This work is part of the Joint Solar Programme (JSP) of Hyet Solar and the Stichting voor Fundamenteel Onderzoek der Materie FOM, which is part of the Netherlands Organization for Scientific Research (NWO).

1. Preparation of a Block for Sectioning
<ol>
 <li>Treat a technical-grade 3" silicon wafer in an air plasma cleaner for 30 sec and then expose it to (tridecafluoro-1,1,2,2,-tetrahydrooctyl)trichlorosilane vapor for one hour. Note: This step is necessary prior to step 1.4 to prevent the epoxy from adhering to the silicon wafer. </li>
 <li>Deposit a layer of gold (usually 100 nm-thick, which defines the width of the wires) through a Teflon master (that defines the length of the resulting wires; 0.5 mm, 1 mm, or 1.5 mm) onto the pre-treated silicon wafer.</li>
 <li>Cover the entire wafer with ~ 8.5 ml of Epofix epoxy pre-polymer and cure for three hours at 60 &deg;C. </li>
 <li>Template-strip the gold layer by carefully peeling the epoxy from the wafer such that the gold remains attached to the cured epoxy. Insert the edge of a razor blade at the interface between the silicon wafer and epoxy and then gently peel the epoxy layer from the silicon wafer. Because of the poor adhesion of gold to the fluorinated silicon wafer (step 1.1) the gold features remain adhered to the epoxy. Note: Be careful not to break the silicon wafer, otherwise the silicon particles will damage the diamond knife in the sectioning step.</li>
 <li>The same Teflon mask is placed back over the gold features, but laterally offset by ~ 80% of the shortest dimension of the gold features and deposit a layer of aluminum or silver through the Teflon master. The thickness of this layer will define the spacing of the nano-gap between the gold wires. The lower limit depends on the metal, but is ~ 5 nm for aluminum and silver, below which the layers become discontinuous. Note: This offset will eventually define the length of the overlap between two gold electrodes and you can measure it with micro-ruler. </li>
 <li>For gaps below 5 nm: Immerse the template-stripped gold on epoxy in a 1 mM solution of an alkanedithiol in ethanol (or any solvent that does not swell the epoxy) overnight in a closed chamber that is purged with nitrogen (to mitigate the spontaneous formation of disulfides). (In this paper we use 1,12-dodecanedithiol, 1,14-tetradecanedithiol or 1,16-hexadecanedithiol to produce gaps of various widths below 3 nm.) Remove the template-stripped gold-on-epoxy substrate from the SAM-forming solution. Rinse it with ethanol and dry it with nitrogen before drying at 60 for 2 min. </li>
 <li>If using a SAM, place back the Teflon mask onto the epoxy substrate but laterally offset by ~ 80% of the shortest dimension of the gold features. If using a metal, the mask is already in position after Step 1.5, so do not change the position of the mask after the deposition of the aluminum or silver. </li>
 <li>Deposit a second layer of gold or any other metal through the mask. This layer will typically be composed of the same metal at the thickness as the first(100 nm-thick in this case).</li>
 <li>Remove the Teflon mask, taking care not to scratch the features, which will result in broken nanowires.</li>
 <li> Re-embed the entire substrate in Epofix pre-polymer (~ 8.5 ml) and cure it for at least three hours at 60. </li>
 <li>Cut the features out using a jeweler's saw (into ~ 4 x 10 mm pieces) and place each into a separate well in a polyethylene ''coffin'' microtome mold. </li>
 <li>Fill the mold with Epofix pre-polymer, and cure it overnight at 60 &deg;C. </li>
</ol>
2. Sectioning
<ol>
 <li>Remove a block from the polyethylene mold and mount it in the sample holder.</li>
 <li>Attach the sample holder to the trimming attachment and mount it in the ultramicrotome.</li>
 <li>Clean a razor blade with ethanol to remove lubricant and metal fragments and inspect the edge of the razor blade under the stereoscope of the ultramicrotome. Any remaining fragments will damage the diamond knife during sectioning. Trim the block to the width of the diamond knife (we use 2 mm or 4 mm Diatome Ultra 35 &deg;) in a trapezoid shape (because it is the most stable shape for sectioning). Note: some ultramicrotomes use trimming attachments that mount to the cutting arm, but we achieve better results with razor blades. </li>
 <li>Align the edge of a glass knife parallel to the bottom edge of the face of the block.</li>
 <li>Start pre-cutting with the ultramicrotome (we used a Leica EMUC-6) equipped with a glass knife to define a smooth surface on the face of the block. </li>
 <li>To fabricate a metallic structure, replace the glass knife with a diamond knife, re-align it, and section the block to either 100 nm at 1 mm/sec or 50 nm at 0.6 mm/sec. Epofix sections are stable down to ~ 30 nm. Note: an easy verification of the thicknesses of the sections is their color, which varies predictable as a function of thickness and does not depend on the resin which has been used; reference cards are available.16</li>
 <li>Collect the epoxy sections containing the structures from the surface of the water in the reservoir of the knife either individually using a Perfect Loop (Electron Microscopy Sciences) or as ribbons of several sections to an Si/SiO2 (for SEM) or SiO2 (for electrical measurements) substrate by placing substrate under the surface of the water and raising it slowly. </li>
 <li>Dry the sections at 60 &deg;C for 3 hr to improve their adhesion to the substrate.</li>
 <li>To ash the epoxy, expose the samples to an oxygen plasma (15 min at 1 mbar is sufficient to remove all traces of the epoxy from 100 or 50 nm-thick sections). Note: If fabricating nanostructures for electrical measurements, this step must be performed after Step 4. </li>
</ol>
3. Etching out the Sacrificial Layer
<ol>
 <li>For aluminum: Place the sections containing aluminum in a 2 M aqueous solution of HCl for 2 hr. For silver: expose the sections to oxygen plasma for 10 min. Note: the selection of materials allows either wet-etching (using HCl) or dry-etching (using oxygen plasma), however silver can be removed by wet etching as well. </li>
 <li>For SAMs: Plasma treatment partially etches the SAMs, but we have been unsuccessful in our efforts to characterize to what degree. </li>
</ol>
4. Electrical Measurements
<ol>
 <li>Place epoxy sections on a SiO2 substrate that has been thoroughly cleaned (e.g. using pira&ntilde;a solution) and dry them (steps 2.7 and 2.8).</li>
 <li>Mount the substrate under a light microscope or the stereoscope attached to the ultramicrotome.</li>
 <li>Apply drops of silver paste (or carbon ink) on two ends of wires in each section. These embedded metallic structures will be visible as either a black line (from the gold/epoxy interface) or, in the case of thicker gold structures (from the deposition steps), directly visible. In either case, the drops should be applied sufficiently far from the center to short the nano-gaps.</li>
</ol>

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No conflicts of interest declared.

In this paper we demonstrated the fabrication of nanogap structures using nanoskiving. This experimentally simple method enables the production of nanostructures at the rate of about one per second, with control over all three dimensions. The gap-size is defined by incorporating either sacrificial layers of aluminum and silver or self-assembled monolayers of dithiols (which affords a resolution as small as &Aring;). The nanostructures can be positioned by hand on any arbitrary substrate and they are directly electrical...

This paper describes the fabrication of electrically addressable, high-aspect-ratio nanowires of gold separated by gaps of single nanometers using vacuum-deposited aluminum and silver as a sacrificial spacer layers for gaps > 5 nm and self-assembled monolayers (SAMs) of alkanedithiols for gaps as small as 1.7 nm. We fabricated these nanostructures without a clean room or any photolithographic processes by sectioning sandwich structures of gold separated by a sacrificial spacer using an ultramicrotome, a form of edge lithography known as nanoskiving.1-3 This method is a combination of the deposition of thin metal films and sectioning using an ultramicrotome. The main step in nanoskiving is slicing thin sections with an ultramicrotome equipped with diamond knife which is attached to a boat full of water to produce slabs that are as thin as ~ 30 nm. Ultramicrotomes are used extensively for the preparation of thin samples for imaging with optical or electron microscopy and many of the most experience practitioners of ultramicrotomy come from a biological or medical background. There are several methods of fabricating nanogaps including mechanical break junctions,4 electron-beam lithography5, electrochemical plating,6, 7 electromigration,8 focused ion beam lithography,9 shadow evaporation,10 scanning probe and atomic force microscopy,11 on-wire lithography,12 and molecular rulers.13 All of these methods have their own characteristics and applications but producing and addressing nanogaps both in useful numbers and with precise control over the dimensions of the gap remains a challenge. In addition these methods have high operating costs, they are limited to the class of materials that can survive the etching processes, and are limited in resolution. Nanoskiving enables the rapid fabrication of electrically-addressable nanowires with spacings of single nanometers on the bench-top. We are interested in the rapid prototyping of nanostructures for Molecular Electronics, for which the nano-fabricated electrodes do not require specialized or time-consuming techniques;14 once a block is made, it can produce hundreds of thousands of nanostructures, (serially) on demand. However, the technique is not limited to SAMs or Molecular Electronics and is a general method for preparing a gap between two nanostructures. In this paper we use silver, aluminum, and SAMs as sacrificial layers to produce gaps of various sizes between gold nanowires, but the technique is not limited to these materials (or to metallic nanowires). The wires are pick-and-place and are compatible with magnetic alignment, thus they can be placed on arbitrary substrates.15 Another strength of nanoskiving is that it affords control over all three dimensions. The dimensions of the samples are determined by the topography of the substrate (X), the thickness of the deposited film (Y) and the thickness of the slab produced by the ultramicrotome (Z). Figure 1 summarizes the procedure used to produce the nanowires with the defined spacing. Gold features (1-2 mm in length) are deposited by evaporation through a Teflon mask onto a silicon substrate. Epofix (Electron Microscopy Sciences) epoxy pre-polymer is poured over the entire wafer, covering the gold features, when the epoxy is cured, the epoxy is separated from the wafer (i.e. via template stripping); the gold features remain adhered to the epoxy. For metallic sacrificial layers, aluminum or silver is evaporated with the desired thickness through the Teflon mask with an offset of 200 - 500 &mu;m over the gold features. To produce sub-5 nm gaps, a SAM is formed by submerging the gold features in a 1 mM ethanolic solution of the appropriate dithiol overnight. A second set of gold (or another metal) is deposited by placing the Teflon shadow mask over the first layer of gold features (covered in silver, aluminum or a SAM) with an offset of 200 - 500 &mu;m with respect to the first evaporation. This offset will eventually define the longest dimension of the gap, and it can be accurately measured using a micro-ruler before embedding the entire structure in epoxy for sectioning. Then the whole structure is embedded in a block of epoxy which then could be ready for sectioning with the ultramicrotome. The sample arm holds the prepared block as the diamond knife advances towards it in controlled steps that will define the thickness of the slabs. The resulting section floats on the water in the boat.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>Epofix epoxy resin </td>
 <td> Electron Microscopy </td>
 <td> 1232 </td>
 <td> </td>
 </tr>
 <tr>
 <td></td>
 <td> Sciences </td>
 <td> </td>
 <td> </td>
 </tr>
 <tr>
 <td>Gold </td>
 <td> Schone Edelmetaal B.V</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Aluminum </td>
 <td> Umicore Materials AG </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Silver </td>
 <td> Umicore Materials AG </td>
 <td> </td>
 <td></td>
 </tr>
 <tr>
 <td>(tridecafluoro-1,1,2,2, </td>
 <td> ABCR GmbH co.KG</td>
 <td> 78560-45-9 </td>
 <td></td>
 </tr>
 <tr>
 <td>-tetrahydrooctyl) </td>
 <td> </td>
 <td> </td>
 <td> </td>
 </tr>
 <tr>
 <td>trichlorosilane </td>
 <td></td>
 <td> </td>
 <td> </td>
 </tr>
 <tr>
 <td>,12-dodecanedithiol </td>
 <td> Home-synthesised </td>
 <td> </td>
 <td>According to: Akkerman et. al., Nature. 441, 69-72 (2006)</td>
 </tr>
 <tr>
 <td>,14-tetradecanedithiol </td>
 <td> synthesized in house </td>
 <td> </td>
 <td> According to: Akkerman et. al., Nature. 441, 69-72 (2006)</td>
 </tr>
 <tr>
 <td>,16-hexadecanedithiol </td>
 <td> synthesized in house </td>
 <td> </td>
 <td>According to: Akkerman et. al., Nature. 441, 69-72 (2006)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Thermal deposition system </td>
 <td> home-built </td>
 <td> </td>
 <td></td>
 </tr>
 <tr>
 <td>Ultramicrotome </td>
 <td> Leica Microsystems </td>
 <td> </td>
 <td></td>
 </tr>
 <tr>
 <td>Dimanod knife ultra 35 </td>
 <td> Diatome </td>
 <td> DU3540</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimanod knife ultra 45 </td>
 <td> Scimed GMBH </td>
 <td> </td>
 <td></td>
 </tr>
 <tr>
 <td>Scanning electron microscope </td>
 <td> JOEL </td>
 <td> </td>
 <td></td>
 </tr>
 <tr>
 <td>Source meter </td>
 <td> Keithley </td>
 <td> </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Tables of Specific Reagents and Equipment.</td>
 </tr>
 </tbody>
</table>

fabricating nanogaps by nanoskiving

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two electrodes-and generating them in practical quantities-is still challenging. The preparation of nanogap electrodes using nanoskiving, which is a form of edge lithography, is a fast, simple and powerful technique. This method is an entirely mechanical process which does not include any photo- or electron-beam lithographic steps and does not require any special equipment or infrastructure such as clean rooms. Nanoskiving is used to fabricate electrically addressable nanogaps with control over all three dimensions; the smallest dimension of these structures is defined by the thickness of the sacrificial layer (Al or Ag) or self-assembled monolayers. These wires can be manually positioned by transporting them on drops of water and are directly electrically-addressable; no further lithography is required to connect them to an electrometer.

We prepared nanogap structures by incorporating two metallic sacrificial layers as the spacer: aluminum and silver. We etched these layers to obtain gaps of the desired thicknesses. As described in the Protocol section, after sectioning we exposed the structures containing silver to oxygen plasma, and those containing aluminum to aqueous HCl. Figure 2 shows scanning electron micrographs (SEMs) of the resulting nanowires with nanometer-scale separation. In both cases gaps are clearly visible and directly ...

Watch this Scientific Journal Video about Fabricating Nanogaps by Nanoskiving at JoVE.com

Fabricating Nanogaps by Nanoskiving

The fabrication of electrically addressable, high-aspect-ratio (> 1000:1) metal nanowires separated by gaps of single nanometers using either sacrificial layers of aluminum and silver or self-assembled monolayers as templates is described. These nanogap structures are fabricated without a clean room or any photo- or electron-beam lithographic processes by a form of edge lithography known as nanoskiving.

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two ...

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11.0K Views.  University of Groningen. The fabrication of electrically addressable, high-aspect-ratio (> 1000:1) metal nanowires separated by gaps of single nanometers using either sacrificial layers of aluminum and silver or self-assembled monolayers as templates is described. These nanogap structures are fabricated without a clean room or any photo- or electron-beam lithographic processes by a form of edge lithography known as nanoskiving.

Video: Fabricating Nanogaps by Nanoskiving

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Channels for the transportation of water molecules in enzymes influence active site solvation and catalysis. Herein we present a protocol for the engineering of these additional catalytic motifs based on in silico computer modeling and experiments. This will enhance our understanding of the influence of solvent dynamics on enzyme catalysis.

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually ...

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, 1,4-benzene-bis(iminoguanidinium) (BBIG), is demonstrated. The ligand is synthesized as the chloride salt (BBIG-Cl) by in situ imine condensation of terephthalaldehyde with aminoguanidinium chloride in water, followed by crystallization as the sulfate salt (BBIG-SO4). Alternatively, BBIG-Cl is synthesized ex situ in larger scale from ethanol. The sulfate separation ability of the BBIG ligand is demonstrated by selective and quantitative crystallization of sulfate from seawater. The ligand can be recycled by neutralization of BBIG-SO4 with aqueous NaOH and crystallization of the neutral bis-iminoguanidine, which can be converted back into BBIG-Cl with aqueous HCl and reused in another separation cycle. Finally, 35S-labeled sulfate and &#946; liquid scintillation counting are employed for monitoring the sulfate concentration in solution. Overall, this protocol will instruct the user in the necessary skills to synthesize a ligand, employ it in the selective crystallization of sulfate from aqueous solutions, and quantify the separation efficiency.

A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion co-flow device to generate uniform high-viscosity droplets in a low-viscosity fluid. The microfluidic capillary device has a common co-flow structure with its exit connecting to a wider tube. Elongated droplets of the low-viscosity fluid are first encapsulated by the high-viscosity fluid in the co-flow structure. As the elongated low-viscosity droplets flow through the exit, which is treated to be wetted by the low-viscosity fluid, phase inversion is then induced by the adhesion of the low viscosity droplets to the tip of the exit, which results in the subsequent inverse encapsulation of the high-viscosity fluid. The size of the resultant high-viscosity droplets can be adjusted by changing the flow rate ratio of the low-viscosity fluid to the high-viscosity fluid. We demonstrate several typical examples of the generation of high-viscosity droplets with a viscosity up to 11.9 Pas, such as glycerol, honey, starch, and polymer solution. The method provides a simple and straightforward approach to generate monodisperse high-viscosity droplets, which may be used in a variety of droplet-based applications, such as materials synthesis, drug delivery, cell assay, bioengineering, and food engineering.

A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics.

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion ...

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

Photodeposition of Pd onto Colloidal Au Nanorods by Surface Plasmon Excitation

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot electrons upon SPR irradiation drive reductive deposition of Pd on colloidal AuNR in the presence of [PdCl4]2-. Plasmon-driven reduction of secondary metals potentiates covalent, sub-wavelength deposition at targeted locations coinciding with electric field &#8220;hot-spots&#8221; of the plasmonic substrate using an external field (e.g., laser). The process described herein details a solution-phase deposition of a catalytically-active noble metal (Pd) from a transition metal halide salt (H2PdCl4) onto aqueously-suspended, anisotropic plasmonic structures (AuNR). The solution-phase process is amenable to making other bimetallic architectures. Transmission UV-vis monitoring of the photochemical reaction, coupled with ex situ XPS and statistical TEM analysis, provide immediate experimental feedback to evaluate properties of the bimetallic structures as they evolve during the photocatalytic reaction. Resonant plasmon irradiation of AuNR in the presence of [PdCl4]2- creates a thin, covalently-bound Pd0 shell without any significant dampening effect on its plasmonic behavior in this representative experiment/batch. Overall, plasmonic photodeposition offers an alternative route for high-volume, economical synthesis of optoelectronic materials with sub-5 nm features (e.g., heterometallic photocatalysts or optoelectronic interconnects).

A protocol for anisotropic photodeposition of Pd onto aqueously-suspended Au nanorods via localized surface plasmon excitation is presented.

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot ...

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Nitrogen Compound Characterization in Fuels by Multidimensional Gas Chromatography

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is crucial. There are significant challenges to overcome when measuring trace compounds in a complex matrix such as fuels. Background interferences and matrix effects can create limitations to routine analytical instrumentation, such as GC-MS. In order to facilitate specific and quantitative measurements of trace nitrogen compounds in fuels, a nitrogen-specific detector is ideal. In this method, a nitrogen chemiluminescence detector (NCD) is used to detect nitrogen compounds in fuels. NCD utilizes a nitrogen-specific reaction that does not involve the hydrocarbon background. Two-dimensional (GCxGC) gas chromatography is a powerful characterization technique as it provides superior separation capabilities to one-dimensional gas chromatography methods. When GCxGC is paired with a NCD, the problematic nitrogen compounds found in fuels can be extensively characterized without background interference. The method presented in this manuscript details the process for measuring different nitrogen-containing compound classes in fuels with little sample preparation. Overall, this GCxGC-NCD method has been shown to be a valuable tool to enhance the understanding of the chemical composition of nitrogen-containing compounds in fuels and their impact on fuel stability. The % RSD for this method is &#60;5% for intraday and &#60;10% for interday analyses; the LOD is 1.7 ppm and the LOQ is 5.5 ppm.

Here, we present a method utilizing two-dimensional gas chromatography and nitrogen chemiluminescence detection (GCxGC-NCD) to extensively characterize the different classes of nitrogen-containing compounds in diesel and jet fuels.

Certain nitrogen-containing compounds can contribute to fuel instability during storage. Hence, detection and characterization of these compounds is ...