Ion transport across vesicle bilayers mediated by an artificial channel compound

A polymer of an isocyanide [R-N=C<]_n (R? = 4′-benzo-18-crown-6) whose pendant crown ether side chains form molecular channels, is able to transport cobalt ions across the bilayer membrane of positively charged vesicles.     

Detection of single ion channel activity on a chip using tethered bilayer membranes

Membrane-bound ion channels are promising biological receptors since they allow for the stochastic detection of analytes at high sensitivity. For stochastic sensing, it is necessary to measure the ion currents associated with single ion channel opening and closing events. However, this calls for stability, high reproducibility, and long lifetimes. A critical issue to overcome is the low stability of the ion channel environment, that is, the bilayer membrane. A promising technique to surmount this is to connect the lower part of the membrane to a surface forming a tethered bilayer membrane. By reconstituting the synthetic ion channel, gramicidin A, into a tethered bilayer as part of a microchip design, we have been able to record the activity of single ion channels. The observed activity was compared with that obtained by a conventional electrophysiology method, tip dipping, to confirm its authenticity. These findings allow for the construction of stable biosensors based on ion channels and provide a novel technique for the characterization of ion channel activity.     

Interaction of cholesterol with artificial bilayer lipid membrane system and development of an electrochemical sensor

Interaction of Cholesterol with the bilayer arrangement of phospholipid molecules was studied using electrochemical impedance spectroscopy in Sodium Chloride (NaCl) bath solutions. The membrane resistance (R_m) was decreased from 3.35 GΩ in 1.0 M NaCl bath to 0.756 GΩ in 0.01 M NaCl bath. The cholesterol molecules were found to penetrate into Bilayer Lipid Membrane (BLM) and fluidized the BLM phase. Due to fluidization, the membrane resistance was decreased. The fluidization effect of cholesterol was dependent on the concentration of bath solutions. In 1.0 M NaCl bath solution, the membrane was stable up to 200 µM concentration of cholesterol. With the addition of cholesterol in NaCl bath solutions, the membrane capacitance was increased. An impedimetric sensor was developed based on the membrane resistance in the presence of cholesterol at various concentrations. The detection limit of cholesterol by impedimetric sensor was dependent on the concentration of NaCl in the bath.     

Quantification of UV-induced cyclobutane pyrimidine dimers using an oligonucleotide chip assay

A lesion-specific enzyme-induced DNA strand break assay was developed for an oligonucleotide chip for the determination of UVB-induced cyclobutane pyrimidine dimers (CPDs). A 20-mer of fluorophore-labeled and biotinylated oligonucleotide was immobilized on the chip. CPDs in DNA on the chip were formed by UVB irradiation (312 nm). T4 endonuclease V (T4N5) was used to excise the CPD site as T4N5 sensitively and specifically detects CPDs. The fluorophore-labeled DNA fragments were detected by a laser-induced fluorescence (LIF) detection system. The number of CPDs induced by UVB was determined based on a mathematical equation obtained from a predetermined calibration curve. The yield of UVB-induced CPDs was 1.73 CPDs per megabase per (kJ/m²). The reliability of this value was proved by its similarity to reference values obtained from gel electrophoresis. The developed assay has strong potential to quantify most kinds of UV-induced DNA lesions.     

Adsorption behavior of microbes on a QCM chip modified with an artificial siderophore-Fe3+ complex

Three hydroxamate-type artificial siderophores with terminal NH(2) groups, tris[2-{3-(N-acyl-N-hydroxamino)propylamido}propyl]aminomethane (1-3, acyl-R group = Me, Et, and Ph, respectively), and their Fe(3+) complexes, 4-6, were prepared. The stability constant (log β) of 4 was estimated to be about 31 by its EDTA titration. The biological activities of 4-6 for Microbacterium flavescens, which is a hydroxamate-type siderophore, auxotrophic gram-positive microbe, clearly indicated that they permeated the cell membrane depending on their terminal bulky acyl-R groups. These artificial siderophore complexes, 4-6, were modified on Au electrode surfaces with the terminal NH(2) group (4-6/Au). The surface modification of 4-6 was confirmed by several electrochemical measurements. The quartz crystal microbalance (QCM) chips were also modified with 4-6. Microbe adsorption measurements using these modified QCM chips for M. flavescens, Pseudomonas putida, and Eschrichia coli were performed. The QCM chips have the ability to adsorb microbes selectively as a result of the differences in the interactions between the structures of Fe(3+)-artificial siderophore complexes and their receptors or binding proteins within the cell membrane.     

Site-specific protein immobilization in a microfluidic chip channel via an IEF-gelation process

A novel strategy for site-specific protein immobilization via combining chip IEF with low-temperature sol-gel technology, called IEF-GEL here, in the channel of a modified poly(methyl methacrylate) (PMMA) microfluidic chip is proposed in this work. The IEF-GEL process involves firstly IEF for homogeneously dissolved protein in PBS containing alumina sol and carrier ampholyte with prearranged pH gradient, and then gelation locally for protein encapsulation. The process and feasibility of proposed IEF-GEL were investigated by EOF measurements, fluorescence microscopic photography, Raman spectrum and further demonstrated by glucose oxidase (GOx) reactors integrated with end-column electrochemical detection. Site-controllable immobilization of protein was realized in a 30 mm long microfluidic chip channel by the strategy to create a approximately 1.7 mm concentrated FITC-BSA band, which leads to great improvement of the elute peak shape, accomplished with remarkably increased sensitivity, approximately 20 times higher than that without IEF-GEL treatment to GOx reactors. The kinetic response of GOx after IEF-GEL treatment was also investigated. The proposed system holds the advantages of IEF and low-temperature sol-gel technologies, i.e. concentrating the protein to be focused and retaining the biological activity for the gel-embedded protein, thus realizes site-specific immobilization of low-concentration protein at nL volume level.     

Automatic transwell assay by an EIS cell chip to monitor cell migration

Here an EIS (electrochemical impedance spectroscopy) biochip to detect cell migration is demonstrated. This biochip has been inspired by a traditional transwell assay/modified Boyden chamber and consists of two compartments separated by a porous membrane. This structure (PDMS-based) is aligned to EIS sensors. Cells are seeded in the upper chamber through microfluidic channels. During migration cells go through the pores of the membrane and get in touch with the electrodes that detect migrated cells. The performance of our cell-chip was tested by investigating the migratory ability of hepatocellular carcinoma (HCC) cells as a function of microenvironment. For this purpose we challenged HCC cells to migrate on different extra-cellular matrix (ECM) components including laminin 1, collagen IV and laminin 5. The results reveal that our cell chip provides reliable results that consistently overlap with those obtained with traditional standardized Boyden chambers. Thus, we demonstrate a new, easy tool to study cell migration and to perform automatic assays. This approach is easier and faster than traditional transwell assays and can be suitable for high-throughput studies in drug discovery applications.     

Microfluidic polymer chip integrated with an ISFET detector for cationic surfactant assay in dental rinses.

A cationic surfactant ion-selective field-effect transistor (cationic surfactant-ISFET) has been developed based on the tetraphenylborate derivative known as sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate. The cationic surfactant-ISFET shows an almost Nernstian response to tetradecyldimethylbenzylammonium chloride (Zephiramine) over a concentration range between 1.0 x 10(-6) M and 1.0 x 10(-3) M, with a slope of 58.5 +/- 1.7 mV/decade. The cationic surfactant-ISFET can be used over a range of pH values, between pH 3 and 9. The cationic surfactant-ISFET shows excellent selectivity for Zephiramine over small inorganic cations, but shows similar selectivity for other cationic surfactants, such as hexadecyltrimethylammonium and stearyltrimethylammonium ions. A microfluidic polymer chip was integrated with the cationic surfactant-ISFET, and this was fabricated using polystyrene plates and stainless wires as a template for the channel. Cationic surfactant-ISFETs used in a batch system and microchips integrated with cationic surfactant-ISFETs showed very similar performance in terms of low detection limits, slope sensitivity and the stability of the potential response. The microfluidic polymer chip was then applied to the determination of cationic surfactants in dental rinses.     

Noncovalent immobilized artificial membrane chromatography, an improved method for describing peptide-lipid bilayer interactions.

A promising approach in assessing hydrophobic peptide-membrane interactions is the use of reversed-phase high-performance liquid chromatography. The present study describes the preparation and properties of a noncovalent immobilized artificial membrane (noncovalent IAM) stationary phase. The noncovalent IAM phase was prepared by coating the C18 chains of a reversed-phase HPLC column with the phospholipid ditetradecanoyl-sn-glycero-3-phosphocholine. Lipid coating was achieved by pumping a lipid solution in water-2-propanol through the column. The formation of a bilayer-like structure on the chromatographic surface was confirmed by calculating the phospholipid surface density of the stationary phase. The surface density was determined to be approximately 1.95 mumol m-2, which is close to that of lipid vesicles. The coating was found to be stable in chromatographic elution systems containing less than 35% of acetonitrile. Employing this new technique, we determined interaction parameters of a set of helical antibacterial magainin-2-amide peptides with pairwise substitutions of adjacent amino acids by their D-enatiomers. The results demonstrate that the chromatographic retention behavior of peptides on noncovalent IAM stationary phase shows an excellent correlation with lipid affinities to phospholipid vesicles.     

Fluorescence-based assay with enzyme amplification on a micro-flow immunosensor chip for monitoring coplanar polychlorinated biphenyls

Detection of pollutants is of significant importance for environmental protection. However, conventional monitoring methods are often time-consuming, and require expensive equipments. Biosensors based on enzyme linked immunosorbent assay (ELISA) provide an alternative method to conventional ones. In this research, the reduction in the size of ELISA utilizing micro-chemical reaction is described in a micro-flow immunosensor chip. The immunosensor chips were fabricated by micro-electromechanical system (MEMS) technology. The quantitative determination of coplanar polychlorinated biphenyls (Co-PCBs) was performed by using a micro-flow immunosensor chip. Polystyrene beads were used as the solid substrate for the immobilization of Co-PCB antibody. The antibody-immobilized beads were introduced into the flow channel. As a competitive ELISA, sample solution mixed with horseradish peroxidase (HRP) conjugated antigen, and non-HRP conjugated antigen was allowed to react in the flow channel. After the antigen-antibody reaction, addition of phosphate buffer solution containing hydrogen peroxide and the fluorogenic substrate produced a fluorescent dye, which was monitored with the resulting change in the fluorescence intensity. By using our micro-flow immunosensor chip, it was possible to determine the sensing range of Co-PCB derivatives up to 0.1 ppt in 30 s. This immunosensor chip had a wide linear range for Co-PCB detection from 0.1 pg/ml to 1.0 μg/ml. The regression analysis provided the correlation coefficients of r = 0.982−0.964 with good reproducibility and precision. In a series of five measurements with immunosensor chips prepared with a new batch of antibody-immobilized polystyrene beads, a relative standard deviation of 21.3% was obtained. Our immunosensor chip design reported here has the potential to be implemented to several different detection methodologies for numerous analytes.     

Molecular dynamics simulation of an archaeal lipid bilayer with sodium chloride

We have performed molecular dynamics simulations of a bilayer formed by the synthetic archaeal lipid, diphytanyl phosphatidylcholine, in NaCl electrolyte solution at four different concentrations (0-4 M) to investigate how structural and dynamic properties of the model archaeal membrane are changed due to the ionic strength of the solution. The archaeal lipid bilayer shows minor changes in their physical properties, indicating the unusual high stability of the membrane against salt, though small reductions of molecular area and lateral diffusion of the lipid are detected at the highest electrolyte concentration of 4 M. Sodium ions penetrate to the ether-rich region, where the ions are likely bound to the ether oxygen in the sn-1 chain rather than to that in the sn-2 chain. The observed salt bridges among two or three neighboring lipids account for the small reduction in the molecular area. The bound ions together with the counter (chloride) ions give rise to a diffusive electric double layer; as a result, the membrane dipole potential is slightly increased with increasing NaCl concentration.     

Examination of the role of intestinal fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay

Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein - intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles.     

Design, synthesis and functional analysis of dansylated polytheonamide mimic: an artificial Peptide ion channel

We report herein the design, total synthesis, and functional analysis of a novel artificial ion channel molecule, designated as dansylated polytheonamide mimic (3). The channel 3 was designed based on an exceptionally potent cytotoxin, polytheonamide B (1). Our strategy for the development of synthetic ion channels, which could be easily derivatized for various functions, involved two key features. First, the structure of 1 was simplified by replacing many of nonproteinogenic amino acid residues which required multistep synthesis by commercially available amino acids while retaining those residues necessary for folding. It significantly reduced the number of synthetic steps and facilitated a practical chemical construction of 3. Second, the introduction of propargyl glycine at residue 44 enabled facile installation of dansyl group as a reporter of the membrane localization of 3. Application of a newly designed protective group strategy provided efficient construction of the 37 amino acid sequence of residues 12-48 through one automatic solid-phase peptide synthesis. After peptide cleavage from the resin, 3 was synthesized via dansyl group introduction and one fragment-coupling reaction with residues 1-11, followed by the global deprotection. The simplified mimic 3 exhibited potent cytotoxicity toward p388 mouse leukemia cells (IC(50) = 12 nM), effectively induced ion transport across the lipid bilayers of liposomes, and displayed H(+) and Na(+) ion channel activities. Because of its simplified yet functional scaffold structure with a potential for diversification, our rationally designed ion channel molecule should be useful as a novel platform for developing various cytotoxic channel molecules with additional desired functions.     

Cytokine assay on a cellular chip by combining collagen gel embedded culture with scanning electrochemical microscopy

A novel cytokine assay has been designed using a cellular chip by combining a collagen gel embedded cell culture technique with scanning electrochemical microscopy-enzyme linked immunosorbent assay (SECM-ELISA). An array of cell-collagen gel mixture (2 μL) was spotted on an antibody-coated chip and incubated for 0.5-24 h. The very small trace amounts of cytokines produced by the activated leukocytes on the chip were effectively entrapped within the collagen gel matrix, and these were collected with the immobilized antibodies on the chip. The chip was further treated with horseradish peroxidase (HRP)-labeled antibodies via the sandwich method after removing the cell-collagen gel spots from the chip. Scanning electrochemical microscopy (SECM) was used to quantitatively evaluate the cytokines from the activated leukocytes produced on the chip, and the SECM images were obtained to visualize the position and concentration of IL-1β secreted from THP-1 and HL-60 cell lines at concentration levels of 10-350 pg mL⁻¹. Based on the chemiluminescence method, the sensitivity of the cytokine assay system in combination with SECM-ELISA is comparable to that of the marketed cytokine assay kit; further, the sample volume required for a single assay is drastically reduced.     

Immobilizing single lipid and channel molecules in artificial lipid bilayers with annexin A5

The effects of annexin A5 on the lateral diffusion of single-molecule lipids and single-molecule proteins were studied in an artificial lipid bilayer membrane. Annexin A5 is a member of the annexin superfamily, which binds preferentially to anionic phospholipids in a Ca2+-dependent manner. In this report, we were able to directly monitor single BODIPY 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and ryanodine receptor type 2 (RyR2) labeled with Cy5 molecules in lipid bilayers containing phosphatidylserine (PS) by using fluorescence microscopy. The diffusion coefficients were calculated at various annexin A5 concentrations. The diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 in the absence of annexin A5 were 4.81 x 10(-8) cm(2)/s and 2.13 x 10(-8) cm(2)/s, respectively. In the presence of 1 microM annexin A5, the diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 were 2.2 x 10(-10) cm(2)/s and 9.5 x 10(-11) cm(2)/s, respectively. Overall, 1 microM of annexin A5 was sufficient to induce a 200-fold decrease in the lateral diffusion coefficient. Additionally, we performed electrophysiological examinations and determined that annexin A5 has little effect on the function of RyR2. This means that annexin A5 can be used to immobilize RyR2 in a lipid bilayer when imaging and analyzing RyR2.     

Unimolecular artificial transmembrane channel with terminal dihydrogen phosphate groups showing transport selectivity for ammonium

A new artificial transmembrane channel molecule bearing dihydrogen phosphate groups has been synthesized.The terminal dihydrogen phosphate groups enable the channel to be highly negatively charged at both ends of the channel structures.The artificial channel could incorporate into the lipid bilayer efficiently under low concentration.The channel displays high NH4+/K+selectivity due to the electrostatic interaction and hydrogen bonding between NH4+and the terminal dihydrogen phosphate groups.