H. Ti Tien

Formation of self-assembled lipid bilayers on solid substrates

Abstract Experimental bilayer lipid membranes (BLM) are usually formed by spreading a lipid solution across an aperture in a hydrophobic septum that separates two aqueous phases. Formed in this manner, a BLM is an extremely fragile structure with a limited lifetime. For protracted studies and for practical applications, as in biosensors and in molecular electronic devices development, a long-lived BLM (or lipid bilayer) is a prerequisite. Here, we report a simple and novel method for forming self-assembled lipid bilayers on solid substrates. The resulting supported BLMs have the desired dynamic properties as well as the requisite mechanical stability. They permit long-term electrochemical and photoelectronic investigations of the bilayer lipid membrane structure. Further, they may provide a foundation for a new class of molecule-based devices and contribute to the development of biosensors in the future.

The lipid bilayer concept and its experimental realization: from soap bubbles, kitchen sink, to bilayer lipid membranes

Abstract The inspiration for lipid bilayer research, without question, comes from the biological world. Although self-assembled bilayer lipid membranes (BLMs) in vitro, were first reported in 1961, experimental scientists have been dealing with BLM-type interfacial adsorption phenomena since Robert Hooke’s time (1672). BLMs (of planar lipid bilayers) have been used in a number of applications ranging from basic membrane biophysics including transport, practical AIDS research, and ‘microchips’ studies, to the conversion of solar energy via water photolysis, to biosensor development using supported bilayer lipid membranes (s-BLMs), and to photobiology comprising apoptosis and photodynamic therapy. This paper presents an overview of the origin of the lipid bilayer concept and its experimental realization, as well as the studies of our laboratory and recent research of others on the use of BLMs as models of certain biomembranes. In addition, we describe briefly our present work on supported BLMs as biosensors and molecular devices; the experiments carried out in close collaboration with colleagues on s-BLMs are delineated.

PHOTOEFFECTS OF PIGMENTED LIPID MEMBRANES IN A MICROPOROUS FILTER

Abstract. The light‐induced voltage and current generated by pigmented lipid membranes have been investigated. The membranes, separating two aqueous solutions, were formed in microporous filters with pore sizes ranging from 0.05 to 8μm in diameter and densities of 105 to 6 × 108 pores/cm2. The structure and some physicochemical properties of these membranes are described and compared with those of planar BLMs. Photopotentials up to 400 mV could be developed across these membranes having an effective current of 21μA‐cm‐2. These and other parameters (redox couples and lifetime) were studied over a period of days. The advantages of this membrane system such as long‐term stability and manipulability are discussed.

Nanostructured platinum-lipid bilayer composite as biosensor.

The present work describes the preparation of supported bilayer lipid membrane (s-BLM) doped with metal nanoparticles for the design of biosensors. Platinum (Pt) nanoparticles were deposited through s-BLM to build a hybrid device of nanoscale electrode array by potential cycling in 1 mM K(2)PtCl(6) solution containing 0.1 M KCl. The properties of Pt nanoparticle-doped s-BLM composite were then characterized by cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). Our results showed that Pt nanoparticles grew in voids of the s-BLMs, through which the underlying glassy carbon (GC) electrode was connected, with maximum length extended out of the lipid membrane around 40 nm. Doping of Pt nanoparticles through s-BLM increased the membrane capacitance and decreased the membrane resistance of s-BLM. Pt nanoparticles array in s-BLM electrocatalyzed the reduction of oxygen (O(2)) in phosphate buffer solution (PBS). Practical application of Pt nanoparticle-doped s-BLM for the construction of glucose biosensor was also demonstrated in terms of its dose-response curve, stability and reproducibility. Thus, lipid membrane doped with Pt nanoparticles is a novel electrode system at nanoscale that can penetrate through the insulating membrane to probe molecular recognition and catalytic events at the lipid membrane-solution interface.

Biophysical aspects of agar-gel supported bilayer lipid nembranes: a new method for forming and studying planar bilayer lipid membranes

Abstract Conventional bilayer lipid membranes (BLMs), formed by either the painting method or the Langmuir-Blodgett technique, lack the desired stability. This paper presents a simple method for forming long-lived BLMs on agar-gel supports. The supported BLM reported has a greatly improved mechanical stability and also has desirable dynamic properties. These self-assembled BLMs are of significant interest, in view of their similarity of biological membranes, their molecular dimension and their spontaneous formation.

Channel-closing activity of porins from Escherichia coli in bilayer lipid membranes

The opening and closing of the ompF porin from Escherichia coli JF 701 was investigated by reconstituting the purified protein into planar bilayer membranes. The electrical conductance changes across the membranes at constant potential were used to analyze the size and aggregate nature of the porin channel complexes and the relative number of opening and closing events. We found that, when measured at pH 5.5, the channel conductance diminished and the number of closing events increased when the voltage was greater than 100 mV. The results suggest that the number of smaller sized conductance channels increases above this potential. There was also an increase in the smaller subunits and in the closing events when the pH was lowered to 3.5, and these changes were further enhanced by increasing the voltage. We propose that both lowering the pH and elevating the potential across the membrane stabilize the porin in a conformation in which the subunits are less tightly associated and the subunits open in a non-cooperative manner. These same conditions also appear to stabilize the closed state of the pore.

Electrochemistry of supported bilayer lipid membranes: background and techniques for biosensor development

Abstract Biomembranes play a pivotal role in signal transduction and information processing. This is due to the fact that most physiological activities involve some kind of lipid bilayer-based receptor-ligand contact interactions. There are many outstanding examples such as ion sensing, antigen-antibody binding, and ligand-gated channels, to name a few. One approach to study these interactions in vitro has been facilitated by employing self-assembled bilayer lipid membranes (BLMs). Our efforts have been focused on ion and/or molecular selectivity and specificity using easy-to-prepare self-assembled BLMs on solid support (i.e., s-BLMs and sb-BLMs) which, with their enhanced stability, greatly aid in research areas of membrane biophysics, biochemistry, and cell biology as well as in biosensor designs and molecular devices development. In this paper, our current work along with the experiments done in close collaboration with others on supported BLMs towards biosensor development will be discussed.

Self-assembled bilayer lipid membranes: from mimicking biomembranes to practical applications

Abstract Since their introduction in the early 1960s, bilayer lipid membranes (BLMs) along with liposomes a few years later, are the most widely used experimental models of biomembranes. The biomembranes enclosing cells and organelles are highly organized, structurally complex, dynamic systems that are the sites of energy conversion, material transport, biosensing, and signal transduction. The major cell membrane constituents are phospholipids, along with proteins, carbohydrates and their complexes, that spontaneously self-assemble into lipid bilayers. Hence, BLMs have been used from basic membrane biophysics studies to the conversion of solar energy. More recently, planar BLMs on various supports have gained considerable importance in the development of biosensors and molecular devices. These supported or s-BLMs have been deposited on a number of substrates such as SnO2, platinum, stainless steel, cooper, gels, and in microporous filters for practical applications. This paper summarizes the past research of our laboratory and others since 1973 on the use of BLMs as models of biological membranes. Further, the paper describes in some detail our current work on s-BLMs as potential biosensors and molecular devices.

A NEW METHOD FOR THE DETERMINATION OF ELECTRICAL PROPERTIES OF SUPPORTED BILAYER LIPID MEMBRANES BY CYCLIC VOLTAMMETRY

Abstract A novel method has been developed for accurate measurements of the membrane capacitance and resistance on the solid-supported bilayer lipid membrane (s-BLM) by the cyclic voltammetry (CV) technique. An improved equivalent circuit for the s-BLM is proposed, which is based on the solution of the coupled equations. This approach greatly facilitates the determination of the s-BLM electrical properties by CV. The curve-fitting procedure for testing the reliability of this method has been elaborated using a newly designed BLM simulator as well as a 50-point goodness-of-fit test. The mechanics and characteristics of formation of different s-BLMs and the acceleration effect of the CV potential on the s-BLM formation have been studied using the newly proposed s-BLM model.

Action of calcium channel and beta-adrenergic blocking agents in bilayer lipid membranes

The action of beta-adrenergic blockers (propranolol, exprenolol, metoprolol, sotalol, atenolol, timolol) and calcium-channel blockers (verapamil, diltiazem) on the electrical properties and fluidity of bilayer lipid membranes (BLM and liposomes) has been investigated. When antibiotic ionophore substances were used as a probe, the electrical measurements showed that many of the drugs inhibited the cation transport across the membrane facilitated by the mobile carrier valinomycin, while having no significant effect on the cation transport through channels formed by gramicidin. The ability of the drugs to decrease the carrier-dependent membrane conductance was correlated to their partition into the lipid bilayer and the magnitude of transmembrane potential induced by them. In the TEMPO ESR spectral measurements, a number of beta-adrenergic and calcium blockers showed the fluidizing effect on liposomes composed of different lipids. The drug concentration required for a detectable change in TEMPO spectra parameter (f) was rather high (0.01 M verapamil), and the variation of pH from 6.5 to 3.0 did not affect the fluidizing effect of the drugs.