The authors would like to thank the Natural Sciences and Engineering Research Council of Canada, Canada Research Chairs Program, Canadian Institutes of Health Research, and Ontario Ministry of Research and Innovation through their Early Researchers Award Program for financial support.

1. Photolithography for Master Mold Fabrication
<ol>
	<li>Bathe a 3 in. silicon wafer in acetone for 30 sec and then methanol for 30 sec. Rinse with dH20 water for 5 min.</li>
	<li>Dry the wafer's surface with a N2 blow gun. Heat the wafer on a hot plate at 140 &deg;C for 2 min.</li>
	<li>Plasma oxidize the surface of the silicon wafer (1 min, 50 W).</li>
	<li>Spin-coat the wafer's surface with 3 ml SU-8 100 photoresist (40 sec; 1,750 rpm).</li>
	<li>Pre-bake the coated wafer on a hot plate at 65 &deg;C for 10 min, then ramp the temperature up to 95 &deg;C over 2 min. Maintain this setting for an additional 1 hr.</li>
	<li>Align a photomask containing the desired channel design. Expose the resist to 550-600 mJ/cm2 of UV light (350-400 nm). Photomasks can be designed in AutoCAD and printed on a transparency with high resolution printing.</li>
	<li>Post-bake the wafer on a hot plate at 65 &deg;C for 1 min and 95 &deg;C for 10 min, ramping the temperature as before.</li>
	<li>Immerse the wafer in SU-8 developer solution for 10-15 min. Check for completion of development by rinsing with isopropanol. If a white precipitate appears, continue developing. The master mold is shown in Figure 2A.</li>
</ol>
2. Soft Lithography for Microchannel Fabrication
<ol>
	<li>Mix 35 ml polydimethylsiloxane (PDMS) elastomer base with 3.5 ml PDMS curing agent.</li>
	<li>Place the fabricated master mold (pattern facing up) and a blank silicon wafer into Petri dishes lined with aluminum foil.</li>
	<li>Pour 20 ml PDMS prepolymer into the master mold dish and 15 ml into the second dish. Eliminate air pockets underneath the wafers by gently pressing on them with a disposable wooden applicator.</li>
	<li>Cover both dishes and set aside for a day to cure. Alternatively, for faster curing, remove air bubbles from the PDMS using a vacuum degasifier and then leave the dishes on a hot plate at 80 &deg;C for 2 hr.</li>
	<li>Remove the foil and peel the PDMS from the wafers.</li>
	<li>Use the Harris Uni-Core (2.5 mm) to punch fluid access ports at both ends of the channel. Cut the channel and blank PDMS into similarly sized strips.</li>
	<li>Load the channel, the blank PDMS strip and a glass slide (75&times;25 mm2) into a plasma oxidizer, likely located in a cleanroom. Expose to oxygen plasma for 40 sec at 40 W power.</li>
	<li>Stick the channel piece and glass slide to opposite sides of the blank strip. Set aside for 2 hr to complete the bonding.</li>
	<li>Place the assembly onto a hot plate at 120 &deg;C. Attach plastic tubing (inner diameter 1/32", outer diameter 3/32"), each at least 6 in. long, to the punched reservoirs using PDMS prepolymer. Affix a fluidic plastic connector to one or both tubes to allow syringe attachment, or use commercially available fittings.</li>
	<li>Allow the PDMS securing the tubing to cure. Insert 3" lengths of 22 gauge insulated copper wire into each reservoir, between the inlet tube and the channel, and secure with PDMS prepolymer. The finished product is shown in Figure 2B.</li>
</ol>
3. Electrotaxis Experiment
<ol>
	<li>Place the microchannel on the stage (preferably XY-movable) of a microscope with a mounted camera connected to a monitor (Figure 1).</li>
	<li>Connect the power supply or amplifier's output wires to the microchannel's electrodes. A simple DC power supply is sufficient if only a DC signal is desired, but an amplifier connected to a function generator allows application of pulsed DC and AC signals as well.</li>
	<li>Attach the microchannel's output tube to a disposable syringe. Submerge the mouth of the inlet tube in M9 physiological buffer and gently aspire liquid into the channel by applying a negative pressure inside the syringe (either manually or using a syringe pump). When the inlet and outlet tubes are both filled with M9, disconnect the syringe from the tube. Level both tubes to the same height to prevent hydrostatically driven flow.</li>
	<li>Apply a DC voltage to the channel and ensure that resistance (R= V/I) is around 0.6 M&Omega; (for a 50 mm long, 0.3 mm wide and ~0.1 mm deep microchannel).</li>
	<li>If satisfied with the channel's integrity, follow the above steps to load worms from a diluted suspension into the channel.</li>
	<li>Disconnect the syringe and hydrostatically manipulate the flow by adjusting the tubes' relative height. Use this method to place a worm in the center of the channel and then lay both tubes flat at the same elevation.</li>
	<li>Set the power supply to the appropriate voltage: 4-12 V/cm for L3 stage animals, 4-10 V/cm for L4s, and 2-4 V/cm for young adults. Activate the electric signal and allow 1 min of pre-exposure for the worm to acclimatize to the field. The worm should begin moving towards the cathode. When the minute has passed, use the camera to begin recording.</li>
	<li>For AC and pulsed DC experiments, the maximum responsive electric field can be adopted from above and frequency and duty cycle of the signal can be modulated as desired 7, 8.</li>
	<li>When experiment is finished, remove all liquid (and worms) from the channel, rinse it with dH20, and leave the device on a hot plate at 125 &deg;C to dry.</li>
	<li>Extract locomotory data from recorded videos manually using NIH ImageJ (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) or custom MATLAB-based worm tracking software.</li>
</ol>

<ol>
	<li>Kuwahara, T., Koyama, A., 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=Familial+Parkinson+mutant+&#945;-synuclein+causes+dopamine+neuron+dysfunction+in+transgenic+Caenorhabditis+elegans.">Familial Parkinson mutant &#945;-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans.</a> J. Biol. Chem. 281 (1), 334-340 (2006).</li><li>Kuwahara, T., Koyama, A., 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=A+systematic+RNAi+screen+reveals+involvement+of+endocytic+pathway+in+neuronal+dysfunction+in+a-synuclein+transgenic.">A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in a-synuclein transgenic.</a> 17 (19), 2997-3009 (2008).</li><li>Su, L. J., Auluck, P. K., 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=Compounds+from+an+unbiased+chemical+screen+reverse+both+ER-to-Golgi+trafficking+defects+and+mitochondrial+dysfunction+in+Parkinson's+disease+models.">Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.</a> Dis. Model Mech. 3 (3-4), 194-208 (2010).</li><li>Rezai, P., Siddiqui, A., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+Caenorhabditis+elegans+in+a+microfluidic+environment.">Electrotaxis of Caenorhabditis elegans in a microfluidic environment.</a> Lab Chip. 10 (2), 220-226 (2010).</li><li>Salam, S., Ansari, A., 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=A+microfluidics+set+up+to+study+neuronal+degeneration+and+identification+of+neuroprotective+compounds+in+C.+elegans.">A microfluidics set up to study neuronal degeneration and identification of neuroprotective compounds in C. elegans.</a> , (2013).</li><li>Rezai, P., Salam, S., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+sorting+of+Caenorhabditis+elegans.">Electrical sorting of Caenorhabditis elegans.</a> Lab Chip. 12 (10), 1831-1840 (2012).</li><li>Rezai, P., Salam, S., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pulse+direct+current+signals+on+electrotactic+movement+of+nematodes+Caenorhabditis+elegans+and+Caenorhabditis+briggsae.">Effect of pulse direct current signals on electrotactic movement of nematodes Caenorhabditis elegans and Caenorhabditis briggsae.</a> Biomicrofluidics. 5 (4), 044116 (2011).</li><li>Rezai, P., Siddiqui, A., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+Caenorhabditis+elegans+in+alternating+electric+field+and+its+application+to+their+localization+and+control.">Behavior of Caenorhabditis elegans in alternating electric field and its application to their localization and control.</a> Appl. Phys. Lett. 96 (15), 153702 (2010).</li><li>van Ham, T. J., Thijssen, K. L., Breitling, R., Hofstra, R. M., Plasterk, R. H., Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+model+identifies+genetic+modifiers+of+alpha-synuclein+inclusion+formation+during+aging.">C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging.</a> PLoS Genet. 4, e1000027 (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><li>Gabel, C. V., Gabel, H., Pavlichin, D., Kao, A., Clark, D. A., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+mediate+electrosensory+behavior+in+Caenorhabditis+elegans.">Neural circuits mediate electrosensory behavior in Caenorhabditis elegans.</a> J. Neurosci. 27 (28), 7586-7596 (2007).</li><li>Chuang, H. -. S., Raizen, D. M., Lamb, A., Dabbish, N., Bau, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoresis+of+Caenorhabditis+elegans.">Dielectrophoresis of Caenorhabditis elegans.</a> Lab Chip. 11 (4), 599-604 (2011).</li><li>Cronin, C. J., Mendel, J. E., Mukhtar, S., Kim, Y. -. M., Stirbl, R. C., Bruck, J., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+system+for+measuring+parameters+of+nematode+sinusoidal+movement.">An automated system for measuring parameters of nematode sinusoidal movement.</a> BMC Genet. 6, 5 (2005).</li><li>Mani&#232;re, X., Lebois, F., Matic, I., Ladoux, B., Meglio, J. -. M. D. i., Hersen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Running+worms:+C.+elegans+self-sorting+by+electrotaxis.">Running worms: C. elegans self-sorting by electrotaxis.</a> PLoS One. 6 (2), e16637 (2011).</li></ol>

The microfluidic electrotaxis assay technology has been filed for patent in the United States of America and Canada.

Taking advantage of the behavioural phenomenon first described by Gabel and colleagues and building on the dielectrophoretic manipulation work of Chuang and colleagues 11,12, our microfluidic-based electrotaxis assay provides an easy, robust and sensitive method to probe neuronal activity in worms using movement as an output. The analysis of movement parameters allows quantitative comparison between different genotypes. The precision of microchannel fabrication and electric field application together provide b...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CALEDON Labs</td>
			<td>1200-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>CALEDON Labs</td>
			<td>6700-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>CALEDON Labs</td>
			<td>8600-1-40</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SU-8</td>
			<td>Microchem Corp.</td>
			<td>Y131273</td>
			<td>SU-8 100</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem Corp.</td>
			<td>Y020100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92x16 mm Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>Contains elastomer base and curing agent</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Tektronix Inc.</td>
			<td>&nbsp;</td>
			<td>Model AFG3022B</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Trek Inc.</td>
			<td>&nbsp;</td>
			<td>Model 2210-CE</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4506</td>
			<td>Model 11 ELITE</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>11675916Q</td>
			<td>Model HP131725Q</td>
		</tr>
	</tbody>
</table>

microfluidic-based electrotaxis for on-demand quantitative analysis of caenorhabditis elegans' locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

A representative video of a wild-type young adult nematode's electrotaxis and its position and velocity outputs from the worm tracking software are shown in Supplementary Video 1 and Figure 3. The movement analysis software itself does not recognize the direction of field polarity and the time of polarity reversal; rather, this information must be obtained from the source video. This could be done using an audio or visual cue in the video or writing down experimental conditions and manip...

Watch this Scientific Journal Video about Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion at JoVE.com

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis

Research

JoVE Journal

Bioengineering

9.8K visualizações.  McMaster University. Um método de micro-electro-fluídico semi-automatizado para induzir a demanda de locomoção em Caenorhabditis elegans É descrito. Este método baseia-se no fenómeno neurophysiologic de vermes respondem a campos eléctricos ligeiros ("electrotaxis") no interior de canais microfluidicos. Microfluidic electrotaxis serve como um baixo custo, e escalável técnica rápida, sensível, para triagem de fatores que afetam a saúde neuronal.

Vídeo: Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.
Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.2
C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

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 photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...