Trends in Biotechnology
ReviewMembrane biosensor platforms using nano- and microporous supports
Introduction
Cell membranes are effective diffusion barriers, upholding concentration gradients of ions and molecules across the membrane. Through their function as scaffolds for membrane proteins, membranes also control the selective transport of molecules and ions across the lipid bilayer to create and modify ion gradients. These signal transduction events guide many important cellular processes, consequently, >50% of all current drugs target membrane proteins [1]. It is thus essential to be able to characterize membrane barrier properties and the changes that occur when these proteins are incorporated into a lipid membrane, activated or blocked.
Creation of robust in vitro membrane systems enabling direct incorporation and integrated electrochemical or voltage clamp measurements of membrane protein function (i.e. ion, charge and liquid transport) could potentially revolutionize current technologies in drug screening. Methods currently used for screening and profiling in the pharmaceutical industry are limited to the monitoring of binding events; but even extraction of binding affinity requires multiple experiments at a range of concentrations. Direct measurement of function in response to (drug) stimuli will enable quicker and better selection of leads and toxic hits. The same kind of platforms could be used for advanced biomimetic sensing. Possible applications would be electronic tongues and noses or light harvesting for energy and biochemical purposes. Important classes of membrane proteins, such as G-protein-coupled receptors (GPCRs) and their analogues, can be exploited for this purpose. From a fundamental scientific point of view, a convenient way of studying membrane constituents down to the single molecule level would help bridge the gap in information and understanding between holistic biological studies of function in whole cells and the expanding knowledge about membrane protein structure and in silico simulation of their function.
These tasks are difficult to carry out directly on native cell membranes with techniques such as the patch clamp. Hence, a host of attempts to find generic ways of achieving integration of biosensors with membranes have been attempted. The main obstacle to taking research on advanced in vitro membrane function from the academic laboratory to commercial applications has been the difficulty in incorporating membrane proteins into biosensor platforms in a manner that allows measurement of the function of the membrane proteins.
Section snippets
Membrane platforms for studying membrane protein function
A selection of proposed membrane sensor platforms of relevance for this review are schematically depicted in Figure 1, and Figure 2 provides brief descriptions of the most common ways to form a lipid membrane on a sensor substrate. For an in-depth review of the many suggested platforms we refer the reader to a recent review by Janshoff and Steinem [2]. Among the in vitro systems, the first developed and most widespread method for studying ion-channel function has been lipid membranes spanning
Demonstration of increased stability for free-spanning membranes
Reducing aperture dimensions for free-spanning membranes on a biosensor chip down to the nanometer range – as depicted in Figure 1c and a typical substrate shown in Figure 3b – has been envisaged as the most straightforward way to increase membrane stability compared with traditional black lipid membranes, which could span apertures of hundreds of μm to several mm. Increase in mechanical stability with reduced aperture size has recently been demonstrated by Simon et al. who compared lipid
Sub-100 nm apertures enable self-assembly of durable solvent-free membranes
Solvent-free solid-supported lipid bilayers that will also be more compatible with sensitive transmembrane proteins can be formed by self-assembly from small and large unilamellar vesicles (Figure 2a) or by the more cumbersome Langmuir-Blodgett deposition techniques (Figure 2b). Homogeneous vesicle fusion is greatly facilitated by the use of unilamellar vesicles with a diameter of 200 nm or smaller. For the creation of free-spanning membranes by vesicle fusion, aperture diameters in the sub-100
Incorporation of membrane proteins into free-spanning membranes
Insertion of transmembrane proteins into a newly formed free-spanning lipid membrane is one of the major challenges for membrane sensing platforms. Usage of apertures in the micron and sub-micron range necessitates control over the insertion and localization of single proteins to correctly analyze the results of single protein activity measurements. The most common method is the insertion of small ion channels directly from solution 2, 4, 27, 30, but this method cannot be applied to the vast
Stabilization of free-spanning membranes
Supported membranes are disrupted by drying. In addition to this problem, free-spanning membranes suffer low temporal stability and typically rupture within hours even when kept hydrated To stabilize aperture-spanning membranes Sleytr et al. have developed a novel approach based on the so-called S-layer proteins found on the exterior of the Bacillus sphaericus bacterium. They formed continuous lipid bilayers by Langmuir-Blodgett deposition over micro-filtration membranes with pores of an
Concluding remarks
The past few years have seen significant developments in nanoporous membrane sensor platforms, up to a point where these platforms seem to be the leading candidate for creating a viable chip format for functional transmembrane protein measurements. This is demonstrated by the current degree of fulfilment of the design criteria summarized in Table 1. The initial key advances have been the reduction of aperture size by nanofabrication to increase stability and the ease of formation of the lipid
Acknowledgements
Swiss Competence Center for Materials Research (CCMX) and Agency for Science, Technology and Research (A*STAR, Singapore) are acknowledged for funding.
References (58)
Orphan G-protein-coupled receptors and natural ligand discovery
Trends Pharmacol. Sci.
(2001)Deposition of highly resistive lipid bilayer on silicon-silicon dioxide electrode and incorporation of gramicidin studied by ac impedance spectroscopy
Electrochim. Acta
(2001)The reaction of cytochrome c from different species with cytochrome c oxidase immobilized in an electrode supported lipid bilayer membrane
J. Electroanal. Chem.
(2002)The protein-tethered lipid bilayer: A novel mimic of the biological membrane
Biophys. J.
(2004)Formation and stability of a suspended biomimetic lipid bilayer on silicon submicrometer-sized pores
J. Colloid Interface Sci.
(2007)- et al.
Photocurrents generated by bacteriorhodopsin adsorbed on nano-black lipid membranes
Biophys. J.
(2005) - et al.
Impedance analysis and single-channel recordings on nano-black lipid membranes based on porous alumina
Biophys. J.
(2004) Channel activity of OmpF monitored in nano-BLMs
Biophys. J.
(2006)Mechanical properties of pore-spanning lipid bilayers probed by atomic force microscopy
Biophys. J.
(2006)Kinetics of valinomycin-mediated K+ ion transport through tethered bilayer lipid membranes
J. Electroanal. Chem.
(2003)