Vitalii Silin

First-Principles Determination of Hybrid Bilayer Membrane Structure by Phase-Sensitive Neutron Reflectometry

The application of a new, phase-sensitive neutron reflectometry method to reveal the compositional depth profiles of biomimetic membranes is reported. Determination of the complex reflection amplitude allows the related scattering length density (SLD) profile to be obtained by a first-principles inversion without the need for fitting or adjustable parameters. The SLD profile so obtained is unique for most membranes and can therefore be directly compared with the SLD profile corresponding to the chemical compositional profile of the film, as predicted, for example, by a molecular dynamics simulation. Knowledge of the real part of the reflection amplitude, in addition to enabling the inversion, makes it possible to assign a spatial resolution to the profile for a given range of wavevector transfer over which the reflectivity data are collected. Furthermore, the imaginary part of the reflection amplitude can be used as a sensitive diagnostic tool for recognizing the existence of certain in-plane inhomogeneities in the sample. Measurements demonstrating the practical realization of this phase-sensitive technique were performed on a hybrid bilayer membrane (self-assembled monolayer of thiahexa (ethylene oxide) alkane on gold and a phospholipid layer) in intimate contact with an aqueous reservoir. Analysis of the experimental results shows that accurate compositional depth profiles can now be obtained with a spatial resolution in the subnanometer range, primarily limited by the background originating from the reservoir and the roughness of the films supporting substrate.

Surface-plasmon-resonance-enhanced cavity ring-down detection.

The cavity ring-down technique is used to probe the absolute optical response of the localized surface plasmon resonance (SPR) of a gold nanoparticle distribution to adsorption of trichloroethylene (TCE) and perchloroethylene (PCE) from the gas phase. Extended Mie theory for a coated sphere with a particle-size-dependent dielectric function is used to elucidate size-dispersion effects, the size-dependence of the SPR sensitivity to adsorption, and the kinetics of adsorption. An approximate Gaussian distribution of nanospheres with a mean diameter of 4.5 nm and a standard deviation of 1.1 nm, as determined by atomic force microscopy, is provided by the intrinsic granularity of an ultrathin, gold film, having a nominal thickness of approximately 0.18 nm. The cavity ring-down measurements employ a linear resonator with an intracavity flow cell, which is formed by a pair of ultrasmooth, fused-silica optical flats at Brewsters angle, where the Au film is present on a single flat. The total system intrinsic loss is dominated by the film extinction, while the angled flats alone contribute only approximately 5 x 10(-5)/flat to the total loss. Based on a relative ring-down time precision of 0.1% for ensembles averages of 25 laser shots from a pulsed optical parametric oscillator, the minimum detectable concentrations of PCE and TCE obtained by probing the SPR response are found to be 2 and 7 x 10(-8) mol/L, respectively, based on a 30 s integration time.

Experimental demonstration of phase determination in neutron reflectometry by variation of the surrounding media

Abstract We have recently shown that it is possible to unambiguously determine the real part of the complex reflection coefficient for a thin film structure by measuring the neutron specular reflectivities for the film in contact with two different backing or two different fronting media, thus simplifying the reference methodology of phase determination and making it more practical. Here we demonstrate the technique in two different experiments. In the first, one backing medium is air, the other, heavy water. In the second, sapphire and silicon serve as two different fronting (incident) media. In addition, we show how the two possible branches of the imaginary part of the reflection coefficient inferred by the real part, while not required for inverting the data to find the film structure, can be a sensitive diagnostic for indicating whether the film of interest is inhomogeneous on a transverse scale comparable to the neutron coherence length. This is especially important in avoiding misinterpretation of data which result from the incoherent average of reflectivities from large-scale heterogeneities.

Biochip for the detection of Bacillus anthracis Lethal Factor and therapeutic agents against anthrax toxins

Tethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer. We characterize the sensor and its properties with electrochemical impedance spectroscopy and surface plasmon resonance. The sensor shows a sensitivity similar to ELISA and can also be used to rapidly screen for molecules that bind to the toxins and potentially inhibit their lethal effects.

The role of human monoacylglycerol lipase (hMAGL) binding pocket in breakup of unsaturated phospholipid membranes

Human monoacylglycerol lipase (hMAGL) plays a key role in homeostatic tuning of the endocannabinoid signaling system and supports aggressive tumorogenesis, making this enzyme a promising therapeutic target. hMAGL features a membrane-associated lid domain that regulates entry of endocannabinoid lipid substrates into the hydrophobic channel accessing the active site, likely from the membrane bilayer. The present work applied simultaneous surface plasmon resonance and electrochemical impedance spectroscopy measurements to show that, in absence of the substrate, hMAGL can remove phospholipid molecules from the membrane and, thereby, disintegrate pre-formed, intact, tethered phospholipid bilayer membrane mimetics (tBLMs) composed of unsaturated phosphatidylcholines. To probe the mechanism of hMAGL-induced on tBLMs compromise, we investigated the effect of wild type and mutant hMAGLs and hMAGL rendered catalytically inactive, as a function of concentration and in the presence of chemically distinct active-site inhibitors. Our data show that hMAGLs lid domain and hydrophobic substrate-binding pocket play important roles in hMAGL-induced bilayer lipid mobilization, whereas hydrolytic activity of the enzyme does not appear to be a factor.

On the Development of New Methods for Ion Channel Structure-function Measurement

Biomimetic interfaces can be constructed on atomically smooth electrode surfaces to provide a measurement platform that allows both high-information structural experiments such as NR, or vibrational spectroscopy as well as allow functional measurements of the protein as well. The true advantage of the robust tethered bilayer is the ability to make many different measurements over a long period of time on the exact same interface.

Integrating biological molecules with electrode surfaces for bioanalytical sensing applications

The integration of complex biological molecules such as proteins with metal and semiconductor interfaces is limited by several inherent incompatibilities. To develop sensors based upon protein-protein interactions at such surfaces, three significant obstacles must be overcome: 1) the sensing protein must retain its native, or native-like structure, 2) the protein must be in electrical (or optical) contact with the metal surface and 3) the reporter protein much be at a sufficiently high concentration to produce a measurable, unambiguous signal. Here, we demonstrate a detection scheme for Anthrax toxins based on the interaction of an ion channel formed by protective antigen 63 (PA63)7, a heptameric ion channel forming toxin, with lethal factor. To prepare a sensor, a biomimetic membrane is formed on an electrode surface through a thiolipid molecule with a custom synthesized poly(ethylene oxide) spacer. Thus a membrane is formed across the electrode with an aqueous space of ∼ 2 nm. This space allows the membrane to retain some fluidity to incorporate transmembrane proteins with separating the protein from a denaturing surface. The high-specificity of the LF-(PA63)7 interaction enables the detection of Anthrax proteins on the order of pmol/L. In addition to highly sensitive detectors, this tethered membrane construct is scaleable and can be extended to devices with micron and sub-micron sensing pads, critical for the miniaturization required for novel bioelectronic devices.