transport, localization and separation of

TRANSPORT, LOCALIZATION AND SEPARATION OF CAENORHABDITIS ELEGANS USING ELECTROTAXIS FOR MOVEMENT BASED BEHAVIORAL ASSAYS IN DRUG DISCOVERY P. Rezai1, S. Salam2, P. R. Selvaganapathy1,*, B. P. Gupta2

1

Department of Mechanical Engineering, McMaster University, ON, CANADA and 2 Department of Biology, McMaster University, ON, CANADA

ABSTRACT Electric field-induced locomotion and transport of worms Caenorhabditis elegans (C. elegans) in a microfluidic device (electrotaxis) is demonstrated in this paper. Direct (DC) and alternating (AC) electric fields inside a microchannel were used to stimulate worms locomotion towards the negative pole and to halt their locomotion while localizing them at desired locations in the channel respectively. This technique promises to facilitate the study of movement-related disorders (e.g., Parkinson’s Disease) and to perform drug discovery assays on a chip. We also demonstrate an application of our system in separating worms based on their age and size from a mixed culture. KEYWORDS: C. elegans, Electrotaxis, Electric Field, Stimulus, Movement Disorder, Drug Discovery, Microfluidic INTRODUCTION AND THEORY Caenorhabditis elegans (worm), with its fully mapped neuronal circuitry [1], ~ 60% human genome homology [2], conserved cellular and molecular processes, short life time, transparent and optically accessible small body, and compatibility with green fluorescent protein imaging technique, is an important model organism for understanding the biology of diseases. For instance, various human diseases such as obesity, depression, hypertension, diabetes, Parkinson’s and Huntington’s have been modeled in C. elegans using mutations. These mutant strains have been used to screen for chemical libraries for gene target identification. However, the traditional laboratory methods used in these screens are expensive, time consuming, tedious and labor-intensive. Recently, high throughput robotic fluid handling systems, used in drug discovery [3], and automated biosorter (COPAS, Union Biometrica; http://www.unionbiometrica.com) have been used to increase the throughput of experimentation. Nevertheless, they are expensive, both to purchase and operate, and are not accessible to many researchers especially in the academic community and in small companies. In recent years, significant efforts have been made to use microfluidics to accelerate, automate, parallelize and lower the cost of manipulation of worms to examine phenotypes and chemical screening on these animals. Microfluidic environments offer significant advantages including high degree of control, accurate dosing, ability to observe and track individual worms, and flexibility to integrate pre- and post-processing of the worms that could be applied both for understanding fundamental biological processes as well as drug screening. This is evident with studies involving phenotypic analysis, imaging, culturing, and microsurgery performed mostly in the past few years [4, 5]. Many of these microfluidic devices use pneumatic forces that is suitable for immobilization, imaging and surgery on these worms. Nevertheless, pneumatics is not ideal for behavioral assays that require stimulation of the natural movement of the worm – on demand – and subsequent characterization of that movement. We have recently developed electrotaxis as a tool to induce natural movement of the worms and have characterized this response in microfluidic channels [6]. Here, we present a comprehensive analysis of C. elegans movement response to electric field (EF) stimulus of different kinds. Direct (DC) and alternating current (AC) EFs have been used to initiate worms’ locomotion towards the cathode and to localize them at designated locations inside a microfluidic channel, respectively. We also demonstrate an application of our system in electrotactically separating worms based on their age and size from a mixed culture. EXPERIMENTAL The schematic of the experimental setup is shown in Fig. 1a. It consists of PDMS microchannels {one for DC and AC electrotaxis assays (Fig.1a) and one for separation application (Fig.1b), see Fig. 1 caption for dimensions}, fabricated using traditional soft lithography techniques, with two copper electrodes at their reservoirs that are connected to an amplifier coupled with a function generator. Synchronized wild type C. elegans of various developmental stages (n=10 each) were delivered from the sample suspension into the channel by applying a suction force at the outlet reservoir using a syringe pump as illustrated in Fig. 1a. In the DC experiments, a constant electric field (1-12 V/cm) was applied across the axial direction of the channel and the response of the worm was recorded in a movie format using the camera connected to the microscope setup (Fig. 1a). In the AC experiments, a DC electric field was initially applied to stimulate the worm’s locomotion for a length of 2-3mm across the channel. When the worm reached the localization region of the microdevice (middle of the channel in Fig. 1b), square waveform AC electric fields (10 mHz to 3 kHz frequency) was subsequently applied and the worm’s response was recorded in a same way. After this, DC electric field was applied again to transport the worm out of localization spot. These videos were then analyzed using the ImageJ software to quantify the worm’s swimming speed under DC electrotaxis locomotion and range of movement under AC electrotaxis localization conditions. ImageJ transforms video files into snapshot images which can then be individually analyzed using the length measurement module of the software with less than 10µm accuracy for our devices. 978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS

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14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands

In the electrotactic separation experiment (Fig. 1c), the applied electric field was higher at the narrow section compared to the wide section at any applied potential across the channel. This enabled us to generate electric fields of different strengths across the channel while the channel widths are still wide enough for the worms to swim through.

Figure 1: Electrotaxis test setup [7], (a) Experimental setup, (b) DC and AC electrotaxis microchannel (50mm×0.3mm×0.1mm), (c) Electrotactic separation microchannel (28mm×0.3mm×0.1mm with a 0.1 mm-wide narrowing section at the middle)

RESULTS AND DISCUSSION DC electric fields stimulated the worm to swim towards the negative pole while square waveform AC electric fields with frequencies higher than 1 Hz localized them for the duration of signal application inside the channel. Fig. 2 illustrates the DC electrotatic swimming (a and c) and AC electrotatic localization (b) of a worm inside the microchannel. DC and AC electrotaxis responses were characterized for all responding developmental stages of worms in Fig. 3a&b respectively. The DC electrotaxis speed and AC electrotaxis range of motion for n=10 worms for each developmental stage was measured. These data were normalized by the average body length of each stage as stated in the top legend parentheses in Fig. 3. It was evident that electrotaxis speed does not vary with the DC electric field strength for each stage. However, Fig. 3a demonstrates that locomotion speed is higher for older nematodes with more sensitivity to lower electric fields. This selective response between different stages was used as a fundamental Figure 2: Electrotaxis response of a worm. design parameter for the separation device discussed later. In the AC The worm’s swimming is initiated by DC localization assay, increasing the frequency to more than 1 Hz resulted electric field in (a) and (c). AC electric field in the worms’ range of motion (normalized by their average body (f=1Hz) is used to localize the animal for 20s length) decrease to less than unity (Fig3b-section III). However, at in the middle of the channel in (b) [7] lower frequencies (10-100 mHz), worms demonstrated a DC-like electrotactic behavior by moving towards the cathode every time the field was switched. Between frequencies of 100 mHz and 1 Hz, except for the L2 stage which became localized, all other stages exhibited a 1D movement towards their initial DC electrotaxis direction. Similar responses were observed by using sinusoidal and triangular wave-shape signals.

Figure 3: Electrotaxis response of various C .elegans developmental stages. (a) Normalized DC electric field response, (b) Square waveform AC electric field response [7]. Normalized traveled range (traveled range/average stage length) decreases with frequency increase for all stages. Regions I, II, and III correspond to Dc-like electrotaxis, one-directional movement, and localization frequency range, respectively, illustrated for wild type (dashed double side arrowed lines) and muscle mutant nematodes (solid double side arrowed lines). Applied electric field was 10 V/cm, 8 V/cm, 6 V/cm, and 3 V/cm for L2, L3, L4, and young adult stages, respectively. Average length for each developmental stage is stated in the legend parentheses. 161

The mutant worms lacking muscle function (unc-54(s74)) responded similarly, but with lower range of motion, to ACelectric field. Symmetric square pulses with duty cycles (ton/tTotal) from 99% to 1% were also tested. Surprisingly, 1% duty cycle with f>1Hz was effective enough to hold the worm localized at one location, lowering the exposure time by 99%. The device depicted in Fig. 1c was designed based on the selectivity of DC electrotaxis response between different C elegans stages (Fig. 3a) for separating worms of varying ages (sizes) in a continuous format (Fig. 4a). We measured the minimum (response initiative) and the maximum (before paralysis) electric fields required for electrotaxis of worms (L2YA) in the wide section (diamonds and squares in Fig.4b) of the channel in a similar experiment to DC electrotaxis. We also measured the voltages applied across the channel that prevented each stage of swimming into the narrow section of the channel. COMSOL multiphysics was used to calculate the corresponding preventing electric fields (triangles in Fig. 4b). Based on this characterization, we applied a 16.8 V potential across the channel resulting in ~6 V/cm and ~16 V/cm electric fields in the wide and narrow sections respectively to separate C. elegans young adult stages from L2 stages (Fig.4a). The electric field inside the narrow section (16 V/cm) is above the maximum threshold (12.5 V/cm) that prevented YAs from swimming through it and hence resulted in their accumulation at the wider entrance of the channel.

Figure 4: Narrowing microchannel for electrotactic separation of adult and L2 stage worms. (a) Adult and L2 stages separation by applying 16.8V potential across the channel resulting in EFwide=6V/cm and EFnarrow=16V/cm. (b) lower and higher EF thresholds in wide section (diamonds and squares respectively) and narrowing section entrance preventing EF (triangles) for each developmental stage, CONCLUSION This is the first demonstration of electric field-based locomotion, transport, and separation of C. elegans in a microfluidic environment. The response is robust, repeatable, and amenable to multiplexing for high throughput assays. Electrotaxis behavior can be used in drug discovery assays for movement-related disorders as well as to carry out age-based activity assays in worms. ACKNOWLEDGEMENTS The authors wish to thank the Natural Sciences and Engineering Research Council of Canada and Ontario Ministry of Research and Innovation for their financial support towards this research. REFERENCES [1] Genome sequence of the nematode C. elegans: a platform for investigating biology. Science, 1998. 282(5396): p. 2012-8. [2] Baumeister, R. and L. Ge, The worm in us - Caenorhabditis elegans as a model of human disease. Trends Biotechnol, 2002. 20(4): p. 147-8. [3] Kwok, T.C., et al., A small-molecule screen in C. elegans yields a new calcium channel antagonist. Nature, 2006. 441(7089): p. 91-5. [4] Ben-Yakar, A., N. Chronis, and H. Lu, Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans. Curr Opin Neurobiol, 2009. [5] Chronis, N., Worm chips: microtools for C. elegans biology. Lab Chip, 2010. 10(4): p. 432-7. [6] Rezai, P., et al., Electrotaxis of Caenorhabditis elegans in a microfluidic environment. Lab Chip, 2010. 10(2): p. 220-6. [7] Rezai, P., et al., Behavior of Caenorhabditis elegans in alternating electric field and its application to their localization and control. Appl Phys Lett, 2010. 96(15): p. 153702Genome sequence of the nematode C. elegans: a platform for investigating biology. Science, 1998. 282(5396): p. 2012-8. CONTACT * Ponnambalam Ravi Selvaganapathy, JHE 212B, Department of Mechanical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4L7, Canada, Tel: (905) 525-9140 ext 27435, Fax: (905) 572-7944, Email: [email protected]

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