We thank the Lindback Foundation for financial support, and Dr. Alexander Sidorenko and Elza Chu for fruitful discussions and technical assistance, and Professor Robert Smith for assistance with the C. elegans assays.

NOTE: In the procedures described below, laboratory safety guidelines must always be followed. Of particular importance is the safety when dealing with high voltage (&#62; 500 V) and handling chemicals.
1. Coating of a Conductive Substrate with Candle Soot
<ol>
	<li>Cut copper metal into rectangles (75 x 43 mm, 0.5 mm thick). Clean each copper substrate by immersion in copper etchant for about 30 sec, wash with tap water for about 20 sec, and dry with paper. 
	NOTE: If using Method 1 be...

<ol>
	<li>Fair, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics:+is+a+true+lab-on-a-chip+possible.">Digital microfluidics: is a true lab-on-a-chip possible.</a> Microfluid Nanofluid. 3 (3), 245-281 (2007).</li><li>Gupta, S., Alargova, R. G., Kilpatrick, P. K., Velev, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-Chip+Dielectrophoretic+Coassembly+of+Live+Cells+and+Particles+into+Responsive+Biomaterials.">On-Chip Dielectrophoretic Coassembly of Live Cells and Particles into Responsive Biomaterials.</a> Langmuir. 26 (5), 3441-3452 (2009).</li><li>Shih, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dried+blood+spot+analysis+by+digital+microfluidics+coupled+to+nanoelectrospray+ionization+mass+spectrometry.">Dried blood spot analysis by digital microfluidics coupled to nanoelectrospray ionization mass spectrometry.</a> Anal Chem. 84 (8), 3731-3738 (2012).</li><li>Gorbatsova, J., Borissova, M., Kaljurand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrowetting-on-dielectric+actuation+of+droplets+with+capillary+electrophoretic+zones+for+off-line+mass+spectrometric+analysis.">Electrowetting-on-dielectric actuation of droplets with capillary electrophoretic zones for off-line mass spectrometric analysis.</a> J Chromatogr. 1234, 9-15 (2012).</li><li>Qin, J., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+exploration+and+learning+in+C.+elegans.">Maze exploration and learning in C. elegans.</a> Lab Chip. 7 (2), 186-192 (2007).</li><li>Koc, Y., de Mello, A. J., McHale, G., Newton, M. I., Roach, P., Shirtcliffe, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-scale+superhydrophobicity:+suppression+of+protein+adsorption+and+promotion+of+flow-induced+detachment.">Nano-scale superhydrophobicity: suppression of protein adsorption and promotion of flow-induced detachment.</a> Lab Chip. 8 (4), 582-586 (2008).</li><li>Perry, G., Thomy, V., Das, M. R., Coffinier, Y., Boukherroub, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+protein+biofouling+using+graphene+oxide+in+droplet-based+microfluidic+microsystems.">Inhibiting protein biofouling using graphene oxide in droplet-based microfluidic microsystems.</a> Lab Chip. 12 (9), 1601-1604 (2012).</li><li>Kumari, N., Garimella, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrowetting-Induced+Dewetting+Transitions+on+Superhydrophobic+Surfaces.">Electrowetting-Induced Dewetting Transitions on Superhydrophobic Surfaces.</a> Langmuir. 27 (17), 10342-10346 (2011).</li><li>Freire, S. L. S., Tanner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive-Free+Digital+Microfluidics.">Additive-Free Digital Microfluidics.</a> Langmuir. 29 (28), 9024-9030 (2013).</li><li>Deng, X., Mammen, L., Butt, H. -. J., Vollmer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candle+Soot+as+a+Template+for+a+Transparent+Robust+Superamphiphobic+Coating.">Candle Soot as a Template for a Transparent Robust Superamphiphobic Coating.</a> Science. 335, 67-70 (2011).</li><li>Kang, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Electrostatic+Fields+Change+Contact+Angle+in+Electrowetting.">How Electrostatic Fields Change Contact Angle in Electrowetting.</a> Langmuir. 18 (26), 10318-10322 (2002).</li><li>Abdelgawad, M., Watson, M. W. L., Young, E. W. K., Mudrik, J. M., Ungrin, M. D., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography:+masters+on+demand.">Soft lithography: masters on demand.</a> Lab Chip. 8 (8), 1379-1385 (2008).</li><li>Barbulovic-Nad, I., Yang, H., Park, P. S., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics+for+cell-based+assays.">Digital microfluidics for cell-based assays.</a> Lab Chip. 8 (4), 519-526 (2008).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li></ol>

The most critical step of the protocol is the protection of the soot layer, directly associated with the success in moving droplets. Metallizing the soot layer (method 1 above) allows close to 100% of fabrication success. However, the maximum operation time is about 10 min; possibly, droplet fractions are wetting the soot through holes in the metal layer. Coating the soot layer with the fluorinated liquid is the easiest and fastest alternative, and requires minimum resources, but only 40&#8211;50% of the fabricated subst...

The miniaturization of devices that work with liquids is of paramount importance for the development of &#8220;lab-on-a-chip&#8221; platforms. In this direction, the last two decades have witnessed a significant progress in the field of microfluidics, with a variety of applications.1-5 Contrasting with the transport of fluid in enclosed channels (channel microfluidics), DMF manipulates droplets on arrays of electrodes. One of the most attractive merits of this technique is the absence of movable parts to transport fluids, and motion is instantly stopped by turning off electric signals.
However, droplet motion is dependent on droplet contents, certainly an undesirable characteristic for a universal &#8220;lab-on-a-chip&#8221; platform. Droplets containing proteins and other analytes stick to device surfaces, becoming unmovable. Arguably, this has been the major limitation for broadening the scope of DMF applications;6-8 alternatives to minimize the unwanted surface fouling involve the addition of extra chemical species to the droplet or its surroundings, which could potentially affect droplet content.
Previously, our group developed a device to allow the transport of cells and proteins in DMF, without extra additives (Field-DW devices).9 This was achieved by combining a surface based on candle soot,10 with a device geometry that favors droplet rolling and leads to an upward force on the droplet, further decreasing droplet-surface interaction. In this approach, droplet motion is not associated with surface wetting.11
The goal of the detailed method described below is to produce a DMF device capable of transporting droplets containing proteins, cells, and entire organisms, without extra additives. The Field-DW devices pave the way for fully controlled platforms working largely independently of droplet chemistry.
Here, we also present simulations showing that, despite the high voltage required for device operation, the voltage drop across the droplet is a small fraction of the applied voltage, indicating negligible effects on bioanalytes inside the droplet. In fact, preliminary tests with Caenorhabditis elegans (C. elegans), a nematode used for a variety of studies in biology, show that worms swim undisturbed as voltages are applied.

<table border="0" cellpadding="0" cellspacing="0" width="1230">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:161px;">Catalog Number</td>
			<td style="width:720px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Paraffin candle</td>
			<td style="width:119px;"></td>
			<td style="width:161px;"></td>
			<td>Any paraffin candle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Sputtering system</td>
			<td style="width:119px;">Denton Vacuum, Moorestown, NJ</td>
			<td style="width:161px;"></td>
			<td>Sputter coater Desk V HP equipped with an Au target.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1-dodecanethiol</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td align="right" style="width:161px;">471364</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Teflon</td>
			<td style="width:119px;">Dupont</td>
			<td style="width:161px;">AF-1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fluorinert FC-40</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:161px;">F9755</td>
			<td>Fluorinated liquid: Prepare Teflon-AF resin in Fluorinert FC-40, 1:100 (w/w), to create the hydrophobic coating.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Graphic design software -Adobe Illustrator</td>
			<td style="width:119px;">Adobe Systems</td>
			<td style="width:161px;"></td>
			<td>Other softwares might be used as well.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:231px;">Copper laminate</td>
			<td style="width:119px;">Dupont</td>
			<td style="width:161px;">LF9110</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:231px;">Laser Printer</td>
			<td style="width:119px;">Xerox</td>
			<td style="width:161px;">Phaser 6360 or similar</td>
			<td>Check for the compatibility with &#34;rich black&#34; or &#34;registration black&#34; (see text).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Copper Etchant</td>
			<td style="width:119px;">Transene</td>
			<td style="width:161px;">CE-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Perfluoroalkoxy (PFA) film</td>
			<td style="width:119px;">McMaster-Carr</td>
			<td style="width:161px;">84955K22</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:231px;">Breadboard</td>
			<td style="width:119px;">Allied Electronics</td>
			<td style="width:161px;">70012450 or similar</td>
			<td>Large enough to allow the assemble of 10 drivers.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:231px;">Universal circuit board</td>
			<td style="width:119px;">Allied Electronics</td>
			<td style="width:161px;">70219535 or similar</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:231px;">Connector</td>
			<td style="width:119px;">Allied Electronics</td>
			<td style="width:161px;">5145154-8 or similar</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Control board and control program (LabView software)</td>
			<td style="width:119px;">National Instruments</td>
			<td style="width:161px;">NI-6229 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">High-voltage amplifier</td>
			<td style="width:119px;">Trek</td>
			<td style="width:161px;">PZD700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Resistor R 27 k&#8486;, 1/4 W</td>
			<td style="width:119px;">Allied&#160;</td>
			<td align="right" style="width:161px;">2964762</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:231px;">Capacitors C and C1, 100 nF, 60 V</td>
			<td style="width:119px;">Allied&#160;</td>
			<td align="right" style="width:161px;">8817183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Transistor T, NPN</td>
			<td style="width:119px;">Allied&#160;</td>
			<td align="right" style="width:161px;">9350289</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Diode D, 1N4007</td>
			<td style="width:119px;">Allied&#160;</td>
			<td align="right" style="width:161px;">2660007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Relay&#160;</td>
			<td style="width:119px;">Allied&#160;</td>
			<td align="right" style="width:161px;">8862527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Visualization system</td>
			<td style="width:119px;">Edmund Optics</td>
			<td style="width:161px;">VZM 200i or similar</td>
			<td>System magnification 24X- 96X. It is combined with a Hitachi KP-D20B 1/2 in CCD Color Camera.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Recorder</td>
			<td style="width:119px;">Sony</td>
			<td style="width:161px;">GV-D1000 NTSC or similar</td>
			<td>It is connected to the camera by an S-video cable.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Simulations</td>
			<td style="width:119px;">COMSOL Multiphysics</td>
			<td style="width:161px;">V. 4.4</td>
			<td></td>
		</tr>
	</tbody>
</table>

taking advantage of reduced droplet-surface interaction to optimize transport of bioanalytes in digital microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

Previously, we have used Field-DW devices to allow the motion of proteins in DMF. In particular, droplets with bovine serum albumin (BSA) could be moved at a concentration 2,000 times higher than previously reported by other authors (without additives). This was due to the reduced interaction between droplet and surface; Figure 4 shows a droplet containing fluorescently-tagged BSA (see Freire et al.9 for more information on the experiments). The first picture on the left shows the dro...

Watch this Scientific Journal Video about Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics at JoVE.com

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

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7.8K ビュー.  University of the Sciences. The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

ビデオ: Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet...

a versatile kit based on digital microfluidics droplet actuation for science education

A Versatile Kit Based on Digital Microfluidics Droplet Actuation for Science Education

We describe an educational kit that allows users to execute multiple experiments and gain hands-on experience on digital...

digital microfluidics for automated proteomic processing

Digital Microfluidics for Automated Proteomic Processing

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing...

droplet digital trap (ddtrap): adaptation of the telomere repeat amplification protocol to droplet digital polymerase chain reaction

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human...

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

plasmid stability analysis with open-source droplet microfluidics

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate...

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

multicolor fluorescence detection for droplet microfluidics using optical fibers

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single...

quantification of circular rnas using digital droplet pcr

Quantification of Circular RNAs Using Digital Droplet PCR

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

electrowetting-based digital microfluidics platform for automated enzyme-linked immunosorbent assay

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay...

quantification of adeno-associated viral genomes in purified vector samples by digital droplet polymerase chain reaction

Author Spotlight: Optimizing Digital Droplet PCR Method for Accurate Adeno-Associated Viral Genome Quantification

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome...

Standardized Protocol for Viral Genome Quantification Using Digital Droplet PCR

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digital-droplet pcr to detect indels mutations in genetically modified anopheline mosquito populations

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant...

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Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

droplet generation and digital droplet pcr for reliable quantification of aav viral genome titers in vector samples

Droplet Generation and Digital Droplet PCR for Reliable Quantification of AAV Viral Genome Titers in Vector Samples

fabrication, operation and flow visualization in surface-acoustic-wave-driven acoustic-counterflow microfluidics

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping...

employing digital droplet pcr to detect braf v600e mutations in formalin-fixed paraffin-embedded reference standard cell lines

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE...

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

single droplet digital polymerase chain reaction for comprehensive and simultaneous detection of mutations in hotspot regions

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

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Author Spotlight: Addressing Regulatory Gaps in Molecular Studies by Quantifying Viral Vectors in Complex Matrices 

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A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears

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Transport of Surface-modified Carbon Nanotubes through a Soil Column

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

digital droplet polymerase chain reaction for indel mutation detection in target genes

Using a 50-microliter multichannel pipette, load 20 microliters of the ddPCR sample mix into the middle row of the cartridge. Then, use a 200-microliter multichannel pipette to load 70 microliters of the oil into the bottom row without generating bubbles throughout the...

Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes

This video demonstrates the role of digital droplet polymerase chain reaction in identifying indel mutation in a DNA sample. The high precision and sensitivity of ddPCR enable accurate and consistent detection of rare mutations present in a sample, even at low levels. It can identify and quantify indel mutations in different regions of the genome, providing valuable insights into the mechanisms of mutation and the functional consequences of these...

Evaluating Plasmonic Transport in Current-carrying Silver Nanowires

Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information support1,2,3. In this context, metal nanowires are especially desirable for realizing dense routing networks4. A prerequisite to operate such shared nanowire-based platform relies on our ability to electrically contact individual metal nanowires and efficiently excite surface plasmon polaritons5 in this information support. In this article, we describe a protocol to bring electrical terminals to chemically-synthesized silver nanowires6 randomly distributed on a glass substrate7. The positions of the nanowire ends with respect to predefined landmarks are precisely located using standard optical transmission microscopy before encapsulation in an electron-sensitive resist. Trenches representing the electrode layout are subsequently designed by electron-beam lithography. Metal electrodes are then fabricated by thermally evaporating a Cr/Au layer followed by a chemical lift-off. The contacted silver nanowires are finally transferred to a leakage radiation microscope for surface plasmon excitation and characterization8,9. Surface plasmons are launched in the nanowires by focusing a near infrared laser beam on a diffraction-limited spot overlapping one nanowire extremity5,9. For sufficiently large nanowires, the surface plasmon mode leaks into the glass substrate9,10. This leakage radiation is readily detected, imaged, and analyzed in the different conjugate planes in leakage radiation microscopy9,11. The electrical terminals do not affect the plasmon propagation. However, a current-induced morphological deterioration of the nanowire drastically degrades the flow of surface plasmons. The combination of surface plasmon leakage radiation microscopy with a simultaneous analysis of the nanowire electrical transport characteristics reveals the intrinsic limitations of such plasmonic circuitry.

Silver nanowires can simultaneously transport electrons and optical information in the form of surface plasmons. A procedure is described here to realize such a shared circuitry and the limitations at propagating both information carriers are evaluated.

Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information ...

Characterization of Thermal Transport in One-dimensional Solid Materials

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials, including conductive, semi-conductive or nonconductive one-dimensional structures. This technique broadens the measurement scope of materials (conductive and nonconductive) and improves the accuracy and stability. If the sample (especially biomaterials, such as human head hair, spider silk, and silkworm silk) is not conductive, it will be coated with a gold layer to make it electronically conductive. The effect of parasitic conduction and radiative losses on the thermal diffusivity can be subtracted during data processing. Then the real thermal conductivity can be calculated with the given value of volume-based specific heat (&#961;cp), which can be obtained from calibration, noncontact photo-thermal technique or measuring the density and specific heat separately. In this work, human head hair samples are used to show how to set up the experiment, process the experimental data, and subtract the effect of parasitic conduction and radiative losses.

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials.

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials, including ...

Real-Time DC-dynamic Biasing Method for Switching Time Improvement in Severely Underdamped Fringing-field Electrostatic MEMS Actuators

Mechanically underdamped electrostatic fringing-field MEMS actuators are well known for their fast switching operation in response to a unit step input bias voltage. However, the tradeoff for the improved switching performance is a relatively long settling time to reach each gap height in response to various applied voltages. Transient applied bias waveforms are employed to facilitate reduced switching times for electrostatic fringing-field MEMS actuators with high mechanical quality factors. Removing the underlying substrate of the fringing-field actuator creates the low mechanical damping environment necessary to effectively test the concept. The removal of the underlying substrate also a has substantial improvement on the reliability performance of the device in regards to failure due to stiction. Although DC-dynamic biasing is useful in improving settling time, the required slew rates for typical MEMS devices may place aggressive requirements on the charge pumps for fully-integrated on-chip designs. Additionally, there may be challenges integrating the substrate removal step into the back-end-of-line commercial CMOS processing steps. Experimental validation of fabricated actuators demonstrates an improvement of 50x in switching time when compared to conventional step biasing results. Compared to theoretical calculations, the experimental results are in good agreement.

The robust device design of fringing-field electrostatic MEMS actuators results in inherently low squeeze-film damping conditions and long settling times when performing switching operations using conventional step biasing. Real-time switching time improvement with DC-dynamic waveforms reduces the settling time of fringing-field MEMS actuators when transitioning between up-to-down and down-to-up states.

Mechanically underdamped electrostatic fringing-field MEMS actuators are well known for their fast switching operation in response to a unit step input ...

How to Build a Vacuum Spring-transport Package for Spinning Rotor Gauges

The spinning rotor gauge (SRG) is a high-vacuum gauge often used as a secondary or transfer standard for vacuum pressures in the range of 1.0 x 10-4 Pa to 1.0 Pa. In this application, the SRGs are frequently transported to laboratories for calibration. Events can occur during transportation that change the rotor surface conditions, thus changing the calibration factor. To assure calibration stability, a spring-transport mechanism is often used to immobilize the rotor and keep it under vacuum during transport. It is also important to transport the spring-transport mechanism using packaging designed to minimize the risk of damage during shipping. In this manuscript, a detailed description is given on how to build a robust spring-transport mechanism and shipping container. Together these form a spring-transport package. The spring-transport package design was tested using drop-tests and the performance was found to be excellent. The present spring-transport mechanism design keeps the rotor immobilized when experiencing shocks of several hundred g (g = 9.8 m/sec2 and is the acceleration due to gravity), while the shipping container assures that the mechanism will not experience shocks greater than about 100 g during common shipping mishaps (as defined by industry standards).

Here we describe how to build a robust spring-transport mechanism for a spinning rotor gauge. This device securely immobilizes the rotor and keeps it under vacuum during transportation. We also describe packaging that minimizes the risk of damage during transport. Tests show our design works for typical shocks during transport.

The spinning rotor gauge (SRG) is a high-vacuum gauge often used as a secondary or transfer standard for vacuum pressures in the range of 1.0 x 10-4 Pa to ...

Digital Printing of Titanium Dioxide for Dye Sensitized Solar Cells

Silicon solar cell manufacturing is an expensive and high energy consuming process. In contrast, dye sensitized solar cell production is less environmentally damaging with lower processing temperatures presenting a viable and low cost alternative to conventional production. This paper further enhances these environmental credentials by evaluating the digital printing and therefore additive production route for these cells. This is achieved here by investigating the formation and performance of a metal oxide photoelectrode using nanoparticle sized titanium dioxide. An ink-jettable material was formulated, characterized and printed with a piezoelectric inkjet head to produce a 2.6 &#181;m thick layer. The resultant printed layer was fabricated into a functioning cell with an active area of 0.25 cm2 and a power conversion efficiency of 3.5%. The binder-free formulation resulted in a reduced processing temperature of 250 &#176;C, compatible with flexible polyamide substrates which are stable up to temperatures of 350 &#730;C. The authors are continuing to develop this process route by investigating inkjet printing of other layers within dye sensitized solar cells.

This paper investigates the suitability of inkjet printing for the manufacturing of dye-sensitized solar cells. A binder-free TiO2 nanoparticle ink was formulated and printed onto a FTO glass substrate. The printed layer was fabricated into a cell with an active area of 0.25 cm2 and an efficiency of 3.5%.

Silicon solar cell manufacturing is an expensive and high energy consuming process. In contrast, dye sensitized solar cell production is less ...

Ultrasonic Fatigue Testing in the Tension-Compression Mode

Ultrasonic fatigue testing is one of a few methods which allow investigating fatigue properties in the ultra-high cycle region. The method is based on exposing the specimen to longitudinal vibrations on its resonance frequency close to 20 kHz. With use of this method, it is possible to significantly decrease the time required for the test, when compared to conventional testing devices usually working at frequencies under 200 Hz. It is also used to simulate loading of material during operation in high speed conditions, such as those experienced by components of jet engines or car turbo pumps. It is necessary to operate only in the high and ultra-high cycle region, due to the possibility of extremely high deformation rates, which can have a significant influence on the test results. Specimen shape and dimensions have to be carefully selected and calculated to fulfill the resonance condition of the ultrasonic system; thus, it is not possible to test the full components or specimens of arbitrary shape. Before each test, it is necessary to harmonize the specimen with the frequency of the ultrasonic system to compensate for deviations of the real shape from the ideal one. It is not possible to run a test until a total fracture of the specimen, since the test is automatically terminated after initiation and propagation of the crack to a certain length, when the stiffness of the system changes enough to shift the system out of the resonance frequency. This manuscript describes the process of evaluation of materials' fatigue properties at high-frequency ultrasonic fatigue loading with use of mechanical resonance at a frequency close to 20 kHz. The protocol includes a detailed description of all steps required for a correct test, including specimen design, stress calculation, harmonizing with the resonance frequency, performing the test, and final static fracture.

A protocol for ultrasonic fatigue testing in the high and ultra-high cycle region in axial tension-compression loading mode.

Ultrasonic fatigue testing is one of a few methods which allow investigating fatigue properties in the ultra-high cycle region. The method is based on ...

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

Crack Monitoring in Resonance Fatigue Testing of Welded Specimens Using Digital Image Correlation

A procedure using digital image correlation (DIC) to detect cracks on welded specimens during fatigue tests on resonance testing machines is presented. It is intended as a practical and reproducible procedure to identify macroscopic cracks at an early stage and monitor crack propagation during fatigue tests. It consists of strain field measurements at the weld using DIC. Images are taken at fixed load cycle intervals. Cracks become visible in the computed strain field as elevated strains. This way, the whole width of a small-scale specimen can be monitored to detect where and when a crack initiates. Subsequently, it is possible to monitor the development of the crack length. Because the resulting images are saved, the results are verifiable and comparable. The procedure is limited to cracks initiating at the surface and is intended for fatigue tests under laboratory conditions. By visualizing the crack, the presented procedure allows direct observation of macrocracks from their formation until rupture of the specimen.

Digital image correlation is used in fatigue tests on a resonance testing machine to detect macroscopic cracks and monitor crack propagation in welded specimens. Cracks on the specimen surface become visible as increased strains.

A procedure using digital image correlation (DIC) to detect cracks on welded specimens during fatigue tests on resonance testing machines is presented. It ...

Automated Delivery of Microfabricated Targets for Intense Laser Irradiation Experiments

Described is an experimental procedure that enables high-power laser irradiation of microfabricated targets. Targets are brought to the laser focus by a closed feedback loop that operates between the target manipulator and a ranging sensor. The target fabrication process is explained in detail. Representative results of MeV-level proton beams generated by irradiation of 600 nm thick gold foils at a rate of 0.2 Hz are given. The method is compared with other replenishable target systems and the prospects of increasing the shot rates to above 10 Hz are discussed.

A protocol is presented for automated irradiation of thin gold foils with high intensity laser pulses. The protocol includes a step-by-step description of the micromachining target fabrication process and a detailed guide for how targets are brought to the laser&#39;s focus at a rate of 0.2 Hz.

Described is an experimental procedure that enables high-power laser irradiation of microfabricated targets. Targets are brought to the laser focus by a ...