CHEMICAL
DANIEL FAGERLUND
ANALYSIS in NANOSCALE
SURFACTANT NETWORKS Newly developed means for transporting materials in nanoconduits between containers and advancements in initiation and control of chemical reactions have opened pathways to devices with single-molecule resolution.
Roger Karlsson Göteborg University
A
lthough the “hard matter” physical sciences (e.g., microelectronics) and the “soft matter” biAnders Karlsson ological sciences (e.g. cell biology and molecuNanoxis AB lar biology) are two distinct areas of research, they are (Sweden) now progressively being combined to create new sysAndrew Ewing tems and devices with applications in analytical chemistry. Miniaturization is one of the important factors Pennsylvania State University here: A major goal is to create devices that can operate Paul Dommersnes with high sensitivity and resolution on length scales and Jean-Francois Joanny time frames relevant to single-molecule studies. Using Institut Curie top-down strategies to fabricate ultrasmall structures is technologically challenging. Therefore, new bottom-up (France) methods of nanoscale fabrication based on self-assembly Aldo Jesorka and self-organization of biological or biomimetic mateOwe Orwar rials are constantly being developed (1–6). The type of Chalmers University of Technology nanoscale engineering described in this article has, at (Sweden) least partly, been derived from our growing understanding of living cells, where many nanomechanical and chemical operations are based on controlled shape transitions in surfactant bilayer membranes that also carry proteins to support important functions. Biological cells have a staggering ability to parallel-process multiple chemical reactions and physical (e.g., transport) processes in nanometer-sized systems and to perform a great number of tasks based on single-molecule processes. Thus, nature has solved many engineering problems on small length scales and achieved truly nanoscale, complex chemical devices that can be used for computational, biophysical, synthetic, and analytical applications (7–11). The philosophy behind the work reviewed here is to produce human-made, biomimetic systems that imitate some key features of small biological systems with the overall goal of providing micro- and nanoscale devices that can perform complex chemical operations. (Sweden)
© 2006 AMERICAN CHEMICAL SOCIETY
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Our lab and other groups have (a) (b) (e) f ~1pN developed concepts and protocols for producing nanoscale devices 1–100 µm and networks based on sponta5–50 µm neous and forced shape transitions in lipid bilayer membranes or other soft materials (12–22). The net100 nm works consist of surface-immobi- (c) lized vesicles (~5–50-µm diam) (d) conjugated by nanotubes (50 – 150-nm radius). From a materials science perspective, lipid bilayer 10 µm membranes have several unique V and attractive properties. They are held together by nonconvalent in- FIGURE 1. Construction of NVNs. teractions and, unlike solid materi- (a) New vesicles are formed by injecting buffer into the nanotube. (b) The interior contents of the vesicles can be als or rigid macromolecules, have differentiated in the process. (c, d) Vesicles can be combined by electrofusion to form more complex networks. features of a 2D fluid. They have (e) Bright-field image of a complex NVN. In the upper right corner, a multilamellar vesicle is attached to a unilamelextraordinary mechanical proper- lar container. ties—a fluid-state lipid membrane When buffer is injected at the rate of tens of femtoliters per has mechanical strength comparable to stainless steel of the same thickness, yet it is capable of undergoing complex shape transi- second, the nanotube expands at the injection tip, and a new tions (23). Networks of nanotubes and vesicles serve as a plat- vesicle is formed that is connected to the original vesicle via a form on which to build nanofluidic devices that operate with sin- lipid nanotube (Figure 1a). The extra lipid material for growth gle molecules and nanoparticles. of the vesicle ultimately comes from the attached multilamellar vesicle; it flows to the unilamellar vesicle and along the nanotube Forming nanotube–vesicle networks by tension-driven Marangoni flow (25, 26). When the expanding Various experimental techniques are now available to control the vesicle growing on the injection tip has reached the desired size, geometry, dimensionality, topology, and functionality in surfac- it can be adhered to a glass surface and the pipette can be retant membranes (12–19). Methods based on self-assembly, self- moved. This procedure can be repeated to create new nanoorganization, forced shape transformations, and micromanipula- tube–vesicle connections until complicated networks are obtion are used to form synthetic or semisynthetic enclosed lipid tained (12). Also, the composition of the internal solution of the bilayer structures with some properties similar to biological com- vesicles can be modified by exchanging the buffer in the mipartments. These flexible, unconventional fabrication procedures cropipette with another solution (Figures 1a and 1b). The topology of the network can also be changed. When yield 3D devices at a scale and complexity that are difficult to transient rectangular dc voltage pulses with field strengths of reach with modern clean-room technology. The process for creating nanotube–vesicle networks (NVNs) is 40–80 V/cm are applied through the micropipette for millisecfairly straightforward (19). A unilamellar vesicle is penetrated with onds, vesicles are electrofused (Figures 1c and 1d). In contrast, a glass pipette by microelectroinjection. The lipid reseals around applying dc voltage pulses directly to a nanotube leads to fission the injection tip, which is retracted from the vesicle. This results in of the containers in the networks and consequently to re-formaa lipid tube that is connected between the injection tip and the tion of individual vesicles. Further manipulations can lead to entangled and knotted networks. Self-tightening knots can funcoriginal vesicle. The force that is required for pulling a tube tion as mechanical torus tweezers and capture high-aspect-ratio objects in solution (27 ). It is also possible to construct hybrid f = 2 p √2 skc networks from a biological cell and a giant unilamellar vesicle is a few piconewtons (24); s is the surface tension, and kc is the (GUV), which are interconnected by a lipid nanotube. –19 Furthermore, networks can be constructed with lipid membending modulus with a characteristic kc value of 10 J. The rafr tube = 2= p √2 sk c dius of the tube rtube is solely dependent 2 s on these two terms and branes derived entirely and directly (without reconstitution) from biological cells (28). Only a single cell is required to build can be written (24) a small network, and rare proteins and lipids can easily be obVLktube c tained by such procedures. A variety of support structures for rttube relax == 2s 2 2pDr tube vesicle formation have also been developed (14, 15). The ultrasmall dimensions of the nanotube enable handling of single molecules the size of a typical protein in the low-nanome- Functionalization of membrane networks VL tube trelax = 2 ter range. The volume of the fluid in such a small Lipid membranes can host a variety of synthetic and biological 2pDrcontained tube channel can be as low as 10–18 L. components; therefore, certain functionalities, especially mem5962
trelax V = t 2V tube
A N A LY T I C A L C H E M I S T R Y / S E P T E M B E R 1 , 2 0 0 6
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brane proteins, can be embedded. Several proteins, such as receptors and ion channels, can be extracted from cellular systems and reconstituted into a synthetic lipid host environment (29–31). Similarly, other proteins and lipids can be directly extracted from biological cells (28). A high degree of compositional complexity can be achieved with both strategies. Davidson et al. demonstrated the successful formation of NVNs with functional, reconstituted membrane protein from red blood cells (17 ). Figures 2a and 2b show a four-container vesicle network with membrane proteins in both the outer and inner shells of the internal submicrometer vesicles. The lateral distribution of protein in the network membrane was determined via monitoring of the fluorescence of labeled, reconstituted band 3 (anion exchanger) protein. Protein distribution, at least for the labeled species, was homogeneous, and protein could diffuse via a nanotube connecting two vesicles. The interior solution of the nanotube–vesicle structures can be differentiated by injecting dyes, colloidal particles, organelles, or small unilamellar vesicles with reconstituted proteins or polymers into different nodes to yield an initially heterogeneous distribution of these species (Figure 2c). Diffusional relaxation will occur for materials that can permeate the nanotubes, although well-defined internal structures can be formed in NVNs to control diffusion. An example is the controlled generation of hydrogels within the internal compartments (32, 33). Polymer solutions or suspensions can be microinjected into vesicles that subsequently undergo controlled sol–gel transitions. Poly(N-isopropyl acrylamide) undergoes a reversible phase transition at 32 8C in pure water and at 27 8C in 100-mM phosphate buffer solution; it allows injection, mixing, and manipulation of clear solutions while enabling reversible solidification and compartmentalization as well as entrapment and release of materials on demand (Figures 2d and 2e). Another material used in GUVs is an aqueous suspension of the electrically conducting “synthetic metal” poly(ethylenedioxythiophene) in a solution of excess poly(styrene sulfonate). This polymer mixture forms hydrogels with high ion conductivity and an electron-conducting polymer backbone upon cross-linking with di-, tri-, or tetravalent metal ions. Vesicles and networks with hydrogel interiors of high density allow, for example, the study of cellular kinetics under simulated macromolecular crowding (cell-like) conditions, controlled drug release, transport, and hindered-diffusion studies. By providing a basic internal structure in a highly controlled manner, this system could serve as a starting point for a model of the biological cell (34, 35).
Transport NVNs can be used as small-scale analytical systems to characterize and quantify small numbers of biological molecules or to study reactions in a crowded, cell-like environment. However, controlled transport of material and solutes through the nanotubes connecting the vesicle containers is essential. Three different modes of
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