Spreading and segregation of lipids in air-stable lipid microarrays

As a result of heterogeneous spreading of distinct lipids within the same microspots of air-stable lipid microarrays, ganglioside GM1 tends to segregate and thus enrich within the center area of microspots where being predetermined by the quill pin used for array fabrication, as indicated by the binding pattern of fluorescein-cholera toxin subunit B.     

MALDI-TOF MS detection of dilute, volume-limited peptide samples with physiological salt levels

This paper presents a highly efficient sample preparation technique for matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The purpose of the research is to use a conventional MALDI support to directly and conveniently detect sub-nM levels of peptides from volume-limited samples with physiological salt levels. In this new method, highly uniform matrix-nitrocellulose spots with a 500 microm diameter were conveniently generated by direct contact of a capillary tip to a stainless steel MALDI plate. An array of 50 microspots can be blotted from 1 microL matrix-nitrocellulose solution within 1 min. It was found that the addition of high concentration nitrocellulose to the alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution is critical for the formation of microspots. Samples are deposited on top of those microspots and incubated for 3 min. The CHCA-nitrocellulose surface shows a significant peptide binding capability for sub-nM levels of peptide. Restricting the matrix spot diameter to 500 microm gives an analyte enrichment effect because the peptides are confined to a small solid-phase surface area. Selective peptide binding is seen even with >0.15 M salt levels. Loading small aliquots of samples with multiple applications allows low level peptide detection down to 100 pM. Push-pull perfusates collected from the rat striatum were successfully analyzed with the microspot method.     

Monitoring the redox cycle of low-molecular peptides using a modified target plate in MALDI-MS

A new method is being proposed for preparing MALDI target plates with a hydrophobic polymer coating and hydrophilic anchors. The particles of the MALDI matrix were pre-mixed with a poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene] solution prior to their placement on a mass-spectrometric sample support. This technique led to the formation of matrix microspots with a diameter of less than 1 mm inside the polymer. The polymer and matrix concentration as well as the amount of suspension placed on the target plate influenced the size and quality of microspots to a great extent. The sensitivity of the mass-spectrometric analysis was confirmed by obtaining the mass spectra of fmole concentrations of an apomyoglobin tryptic digest. The potential proteomic application of this type of MALDI surface preparation was demonstrated by performing the redox cycle using glutathione and its analogue. All reactions were carried out directly on a MALDI plate, which accommodates low volumes of reagents and prevents sample loss.     

Quantification of the surface diffusion of tripodal binding motifs on graphene using scanning electrochemical microscopy

The surface diffusion of a cobalt bis-terpyridine, Co(tpy)(2)-containing tripodal compound (1·2PF(6)), designed to noncovalently adsorb to graphene through three pyrene moieties, has been studied by scanning electrochemical microscopy (SECM) on single-layer graphene (SLG). An initial boundary approach was designed in which picoliter droplets (radii ~15-50 μm) of the tripodal compound were deposited on an SLG electrode, yielding microspots in which a monolayer of the tripodal molecules is initially confined. The time evolution of the electrochemical activity of these spots was detected at the aqueous phosphate buffer/SLG interface by SECM, in both generation/collection (G/C) and feedback modes. The tripodal compound microspots exhibit differential reactivity with respect to the underlying graphene substrate in two different electrochemical processes. For example, during the oxygen reduction reaction, adsorbed 1·2PF(6) tripodal molecules generate more H(2)O(2) than the bare graphene surface. This product was detected with spatial and temporal resolution using the SECM tip. The tripodal compound also mediates the oxidation of a Fe(II) species, generated at the SECM tip, under conditions in which SLG shows slow interfacial charge transfer. In each case, SECM images, obtained at increasing times, show a gradual decrease in the electrochemical response due to radial diffusion of the adsorbed molecules outward from the microspots onto the unfunctionalized areas of the SLG surface. This response was fit to a simple surface diffusion model, which yielded excellent agreement between the two experiments for the effective diffusion coefficients: D(eff) = 1.6 (±0.9) × 10(-9) cm(2)/s and D(eff) = 1.5 (±0.6) × 10(-9) cm(2)/s for G/C and feedback modes, respectively. Control experiments ruled out alternative explanations for the observed behavior, such as deactivation of the Co(II/III) species or of the SLG, and verified that the molecules do not diffuse when confined to obstructed areas. The noncovalent nature of the surface functionalization, together with the surface reactivity and mobility of these molecules, provides a means to couple the superior electronic properties of graphene to compounds with enhanced electrochemical performance, a key step toward developing dynamic electrode surfaces for sensing, electrocatalysis, and electronic applications.     

Development of microspot multi-analyte ratiometric immunoassay using dual fluorescent-labelled antibodies

Radioisotopes are progressively being replaced by non-isotopic labels as reagent markers for immunoassay purposes. The reasons for this development include an avoidance of the practical and environmental disadvantages associated with the use of radioisotopes and the need for higher assay sensitivity. A further stimulus is the increasing requirement to measure multiple analytes simultaneously in the same sample. This paper outlines the general principles underlying multi-analyte “microspot” immunoassay methodology. It relies on the measurement of the ratio of fluorescent signals from individual antibody “microspots” forming a microspot array on a suitable plastic surface. In principle, the methodology is capable of measuring 10⁶ different substances in a sample volume of 100 μl.     

细胞无机化学中值得研究的问题

在探索生命奥秘的过程中,生物无机化学研究由分子层次上升到细胞层次是一个必然的趋势,也是解决实际问题的需要.细胞无机化学研究在细胞生命体系中的无机化学反应和探索无机物对生命过程调节或干预的作用和机理,是探索生命体系复杂性研究的重要部分.细胞是保留完整生命活动特征的最小单位,存在周期、分化和受激等状态的不同.从化学的观点,细胞是一个严密设计的分子有序组装体,为一个多靶分子系统,细胞应答表现为由相关反应组合成的复杂过程.细胞无机化学研究包括无机物种在细胞膜上的结合和随后发生的膜结构和功能改变、跨细胞膜和跨生物组织屏障的转运和细胞代谢、细胞中无机化学反应同细胞信号系统的偶联、无机离子与自由基的相互代谢关系以及细胞-无机物固相的相互作用等方面.本文对当前细胞无机化学研究的重点问题进行了讨论.     

Membrane protein microarrays

This paper describes the fabrication of microarrays consisting of G protein-coupled receptors (GPCRs) on surfaces coated with gamma-aminopropylsilane (GAPS). Microspots of model membranes on GAPS-coated surfaces were observed to have several desired properties-high mechanical stability, long range lateral fluidity, and a thickness corresponding to a lipid bilayer in the bulk of the microspot. GPCR arrays were obtained by printing membrane preparations containing GPCRs using a quill-pin printer. To demonstrate specific binding of ligands, arrays presenting neurotensin (NTR1), adrenergic (beta1), and dopamine (D1) receptors were treated with fluorescently labeled neurotensin (BT-NT). Fluorescence images revealed binding only to microspots corresponding to the neurotensin receptor; this specificity was further demonstrated by the inhibition of binding in the presence of excess unlabeled neurotensin. The ability of GPCR arrays to enable selectivity studies between the different subtypes of a receptor was examined by printing arrays consisting of three subtypes of the adrenergic receptor: beta1, beta2, and alpha2A. When treated with fluorescently labeled CGP 12177, a cognate antagonist analogue specific to beta-adrenergic receptors, binding was only observed to microspots of the beta1 and beta2 receptors. Furthermore, binding of labeled CGP 12177 was inhibited when the arrays were incubated with solutions also containing ICI 118551, and in a manner consistent with the higher affinity of ICI 118551 for the beta2 receptor relative to that for the beta1 receptor. The ability to estimate binding affinities of compounds using GPCR arrays was examined using a competitive binding assay with BT-NT and unlabeled neurotensin on NTR1 arrays. The estimated IC(50) value (2 nM) for neurotensin is in agreement with the literature; this agreement suggests that the receptor -G protein complex is preserved in the microspot. This first ever demonstration of direct pin-printing of membrane proteins and ligand-binding assays thereof fills a significant void in protein microchip technology--the lack of practical microarray-based methods for membrane proteins.     

Immunoassay and other ligand assays: present status and future trends.

Immunoassay and other ligand assays have made a major impact on medical research and diagnosis since the first modern (radioisotopically-based) methods emerged. These ubiquitous microanalytic techniques are broadly classifiable as first generation (generally of "competitive" design, e.g., radioimmunoassay), and second generation (generally "noncompetitive," and relying on nonisotopic labels) these (often described as "ultrasensitive") being distinguished by dramatic improvements in sensitivity and performance time. A third generation is now in prospect (based on microarrays of antibody microspots) capable of ultrasensitive determination of hundreds of analytes in a drop of blood. Analogous technology (based on oligonucleotide arrays) is under intensive development for DNA analysis. Array technologies are likely to transform diagnostic medicine in the next decade.     

Flow‐Through Synthesis on Teflon‐Patterned Paper To Produce Peptide Arrays for Cell‐Based Assays

A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell‐based assays. Solvent‐repelling barriers made of Teflon‐impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow‐through syntheses on paper. The confinement and flow‐through mixing significantly improves the peptide yield and simplifies the automation of this synthesis. The synthesis of 100 peptides ranging from 7 to 14 amino acids in length gave over 60 % purity for the majority of the peptides (>95 % yield per coupling/deprotection cycle). The resulting peptide arrays were used in cell‐based screening to identify 14 potent bioactive peptides that support the adhesion or proliferation of breast cancer cells in a 3D environment. In the future, this technology could be used for the screening of more complex phenotypic responses, such as cell migration or differentiation.     

Polymer microfluidic chips for electrochemical and biochemical analyses

Our recent developments concerning the fabrication of polymer microchips and their applications for biochemical analyses are reviewed. We first describe two methods of fabrication of polymer microfluidic chips, namely UV-laser photoablation and plasma etching that are well suited for the prototyping and mass fabrication of microchannel networks with integrated microelectrodes. These microanalytical systems can be coupled with various detection means including mass spectrometry, and their applications in capillary electrophoresis are presented here. We also present how UV laser photoablation can be used for the patterning of biomolecules on polymer surfaces for generating two-dimensional arrays of microspots to carry out affinity assays. Finally, the use of the microchips for the development of fast affinity and immunological assays with electrochemical detection is presented, demonstrating the potential of these polymer microchips for medical diagnostics and drug discovery.     

贫PbI2基体的胶体量子点固体用于高效红外太阳能电池(英文)

胶体量子点(CQD)具有优异的红外光吸收能力和光谱可调特性,是用于制备高效太阳能电池最有前途的红外光电材料之一。然而,以醋酸铵(AA)为添加剂的液相配体交换会导致CQD固体中产生宽带隙PbI₂基质,其将作为电荷传输势垒,在很大程度上影响了CQD太阳能电池(CQDSC)中载流子的提取,从而影响了光伏性能。本文报道利用二甲基碘化铵(DMAI)调节CQD配体交换过程,使载流子在CQD固体中的传输势垒大幅降低。通过对CQD固体进行全面的表征和理论计算,充分揭示了DMAI和CQD之间的相互作用。结果表明,通过DMAI调节CQD配体交换过程,使CQD固体均匀堆积,提高了载流子输运性能,并且陷阱辅助复合受到显著抑制。因此,CQDSC器件中的载流子提取得到了大幅提高,能量转换效率(PCE)比用AA制备的CQDSC器件提高了17.8%。此工作为调控CQD表面化学特性提供了新的研究思路,并为降低CQD固体中载流子输运的势垒提供了可行的方法。     

Ion transfer across ion-exchange membranes with homogeneous and heterogeneous surfaces

A homogeneous (AMX) and two heterogeneous (MA-40, MA-41) anion-exchange membranes, as well as a heterogeneous cation-exchange membrane (MK-40), are studied by electronic scanning microscopy, voltammetry, and chronopotentiometry. The presence of conducting and nonconducting regions on the surfaces of heterogeneous membranes is established by means of element analysis. The fraction of conducting regions is found by an image treatment. The surface of the AMX membrane was partially coated with microspots of a paint to make it heterogeneous (AMXheter). Voltammetric and chronopotentiometric measurements for AMX, AMXheter, and MA-41 membranes in NaCl solutions are carried out and the pH changes in the solution layers adjoining to these membranes are recorded. Analysis of obtained results shows that the concentration polarization of studied membranes characterized by the potential drop and the rate of water dissociation at the interface is mainly governed by the properties of their surfaces. It is found that the local limiting current density through conducting regions of a heterogeneous membrane is several times higher than the average limiting current through a homogeneous membrane.     

A multicellular spheroid formation and extraction chip using removable cell trapping barriers

This paper presents a multicellular spheroid chip capable of forming and extracting three-dimensional (3D) spheroids using removable cell trapping barriers. Compared to the conventional macro-scale spheroid formation methods, including spinning, hanging-drop, and liquid-overlay methods, the recent micro-scale spheroid chips have the advantage of forming smaller spheroids with better uniformity. The recent micro spheroid chips, however, have difficulties in extracting the spheroids due to fixed cell trapping barriers. The present spheroid chip, having two PDMS layers, uses removable cell trapping barriers, thereby making it easy to form and extract uniform and small-sized spheroids. We have designed, fabricated and characterized a 4 × 1 spheroid chip, where membrane cell trapping barriers are inflated at a pressure of 50 kPa for spheroid formation and are deflated at zero gauge pressure for simple and safe extraction of the spheroids formed. In this experimental study, the cell suspension of non-small lung cancer cells, H1650, is supplied to the fabricated spheroid chip in the pressure range 145-155 Pa. The fabricated spheroid chips collect the cancer cells in the cell trapping regions from the cell suspension at a concentration of 2 × 10(6) ml(-1), thus forming uniform 3D spheroids with a diameter of 197.2 ± 11.7 μm, after 24 h incubation at 5% CO(2) and 37°C environment. After the removal of the cell trapping barriers, the spheroids formed were extracted through the outlet ports at a cell inlet pressure of 5 kPa. The cells in the extracted spheroids showed a viability of 80.3 ± 7.7%. The present spheroid chip offers a simple and effective method of obtaining uniform and small-sized 3D spheroids for the next stage of cell-based biomedical research, such as gene expression analysis and spheroid inoculation in animal models.     

ESI–MS method for in vitro investigation of skin penetration by copper–amino acid complexes: from an emulsion through a model membrane

Copper can be found in many cosmetic formulations, mainly as complexes with peptides, hydroxyacids or amino acids. The main reason that the usage of this element in this context is still increasing is its beneficial biochemical activity, although the mechanism that enables its complexes to permeate through skin barriers is largely unknown. The ability of copper complexes with amino acids to penetrate through the stratum corneum and participate in copper ion transport processes is key to their cosmetic and pharmaceutical activities. The penetration process was studied in vitro in a model system, a Franz diffusion cell with a liposome membrane, where a liquid crystalline system with physicochemical properties similar to those of the intercellular cement of stratum corneum was used to model the skin barrier. The influences of various ligands on the model membrane migration rate of copper ions was studied, and the results highlighted the crucial roles of metal ion complex structure and stability in this process.     

The progress and perspective of strategies to improve tumor penetration of nanomedicines

Tumor penetration is important fo r effectively tumor targeting drug delivery.Recently,many researches are published to overcome the barriers that restrict tumor penetration and improve drug delivery efficiency.In the mini review,we first analyzed the barriers influence the tumor penetration,including tumor microenvironment barriers,nanoparticle properties,and interaction barriers between tumor and nanoparticles.To overcome the barrier,several strategies are developed,including modulating tumor microenvironment,changing particle size,transcytosis enabled tumor penetration,cell penetrating peptide modification and overcoming binding site barrier,which could effectively improve tumor penetration,and finally enhance tumor treatment outcome.     

Molecular Basis of the Dynamic Strength of the Sialyl Lewis X—Selectin Interaction

The selectins are Ca²⁺‐dependent cell adhesion molecules that facilitate the initial attachment of leukocytes to the vascular endothelium by binding to a carbohydrate moiety as exemplified by the tetrasaccharide, sialyl Lewis X (sLeX). An important property of the selectin‐sLeX interaction is its ability to withstand the hydrodynamic force of the blood flow. Herein, we used single‐molecule dynamic force spectroscopy (DFS) to identify the molecular determinants within sLeX that give rise to the dynamic properties of the selectin/sLeX interaction. Our atomic force microscopy (AFM) measurements revealed that the unbinding of the selectin/sLeX complexes involves overcoming at least two activation barriers. The inner barrier, which determines the dynamic response of the complex at high forces, is governed by the interaction between the Fuc residue of sLeX and a Ca²⁺ ion chelated to the lectin domain of the selectin molecule, whereas the outer activation barrier can be attributed to interactions involving the sialic acid residue of sLeX. Due to their steep inner activation barriers, the selectin‐sLeX complexes are less sensitive to high pulling forces. Hence, besides its contribution to the bond energy, the Ca²⁺ ion also grants the selectin–sLeX complexes a tensile strength that is crucial for the selectin‐mediated rolling of leukocytes.     

Reactivity of gas barrier membranes filled with reactive particulates

Gas barrier plastic films and structures capable of removing a permeating solute via irreversible chemical reactions in the polymer matrix are investigated in applications of active packaging for controlling contained environments. To significantly reduce permeation rates through such active barriers, high barrier reactivity is required. When the reactive species take the form of fine particulates sparingly dispersed in the matrix, the reactivity of a composite membrane is affected by a lower frequency of collisions of dissolved gas molecules with the distributed reactive particles. The mean size of the particles and their volume fraction in the matrix are identified as the critical factors affecting the overall reactivity of uniformly filled composite barriers. A two-scale coarse-grained model of reaction-diffusion in a matrix is developed by introducing a unit cell approach to estimate the reaction rate upon collision with the particles and by choosing the unit length of diffusive molecular displacement equal to the mean diameter of the reactive particles to describe diffusion across the barrier and statistics of molecule-particle collisions. The corresponding method for evaluating the barrier reactivity is described. The effects of the system parameters on the effective gas transport rates across the reactive composite barriers including polymer nanocomposite barriers with reactive additives are quantified.     

Current status of automotive fuel cells for sustainable transport

Automotive proton-exchange membrane fuel cells (PEMFCs) have finally reached a state of technological readiness where several major automotive companies are commercially leasing and selling fuel cell electric vehicles, including Toyota, Honda, and Hyundai. These now claim vehicle speed and acceleration, refueling time, driving range, and durability that rival conventional internal combustion engines and in most cases outperform battery electric vehicles. The residual challenges and areas of improvement which remain for PEMFCs are performance at high current density, durability, and cost. These are expected to be resolved over the coming decade while hydrogen infrastructure needs to become widely available. Here, we briefly discuss the status of automotive PEMFCs, misconceptions about the barriers that platinum usage creates, and the remaining hurdles for the technology to become broadly accepted and implemented.     

The effect of octahedral tilting on proton binding sites and transition states in pseudo-cubic perovskite oxides

Proton conducting oxide ceramics have shown potential for use in fuel cell technologies. Understanding the energy pathways for proton conduction could help us design more efficient fuel cell materials. This paper describes how octahedral tilting affects the relative energies of proton binding sites, transition states, and conduction pathways in cubic and pseudo-cubic perovskites. First, the structure for cubic and pseudo-cubic forms of BaTiO(3), BaZrO(3), CaTiO(3), and CaZrO(3), is found. Even when cubic symmetry is enforced, CaTiO(3), and CaZrO(3) exhibit octahedral tilting distortions characteristic of orthorhombic phases while BaTiO(3) and BaZrO(3) remain undistorted. Octahedral tilting gives rise to proton binding sites facilitating inter- and intra-octahedral proton transfer while the proton binding sites of undistorted perovskites facilitate only intra-octahedral proton transfer. The nudged elastic band method is used to find minimum energy paths between the proton binding sites. As distortions increase, inter-octahedral proton transfer barriers decrease while intra-octahedral proton transfer barriers increase. Concurrently, rotational barriers from oxygens facilitating inter-octahedral proton transfer increase while rotational barriers from oxygens facilitating intra-octahedral proton transfer decrease. Intra-octahedral transfer is the rate-limiting step to the lowest energy extended proton conduction pathway in all the perovskites considered.     

Recent developments in catalyst-related PEM fuel cell durability

Cost and durability remain the two major barriers to the widespread commercialization of polymer electrolyte membrane fuel cell (PEMFC)-based power systems, especially for the most impactful but challenging fuel cell electric vehicle (FCEV) application. Commercial FCEVs are now on the road; however, their PEMFC systems do not meet the cost targets established by the U.S. Department of Energy, primarily due to the high platinum loading needed on the cathode to achieve the requisite performance and lifetime. While the activities of a number of commercial Pt-based alloy cathode catalysts exceed the beginning-of-life (BOL) targets, these activities, and the overall cathode performance, degrade via a variety of mechanisms described herein. Degradation is mitigated in current FCEVs by utilizing a cathode catalyst with a lower BOL activity (e.g., much lower transition metal alloy content and larger BOL nanoparticle size), necessitating higher catalyst loadings, and through the utilization of system controls that avoid conditions known to exacerbate degradation processes, such as limiting the fuel cell stack voltage range. The design and development of active and robust materials and eliminating the need for vehicle mitigation strategies would greatly simplify the operating system, allowing for greater transient operation, avoiding large hybridization, and curtailing of fuel cell power. Although system mitigation strategies have provided the near-term pathway for FCEV commercialization, material-specific solutions are required to further reduce costs and improve operability and efficiency. Future material developments should focus on stabilization of the electrode structure and minimization of the catalyst particle susceptibility to dissolution caused by oxide formation and reduction over PEMFC cathode-relevant operating potentials plus minimization of support corrosion. Ex situ accelerated stress tests have provided insight into the processes responsible for material and performance degradation and will continue to provide useful information on the relative stability of materials and benchmarks for robust and stable materials-based solutions not requiring system mitigation strategies to achieve adequate lifetime.