Hierarchical Superhydrophobic Device to Concentrate and Precisely Localize Water-Soluble Analytes: A Route to Environmental Analysis.

An efficient superhydrophobic concentrator is developed using a hierarchical superhydrophobic surface on which the evaporation of a sessile droplet (6 µL) drives the nonvolatile elements it contains on a predefined micrometric analytical surface (pedestal of 80 µm diameter). This hierarchical silicon surface exhibits a surface texture made of etched nanopillars and consists of micropillars and guiding lines, arranged in radial symmetry around the central pedestal. The guiding lines ensure the overall convergence of the sessile droplet toward the central pedestal during evaporation. The nanopillar texturing induced a delay in the Cassie-Baxter to Wenzel regime transition, until the edge of the droplet reaches the periphery of the pedestal. Experiments performed with polymer microparticles suspended in ultrapure water or with DNA molecules solubilized in ultrapure water at sub-fM concentrations demonstrated that the totality of the nonvolatile elements in the liquid microvolume is delivered on or close to the pedestal area, in a very reproducible manner. The very high concentration capacity of the device enabled the discrimination of the degree of purity of ultrapure water samples from different origins. The concentrator also turned out to be functional for raw water samples, opening possible applications to environmental analysis.

Integration of solid-state nanopores into a functional device designed for electrical and optical cross-monitoring.

We present a new strategy for fabricating a silicon nanopore device allowing straightforward fluidic integration and electrical as well as optical monitoring. The device presents nanopores of diameters 10 nm to 160 nm, and could therefore be used to obtain solvent-free free-standing lipid bilayers from small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV). The silicon chip fabrication process only requires front side processing of a silicon-on-insulator (SOI) substrate. A polydimethylsiloxane (PDMS) microfluidic interface is assembled on the silicon chip for fluidic handling and electrical addressing. We detail the electrical specifications of our device and some perspectives showing that the use of an SOI substrate is a convenient way to reduce the electrical noise in a silicon nanopore device without the need of a photolitographic patterned passivation layer. We then demonstrate simultaneous electrical and optical monitoring by capturing negatively charged fluorescent nanoparticles. Finally, in the perspective of solvent-free free-standing lipid bilayers, we show that incubation of SUV results in a drastic increase of the device electrical resistance, which is likely due to the formation of a free-standing lipid bilayer sealing the nanopores. Graphical abstract ᅟ.

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.

Indentation of living cells by AFM tips may not be what we thought!

The models used to calculate Young's moduli from atomic force microscopy (AFM) force curves consider the shape of the indentation. It is then assumed that the geometry of the indentation is identical to the geometry of the indenter, which has been verified for hard materials (E > 1 MPa). Based on this assumption, the force curves calculated by these models, for the same object with a given Young's modulus, are different if the indenter geometry is different. On the contrary, we observe experimentally that the force curves recorded on soft living cells, with pyramidal, spherical, or tipless indenters, are almost similar. This indicates that this basic assumption on the indentation geometry does not work for soft materials (E of the order of 5 kPa or less). This means that, in this case, the shape of the indentation is therefore different from the shape of the indenter. Indentation of living cells by AFM is not what we thought!

Cell Aggregate Assembly through Microengineering for Functional Tissue Emergence.

Compared to cell suspensions or monolayers, 3D cell aggregates provide cellular interactions organized in space and heterogeneity that better resume the real organization of native tissues. They represent powerful tools to narrow down the gap between in vitro and in vivo models, thanks to their self-evolving capabilities. Recent strategies have demonstrated their potential as building blocks to generate microtissues. Developing specific methodologies capable of organizing these cell aggregates into 3D architectures and environments has become essential to convert them into functional microtissues adapted for regenerative medicine or pharmaceutical screening purposes. Although the techniques for producing individual cell aggregates have been on the market for over a decade, the methodology for engineering functional tissues starting from them is still a young and quickly evolving field of research. In this review, we first present a panorama of emerging cell aggregates microfabrication and assembly technologies. We further discuss the perspectives opened in the establishment of functional tissues with a specific focus on controlled architecture and heterogeneity to favor cell differentiation and proliferation.

Atomic force microscopy-single-molecule force spectroscopy unveils GPCR cell surface architecture.

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. Despite considerable insights into their pharmacology, the GPCR architecture at the cell surface still remains largely unexplored. Herein, we present the specific unfolding of different GPCRs at the surface of living mammalian cells by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). Mathematical analysis of the GPCR unfolding distances at resting state revealed the presence of different receptor populations relying on distinct oligomeric states which are receptor-specific and receptor expression-dependent. Moreover, we show that the oligomer size dictates the receptor spatial organization with nanoclusters of high-order oligomers while lower-order complexes spread over the whole cell surface. Finally, the receptor activity reshapes both the oligomeric populations and their spatial arrangement. These results add an additional level of complexity to the GPCR pharmacology until now considered to arise from a single receptor population at the cell surface.

AFM dendritips functionalized with molecular probes specific to cell wall polysaccharides as a tool to investigate cell surface structure and organization.

The yeast cell wall is composed of mannoproteins, ß-1,3/ß-1, 6-glucans and chitin. Each of these components has technological properties that are relevant for industrial and medical applications. To address issues related to cell wall structure and alteration in response to stress or conditioning processes, AFM dendritips were functionalized with biomolecules that are specific for each of the wall components, which was wheat germ agglutinin (WGA) for chitin, concanavalin A (ConA) for mannans and anti-ß-1,3/anti-ß-1,6-glucan antibodies for ß-1,3/ß-1,6-glucans. Binding specificity of these biomolecules were validated using penta-N-acetylchitopentaose, α-mannans, laminarin (short ß-1,3-glucan chain) and gentiobiose (2 glucose units linked in ß 1→6) immobilized on epoxy glass slides. Dynamic force spectroscopy was employed to obtain kinetic and thermodynamic information on the intermolecular interaction of the binary complexes using the model of Friddle-Noy-de Yoreo. Using this model, transition state distance xt, dissociate rate koff and the lowest force (feq ) required to break the intermolecular bond of the complexes were approximated. These functionalized dendritips were then used to probe the yeast cell surface treated with a bacterial protease. As expected, this treatment, which removed the outer layer of the cell wall, gave accessibility to the inner layer composed of ß-glucans. Likewise, bud scars were nicely localized using AFM dendritip bearing the WGA probe. To conclude, these functionalized AFM dendritips constitute a new toolbox that can be used to investigate cell surface structure and organization in response to a wide arrays of cultures and process conditions.

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.

Dendrimeric coating of glass slides for sensitive DNA microarrays analysis.

Successful use and reliability of microarray technology is highly dependent on several factors, including surface chemistry parameters and accessibility of cDNA targets to the DNA probes fixed onto the surface. Here, we show that functionalisation of glass slides with homemade dendrimers allow production of more sensitive and reliable DNA microarrays. The dendrimers are nanometric structures of size-controlled diameter with aldehyde function at their periphery. Covalent attachment of these spherical reactive chemical structures on amino-silanised glass slides generates a reactive approximately 100 A layer onto which amino-modified DNA probes are covalently bound. This new grafting chemistry leads to the formation of uniform and homogenous spots. More over, probe concentration before spotting could be reduced from 0.2 to 0.02 mg/ml with PCR products and from 20 to 5 micro M with 70mer oligonucleotides without affecting signal intensities after hybridisation with Cy3- and Cy5-labelled targets. More interestingly, while the binding capacity of captured probes on dendrimer-activated glass surface (named dendrislides) is roughly similar to other functionalised glass slides from commercial sources, detection sensitivity was 2-fold higher than with other available DNA microarrays. This detection limit was estimated to 0.1 pM of cDNA targets. Altogether, these features make dendrimer-activated slides ideal for manufacturing cost-effective DNA arrays applicable for gene expression and detection of mutations.

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 MacroStamp(TM) bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStamp(TM) 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.

Aryldiazomethane derivatives as reagents for site specific labeling of nucleic acids at phosphate.

An efficient and direct labeling method based on direct alkylation of nucleic acids at phosphates by aryldiazomethane derivatives is described.

Hybridisation of N4-methylcytosine-containing amplicons on DNA microarrays.

5'-Cy5-labelled PCR amplicons containing the analogue base, N(4)-methylcytosine, instead of cytosines were compared in microarray hybridisation experiments with the corresponding amplicons containing the canonical set of bases, with respect to the intensity of the fluorescence signal obtained, and cross hybridisation to non-corresponding probes. In general, higher hybridisation temperatures resulted in reduced signal intensities, particularly in the case of the N(4)-methylcytosine containing amplicons. At the lower hybridisation temperatures tested (40 °C, 30 °C), these modified amplicons gave about equal or stronger fluorescence signal than the corresponding regular amplicons. With the two GC-richest amplicons tested, in one instance the N(4)-methylated target gave a dramatically higher signal intensity than the unmodified amplicon, interpreted as reflecting the reduced formation of hairpin structures in the target sequence, due to the lower thermodynamic stability of the G:N(4)-methylC base pair, making the target more accessible, while in the other case no hybridisation was observed with either version of the amplicon, probably due to interference from a G-tetrad structure. Both for the regular and the N(4)-methylated amplicons, no significant cross hybridisation was seen in these experiments.

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.

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-microm 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.

Microstructured liposome array.

Conversion of a DNA chip to a nanocapsule array was performed by grafting on a liposome an oligonucleotide complementary to an oligonucleotide bound to the array. Each liposome may be loaded by a soluble molecule or may present a hydrophobic or amphiphilic molecule inserted in its wall. To detect liposomes on the chip, we used fluorescent dyes encapsulated in the liposome internal volume or fluorescent lipids. We observed that an oligonucleotide-grafted liposome containing a defined dye specifically accumulated on the area where its complementary oligonucleotide had been spotted on the array. The virtually unlimited amount of addresses allows the specific binding of large amounts of liposomes in one single batch.

Biotin-phenyldiazomethane conjugates as labeling reagents at phosphate in mono and polynucleotides.

Molecules 2-5 that include in their structure a biotin moiety as detectable unit and differently substituted phenyl diazo functions as reactive group were prepared as reagents for labeling the phosphate group in mono and polynucleotides. These molecules were shown to react selectively and quantitatively with the model nucleotide 3'-UMP. They were used successfully in the labeling step of DNA and RNA analysis using high-density DNA-chips (or microarrays) technology.

Aryldiazomethanes for universal labeling of nucleic acids and analysis on DNA chips.

DNA and RNA labeling and detection are key steps in nucleic acid-based technologies, used in medical research and molecular diagnostics. We report here the synthesis, reactivity, and potential of a new type of labeling molecule, m-(N-Biotinoylamino)phenylmethyldiazomethane (m-BioPMDAM), that reacts selectively and efficiently with phosphates in nucleotide monomers, oligonucleotides, DNA, and RNA. This molecule contains a biotin as detectable unit and a diazomethyl function as reactive moiety. We demonstrate that this label fulfills the requirements of stability, solubility, reactivity, and selectivity for hybridization-based analysis and especially for detection on high-density DNA chips.