Membrane-mediated capillary electrophoresis: interaction of cationic peptides with bicelles

Electrokinetic capillary chromatography is applied to determine the membrane affinity of peptides using both 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles and DHPC/1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bicelles under controlled conditions. The effect of temperature and the bicelle q value in surface association with cationic peptides is studied. The cationic peptides selected have a well-defined membrane structure (indolicidin), induced secondary structure (melittin, magainin 2), or do not possess classical secondary structure (atrial natriuretic peptide (ANP) 1-28, 4-28, 5-27). Electrokinetic capillary chromatography facilitated by DMPC and DHPC additives provides a rapid means of estimating lipophilicity and screening for peptides that have membrane affinity.     

Membrane-mediated ultrafast restriction digestion and subsequent rapid gel microchip electrophoresis of DNA

Ultrafast, membrane-mediated restriction digestion of DNA molecules followed by rapid gel microchip electrophoresis of the resulting fragments is described. Combination of restriction endonuclease digestion on small pore-size microfibrous membranes with sample loading and electrophoresis analysis in a multilane (up to 96) format resulted in very fast restriction digest based microscale DNA analysis. Complete digestion of several nanogram target DNA was accomplished on the microporous membrane at room temperature just in a few minutes with a single or a combination of various restriction enzymes, using only submicroliter quantities of samples and reagents. The reaction mixture containing membrane also served as sample loading device for the subsequent gel microchip electrophoresis based analysis. This work establishes methods for high-speed, high-throughput DNA analysis, featuring extremely low sample and reagent consumption, and fast restriction digestion in combination with sample loading and rapid gel microchip analysis of the resulting fragments. The entire restriction digestion, sample loading and electrophoresis analysis process required less than 20 min.     

A Guanidyl-Based Bivalent Peptidomimetic Inhibits K-Ras Prenylation and Association with c-Raf

Unusual lipid modification of K-Ras makes Ras-directed cancer therapy a challenging task. Aiming to disrupt electrostatic-driven protein–protein interactions (PPIs) of K-Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure-activity-relationship study resulted in 8 b , in which a biphenyl-based peptidomimetic FTI-277 was attached to a guanidyl-containing gallate moiety through an alkyl linker. Cell-based evaluation demonstrated that 8 b exhibited substantial inhibition of K-Ras processing without apparent interference with Rap-1A processing. Fluorescent imaging showed that 8 b disrupts localization of K-Ras to the plasma membrane and impairs interaction with c-Raf, whereas only FTI-277 was found to be inactive. These results suggest that targeting the PPI interface of K-Ras may provide an alternative method of inhibiting K-Ras.     

Paper-based diagnostic platforms and devices

Paper-based analytical devices have become lately “must have” components in equipment and instrumental designed for point-of-care applications, especially when they are used in tandem with microfluidic platforms. Nowadays, paper-based electrochemical devices (PEDs) represent the first choice in the development of lab-on-a-chip biosensors because of their benefits in biomedical diagnosis in terms of simplicity, affordability, portability, and disposability. Moreover, cellulose is a biodegradable and biocompatible substrate, ideal for building disposable devices for use in remote locations or low-resource settings. Despite their low costs and simplicity, PEDs must face a tough challenge—meeting the affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users criteria. The latest achievements in microfluidic PEDs for clinical diagnosis will be critically discussed, putting emphasis on innovative assay formats and methods for surface modification.     

Effect of microdomain structure on the mechanical behavior of binary blends

The relationship between microphase structure and mechanical response of the binary blends consisting of polystyrene-block-polyisoprene-block-polystyrene copolymer and low molecular weight polystyrene has been investigated. Low molecular weight polystyrene was chosen to obtain uniformly solubilized nano-blends without macrophase separation. The specimens were solution-cast by adding different amounts of homo-polystyrene to acquire different microphase structures. Small angle X-ray scattering(SAXS), transmission electron microscopy(TEM) and atom force microscopy(AFM) have been used to study the microdomain and grain structure. It is observed that the structural changes in d-spacing and grain size on account of different amounts of polystyrene alter the mechanical behavior in both monotonic tensile and cyclic tests. The elastic and the Mullins effects are strongly sensitive to the changes in d-spacing and grain sizes. Moreover, the sample with bi-continuous structure shows the largest tensile strength and Mullins effect. In addition, the Mooneye-Rivlin phenomenological model was used to evaluate and explore the relationship between the polymer topological networks and the rubber elasticity of these styrenic nano-blends.     

Small-angle X-ray scattering studies of microdomain structure in segmented polyurethane elastomers

The small-angle x-ray scattering (SAXS) technique has been used to characterize the detailed microphase structure of two crosslinked segmented polyurethane elastomers. Both copolymers contain trifunctional polypropylene ether triols in the rubbery elastomeric block, but are synthesized with different hard segments: a symmetric 4,4′-diphenylmethane diisocyanate (MDI) chain extended with butanediol (BD); and an 80/20 mixture of asymmetric 2,4-toluene diisocyanate (TDI) and symmetric 2,6-toluene diisocyanate (TDI) chain extended with ethylene glycol (EG). Calculations of SAXS invariants and determinations of deviations from Porod's law are used to examine the degree of phase segregation of the hard- and soft-segment domains. Results show that the overall degree of phase separation is poorer in the asymmetric TDI/EG-based copolymer than in the symmetric MDI/BD-based copolymer. Determination of diffuse phase boundary thicknesses, however, reveals that the domain boundaries are sharper in the asymmetric TDI/EG system. The contrasting morphologies found in the two systems are interpreted in terms of differences in hard-soft segment compatibility, diisocyanate symmetry, and diisocyanate length. Coupled with conformational considerations, this information is used to construct a new model for polyurethane hard-segment microdomain structure. Important features of the model are that it takes into account the effects of hard-segment sequence length distribution and allows for folding of the longer hard-segment sequences back into the hard-segment domain.     

Optoelectrofluidic platforms for chemistry and biology

Extraordinary advances in lab on a chip systems have been made on the basis of the development of micro/nanofluidics and its fusion with other technologies based on electrokinetics and optics. Optoelectrofluidic technology, which has been recently introduced as a new manipulation scheme, allows programmable manipulation of particles or fluids in microenvironments based on optically induced electrokinetics. Herein, the behaviour of particles or fluids can be controlled by inducing or perturbing electric fields on demand in an optical manner, which includes photochemical, photoconductive, and photothermal effects. This elegant scheme of the optoelectrofluidic platform has attracted attention in various fields of science and engineering. A lot of research on optoelectrofluidic manipulation technologies has been reported and the field has advanced rapidly, although some technical hurdles still remain. This review describes recent developments and future perspectives of optoelectrofluidic platforms for chemical and biological applications.     

A hybrid dielectrophoretic and hydrophoretic microchip for particle sorting using integrated prefocusing and sorting steps

This work explores dielectrophoresis (DEP)‐active hydrophoresis in sorting particles and cells. The device consists of prefocusing region and sorting region with great potential to be integrated into advanced lab‐on‐a‐chip bioanalysis devices. Particles or cells can be focused in the prefocusing region and then sorted in the sorting region. The DEP‐active hydrophoretic sorting is not only based on size but also on dielectric properties of the particles or cells of interest without any labelling. A mixture of 3 and 10 μm particles were sorted and collected from corresponding outlets with high separation efficiency. According to the different dielectric properties of viable and nonviable Chinese Hamster Ovary (CHO) cells at the medium conductivity of 0.03 S/m, the viable CHO cells were focused well and sorted from cell sample with a high purity.     

Inhibition of K-Ras4B-plasma membrane association with a membrane microdomain-targeting peptide

The association of K-Ras4B protein with plasma membrane (PM) is required for its signaling activity. Thus, direct inhibition of K-Ras4B–PM interaction could be a potential anti-Ras therapeutic strategy. However, it remains challenging to modulate such protein–PM interaction. Based on Ras isoform-specific PM microdomain localization patterns, we have developed a potent and isoform-selective peptide inhibitor, Memrasin, for detachment of K-Ras4B from the PM. Memrasin is one of the first direct inhibitors of K-Ras4B–PM interaction, and consists of a membrane l_d region-binding sequence derived from the C-terminal region of K-Ras4B and an endosome-escape enhancing motif that can aggregate on membrane. It forms peptide-enriched domains in the l_d region, abrogates the tethering of K-Ras4B to the PM and accordingly impairs Ras signaling activity, thereby efficiently decreasing the viability of several human lung cancer cells in a dose-responsive and K-Ras dependent manner. Memrasin provides a useful tool for exploring the biological function of K-Ras4B on or off the PM and a potential starting point for further development into anti-Ras therapeutics.

A membrane l_d microdomain-targeting hybrid peptide displays potent inhibition effect toward K-Ras4B-plasma membrane interaction and impairs Ras signaling output.     

Microarray platforms for enzymatic and cell-based assays

This tutorial review introduces the uninitiated to the world of microarrays (or so-called chips) and covers a number of basic concepts such as substrates and surfaces, printing and analysis. It then moves on to look at some newer applications of microarray technology, which include enzyme analysis (notably kinases and proteases) as well as the growing enchantment with so-called cell-based microarrays that offer a unique approach to high-throughput cellular analysis. Finally, it looks forwards and highlights future possible trends and directions in the microarray arena.     

Verification of the microdomain model of crystallinity in polyvinyl chloride by electron microscopy

A study of the fine scale microstructure of PVC was carried out using a combination of high resolution transmission electron microscopy and digital image analysis techniques. The images obtained contained a degree of order of the approximate size and distribution as predicted by the microdomain model of crystallinity in PVC. The microdomain model for crystallinity in PVC has been built up from previous studies using various analytical techniques including wide- and small-angle x-ray diffraction and differential scanning calorimetry. Earlier studies using transmission electron microscopy did not find any direct evidence supporting this model. Significant advances in both electron microscope and image processing technology had taken place since the earlier microscopic studies. The TEM imaging and image analysis procedures that have been utilized in this research may be applicable to the imaging of very fine scale ordering in other polymers. © 1996 John Wiley & Sons, Inc.     

Electrical sorting of Caenorhabditis elegans

The nematode (worm) C. elegans is one of the widely studied animal model organisms in biology. It develops through 4 larval stages (L1-L4) in 2 to 3 days before becoming a young adult. Biological assays involving C. elegans frequently require a large number of animals that are appropriately staged and exhibit a similar behaviour. We have developed a new method to synchronize animals that relies on the electrotactic response (electric field-induced motion) of C. elegans to sort them in parallel based on their age, size and phenotype. By using local electric field traps in a microfluidic device, we can efficiently sort worms from a mixed culture in a semi-continuous flow manner (with a minimum throughput of 78 worms per minute per load-run) and obtain synchronized populations of animals. In addition to sorting larvae, our device can also distinguish between young and old adults efficiently. Unlike fluorescent based sorting systems that use active imaging based feedback, this method is passive and automatic and uses the innate behaviour of the worm. Considering that the entire procedure takes only a few minutes to run and is cost-effective, it promises to simplify and accelerate experiments requiring homogeneous cultures of worms as well as to facilitate isolation of mutants that have abnormal electrotaxis. More importantly, our method of isolating and separating worms using locomotion as a defining characteristic promises development of advanced microfluidics-based systems to study the neuronal basis of movement-related defects in worms and facilitate high-throughput chemical screening and drug discovery.     

Influence of a liquid crystalline block on the microdomain structure of block copolymers

We report on the phase behavior and microdomain structure of two types of diblock copolymers containing a liquid crystal (LC) block joined to a flexible coil block. Consideration of the symmetry groups of the liquid crystalline phases and of the block copolymer microdomain structures provides a rationale for predicting the possible types of liquid crystalline block copolymer morphologies. Both previously reported and newly discovered structural types are identified. Possible organizational schemes are developed for the mesogens and periodic disclination defects with respect to the intermaterial dividing surfaces separating the liquid crystalline and flexible coil domains. The first type of copolymer investigated has a rod-like LC block whereas the second type copolymer has a side chain LC block. Five different rod-coil diblocks based on poly(hexyl isocyanate-b-styrene) P(HIC-b-S) were synthesized by anionic polymerization. Wavy lamellae, zig-zag and arrowhead microdomain morphologies corresponding to smectic-C and smectic-O structures were observed depending on the composition. These layered phases have the director (PHIC chain axis) tilted at various orientations with respect to the layer normal. Side-chain LC diblocks based on functionalized poly(isoprene-b-styrene) P(I-b-S) were also investigated. These polymers were synthesized using polymer analogous chemistry from P(I-b-S) precursors. Three different mesogenic groups were attached to the PI blocks: one based on biphenyl benzoate and two based on azobenzene. The microdomain structures found for the functionalized poly(isoprene side-chain LC-b-styrene) P(ILC-b-S) diblocks are typical of traditional coil-coil diblocks (lamellae and cylinders). However, these morphologies possess an additional smectic layering of the mesogens within the microdomains of the LC block. In the case of the rod-coil diblocks, the transformation from an initially isotropic state to the final microphase separated solid state occurs via nematic and then smectic liquid crystalline states, whereas for the side-chain LC-coil cases, the microphase separation transition occurs prior to development of orientational order. The long-range microdomain order of LC block-coil block copolymers can extend over very large distances due to the influence of the orientational ordering of the LC block.     

Correlation between crystallization kinetics and microdomain morphology in block copolymer blends exhibiting confined crystallization

The crystallization kinetics of poly(ethylene oxide) (PEO) blocks in poly(ethylene oxide)‐block‐poly(1,4‐butadiene) (PEO‐b‐PB)/poly(1,4‐butadiene) (PB) blends were previously found to display a one‐to‐one correlation with the microdomain morphology. The distinct correlation was postulated to stem from the homogeneous nucleation‐controlled crystallization in the cylindrical and spherical PEO microdomains, where there existed a direct proportionality between the nucleation rate and the individual domain volume. This criterion was valid for confined crystallization in which the crystallization was spatially restricted within the individual domains. However, it was possibly not applicable to PEO‐b‐PB/PB, in that the melt mesophase was strongly perturbed upon crystallization. Therefore, it may be speculated that the crystal growth front developed in a given microdomain could intrude into the nearby noncrystalline domains, yielding the condition of cooperative crystallization. To establish an unambiguous model system for verifying the existence of microdomain‐tailored kinetics in confined crystallization, we crosslinked amorphous PB blocks in PEO‐b‐PB/PB with a photoinitiated crosslinking reaction to effectively suppress the cooperative crystallization. Small‐angle X‐ray scattering revealed that, in contrast to the noncrosslinked systems, the pre‐existing domain morphology in the melt was retained upon crystallization. The crystallization kinetics in the crosslinked system also exhibited a parallel transition with the morphological transformation, thereby verifying the existence of microdomain‐tailored kinetics in the confined crystallization of block copolymers. Homogeneous nucleation‐controlled crystallizations in cylindrical and spherical morphologies were demonstrated in an isothermal crystallization study in which the corresponding crystallinity developments followed a simple exponential rule not prescribed by conventional spherulitic crystallization. Despite the effective confinement imposed by the crosslinked PB phase, crystallization in the lamellar phase still proceeded through a mechanism analogous to the spherulitic crystallization of homopolymers. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 519–529, 2002; DOI 10.1002/polb.10121     

Fluorine-hydrocarbon separation and microdomain formation in N-(2,3,5,6-Tetrafluoropyridin-4-yl)benzene-diamines

Compounds with both aromatic and perfluoro aromatic moieties often assemble in the form of π-stacks with alternating arene perfluoroarene arrangements. The 1,4 and 1,3-diamines of N-(2,3,5,6-tetrafluoropyridin-4-yl)benzene (1) and (2), C₁₁H₇F₄N₃, however, show a behavior more commonly observed for diblock perfluoro alkyl derivatives: the fluorinated and the hydrocarbon moieties of the molecules segregate from each other and form fluoro and hydrocarbon microdomains with parallel layers of π-stacked tetrafluoropyridine rings and diamino-phenyl moieties. The structural features of the two derivatives are, despite the different substitution patterns and cell settings, strikingly similar. The main feature in the non-fluorinated domains of 1 and 2 is a set of N–H···H and N–H···π interactions that connect the amino groups of four molecules with each other, which are augmented by some weaker C–H···π and C–H···F interactions, but no π–π stacking is observed. The fluoro microdomains in both 1 and 2 are characterized by extremely short interplanar distances between the tetrafluoropyridine rings of only about 3 Å. The molecules in the layers are shifted sideways against each other by half a pyridyl ring thus bringing the fluorine atoms to atop of the aromatic carbon atoms and vice versa.     

Robust control of microdomain orientation in thin films of block copolymers by zone casting

Block copolymers with chemically immiscible segments exhibit a variety of microphase-separated nanostructures on the scale of 10-100 nm. Controlling the orientation of these microphase separated nanostructures is vital in many applications such as lithography, membranes, data storage, and so forth. Typical strategies involve the use of external fields or patterned substrates. Here, we report a robust zone casting technique to achieve highly ordered thin films of block copolymers on centimeter-scale substrates. The robustness of this technique is its powerful control on diverse morphologies and exceptional tolerance on versatility of block copolymer chemistry as well as allowance of a wide spectrum of substrates. We demonstrate that perpendicular orientations with respect to the surface are achieved for block copolymers with both lamellar and cylindrical morphologies by controlling solution casting rate, temperatures, and block copolymer chemical structures. Thin films of both noncrystalline and crystalline block copolymers exhibit excellent orientational order and lateral order. However, the lateral order in the thin films of crystalline block copolymers shows dependence on casting temperature and melting temperature of the crystalline segment. Remarkably, all the ordering is independent of the substrates on which the block copolymer films are cast.     

Natural and artificial ion channels for biosensing platforms

The single-molecule selectivity and specificity of the binding process together with the expected intrinsic gain factor obtained when utilizing flow through a channel have attracted the attention of analytical chemists for two decades. Sensitive and selective ion channel biosensors for high-throughput screening are having an increasing impact on modern medical care, drug screening, environmental monitoring, food safety, and biowarefare control. Even virus antigens can be detected by ion channel biosensors. The study of ion channels and other transmembrane proteins is expected to lead to the development of new medications and therapies for a wide range of illnesses. From the first attempts to use membrane proteins as the receptive part of a sensor, ion channels have been engineered as chemical sensors. Several other types of peptidic or nonpeptidic channels have been investigated. Various gating mechanisms have been implemented in their pores. Three technical problems had to be solved to achieve practical biosensors based on ion channels: the fabrication of stable lipid bilayer membranes, the incorporation of a receptor into such a structure, and the marriage of the modified membrane to a transducer. The current status of these three areas of research, together with typical applications of ion-channel biosensors, are discussed in this review.     

On-chip fluorescence-activated particle counting and sorting system

A fluorescence-activated particle counting and sorting system is developed for lab-on-a-chip applications. This system integrates the microfluidic chip, fluorescence excitation and detection, electronic power switch control, and optical visualization. The automatic sorting function is achieved by electrokinetic flow switching, which is triggered by a pre-set fluorescent threshold. A direct current electric pulse is generated to dispense the fluorescent particles to the collection reservoir. A user-friendly software interface is developed for automatic real-time counting, sorting and visualization. The design of the disposable microfluidic chip is simple and easy for integration. This system represents a promising prototype for development of affordable and portable flow cytometric instruments.