Biomimetic nanoscale reactors

Biomimetic nanoscale reactors

(Parte 1 de 8)

17 Apr 2004 17:27 AR AR212-PC55-20.tex AR212-PC55-20.sgm LaTeX2e(2002/01/18) P1: FHD 10.1146/annurev.physchem.5.091602.094319

Annu. Rev. Phys. Chem. 2004. 5:613–49 doi: 10.1146/annurev.physchem.5.091602.094319

Copyright c° 2004 by Annual Reviews. All rights reserved First published online as a Review in Advance on February 26, 2004

Mattias Karlsson,1 Max Davidson,1 Roger Karlsson,2 Anders Karlsson,2 Johan Bergenholtz,2 Zoran Konkoli,3 Aldo Jesorka,1 Tatsiana Lobovkina,1 Johan Hurtig,1

Marina Voinova,3 andOwe Orwar1 1Department of Chemistry and Bioscience, and Microtechnology Center at Chalmers, 2Department of Chemistry, Goteborg University, 3Department of Applied Physics, Chalmers University of Technology, SE-41296 Goteborg, Sweden; email:

Key Words biomimetic, liposome, nanotube, network, reaction, nanofluidic n Abstract Methods based on self-assembly, self-organization, and forced shape transformations to form synthetic or semisynthetic enclosed lipid bilayer structures with several properties similar to biological nanocompartments are reviewed. The procedures offer unconventional micro- and nanofabrication routes to yield complex soft-matter devices for a variety of applications for example, in physical chemistry and nanotechnology. In particular, we describe novel micromanipulation methods for producingfluid-statelipidbilayernetworksofnanotubesandsurface-immobilizedvesicles with controlled geometry, topology, membrane composition, and interior contents. Mass transport in nanotubes and materials exchange, for example, between conjugated containers, can be controlled by creating a surface tension gradient that gives rise to a moving boundary or by induced shape transformations. The network devices can operate with extremely small volume elements and low mass, to the limit of single molecules and particles at a length scale where a continuum mechanics approximation may break down. Thus, we also describe some concepts of anomalous fluctuationdominated kinetics and anomalous diffusive behaviours, including hindered transport, as they might become important in studying chemistry and transport phenomena in these confined systems. The networks are suitable for initiating and controlling chemical reactions in confined biomimetic compartments for rationalizing, for example, enzyme behaviors, as well as for applications in nanofluidics, bioanalytical devices, and to construct computational and complex sensor systems with operations building on chemical kinetics, coupled reactions and controlled mass transport.

06-426X/04/0601-0613$14.0 613

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Several important breakthroughs in optical and electrochemical methods for probing chemical reactions even down to the single-molecule limit in condensed media have been made during the past several years (1, 2). With these powerful methods at hand, experimental methods for controlled reaction initiation in a well-defined confined reactor of nanometer-to-micrometer dimensions involving few reactants are currently under development. The escalating interest in such systems is partly based on (a) the basic need from a pure physicochemical perspective to understand how chemical reactions proceed, that is, kinetics and mechanisms in a small-scale environment (for a general review see Reference 3); (b) the outstanding significance such methods might have to understanding chemistry in biological systems that take place in compartments of short length scales (for a general review see Reference 4); and (c) the importance of such methods in the rapidly emerging field of nanoscience and technology, where some problems and applications critically rely on performing chemistry in nanoscale devices with few molecules (5–7). In particular, methods for controlling reaction initiation, mixing, and mass transport in biomimetic nanoscale compartments with tailored surfaces and functionalities is of considerable relevance in understanding, for example, the actions of enzymes, protein transporters, and other biomolecules as well as properties of signaling pathways and other reaction networks. This article reviews unconventional fabrication methods that use surfactant bilayer membranes to build biomimetic reactors and complex network devices for studying, for example, chemical reactions, polymer dynamics, and transport phenomena at the micrometer and nanometer level. As an introduction to this multidisciplinary and multifaceted field, we address these systems from some different angles that hopefully will converge into a unifying view of what this is all about.

The desire to control solution chemistry to the limit where singular reacting species can be selected and monitored in a controlled environment is a daunting engineeringchallenge.Itpertainstotheproblemofconstructingnanoscalereactors with flow or transport control and particular functionalities for initiating and controlling reactions, as well as some desired properties such as efficient dissipation of Joule heating. Previously, we have seen a strong development in miniaturization of fluidic devices to the micrometer scale (for a general review see References 8 and 9). These systems are mainly based on plumbing-and-valving principles and are therefore linear down-scaled equivalents to the traditional fluidic apparatus. If we wish to extend these systems to the low-nanoscale regime with channels of dimensions between 1 and 100 nm in diameter that have the potential to handle down to single molecules, nanoscale fluidic devices offering unprecedented control over transport, manipulation as well as detection are required. Even though methods of fabrication can produce nanochannels and nanocontainers in hard materials using e-beam lithography, sacrificial layers of carbon nanotubes, colloidal lithography, and many more (10) methods of fabrication at the nanoscale are not at all as readily available and mature as those applicable in the micrometer range.

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Clearly, novel materials and novel principles of fabrication are required to achieve these goals. Ideally, materials should be used that naturally adopt—or with very little force are made to adopt—certain three-dimensional shapes on the nanometer scale that are useful in building, for example, reaction containers of particular geometries, nanofluidic channels, and bifurcating networks. Such selfassembly and low-energy-barrier forced shape transformations should be matched with desired materials properties and desired functionalities. Furthermore, because small-scale systems are dominated by surface interactions, controlling surface properties are of prime importance. Therefore the materials used should offer the ability to control wettability, charge, specific binding interactions, and other interactionsthathaveabearingonwaterstructure,solvation,depletioninteractions, and many more phenomena.

Materials are readily available that can aid in this development, but they are usually not associated with fabrication procedures because of their soft properties.

Several interesting molecular systems aggregate or self-organize through noncovalent interactions into micro- and nanoscale structures with controlled geometries. Fabrication in this instance is based on the natural tendency of the material to adopt certain shapes and geometries. Systems represented by, for example, microemulsions(1,12),surfactantmembranes(13),andsomepolymersystems(14) areallapplicabletothesemethodsoffabrication.Notonlydosomeofthesematerials have a strong tendency to self-assemble into nanoscale structures, but they also sometimes possess remarkable mechanical properties, as well as programmable surface properties. Some of these materials are also heterogeneous, with local variations in composition. Interestingly, biological systems completely rely on these dynamic and soft materials and have used them to solve a number of challenging nanoengineering problems, for example, to perform controlled transport, store chemical information, and perform computations and operations on single or few molecules. In living cells we find confined reaction compartments of nanometer dimensions made in lipid bilayer materials. The nanoscale compartments are integrated in reaction networks, which as well as producing a chemical species such as anessentialmetabolite,canbeusedtotransferinformationintheformofachemical compound with concentration varying in time and space to make biological sense (15). In a technology context, utilization of solution chemistry with the complexity, flexibility,andfinecontrolfoundinbiologicalsystemswouldbeofimmensevalue.

Inspired by biological systems, we as well as other groups wish to develop concepts for nanoscale network and device design based on surfactant membranes (16–20). In particular, methods based on self-assembly, self-organization, and forced shape transformations using novel micromanipulation tools can be used to form synthetic or semisynthetic enclosed lipid bilayer structures with several properties similar to biological nanocompartments. These procedures offer unconventional fabrication routes with great flexibility to yield three-dimensional soft-matter devices of different function and geometries at a length scale that is difficult to reach with modern clean room technology. Figure 1 shows a flowchart type of diagram that summarizes the fabrication strategy and

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procedures. In their simplest configuration, these systems can be used to study and control chemical reactions in a well-defined nanoscale environment, but they have the potential to extend into advanced devices for nanofluidics and nanochemistry applications. In a more complex context, we envison biomimetic sensor devices potentially with computational capabilities based on reconstitution of biomolecular functionalities such as mechanical, electrical, optical, and chemical molecular sensors and transducers.

Given the unique biomimetic materials and the particular topologies used in these devices, it is tempting to think of them as primitive models of biological cells or cell networks. Liposomes, for example, have previously earned much attention as reductionistic artificial cell models. Plausibly, since all cells today are composed of lipid bilayer assemblies, early structures for compartmentalization, which are necessary for maintaining integrity and separation of reactants and for controlled transport of reactants and protection from outer environmental changes, lipidbilayermembranesmayhaveplayedanimportantroleintheearlydaysofevolution (21). There are different approaches to construct a minimalistic cell model. The bottom-up approach involves the starting point where monomers and simple low-molecular-weight molecules assemble into specific sequences and structures such as nucleic acids and proteins inside a liposome. The top-down approach involves the incorporation of extant DNA, RNA, and/or enzymes into liposomes by various methods (2–26).

This article is outlined as follows. Sections 2 to 4 give a brief background of bilayer membranes, paying special attention to liposomes and mechanical properties as well as adhesion of vesicles to surfaces. These chapters are somewhat basic but provide essential information for the nonspecialist to understand why nanotube-vesicle networks can be formed and some of their important chemical and physical properties. Section 5 discusses production of tethers or nanotubes from surface-immobilized vesicles by application of point-loads. In Section 6, we present micromanipulation methods for production of nanotube-vesicle networks. In Section 7, we present methods for functionalizing the membranes with transporters to render the networks controlled multiple-input, multiple-output capabilities, as well as methods to internalize catalytic colloidal particles to perform compartmentalized chemistry in the nodes. Section 8 discusses transport control in nanotube-vesicle networks, as well as theoretical considerations on hindered transport and diffusion in confined media. In Section 9 we provide different experimental protocols to initiate reactions in lipid membrane vesicles and nanotube-vesicle networks, and Section 10 discusses effects of confinement on reaction kinetics. Finally, Section 1 gives some applications of these networks to different problems in chemistry, physics, and biotechnology.


Fluid-state lipid bilayer membranes play a central role in the structure and function of living matter. Formed by amphiphilic molecules that associate into a well-defined bilayer of only 5-nm thickness, this membrane defines the cellular

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BIOMIMETICNANOSCALEREACTORSANDNETWORKS 617 boundary and its subcompartments. Thus, the lipid bilayer membrane is one of the most important structural and functional components in biological cells (27).

Synthetic or semisynthetic bilayer structures with several properties similar to biological membranes can be formed in the laboratory. Most often, the preparation methods used build on the capability of these membranes to self-assemble under certain conditions. Self-assembly is typically based on a series of molecular recognition events involving interactions that are mainly noncovalent in nature and where the information that guides the assembly is contained within the subunits (28). Consequently, careful engineering of these properties in the subunits (in this instance the molecular properties of phospholipids) will define the composition, shape, and properties (function) of the formed suprastructure.

From a materials science perspective, lipid bilayer membranes have several unique and attractive properties. They are held together by nonconvalent interactions, and unlike solid particles or rigid macromolecules, these supramolecular aggregates are fluid-like. They have extraordinary mechanical properties, and a fluid-state lipid membrane has a mechanical strength comparable to stainless steel of the same thickness; yet it behaves as a two-dimensional liquid capable of undergoing complex shape transitions (13).

Amphiphiles that have two hydrocarbon tails are very likely to form bilayer membranes, and the most common type is the 1,2-dialkylphosphoglycerides (27). The doubling of the hydrocarbon chains largely affects both the static and dynamic properties of the assembled aggregates. The water solubility of the amphiphiles is drastically decreased; bilayer-forming lipids typically have critical micelle concentrations (CMCs) of 10¡7 to 10¡10 M. This extremely low solubility affects the temporal stability of the membrane (29). Typically, the residence time for bilayer membrane-forming amphiphiles such as glycerophospholipids is approximately 104 s. Another dynamic feature displayed by bilayer membranes is the transverse exchange of monomers between the two monolayers, commonly referred to as “flip-flop” of amphiphiles. Because the energy required to induce a flip-flop transition is quite high, the corresponding timescale is long, typically in the range of hours to days. Single-component bilayers often have a very distinct transition temperature,

Tc, between the crystalline, or frozen, state and the fluid state. It is, however, important to point out that bilayers below Tc do not always transform directly into the crystalline phase. Sometimes the bilayer retains some of its fluid-like behavior; this is often referred to as the gel state.


Because of the extremely low solubility of membrane-forming lipids in aqueous solutions, the corresponding lipid bilayer aggregate may be considered a fully stable and static material during experimental timescales. Consequently, the lipid bilayer membrane can be approximated as an infinitesimally thin monolithic sheet that can be described by a continuum mechanical approach.

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17 Apr 2004 17:27 AR AR212-PC55-20.tex AR212-PC55-20.sgm LaTeX2e(2002/01/18) P1: FHD 618 KARLSSON ET AL.

Figure 2 Schematic drawing showing the three basic shape deformations of a membrane element. In (a) the membrane element is unperturbed, with a unit area corre- spondingtoA0.In(b)tangentialforcesactingperpendiculartothesideofthemembrane are stretching or compressing the element. In (c) and (d) forces are acting normal to the plane of the membrane element, causing a bending deformation of the unit area. (c) and (d) represent the cases of positive and negative Gaussian curvature, respectively. In (e) tangential forces are acting parallel to the side of the membrane element without a change in surface area, thus causing a shear deformation of the membrane.

The idea of applying the thin elastic shell theory to membrane mechanics was first introduced by Helfrich in the early 1970s (30). The basic concept is that the surface free energy of a lipid membrane, reflecting the intramolecular forces acting within the membrane and at the water interfaces, can be described by a series of simple independent shape deformations in local regions of the membrane surface. These basic deformations are area dilation or condensation, shear deformation (inplane extension at constant surface density), and curvature change. The different modes of deformation are illustrated in Figure 2. Any external forces applied to a membrane will be distributed throughout the entire surface element as a force, or moment per unit length along the membrane contour. Correspondingly, there are three first-order elastic relations that reflect these basic deformations. However, frozen bilayers appear to exhibit a limited elastic response to shear deformations; often a “yield and surface flow”–type behavior is observed instead because of crystal defects (31).

Following the reasoning of Helfrich, the total surface free energy of a fluid-state membrane is a linear combination of the elastic relations representing bending and stretching integrated over the entire membrane surface:

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The first term represents area dilation where A is the area of the stretched mem- brane, A0 is the area of the unperturbed surface, and Ka, is the elastic modulus, typically having a characteristic value of 240 mN/m for lipid bilayers (32). The second and third terms represent bending deformations where •c is the bending modulus, which has a characteristic value of 10¡19 J, c1 and c2 are the two princi- pal curvatures, and c0 is the spontaneous curvature. The last term is the so-called Gaussian curvature contribution, adjusting the bending energy in saddle points on a membrane element, where •g is the Gaussian bending modulus. Anotherparameterthataffectsthesurfacefreeenergyofmembranestructuresis adhesion to surfaces. The adhesive behavior is promoted by the attractive potential between the two surfaces and is balanced by the elastic energy of the membrane (3). In the case of immobilized vesicles, we can write the adhesive contribution to the free energy as where istheeffectivecontactpotentialandA⁄ isthecontactareaoftheboundpart of the vesicle membrane. Because of the Brownian motion of a flaccid membrane surface, the contact area of an adhering liposome will grow progressively until it reaches equilibrium. The surface free energy of a bound vesicle will then be given by the balance between the adhesive forces, lateral tension, and bending rigidity;

The original Helfrich equation treats the lipid membrane as a thin monolithic shell. In reality, however, the lipid membrane is composed of two monolayers that behave as two coupled physical entities with the capability of dissipating energy separately. The total elastic energy of a bilayer membrane is therefore the sum of the elastic energies in the two monolayers. In the thin shell theory this behavior is accounted for by introducing a nonlocal bending term (expansion/compression of the inner and outer monolayers when the bilayer is curved) (34).


Liposomes, or lipid vesicles, are fully enclosed spherical compartments of bilayer membranes entrapping a part of the aqueous medium in which they were formed. Liposomes are formed from phospholipid monomer dispersions that can also include, for example, membrane proteins by self-assembly in aqueous solution, and the choice of monomer composition has direct bearing on surface properties and functionality, that is, the surfaces and interior contents of liposomes can be tailored in a single one-step synthesis. The discovery of lipid vesicles, or liposomes,

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(Parte 1 de 8)