Hydrophilic Spots Research Articles

How does Thermosensor Desk Measure Membrane Thickness? Tryptophan Fluorescence and Mutagenesis Analysis

The Bacillus subtilis histidine kinase DesK is a 5 transmembrane stretches- thermosensor suited to remodel membrane fluidity when temperature drops below 30 oC. We have recently designed a hybrid nanosensor with one transmembrane domain which is 5 times smaller than the parental protein. This chimerical protein fully retains, in vivo and in vitro, the sensing properties of the parental system and was called Minimal Sensor-DesK (MS-DesK). A recent paper (1) provides evidence that this perfectly simplified system could serve as a model to study a complex biological phenomena. Thus the MS-DesK is used here to study the conversion of a physical stimulus into a biological response that involves a change in the signaling transduction state.The N-terminus of TMS1 contains three hydrophilic aminoacids near the lipid-water interface creating an instability hot spot. We show that this boundary-sensitive motif controls the sensing and transmission activity by mutagenesis and in vitro reconstitution experiments. Accordingly, we hypothesize that membrane thickness is the temperature agent that determines the signaling state of the cold sensor by dictating the hydration level of the meta-stable hydrophilic spot. Tryptophan-labeled peptides corresponding to the sensor (functional and non-functional) transmembrane domain were synthesized and incorporated into membrane vesicles. Fluorescence spectroscopy and Circular Dichroism data of the peptides incorporated in liposomes of varying fatty acid chain length and different melting temperatures is presented.[1] Cybulski LE, Martin M, Mansilla MC, Fernandez A, de Mendoza D. Membrane thickness cue for cold sensing in a bacterium. Curr Biol. 20: 1539-1544, 2010

Passive water control at the surface of a superhydrophobic lichen

Some lichens have a super-hydrophobic upper surface, which repels water drops, keeping the surface dry but probably preventing water uptake. Spore ejection requires water and is most efficient just after rainfall. This study was carried out to investigate how super-hydrophobic lichens manage water uptake and repellence at their fruiting bodies, or podetia. Drops of water were placed onto separate podetia of Cladonia chlorophaea and observed using optical microscopy and cryo-scanning-electron microscopy (cryo-SEM) techniques to determine the structure of podetia and to visualise their interaction with water droplets. SEM and optical microscopy studies revealed that the surface of the podetia was constructed in a three-level structural hierarchy. By cryo-SEM of water-glycerol droplets placed on the upper part of the podetium, pinning of the droplet to specific, hydrophilic spots (pycnidia/apothecia) was observed. The results suggest a mechanism for water uptake, which is highly sophisticated, using surface wettability to generate a passive response to different types of precipitation in a manner similar to the Namib Desert beetle. This mechanism is likely to be found in other organisms as it offers passive but selective water control.

Thermosensor Desk Measures Membrane Thickness

The bacterium Bacillus subtilis adjusts the composition of membrane lipids to cope with temperature variations. The histidine kinase DesK is a five-pass transmembrane thermosensor suited to remodel membrane fluidity in B. subtilis according to temperature. To understand the mechanism of sensing, individual transmembrane segments (TMS) were fused to the cytoplasmic catalytic domain of DesK (DesKC) and the ability to respond to temperature analyzed. Surprisingly, a hybrid TMS composed of 17 aminoacids of the first TMS and the C-terminal 14-residue portion of TM5 fused to DesKC, fully retains in vivo and in vitro the sensing properties of full-length DesK [1]. Besides, when chimerical fusions of DesKC to either TMS1 or TMS5 are expressed in vivo individually in a desK- strain, thermosensing is lost. Nevertheless, when they are co-expressed thermosensing is recovered; suggesting that interactions between TMS1 and TMS5 are needed for signaling.The N-terminus of TMS1 contains three hydrophilic aminoacids near the lipid-water interface creating an instability hot spot. We showed that this boundary-sensitive motif controls the sensing and transmission activity. Accordingly, we hypothesize that membrane thickness is the temperature agent that determines the signaling state of the cold sensor by dictating the hydration level of the meta-stable hydrophilic spot. This hypothesis is supported through biochemical studies including in vitro reconstitution of the MS in liposomes with different chain length.[1] Cybulski LE, Martin M, Mansilla MC, Fernandez A, de Mendoza D. Membrane thickness cue for cold sensing in a bacterium. Curr Biol. 20: 1539-1544, 2010

Membrane Thickness Cue for Cold Sensing in a Bacterium

Thermosensors are ubiquitous integral membrane proteins found in all kinds of life. They are involved in many physiological roles, including membrane remodeling, chemotaxis, touch, and pain [1-3], but, the mechanism by which their transmembrane (TM) domains transmit temperature signals is largely unknown. The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below approximately 30°C [1, 4] providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. Here we show that the multimembrane-spanning domain from DesK can be simplified into a chimerical single-membrane-spanning minimal sensor (MS) that fully retains, in vivo and in vitro, the sensing properties of the parental system. The MS N terminus contains three hydrophilic amino acids near the lipid-water interface creating an instability hot spot. Mutational analysis of this boundary-sensitive beacon revealed that membrane thickness controls the signaling state of the sensor by dictating the hydration level of the metastable hydrophilic spot. Guided by these results we biochemically demonstrated that the MS signal transmission activity is sensitive to bilayer thickness. Membrane thickness could be a general cue for sensing temperature in many organisms.

Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Signal Enhancement of Peptides after Selective Extraction with Polymeric Reverse Micelles

Extraction of peptides by reverse micelle-forming amphiphilic homopolymers and subsequent matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) detection of these peptides in the presence of these polymers can significantly enhance peptide ion signals. Here, the mechanism of this MALDI signal enhancement is investigated. We find that the signal enhancement is caused by coalescence of polymer-peptide conjugates into "hotspots" on the MALDI target. Hotspot formation is observed only on hydrophilic surfaces and not hydrophobic surfaces. With the use of an Anchorchip MALDI target, which contains very small hydrophilic spots surrounded by a larger hydrophobic area, we find that this hotspot formation can be further exploited for ultrasensitive MALDI-MS analyses of peptides and peptide mixtures. MALDI-MS signals can be enhanced by 3-5 orders of magnitude when peptides are extracted by the amphiphilic homopolymers and detected on the Anchorchip MALDI target. This signal enhancement combined with the extraction selectivity of these reverse micelle-forming homopolymers makes these materials promising tools for sensitive detection of peptides in complex mixtures.

Engineering a Thermosensor To Dissect a Transmembrane Signaling System

The DesK-DesR two-component system regulates the order of membrane lipids in the bacterium Bacillus subtilis by controlling the expression of the des gene coding for the delta 5-acyl-lipid desaturase. In this work, we address the process by which DesK transmembrane segments (TMS) transmit temperature signals across the membrane by engineering the 5 TMS domain of the DesK into a single-TMS chimeric sensor. This so-called Minimal Sensor (MS) fully retains in vivo and in vitro the sensing input and transmission output of the parental system. Progressive deletions of TM segments revealed that only the first TM segment (TM1) is essential to regulate the kinase activity. Therefore, our engineered MS combines the N-terminal 17-residue portion of TM1 with the C-terminal 14-residue portion of TM5 which is naturally fused to the cytosolic catalytic domain. The MS N-terminus contains three hydrophilic aminoacids near the lipid-water interface creating an instability hot spot. This boundary-sensitive motif controls the sensing and transmission activity. Accordingly, we hypothesize that membrane thickness is the temperature agent that determines the signaling state of the cold sensor by dictating the hydration level of the meta-stable hydrophilic spot. This hypothesis is supported through the study of the signaling behavior of MS variants purposely constructed.

A thermoresponsive hydrogel poly(N-isopropylacrylamide) micropatterning method using microfluidic techniques

Poly(N-isopropylacrylamide) (PNIPAAm) is a thermoresponsive hydrogel that has been widely used in various biomedical applications, including tissue engineering. Making PNIPAAm into a microscale structure is an effective method of increasing its thermoresponsiveness and modulating surface properties compared to bulk PNIPAAm. The commonly used method of direct photolithography combined with a photomask is challenging in creating pure PNIPAAm patterns smaller than 10 µm. Also, each time when there is a need to change the sizes of resulting PNIPAAm patterns, a new photomask is required. Here, a microfluidically controlled micropatterning method utilizing hydrophilic spots on a hydrophobic substrate was developed to create pure PNIPAAm microstructures. This method enabled the fabrication of pure PNIPAAm microstructures with a wide range of sizes from 70 µm to sub-10 µm out of the same substrate preparation by simply varying the flow speed of the hydrogel precursor solution through a microfluidic channel. Hydrogel microstructures with diameters of 12 to 59% of the hydrophilic pattern diameters were successfully fabricated using this fabrication scheme. The smallest hydrogel pattern fabricated using this method was 2.3 µm in diameter using a 5 µm diameter hydrophilic pattern at a flow speed of 100 mm s−1. This simple and versatile fabrication scheme could be an ideal method for creating large arrays of hydrogel micropatterns with varying sizes.

An Analytical System for Single Nanomaterials: Combination of Capillary Electrophoresis with Raman Spectroscopy or with Scanning Probe Microscopy for Individual Single-Walled Carbon Nanotube Analysis

Nanomaterials continue to attract widespread attention in many scientific and technological fields. The sizes and shapes of nanomaterials determine their physical and chemical properties. We have developed an analytical system for single nanomaterials that combines capillary electrophoresis (CE) with a highly sensitive detection method. In this manuscript, we combined CE with Raman spectroscopy or with scanning probe microscopy (SPM) for the analysis of individual single-walled carbon nanotubes (SWNTs). To combine CE with these detection techniques, we fabricated a fraction collection system that can collect droplets of small volume (<300 nL) in a small hydrophilic spot on a fractionation glass plate. The CE-separated fractions were concentrated by the evaporation of effluent, thus increasing the sensitivity by more than a factor of 10 in the case of Raman spectroscopic analysis. We characterized the fractionated SWNTs by means of Raman spectroscopy and SPM, both of which detected single SWNTs. Raman analysis enabled us to recognize a diameter difference of only 0.02 nm between SWNTs, and it was supposed that the separation by CE occurred based on the diameters of the SWNTs. We also observed a fibrous SWNT structure 1 nm high via SPM, and this structure was thought to be a single SWNT. These combined analytical systems enable the precise separation and characterization of individual SWNTs. We expect that methods developed herein can be applied to the analysis of many nanomaterials, because these methods offer separation and analysis with nanometer-scale precision. The characterization of nanomaterials at the single-compound level will be a necessity as the field of nanomaterials continues to evolve, and these combined methods may become indispensable techniques for the analysis of widely available nanomaterials.

Microscopic Wetting of Mixed Self-assembled Monolayers: A Molecular Dynamics Study

Molecular dynamics simulations are used to study the evolution of the organization of water molecules on the flat surface of well-ordered self-assembled monolayers (SAMs) of eight-carbon alkanethiolate chains bound to a gold substrate, as the character of the surface is finely tuned from completely hydrophobic to completely hydrophilic, and as the level of hydration is increased from submonolayer to the equivalent of about two monolayers of water. The hydrophilicity of the SAM surfaces is increased by randomly replacing methyl-terminated alkanethiolate chains with carboxylic acid-terminated chains. We report on the evolution of the structure of the surfaces of the SAMs, both in the absence and presence of water, and the organization of water molecules and the extent of wetting of the surfaces, as the fraction of hydrophilic groups is increased. The results suggest that on the flat organic surfaces with a small fraction of the hydrophilic components the hydrophilic spots serve as nucleation sites, resulting in the growth of a larger number of (smaller) water droplets compared to the completely hydrophobic surface, whereas on the surfaces with a large fraction of the hydrophilic component the uptake of water proceeds via a water film growing, at first, over the hydrophilic domains and, eventually, bridging over the hydrophobic patches, and spreading out over the entire surface. We discuss the implications of these processes on the properties of the organic aerosols in the atmosphere.

Controlled Pattern Formation in Thin Liquid Layers

We examine the fully nonlinear behavior of a thin liquid film on a hydrophobic/hydrophilic solid support in three dimensions using a phase field model. For flat homogeneous substrates, the stability of thin liquid layers is investigated under the action of gravity. The coarsening process at the solid boundary can be controlled on inhomogeneous substrates. On substrates chemically patterned in an adequate way with hydrophobic and hydrophilic spots, one can obtain stable, regular liquid droplets and even design liquid structures (PACS numbers: 47.54.-r, 68.18.Jk, and 05.70.Np).

Protein Arrays on Patterned Porous Gold Substrates Interrogated with Mass Spectrometry: Detection of Peptides in Plasma

Methyl- and carboxy-terminated self-assembled monolayers (SAMs) were custom-patterned on porous gold substrates with equipment commonly used to print protein arrays, without complex surface chemistry protocols. Proteins were covalently immobilized on hydrophilic carboxy-terminated SAM spots, while the remainder of the surface was superhydrophobic due to the roughened gold surface and the methyl-terminated SAM. The resistance of these patterns to biofouling and the effective containment of MALDI matrix solution within the hydrophilic spot made these surfaces amenable to analyzing protein-peptide binding with mass spectrometry. A model system of the affinity peptides HA, cmyc, and V5 and their corresponding antibodies was used to demonstrate the utility of the patterned porous gold. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectra and images obtained reflected the effective capture of the affinity peptides directly from spiked bovine plasma.

Imbibition by polygonal spreading on microdecorated surfaces

Micropatterned surfaces have been studied extensively as model systems to understand influences of topographic or chemical heterogeneities on wetting phenomena. Such surfaces yield specific wetting or hydrodynamic effects, for example, ultrahydrophobic surfaces, 'fakir' droplets, tunable electrowetting, slip in the presence of surface heterogeneities and so on. In addition, chemical patterns allow control of the locus, size and shape of droplets by pinning the contact lines at predetermined locations. Applications include the design of 'self-cleaning' surfaces and hydrophilic spots to automate the deposition of probes on DNA chips. Here, we discuss wetting on topographically patterned but chemically homogeneous surfaces and demonstrate mechanisms of shape selection during imbibition of the texture. We obtain different deterministic final shapes of the spreading droplets, including octagons, squares, hexagons and circles. The shape selection depends on the topographic features and the liquid through its equilibrium contact angle. Considerations of the dynamics provide a 'shape' diagram that summarizes our observations and suggest rules for a designer's tool box.

Hierarchical roughness optimization for biomimetic superhydrophobic surfaces

Superhydrophobic surfaces should have high contact angles (CA) with water and low contact angle hysteresis (CAH). High CA may be achieved by increasing surface roughness, while in order to have low CAH, superhydrophobic surfaces should be able to form a stable composite interface with air pockets between solid and liquid. Capillary waves, nanodroplets condensation, hydrophilic spots due to chemical surface inhomogeneity, and liquid pressure can destroy the composite interface. These destabilizing factors have different characteristic length scales, so a hierarchical roughness is required to resist them. It is shown that convex rather than concave profile enhances stability, so nanoscale convex bumps should be superimposed over microasperities, in order to pin the liquid–air interface. In addition, the nanoroughness is required to support nanodroplets. The ability of the interface to support high pressure requires high asperity density and size, so it is in conflict with the requirement of low fractional solid–liquid contact area for low CAH and slip length. The new parameter, spacing factor for asperities, is proposed, and requirements for optimum design, which combines conflicting conditions, are formulated and discussed. Remarkably, biological superhydrophobic surfaces satisfy these requirements.

Structured R2R Functionalisation of Polymer Film Surfaces by a Xenon Excimer Lamp

Films of a polyolefin (COC) and polyester (PET) were photo-oxidised using a 172 nm excimer lamp. The irradiation through a mask created hydrophilic spots on the hydrophobic material which are stable for at least several months. Treating the films with diaminoethane (DAE) vapour in a gas flow channel grafts primary amino groups to the exposed areas. Then DNA fragments were coupled to the amines by spotting reaction solutions to the hydrophilic parts of the surface, which served as reaction compartments. This is the basic technology for producing micro-arrays (bio-chips) very efficiently in a roll-to-roll (R2R) approach.

Evaporative Properties and Pinning Strength of Laser-Ablated, Hydrophilic Sites on Lotus-Leaf-like, Nanostructured Surfaces

Wetting, evaporative, and pinning strength properties of hydrophilic sites on superhydrophobic, nanostructured surfaces were examined. Understanding these properties is important for surface characterization and designing features in self-cleaning, lotus-leaf-like surfaces. Laser-ablated, hydrophilic spots between 250 mum and 2 mm in diameter were prepared on silicon nanowire (NW) superhydrophobic surfaces. For larger circumference pinning sites, initial contact angle measurements resemble the contact angle of the surface within the pinning site: 65-69 degrees . As the drop volume is increased, the contact angles approach the contact angle of the NW surface without pinning sites: 171-176 degrees . The behavior of water droplets on the pinning sites is governed by how much of the water droplet is being influenced by the superhydrophobic NW surfaces versus the hydrophilic areas. During the evaporation of sinapic acid solution, drops are pinned by the spots except for the smaller circumference sites. Pinning strengths of the hydrophilic sites are a linear function of the pinning spot circumference. Protein samples prepared and deposited on the pinning sites for analysis by matrix-assisted laser desorption ionization indicate an improvement in sensitivity from that of a standard plate analysis by a factor of 5.

How Wenzel and Cassie Were Wrong

We argue using experimental data that contact lines and not contact areas are important in determining wettability. Three types of two-component surfaces were prepared that contain "spots" in a surrounding field: a hydrophilic spot in a hydrophobic field, a rough spot in a smooth field, and a smooth spot in a rough field. Water contact angles were measured within the spots and with the spot confined to within the contact line of the sessile drop. Spot diameter and contact line diameter were varied. All of the data indicate that contact angle behavior (advancing, receding, and hysteresis) is determined by interactions of the liquid and the solid at the three-phase contact line alone and that the interfacial area within the contact perimeter is irrelevant. The point is made that Wenzel's and Cassie's equations are valid only to the extent that the structure of the contact area reflects the ground state energies of contact lines and the transition states between them.

Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI‐MS

A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a approximately 200-500-microm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-microm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-microl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-microl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS.

Drop impact on chemically structured arrays

We describe recent investigations on the impact behaviour of liquid drops onto chemicallystructured surfaces. The surface patterns were prepared via photochemical attachment ofpolymer molecules with different hydrophilicities using self-assembled monolayers ofbenzophenone bearing silanes. Immobilization of the polymer monolayers was followed byan ablation process to generate a chemical surface pattern. Impact experiments onsystems consisting of very hydrophobic poly(perfluoroalkylethyl)acrylate coatingsand hydrophilic areas show that within certain limitations the water drop hasa strong tendency to reach the hydrophilic spots, even for inclined substrates.Impact experiments of drops on arrays of hydrophilic spots on the backgroundof a perfluorinated polymer show that the drops spontaneously self-centre onthe lithographically generated pattern. The obtained results suggest that theprocess can be used to circumvent some of the current problems in micro-arrayfabrication.

Patterned monolayer/polymer films for analysis of dilute or salt-contaminated protein samples by MALDI-MS.

This paper describes a surface science/mass spectrometry effort to develop and characterize a patterned gold surface that serves as a MALDI sample platform capable of concentrating and purifying proteins. Using microcontact printing, small (200-microm diameter) hydrophilic spots of bare gold or chemically anchored poly(acrylic acid) (PAA) are patterned at 5-mm intervals in a hydrophobic field consisting of a self-assembled monolayer of hexadecanethiol. Building on recent innovations by others, the small hydrophilic spots concentrate the sample to achieve good reproducibility and high sensitivity in the MALDI signal. One of the key features in this work is the combination of the high density of carboxylate groups in PAA with a small spot size to afford both concentration and purification of proteins via ionic interactions. This translates into detection limits for salt-contaminated proteins that are 20-100 times lower (low femtomole) than those reported for previous polymer- or monolayer-modified MALDI probes (using proteins in the 3-15-kDa range). Reflectance FT-IR spectroscopy and ellipsometry were used to determine the amount of protein adsorbed to a PAA-modified sample plate as a function of pH and salt concentration. Amide absorbances in IR spectra correlate well with MALDI-MS signals measured after addition of 2,5-dihydroxybenzoic acid as a matrix.

DNASER. II. Novel surface patterning for biomolecular microarray

A new matrix-integral part of the new DNA microarray instrumentation DNA analyzer (DNASER) is introduced based on a novel DNA patterning on the solid support surface. Such patterning found the way to modify a glass surface for a precise positioning of small droplets of aqueous DNA solutions, without special robots (arrayers), within the boundaries of the modified regions. The physically heterogeneous surface consists of highly hydrophilic spots surrounded by a highly hydrophobic area leading to the surface patterning needed for a DNA microarray: a matrix of hydrophilic spots properly activated for immobilization of oligonucleotides has been fabricated on absolutely passive hydrophobic surface. The optimal efficiency of the above functionalitation technology of a glass-substrate in obtaining DNA microarray was confirmed by the Cy3-dCTP-labeled DNA sample, as shown by charge-coupled device images of the DNASER previously described.