Scenario: Robot Production

Exploration of vesicular properties allowing to test experimentally partial solutions for a nano-robot

This section describes the experimental results, which relates directly to the road-map for the construction of a nano-robots. Based on these facts, in the application section, a plausible roadmap for the construction of such robots is developed. Methods for the production of vesicles were developed and these methods were adjusted that makes it easy to produce vesicles as well as to explore a large number of their properties. The developed system allows the production of differently sized vesicles with different contents. In a single production step up to 384 (in the current system with the use of four micro-well plates with 96 wells) different vesicles can be produced.



Left: Vesicles are produced in a two phase system. A water-in-oil-emulsion stabilized by a monolayer of phospholipids is added on top of an aquous solution. The oil-water interface is also stabilized by a monolayer of phospholipids. Vesicles are formed when the droplets pass the interface due to centrifugation. Right: For parallel productin of different types of vesicles, 96-well microplates are used.

Due the production method described above it is easy to determine the content of vesicles. In the following Figure three examples are illustrated in which the content of the vesicles was filled with a colorant.



Vesicles filled by Eosin (right) and malachit green (middle), and fluorescently labeled DNA.

In order to get programmable self-assembly specific linkers of different type had to be constructed. First, ssDNA molecules were directly bound to the phospholipids. Second, specific bonding between biotinylated phospholipids, strepatvidin and biotinylated ssDNA was used. In order to get a structured oil droplet-vesicle assembly (OVA), we need the permanent and selective adhesion of oil droplets and vesicles. It is conceivable that this could be achieved by linking vesicles with DNA-DNA linkers. We identified two different problems related to a highly specific linking of vesicles via hybridization of cholesterol-tagged membrane-anchored: 1. Because the processes of vesicle formation and vesicle functionalization (linking molecules) are not separated, the formation procedure has to be adjusted anew for each change in the vesicle modification - exacerbating the experimental procedure. 2. The cholesterol anchors of the cholesterol-tagged DNA spontaneously leave the lipid bilayer and incorporate randomly into (other) lipid bilayers. Thus, therefore the specificity of the linking system gets lost over time. Therefore, we propose the following procedure to avoid these problems associated with cholesterol-tagged membrane-anchored DNA-DNA-hybridization. The processes of vesicle formation and their functionalization are separated by a constant modification of the lipid bilayer during vesicle formation followed by a two-step functionalization process. By incorporating biotinylated phospholipids into the lipid bilayer during vesicle formation a functionalization process independent of the formation process is provided. After formation and incorporation of biotinylated phospholipids to the lipid bilayer the vesicles are functionalized by decoration of their surface with (strept)avidin first followed by a decoration with biotinylated DNA (btnDNA). Thus, linking of vesicles is established via a complex of biotinylated phospholipid-(strept)avidin-btnDNA hybrid-(strept)avidin- biotinylated phospholipid (Brigitte Städler, The anchorage of DNA strands using biotinylated phospholipid-(strept)avidin-btnDNA is stronger than cholesterol-tagged ssDNA. Thus, we expect no loss of specificity due to spontaneously leaving the lipid bilayer of the linkers and incorporate randomly into (other) lipid bilayers.



Concepts of how to equip vesicles with specific stickers. The linking is done by biotinylated phospholipids, streptavidin and biotinylated ssDNA.

DNA directly linked with phospholipids can exchange places with their neighbors. This exchange of linkers will lead to unspecific clustering of vesicles due to the distribution of the linkers on all partner vesicles. Therefore the second method with biotin-streptavidin-ssDNA-linkers proved to be superior due to anchoring of the sticker with two phospholipids and therefore a diminished probability of an exchange of linkers between two vesicles jeopardizing the specificity of linking between them. In the final step linking of vesicles was finally achieved. Only by combined efforts of SDU and UZH groups make the spontaneous linkage of vesicles by a hybridization of biotin-streptavidn-ssDNA-linkers possible.



Spontaneous linkage of vesicles by a hybridization of biotin-streptavidin-ssDNA-linkers. The fluorescently labeled streptavidin accumulates in the contact zone of the linked vesicles. During the development of vesicles able to bind specifically, alternative experiments using beads to explore self-assembling mechanisms were developed. Different types of beads with different sizes (250 nm, 1 µm, 100 µm) coated with biotin or streptavidin were used. These beads were used to thest self-assembly concepts. Control of unspecific adhesion of membrane-coated vesicles to surfaces is difficult. Beads and DNA-coated vesicles bind to surfaces and it is hard to find coatings which inhibit this unspecific binding but do not affect the stability of the vesicles themselves. One of the promising approaches was to use giant unilamellar vesicles (GUVs) as self-assembly chambers for vesicle-vesicle-, vesicle-bead-, and bead-bead-assemblies,, because neither beads nor vesicles bind spontaneously to the membrane of unmodified GUVs. The vesicular production methods developed during this project make it easy to put substances, and even beads or vesicles inside them. Due to the restricted temporal and spatial resolution of the laser scanning confocal microscope, the number of vesicles and beads within a single GUV had to be small to trace them properly.



Giant vesicles as self-assembly chambers for smaller units such as vesicles and beads. The examples show two different beads and a smaller vesicles inside a giant vesicle. The experimental results achieved in the PACE-consortium up to the present state give us the opportunity to interlace, anchor and explore theoretical IT-concepts directly with and in experimental work. Thereby, the IT-potential of multi-vesicular compounds can be exploited over a smooth range of increasing complexity. The information processing results from the interplay of only a few types of basic processes: a) self-assembly b) fusion and/or material exchange between adjacent vesicles c) chemical reactions in vesicles and d) material uptake. The basic idea is to implement these processes in a manner susceptible to external conditions and/or signals. Applications start at vesicles of different types assembling in patterns governed by external conditions. An intermediate level of sophistication is reached by self-assembled compounds of vesicles of different types and contents, where the assembly is controlled by specific surface interactions between the cells. A next step could be reached by controlled fusion of smaller, but specific vesicles with larger ones. By preparing vesicles with different surface properties as well as different contents, the controlled fusion allows to produce functionally differentiated vesicles. Furthermore, all components, which the current state of the artificial cells is not yet able to reproduce, could be replenished by controlling the fusion externally. This technique would allow exploring the potential of the artificial cell without having a fully self-contained one and would allow testing the IT potential experimentally. A concrete example of a working scenario would be the creation of a “chemical robot” in which functionally differentiated vesicles are seen as the robot’s building blocks specialized for structuring, sensing and moving. At the far end of this route stand fully metabolizing systems, whereby the metabolism lead to replication.

Actuation

We recently demonstrated autonomous and controlled movement of nano- and micro-scale oil droplets in aqueous media (Martin M. Hanczyc, Taro Toyota, Takashi Ikegami, Norman Packard, and Tadashi Sugawara. Fatty acid chemistry at the oil-water interface: Self-propelled oil droplets. JACS, 129:9386-9391, 2007). In this system the oil droplet agent can be thought of as a very small, chemically embodied soft matter robot. The agent is a synthesis of the following components: a motor based on an internal convection cell within the oil phase triggered by a Marangoni type of instability; the oil water interface acts as a very sensitive and dynamic sensor for the system; added surfactants that self-assemble at the interface between the oil droplet and water act as a link between the motor and the sensor, embedded chemistry within the oil droplet will fuel both autonomous movement and the processing of external chemical signals.

Robot Scenario

As stated in section (2.1) the question of how difficult the production of replicating artificial cells will be, has also to be investigated on the molecular level and not just on an abstract theoretical one. Therefore, efforts were undertaken to see how molecular interactions and material properties could be used to program vesicles to assemble to a functioning unit: a modular robot built on vesicles. An incremental approach was chosen. First, the vesicle should be linked in a programmable way allowing the positioning of vesicles with different content (functions, sensors, actuators). Then mechanisms for actuation, sensing and coupling of the two were investigated.

First, different physical constraints due to the small size of vesicles have to be fulfilled.

To self-assemble vesicles, which are dispersed in an aqueous solution or settled on a surface, they have, trivially so, to encounter each other. Vesicles of a diameter smaller than 500 nm could self-assemble by Brownian motion within a convenient time. The time span until two vesicles encounter depends on how fast the vesicles travel. The distance traveled follows the well known relation:



Using the Stokes formula and the Einstein relation, we get the following formula for the distance traveled depending on the size of the vesicles:



T is the temperature, k is the Boltzmann constant, η is the viscosity of water, distance is the mean-square distance traveled by a sphere with radius R . To give an example : 1 μm sphere in water would take about a minute to wander a mean-square distance of 5 μm. With a size of 10 μm and a traveled distance of 10 μm it would take 12 minutes already: a very slow exploration of the available space. Therefore, the 1 µm size is approximately an upper limit to get convenient waiting times for self-assembly. Therefore, we had to aim for smaller vesicles, preferably 250-500 nm. A further sine qua non for self-assembly, is the demand of a specific linkage of building units when they encounter each other. As vesicles are very soft, new forces between them arise. The softness of interfaces in general and membranes in particular has some striking consequences for the colloidal forces that act between them. A colloidal force is an entropic force. The origin of this force is to due to the thermal undulations of membranes. If a membrane is confined not to be able to wander further than a certain distance d from another vesicle, then a very large number of configurations that otherwise the membrane would have explored would be forbidden to it, with a consequent loss of entropy. This leads to a repulsive force, the so-called Helfrich force, which is

ΔF ~ (kB T)2 /(κ d3),

where κ is the bending rigidity, d the distance, T the temperature and kB the Boltzmann constant. This force has a quite large range of interactions. By changing the rigidity of the vesicles (osmotic pressure) or by using surface charges, this force can be reduced that it no longer inhibits spontaneous adhesion. Unspecific linking between vesicles can be avoided by using stickers, which were rather long. Overlap of stickers will create a repulsive entropic potential. We used biotinylated PEG2000 molecules, streptavidin and a biotinylated ssDNA to get a length of the sticker of approximately 10-15nm, which give a total distance of the vesicles of about 20-30 nm, a range in which the Helfrich force gets rather small. Once vesicle encounter, they should adhere only in a pre-programmed way. This programming was done by using specific, addressable stickers on the vesicular surface. The vesicle which contained biotinylated PEG linked to phospholipids were incubated with specific DNA-linkers bound to streptavidin. The resulting vesicles contained specific tags on their surfaces (single stranded sense DNA) were able to link to other vesicles having corresponding antisense-DNA-linkers on their membranes. Note that this approach allows to separate the production process of vesicles from the coating of their surface. This makes the production easier and avoids damaging interactions during the self-assembly of the vesicles. The linking process was investigated by fluorescently tagged molecules as outlined in the experimental section. The main result was that specific linking of vesicles could be established. DNA strands are negatively charged and bind only to each other in the presence of additional ions. By adjusting the additional ion-concentration we prepared a system in which the vesicles still could bind, but no hemifusion was observed. The distinction between hemifusion and specifically linked vesicles is rather simple: in the hemifused state the vesicles share a common membrane: thus, the adhesion surface gets void of the fluorescence signal. Whereas in the case of the specific linker the fluorescent signal gets stronger in the adhesion surface. Depending on the size of the used vesicles, different assembly methods were tried out. For pure self-assembly assisted by Brownian motion, small vesicles have to be used. For bigger vesicles one has to increase the encounter probability by inducing relaive movement of vesicle



Vesicles or beads with specific linkers on their surface are fixed in space by holding pipettes (gray cylinders). The vesicles are dragged towards the seeds by an induced flow. If the linkers of two encountering vesicles are compatible (sense and anti-sense DNA tags), the two vesicles will establish a link and stay together. The color of the vesicles indicates their tags of the vesicles, which are designer specified.

One of the disadvantages of this approach is that beads, vesicles, proteins and DNA stick to surfaces. During the development of the specific addresses for the vesicles, we performed also self-assembly experiments with beads. The first experiments failed, because in the moment the beads reached the glass slide of the microscope, they got stuck to this surface (a nice example of a random walk with absorption). Although different surface modifications were tried (silanisation, proteins, modified micro-well plates etc.), no substance could be found to inhibit the adsorption of the beads to such surfaces. These findings lead to the idea of using giant unilamellar vesicles as self-assembly chambers in which we did not observe any unspecific adhesion neither of beads nor of vesicles. The advantages of such small self-assembly chambers are at least twofold: first, the repulsive Helfrich force reduces unspecific interactions with surfaces and second, the small volumes of vesicles increase the encounter probability of the self-assembling partners (see the experimental part for an example). During the PACE project actuated oil droplets (see the experimental section about actuation) were built able to propagate (Martin M. Hanczyc, Taro Toyota, Takashi Ikegami, Norman Packard, and Tadashi Sugawara. Fatty acid chemistry at the oil-water interface: Self-propelled oil droplets. JACS, 129:9386-9391, 2007). Until now we had not enough time to find a way to link these oil droplets with the vesicles. An obvious way would be to cover the oil droplets with tagged phospholipids and link them to vesicles by the same self-assembly mechanism according to the following scenario:. An oil-in-water emulsion is stabilized by the addition of phospholipids partly linked to a biotinylated spacer. After coating of these biotinylated oil-droplets with biotinylated sense ssDNA (sense), vesicles coated with the corresponding antisense ssDNA pass through the oil-in-wate emulsion by centrifugation. On the path the vesicles will encounter the stabilized oil-droplets and specifically link to them. As the density of oil droplets is lower than the density of the aqueous solution as well as the vesicles, all unbound droplets will accumulate at the surface of the aqueous solution over time, whereas the vesicles (partly covered by oil droplets) will sink to the bottom of the solution. Thus, separating two populations of oil droplets: bound and unbound. In such a way the vesicles and their actuators could be linked in the near future. As in PACE no proteins as molecular machines are allowed (because its hard to replicate all the necessary protein-producing molecules), What would be a simple sensory system for a sensory-motor coupling? We propose to implement a temperature sensor within vesicles (by the generation of temperature dependent pores), which then will trigger the chemical actuation of the oil droplets. Such temperature-dependent porous membranes can be produced by mixing 1,2-di-myristoyl-sn-glycero-3-phospocholine (DMPC), 1,2-di-palmitoyl-sn-gylero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Using the different phase transition temperatures of these PCs, an efflux of H+-ions out of the vesicles to the ambient space around the vesicle-oil-droplet-hybrids will trigger these actuators.

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