Name: aqueous droplets used as enzymatic microreactors and their electromagnetic actuation
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The authors would like to acknowledge the DFG for the support.

1. Hydrophobization of Magnetic Nanoparticles
<ol>
	<li>For the synthesis of the hydrophobic magnetic particles, add 0.85 g FeCl3 hexahydrate (3.14 mmol) and 0.30 g FeCl2 tetrahydrate (1.51 mmol) to 200 mL water/ethanol solution (4:1 v/v).</li>
	<li>To this mixture, add 0.20 mL 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (PFOTES) (5.23 mmol) with vigorous stirring by a magnetic stirrer (500 rpm). Carry out the synthesis in an inert gas atmosphere (N2) by using a round-bottom flask with a ...

<ol>
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For the successful use of microfluidic technologies, it is important to move the reaction volume corresponding to the requirements of the biotechnological synthesis and analyses. The actuation platform presented here makes it possible to move microfluidic droplets by magnetic force. The movement can be performed freely in two dimensions on a planar surface of a reaction platform by enclosing the liquid drop with a magnetic superhydrophobic shell. Thus an alternative system to predefined microfluidic channels with complex...

Technical applications with micro reactions are predominantly carried out in predefined microchannel chips. These systems are widely established and comprehensively described in the literature (inter alia 1,2,3). In 2011, the turnover of microfluidic technologies worldwide totaled 6.2 billion euro 4. In contrast, the use of freely movable micro reactor compartments was previously only examined and published to a limited extent. The most common method for moving aqueous micro droplets is electrowetting 5. Other methods for the motion of drops on surfaces are based on electric fields 6, magnetic force 7 or acoustic actuation 8. Due to their unfavorable surface to volume ratio, these droplet-based microreactor systems are exposed to strong evaporation effects. Thus, the drop motion is usually established as a liquid two-phase system, where the upper phase has a high boiling point protecting the aqueous phase from evaporation. Nevertheless, this approach involves a high risk of contaminating the reaction droplet by uncontrolled diffusion. This is a significant obstacle for the technical establishment of the mentioned systems.
Recent work is concerned with non-adherent liquid-solid phase transitions. A highly effective approach is the use of superhydrophobic surfaces, allowing the formation of spherical aqueous droplets. An extension of this reaction concept is the use of micro reaction compartments with a superhydrophobic surface or shell, which may for example consist of polytetrafluoroethylene (PTFE) particles 9. Their contact angles on surfaces are usually in the range of 160&#176; (depending on the surface roughness). The spherical compartments thus provide minimal resistance to movement on a surface and simultaneously provide protection against water evaporation.
Aqueous drops coated with micro sized PTFE particles may maintain their spherical shape up to a diameter of around 2 mm. At higher volumes, the hydrophobic shell is usually not completely closed anymore 10. The influence of other shell materials and the expansion of the field of application of the liquid marble to nonpolar solvents was implemented by Gao and McCarthy by using ionic liquids 12. For the formation of hydrophobic particle-based shells, so far particle diameters in sizes of 10 nm- 30 &#181;m have been described 11,14,16. New studies showed that hydrophobic nanoparticles as shell material are of even better use than that of microparticles 13. First stability studies confirmed an increase in stability when the particle size is reduced from ca. 600 nm to ca. 100 nm. This likely results from the denser particle distribution around the aqueous sphere 15.
The protection of aqueous reaction compartments by a hydrophobic shell and their designation as liquid marbles was first described in 2001 by Aussillous et al. and Mahadevan et al. 17,18. Since then, few applications of these defined reaction compartments have been described. For example, a gas sensor based on liquid marbles 19 and a detection method for water contamination based on an optically qualitative basis have been developed 20. The authors distinguish the advantages of high reaction rates and the low consumption of chemicals of their micro reaction systems. Recent publications deal with the production of pH-sensitive liquid marbles 16 or the representation of &#39;Janus particles&#39; with two different coatings of different functionality. For example, Bormashenko et al. could synthesize a microreactor with shells made of Teflon and semiconducting Carbon Black 21. Furthermore it was demonstrated that microreactors can efficiently and convenient synthesize polyperoxides by absorbing external oxygen as comonomer through the permeable gas-liquid interface 24. In another approach the shell of silica-particle-based liquid marbles provide the reactive substrate surfaces to regulate the classical silver mirror reaction 26. Current problems for research and development in the field of hydrophilic-core-hydrophobic-shell droplets are the particle size adjustment, the reproducible production of monodisperse droplets, the wettability of surfaces and the effect of a second hydrophilic shell on the micro reaction compartments 22, as well as a better control of the droplet trajectories, e.g. for the development of continuous microPCR-systems 4.
A magnetic actuation of these microreactors offers the advantage of relatively high movement ranges and a good selectivity of the force when working in biochemical systems. When using hydrophobic magnetite particles, they fulfil both the function of the magnetic force transmission to the movement of the microreactors, as well as the function of a hydrophobic shell. The magnetic movement of droplets with magnetic particles inside a droplet was postulated for the first time in 2006 by Lehmann et al. 23 and Shikida et al. 25, who used manually moved permanent magnets as actuators for the mobilization of a single droplet. Another approach to move a small amount of liquid was realized by Zhao et al., who used the hydrophobic Fe3O4 particles as magnetic shell. The shell of the magnetic liquid marble was opened on the upper side of the drop by a vertical reverse magnetic field 27. Based on this concept, Xue et al. were able to develop particles which form a microreactor with a surface tension of 20.1 dyne cm&#8722;1 28. Lin et al. fabricated novel cellulose-based micro/nano hierarchical spheres with both superparamagnetism and superhydrophobicity which provide god stability for magnetic liquid droplet transportation and manipulation 31.This was so far only released as a proof-of-principle study and not used for any application. The magnetic and electrical control of the liquid marbles is currently pursued in first approaches. Zhao et al. in 2010 15 and Zhang et al. 2012 29 were able to develop a droplet manipulation by the manual (hand-operated) movement of a permanent magnet beneath core-shell droplets. Bormashenko et al. 11 achieved the acceleration of a ferromagnetic liquid marble to a speed of 25 cm s-1 by approaching a neodymium magnet. The above mentioned principle studies were carried out exclusively by the manual movement of a small permanent magnet. As a next development step, Zhao et al. were recently able to estimate the required magnetic flux density for the movement of magnetic liquid marble by varying the distance of a permanent magnet 30. For a reaction control comparable to that of common lab-on-a-chip systems, it seems inevitable to provide the means of automated control of the discrete liquid volumes. To satisfy this need, we developed a new control system based on variable field gradients to fixate, move and open the magnetic microreactors.

<table border="0" cellpadding="0" cellspacing="0" width="903">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">3D-printer</td>
			<td style="width:163px;">FelixPrinters</td>
			<td style="width:211px;">Pro1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red)</td>
			<td>Life Technologies</td>
			<td>A12222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium hydroxide</td>
			<td style="width:163px;">TU-KL</td>
			<td>1072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CAD software</td>
			<td style="width:163px;">Siemens</td>
			<td style="width:211px;">Soled edge</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Contact angle measuring device</td>
			<td style="width:163px;">Dataphysics</td>
			<td style="width:211px;">OCA 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Cylinder magnet</td>
			<td style="width:163px;">Webcraft GmbH</td>
			<td style="width:211px;">S-04-13-N</td>
			<td>https://www.supermagnete.de/stabmagnete-neodym-rund/stabmagnet-durchmesser-4mm-hoehe-12.5mm-neodym-n42-vernickelt_S-04-13-N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dipotassium phosphate</td>
			<td>Bernd Kraft</td>
			<td>7758-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drying oven</td>
			<td>Binder</td>
			<td>FD 115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>68-17-5</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">FeCl2 tetrahydrate</td>
			<td style="width:163px;">TU-KL</td>
			<td>1625</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">FeCl3 hexahydrate</td>
			<td style="width:163px;">TU-KL</td>
			<td>1622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence probe</td>
			<td style="width:163px;">PerkinElmer</td>
			<td style="width:211px;">LS 55</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horseradish peroxidase</td>
			<td>Carl Roth</td>
			<td>9003-99-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen peroxide</td>
			<td>Th.Geyer GmbH &#38; Co</td>
			<td>7722-84-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monopotassium phosphate</td>
			<td>Bernd Kraft</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peltier element</td>
			<td style="width:163px;">Conrad&#160;</td>
			<td style="width:211px;">193569</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfluoroctyltriethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>51851-37-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanning Electron Microscope</td>
			<td style="width:163px;">FEI</td>
			<td style="width:211px;">Helios NanoLab 650 DualBeam</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Separation bar magnet</td>
			<td style="width:163px;">Webcraft GmbH</td>
			<td style="width:211px;">Q-40-20-10-N,&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Winding machine</td>
			<td style="width:163px;">IWT GmbH</td>
			<td style="width:211px;">FW122</td>
			<td></td>
		</tr>
	</tbody>
</table>

aqueous droplets used as enzymatic microreactors and their electromagnetic actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

The shell particles have a diameter of around 640 nm. The magnetizable nanoparticles enclosed in this fluorosilane shell particles have diameters in a range between 22 nm and 37 nm. A 5 &#181;L microreactor with water as a liquid core had a contact angle of around 160&#176;.
The force needed to move a 10 &#181;L microreactor as described above is 1.34 &#177; 0.08 &#181;N. Figure 1 shows the electrom...

Watch this Scientific Journal Video about Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation at JoVE.com

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

The overall purpose of this device is to provide a new microfluidic platform technology that realizes automated cascade reactions in small aqueous droplets. This method could help answer key questions to the field of synthetic biology by reproducing reaction sequences of microorganisms performing complex reaction sequences with enzymes and screening new catalysts. The main advantage of our lab-in-a-drop approach is that it is very adaptable.As opposed to channel-based microfluidic chips, the only prerequisite is a hydrophilic-reaction environment. Though this method can provide insight into enzymatic reactions, it can also be applied to other systems, such as inorganic catalysts or stringing of ligands. I first had the idea for this method when I tried to design a microfluidic device with integrated product separation but without a complex system of valves and separators.By moving the reaction solution as a droplet, reaction control becomes very simple. First, design in 3D print the coil bodies. Then, use a winding machine to wrap the bodies with a 08-millimeter copper wire 45 hundred times each.Place a Peltier element on an electric board. Screw the first layer of coils onto the Peltier element to form a square. Then, secure the second layer of coils on top with the 3D-printed plastic frame or other non-conductive material.Connect the electric board to the magnet control with a ribbon cable. Insert neodymium magnets into the coils. Place a one-millimeter thick quartz glass or plastic plate on top of the coil matrix.Secure the cover in place with screws to finish assembling the actuation platform. To begin the nanoparticle synthesis under an inert atmosphere, first suspend 85 grams Iron(III)chloride hexahydrate and 3 grams Iron(II)chloride tetrahydrate in 200 milliliters of a four-to-one water-to-ethanol solution. Then, add 0.2 milliliters PFOTES and stir the mixture at 500 RPM with a magnetic stir bar.Add 1.5 molar ammonium hydroxide to the mixture to achieve a pH of 8.0. And then continue stirring the solution for 24 hours to obtain the hydrophobic magnetic nanoparticles. Affix a bar magnet with an adhesive force of 25 kilograms to the bottom of the reaction flask.Pour off the solution and wash the particles three times with a four-to-one water-to-ethanol solution. Remove the magnet and dry the particles at 60 degree Celsius for 24 hours. Characterize the particles with scanning electron microscopy.Prepare a solution of 0.1 micrograms per milliliter horseradish peroxidase in 0.1 molar pH 6.5 potassium phosphate buffer with 10 millimolar hydrogen peroxide. Then, prepare a 20-micromolar solution of the fluorescent probe 10-Acetyl-3, 7-dihydroxyphenoxazine in potassium phosphate buffer. Then, gently grind the dry nanoparticles using a glass mortar and pestle.Transfer the particles to a polystyrene weighing pan and add 10 microliters of the peroxidase solution. Gently swirl the pan for about 10 seconds to achieve nanoparticle self assembly around the peroxidase solution. Transfer this microreactor to the actuation platform.Repeat this process with 10 microliters of the fluorescent probe solution and place the second microreactor on the platform. Store the remaining particles at room temperature. Use the Peltier element to keep the reaction solutions in the microreactors at 25 degree Celsius.Mount a fluorescence microscope about 10 millimeters above the microreactors and connect the microscope to a computer. Using the magnet control activate the magnets and turn to position the microreactor of fluorescent probe solution under the microscope. Turn on the excitation light.Raise the coil magnet to open the microreactor and begin recording the fluorescence microscopy image. After two-to-five seconds, lower the magnet to close the microreactor. Then, use the magnet control to move the peroxidase microreactor adjacent to the fluorescence microreactor.Open the fluorescent probe microreactor. The peroxidase microreactor is pulled into the probe microreactor and opened, initiating the reaction. Monitor the reaction by fluorescence microscopy opening and closing the merged microreactor as needed.Microreactors were created by coding droplets of solution with hydrophobic iron nanoparticles. These microreactors can be moved, opened, closed, and merged by manipulation of neodymium magnets under an actuation platform. The actuation platform can also be constructed with iron cores in the coil bodies.This provides enough magnetic force to move a microreactor by more than 10 millimeters but is insufficent to open the microreactor. Thus, a neodymium magnet must be used to perform reactions in small solution volumes. A representative enzymatic reaction between a peroxidase and a fluorescent label was performed and monitored by fluorescent microscopy.The Michaelis-mentioned kinetics observed during the reaction agreed well with literature values, indicating the the microreactor setup does not influence the reaction progress. After watching this video, you should have a good understanding of how to synthesize hydrophobic microparticles and to generate aqueous microdroplets with them. The platform technology can be used for automated reaction control by droplet merging.Additionally, single microreactors filled with substrate can moved on top of small surface zones with mobilized enzymes. Using multiple immobilzation zones with different enzymes enables a sequential reaction cascade with transportable and immediate products. This prototype development is just the beginning.Currently, we are implementing an automated dispenser system above the reaction platform to reposit immobilized enzymes at any position. The coil matrix will be increased to 10 times 10 positions. This technique can pave the way for computer-controlled planning and execution of biochemical reaction sequences.

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Research

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Bioengineering

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.