Proton transport into a tethered bilayer lipid membrane

Tethered bilayer lipid membranes (tBLMs) are increasingly used to study biological membranes, membrane proteins and a variety of related topics. A tBLM is formed by binding a lipid bilayer to a metal surface (usually gold) via a hydrophilic tether (usually an ethyleneoxy chain). In this report we present an electrochemical study on ubiquinone in a tBLM which has provided insights into the properties of this hydrophilic layer, which has a very limited capability of storing and releasing protons. It is concluded that the often observed decrease in tBLM resistance upon addition of ionophores (or protonophores) could be due to the penetration of ions (or protons) into the membrane rather than transport through the membrane.     

Tethered bilayer lipid membranes based on monolayers of thiolipids mixed with a complementary dilution molecule. 1. Incorporation of channel peptides

Tethered bilayer lipid membranes (tBLMs) are described based on the self-assembly of a monolayer on template stripped gold of an archea analogue thiolipid, 2,3-di-o-phytanyl-sn-glycerol-1-tetraethylene glycol-d,l-alpha-lipoic acid ester lipid (DPTL), and a newly designed dilution molecule, tetraethylene glycol-d,l-alpha-lipoic acid ester (TEGL). The tBLM is completed by fusion of liposomes made from a mixture of diphytanoylphosphatidyl choline (DPhyPC), cholesterol, and 1,2-diphytanoyl-sn-glycero-3-phosphate (DPhyPG) in a molar ratio of 6:3:1. Melittin and gramicidin are incorporated into these tBLMs as shown by surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) studies. Ionic conductivity at 0 V vs Ag|AgCl, 3 M KCl, measured by EIS measurements are comparable to the results obtained by other research groups. Admittance plots as a function of potential are discussed on a qualitative basis in terms of the kinetics of ion transport through the channels.     

Monte Carlo simulation of ion transport in ion mobility spectrometry

A program for simulation of ion trajectories in ion mobility spectrometry (IMS) instruments has been developed and incorporated into SIMION 7.0 [Int. J. Mass Spectrom. 200 (2000) 3–25]. Simulations were based on elastic collisions between ions and gas particles and conducted for an IMS drift tube. The program was validated by comparing the reduced mobility of helium ions derived from the simulation with the experimental data for helium ions in neon drift gas in low electric fields. Typical IMS parameters, including pressure, temperature, and flow rate of the drift gas were taken into account in the simulations. The program demonstrates capabilities of generating IMS spectra and predicting ion transport efficiency and separating ions. For the IMS drift tube studied, a correlation between imperfection of the electric field distribution and low resolution has been observed.     

Voltammetric study on ion transport across a bilayer lipid membrane in the presence of a hydrophobic ion or an ionophore

This review describes voltammetric studies on ion transport from one aqueous phase (W1) to another (W2) across a bilayer lipid membrane (BLM) containing a hydrophobic ion, valinomycin (Val) or gramicidin A (GA). In particular, the ion transport mechanisms are discussed in terms of the distribution of a pair of ions between aqueous and BLM phases. By addition of a small amount of hydrophobic ion into W1 and/or W2 containing a hydrophilic salt as a supporting electrolyte, the hydrophobic ion was distributed into the BLM with the counter ion to maintain electroneutrality within the BLM phase. It was found that the counter ion was transferred between W1 and W2 across the BLM by applying a membrane potential. Facilitated transport of alkali ions across a BLM containing Val as an ion carrier compound, could be interpreted by considering not only the formation of the alkali metal ion–Val complex but also the distribution of both the objective cation and the counter ion. In the case of addition of GA as a channel-forming compound into the BLM, the facilitated transport of alkali ions across the BLM depended on the ionic species of the counter ions. It was discovered that the influence of the counter ion on the facilitated transport of alkali ions across the BLM could be explained in terms of the hydrophobicity and the ionic radius of the counter ion.     

Characterizing the transmission properties of an ion funnel

The characteristic features of ion transmission properties in a radio-frequency (RF) electric-field driven ion funnel using the SIMION ion trajectory simulation package is presented. A user program applying the Douglas ion-neutral collisional drag coefficient model is incorporated to properly account for the ion focusing and transport effect of the background gas under the effect of the driving RF and a superimposed DC field. The simulated m/z transmission window compares favorably with the experimental results reported by Smith et al. RF amplitude and pressure dependence of experimentally observed m/z transmission windows are also examined, and an approximated effective potential model based on Gerlich's equation is proposed to interpret the low-m/z cutoff behavior. A modified ion funnel configuration is described.     

Ion trajectory simulation for electrode configurations with arbitrary geometries

A multi-particle ion trajectory simulation program ITSIM 6.0 is described, which is capable of ion trajectory simulations for electrode configurations with arbitrary geometries. The electrode structures are input from a 3D drawing program AutoCAD and the electric field is calculated using a 3D field solver COMSOL. The program CreatePot acts as interface between the field solver and ITSIM 6.0. It converts the calculated electric field into a field array file readable by ITSIM 6.0 and ion trajectories are calculated by solving Newton's equation using Runge-Kutta integration methods. The accuracy of the field calculation is discussed for the ideal quadrupole ion trap in terms of applied mesh density. Electric fields of several different types of devices with 3D geometry are simulated, including ion transport through an ion optical system as a function of pressure. Ion spatial distributions, including the storage of positively charged ions only and simultaneous storage of positively/negatively charged ions in commercial linear ion traps with various geometries, are investigated using different trapping modes. Inelastic collisions and collision induced dissociation modeled using RRKM theory are studied, with emphasis on the fragmentation of n-butylbenzene inside an ideal quadrupole ion trap. The mass spectrum of 1,3-dichlorobenzene is simulated for the rectilinear ion trap device and good agreement is observed between the simulated and the experimental mass spectra. Collisional cooling using helium at different pressures is found to affect mass resolution in the rectilinear ion trap.     

Monte Carlo simulation of ion transport in non-linear ion mobility spectrometry

A program for Monte Carlo simulation of ion transport in non-linear ion mobility spectrometry, also known as field asymmetric ion mobility spectrometry (FAIMS) or differential mobility spectrometry (DMS), has been developed. Simulations are based on elastic collisions between the ions and the gas particles, and take into account the effects of flow dynamics and asymmetric electric fields. Using this program, the separation and diffusion of the ions moving in a planar DMS filtration gap are demonstrated. Ion focusing in a cylindrical filtration gap is also confirmed. A characteristic compensation voltage is found to provide insight for understanding separation in non-linear ion mobility spectrometry. The simulation program is used to study the characteristics of non-linear ion mobility spectrometry, the effect of the carrier gas flow, and the dependence of the compensation voltage and nonlinear mobility coefficient (α) on the applied asymmetric electric field.     

Tethered bilayer lipid membranes on mixed self-assembled monolayers of a novel anchoring thiol: impact of the anchoring thiol density on bilayer formation

Tethered bilayer lipid membranes (tBLMs) are designed on mixed self-assembled monolayers (SAMs) of a novel synthetic anchoring thiol, 2,3-di-o-palmitoylglycerol-1-tetraethylene glycol mercaptopropanoic acid ester (TEG-DP), and a new short dilution thiol molecule, tetraethylene glycol mercaptopropanoic acid ester (TEG). tBLM formation was accomplished by self-directed fusion of small unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. The influence of the dilution of the anchoring thiol molecule in the SAM on the vesicle fusion process and on the properties of the resulting tBLMs is studied. It is observed by quartz crystal microbalance that vesicle fusion is a one-step process for a pure TEG-DP SAM as well as for mixed SAMs containing a high concentration of the anchoring thiol. However, upon dilution of the anchoring thiol to moderate concentrations, this process is decelerated and possibly follows a pathway different from that observed on a pure TEG-DP SAM. Electrochemical impedance spectroscopy is used to qualitatively correlate the composition of the SAM to the electrical properties of the tBLM. In this paper we also delineate the necessity of a critical concentration of this anchoring TEG-DP thiol as a requisite for inducing the fusion of vesicles to form a tBLM.     

The Impact of an Anchoring Layer on the Formation of Tethered Bilayer Lipid Membranes on Silver Substrates

Tethered bilayer lipid membranes (tBLMs) have been known as stable and versatile experimental platforms for protein–membrane interaction studies. In this work, the assembly of functional tBLMs on silver substrates and the effect of the molecular chain-length of backfiller molecules on their properties were investigated. The following backfillers 3-mercapto-1-propanol (3M1P), 4-mercapto-1-butanol (4M1B), 6-mercapto-1-hexanol (6M1H), and 9-mercapto-1-nonanol (9M1N) mixed with the molecular anchor WC14 (20-tetradecyloxy-3,6,9,12,15,18,22 heptaoxahexatricontane-1-thiol) were used to form self-assembled monolayers (SAMs) on silver, which influenced a fusion of multilamellar vesicles and the formation of tBLMs. Spectroscopic analysis by SERS and RAIRS has shown that by using different-length backfiller molecules, it is possible to control WC14 anchor molecules orientation on the surface. An introduction of increasingly longer surface backfillers in the mixed SAM may be related to the increasing SAMs molecular order and more vertical orientation of WC14 at both the hydrophilic ethylenoxide segment and the hydrophobic lipid bilayer anchoring alkane chains. Since no clustering of WC14 alkane chains, which is deleterious for tBLM integrity, was observed on dry samples, the suitability of mixed-component SAMs for subsequent tBLM formation was further interrogated by electrochemical impedance spectroscopy (EIS). EIS showed the arrangement of well-insulating tBLMs if 3M1P was used as a backfiller. An increase in the length of the backfiller led to increased defectiveness of tBLMs. Despite variable defectiveness, all tBLMs responded to the pore-forming cholesterol-dependent cytolysin, vaginolysin in a manner consistent with the functional reconstitution of the toxin into phospholipid bilayer. This experiment demonstrates the biological relevance of tBLMs assembled on silver surfaces and indicates their utility as biosensing elements for the detection of pore-forming toxins in liquid samples.     

Realistic modeling of ion cloud motion in a Fourier transform ion cyclotron resonance cell by use of a particle-in-cell approach

Using a 'Particle-In-Cell' approach taken from plasma physics we have developed a new three-dimensional (3D) parallel computer code that today yields the highest possible accuracy of ion trajectory calculations in electromagnetic fields. This approach incorporates coulombic ion-ion and ion-image charge interactions into the calculation. The accuracy is achieved through the implementation of an improved algorithm (the so-called Boris algorithm) that mathematically eliminates cyclotron motion in a magnetic field from digital equations for ion motion dynamics. It facilitates the calculation of the cyclotron motion without numerical errors. At every time-step in the simulation the electric potential inside the cell is calculated by direct solution of Poisson's equation. Calculations are performed on a computational grid with up to 128 x 128 x 128 nodes using a fast Fourier transform algorithm. The ion populations in these simulations ranged from 1000 up to 1,000,000 ions. A maximum of 3,000,000 time-steps were employed in the ion trajectory calculations. This corresponds to an experimental detection time-scale of seconds. In addition to the ion trajectories integral time-domain signals and mass spectra were calculated. The phenomena observed include phase locking of particular m/z ions (high-resolution regime) inside larger ion clouds. A focus was placed on behavior of a cloud of ions of a single m/z value to understand the nature of Fourier transform ion cyclotron resonance (FTICR) resolution and mass accuracy in selected ion mode detection. The behavior of two and three ion clouds of different but close m/z was investigated as well. Peak coalescence effects were observed in both cases. Very complicated ion cloud dynamics in the case of three ion clouds was demonstrated. It was found that magnetic field does not influence phase locking for a cloud of ions of a single m/z. The ion cloud evolution time-scale is inversely proportional to magnetic field. The number of ions needed for peak coalescence depends quadratically on the magnetic field.     

Formation and finite element analysis of tethered bilayer lipid structures

Rapid solvent exchange of an ethanolic solution of diphytanoyl phosphatidylcholine (DPhyPC) in the presence of a mixed self-assembled monolayer (SAM) [thiolipid/β-mercaptoethanol (βME) (3/7 mol/mol) on Au] shows a transition from densely packed tethered bilayer lipid membranes [(dp)tBLMs], to loosely packed tethered bilayer lipid membranes [(lp)tBLMs], and tethered bilayer liposome nanoparticles (tBLNs) with decreasing DPhyPC concentration. The tethered lipidic constructs in the aqueous medium were analyzed by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Finite element analysis (FEA) was applied to interpret spectral EIS features without referring to equivalent circuit modeling. Using structural data obtained earlier from neutron reflectometry and dielectric constants of lipid bilayers, we reproduced experimentally observed features of the electrochemical impedance (EI) spectra of complex surface constructs involving small pinhole defects, large membrane-free patches, and bound liposomes. We demonstrated by FEA that highly insulating (dp)tBLMs with low-defect density exhibit EI spectra in the shape of a perfect semicircle with or without low-frequency upward "tails" in the Cole-Cole representation. Such EI spectra were observed at DPhyPC concentrations of >5 × 10(-3) mol L(-1). While AFM was not able to visualize very small lateral defects in such films, EI spectra unambiguously signaled their presence by increased low frequency "tails". Using FEA we demonstrate that films with large diameter visible defects (>25 nm by AFM) produce EI spectral features consisting of two semicircles of comparable size. Such films were typically obtained at DPhyPC concentrations of <5 × 10(-3) mol L(-1). At DPhyPC concentrations of <1.0 × 10(-3) mol L(-1) the planar bilayer structures were replaced by ellipsoidal liposomes with diameters ranging from 50 to 500 nm as observed in AFM images. Despite the distinct surface morphology change, the EI curves exhibited two semicircle spectral features typical for the large size defects in planar tBLMs. FEA revealed that, to account for these EI features for bound liposome systems (50-500 nm diameter), one needs to assume much lower tBLM conductivities of the submembrane space, which separates the electrode surface and the phospholipid bilayer. Alternatively, FEA indicates that such features may also be observed on composite surfaces containing both bound liposomes and patches of planar bilayers. Triple semicircular features, observed in some of the experimental EI curves, were attributed to an increased complexity of the real tBLMs. The modeling demonstrated that such features are typical for heterogeneous tBLM surfaces containing large patches of different defectiveness levels. By integrating AFM, EIS, and FEA data, our work provides diagnostic criteria allowing the precise characterization of the properties and the morphology of surface supported bilayer systems.     

Artificial ion channel formed by cucurbit[n]uril derivatives with a carbonyl group fringed portal reminiscent of the selectivity filter of K+ channels

Novel artificial ion channels (1 and 2) based on CB[n] (n = 6 and 5, respectively) synthetic receptors with carbonyl-fringed portals (diameter 3.9 and 2.4 A, respectively) can transport proton and alkali metal ions across a lipid membrane with ion selectivity. Fluorometric experiments using large unilamellar vesicles showed that 1 mediates proton transport across the membranes, which can be blocked by a neurotransmitter, acetylcholine, reminiscent of the blocking of the K+ channels by polyamines. The alkali metal ion transport activity of 1 follows the order of Li+ > Cs+ approximately Rb+ > K+ > Na+, which is opposite to the binding affinity of CB[6] toward alkali metal ions. On the other hand, the transport activity of 2 follows the order of Li+ > Na+, which is also opposite to the binding affinity of 2 toward these metal ions, but virtually no transport was observed for K+, Rb+, and Cs+. It is presumably because the carbonyl-fringed portal size of 2 (diameter 2.4 A) is smaller than the diameters of these alkali metal ions. To determine the transport mechanism, voltage-clamp experiments on planar bilayer lipid membranes were carried out. The experiments showed that a single-channel current of 1 for Cs+ transport is approximately 5 pA, which corresponds to an ion flux of approximately 3 x 107 ions/s. These results are consistent with an ion channel mechanism. Not only the structural resemblance to the selectivity filter of K+ channels but also the remarkable ion selectivity makes this model system unique.     

Kinetic considerations of ion selectivity coefficients of ion-selective electrodes based on neutral carriers

Ion selectivity coefficients of ion-selective electrodes based on neutral carriers are described by means of a mixed potential model of ion transport reactions at the aqueous solution/ion-sensitive membrane interface. The decrease in ion selectivity can be explained by the deviations from the equilibrium conditions, which arise from the ionic partial current across the interface, but the proposed correspondence of the exchange current density of ion transfer reactions with the ion selectivity coefficients is rationalized only for certain conditions of the kinetic parameters. The ion selectivity for liquid membrane transport is discussed starting from three different rate-determining steps. It is shown that the potentiometric selectivities of ion-selective electrodes and the transport selectivities are correlated when the ionic transfer across the aqueous solution/ membrane interface is fast compared with the complex ion transport through the membrane. The significance of a kinetic approach for the design of neutral carriers for ion-selective electrodes is stressed.     

Kinetic energy effects in an ion ensemble subjected to mass-selective isolation and resonance excitation: A simulation study

A simulation study is described of the behaviour of ions confined in a quadrupole ion trap during each of two separate operations of a tandem mass spectrometric experiment. The two operations are those of mass-selective ion isolation and mass-selective resonance excitation to the point of ion ejection from the ion trap. The method of mass-selective ion isolation simulated is that of consecutive ion isolation. Simulation data indicate that the collisional history of the ions prior to the isolation process can greatly influence the degree to which ions survive this process. Simulation data for mass-selective resonance ejection are compared with experimental data obtained with a Finnigan-MAT ion trap mass spectrometer. In each operation, the facility with which ions absorb energy from the field within the ion trap, whether this field is derived from the R.F. drive potential or a supplementary potential, can determine the extent to which ions are retained within the ion trap during the two mass-selective operations described.     

A study of the transport of ions against their concentration gradient across ion-exchange membranes using the network method

The network method has been used to analyze the conditions that favour the uphill transport across ion-exchange membranes. A model for the Nernst-Planck-Poisson equations describing the ionic transport in such system is proposed, including the Donnan equilibrium relations at the membrane/solution interfaces. With this model and the electric circuit simulation program PSPICE, the transient response of the system under open circuit conditions (I=0) and the response of the system subject to an applied potential difference are simulated. The ionic concentrations and electric potential profiles, as well as the electric current density, the ionic fluxes and the charge density, have been obtained as a function of time.     

Incorporation of inhomogeneous ion diffusion coefficients into kinetic lattice grand canonical monte carlo simulations and application to ion current calculations in a simple model ion channel

To deal with inhomogeneous diffusion coefficients of ions without altering the lattice spacing in the kinetic lattice grand canonical Monte Carlo (KLGCMC) simulation, an algorithm that incorporates diffusion coefficient variation into move probabilities is proposed and implemented into KLGCMC calculations. Using this algorithm, the KLGCMC simulation method is applied to the calculation of ion currents in a simple model ion channel system. Comparisons of ion currents and ion concentrations from these simulations with Poisson-Nernst-Planck (PNP) results show good agreement between the two methods for parameters where the latter method is expected to be accurate.     

Influence of inhalation anesthetics on ion transport across a planar bilayer lipid membrane

Ion transport from one aqueous phase (W1) to another (W2) across a planar bilayer lipid membrane (BLM) in the presence of inhalation anesthetics was electrochemically investigated. In the absence of inhalation anesthetics in the BLM system, no ion transport current flowed between W1 and W2 across the BLM. When inhalation anesthetics such as halothane, chloroform, diethyl ether and trichloroethylene were added to the two aqueous phases or the BLM, the ion transport current quite clearly appeared. When the ratio of the concentration of KCl or NaCl in W1 to that in W2 was varied, the zero current potential across the BLM was shifted. By considering the magnitude of the potential shift, we concluded that the ion transport current can be predominantly ascribed to the transport of Cl(-) across the BLM. Since the dielectric constants of these anesthetics are larger than that of the inner hydrophobic domain of the BLM, the concentration of hydrophilic electrolyte ions in the BLM increases with the increase in the dielectric constant of the inner hydrophobic domain caused by addition of these anesthetics. These situations lead to an increase in the ion permeability coefficient.     

Product ion spectral simplification using time-delayed fragment ion capture with tandem linear ion traps

A new technique to generate product ion spectra as the internal energy of a collisionally activated precursor ion evolves is described. The precursor ion is activated by acceleration into a mass-selective linear ion trap under conditions whereby some of the fragment ions formed are unstable within the trap. After a time delay the stability parameters of the ion trap are changed to allow capture of fragments that that were previously unstable. The result is a product ion spectrum that originates from precursor ions with a modified internal energy distribution. It is possible to follow the evolution of the precursor internal energy distribution for many milliseconds after admittance of the precursor ions into the linear ion trap. Time-delayed fragmentation product ion spectra typically display reduced sequential fragmentation products leading to spectra that are more easily interpreted. Several important experimental parameters important to time-delayed fragmentation have been identified and are discussed. The technique has applications for both small precursor ions and multiply charged peptides.     

Automated determination of precursor ion, product ion, and neutral loss compositions and deconvolution of composite mass spectra using ion correlation based on exact masses and relative isotopic abundances

After an accidental, deliberate, or weather-related dispersion of chemicals (dispersive event), rapid determination of elemental compositions of ions in mass spectra is essential for tentatively identifying compounds. A direct analysis in real time (DART)ion source interfaced to a JEOL AccuTOFmass spectrometer provided exact masses accurate to within 2 mDa for most ions in full scan mass spectra and relative isotopic abundances (RIAs) accurate to within 15-20% for abundant isotopic ions. To speed determination of the correct composition for precursor ions and most product ions and neutral losses, a three-part software suite was developed. Starting with text files of m/z ratios and their ion abundances from mass spectra acquired at low, moderate, and high collision energies, the ion extraction program (IEP) compiled lists for the most abundant monoisotopic ions of their exact masses and the RIAs of the +1 and +2 isotopic peaks when abundance thresholds were met; precursor ions; and higher-mass, precursor-related species. The ion correlation program (ICP) determined if a precursor ion composition could yield a product ion and corresponding neutral loss compositions for each product ion in turn. The input and output program (IOP) provided the ICP with each precursor ion:product ion pair for multiple sets of error limits and prepared correlation lists for single or multiple precursor ions. The software determined the correct precursor ion compositions for 21 individual standards and for three- and seven-component mixtures. Partial deconvolution of composite mass spectra was achieved based on exact masses and RIAs, rather than on chromatography.     

Two-species ion clusters in a Paul trap

The ordered structures of two-species ions stored in a Paul trap are simulated by minimizing the total effective potential energy of the ion system. It is shown that the homo-charged two-species ion clusters display separating and mixing structures for different mass and charge ratios. The hetero-charged ion clusters consisting of one light negatively (positively) charged ion and three to six heavy positively (negatively) ions are found to exist stably, and the stability conditions obtained through the perturbative analysis indicate that the ion cluster consisting of more heavy ions has a looser limitation on mass and charge ratios of two-species ions.