. In this paper, we present a large-scale and multiplexed microcontact printing process of extracellular matrix proteins for the fabrication of cell microarrays. We have developed a microfluidic inking approach combined with a magnetic clamping technology that can be adapted to most standard substrates used in biology. We have demonstrated a significant improvement of homogeneity of printed protein patterns on surfaces larger than 1 cm2 through the control of both the flow rate and the wetting mechanism of the stamp surface during microfluidic inking. Thanks to the reproducibility and integration capabilities provided by microfluidics, we have achieved the printing of three different adhesion proteins in one-step transfer. Selective cell adhesion and cell shape adaptation on the produced patterns were observed, showing the suitability of this approach for producing on-demand large-scale cell microarrays.

Fabrication of biomolecule microarrays for cell immobilization using automated microcontact printing

Biomolecule microarrays are generally produced by conventional microarrayer, i.e., by contact or inkjet printing. Microcontact printing represents an alternative way of deposition of biomolecules on solid supports but even if various biomolecules have been successfully microcontact printed, the production of biomolecule microarrays in routine by microcontact printing remains a challenging task and needs an effective, fast, robust, and low-cost automation process. Here, we describe the production of biomolecule microarrays composed of extracellular matrix protein for the fabrication of cell microarrays by using an automated microcontact printing device. Large scale cell microarrays can be reproducibly obtained by this method.