Pouya Rezai

Behavior of Caenorhabditis elegans in alternating electric field and its application to their localization and control

Caenorhabditis elegans is an attractive model organism because of its genetic similarity to humans and the ease of its manipulation in the laboratory. Recently, it was shown that a direct current electric field inside microfluidic channel induces directed movement that is highly sensitive, reliable, and benign. In this letter, we describe the worm’s movement response to alternating electric fields in a similar channel setup. We demonstrate that the 1 Hz and higher frequency of alternating current field can effectively localize worms in the channel. This discovery could potentially help design microfluidic devices for high throughput automated analysis of worms.

Microfluidic Approaches for Manipulating, Imaging, and Screening C. elegans

The nematode C. elegans (worm) is a small invertebrate animal widely used in studies related to fundamental biological processes, disease modelling, and drug discovery. Due to their small size and transparent body, these worms are highly suitable for experimental manipulations. In recent years several microfluidic devices and platforms have been developed to accelerate worm handling, phenotypic studies and screens. Here we review major tools and briefly discuss their usage in C. elegans research.

Plasma enhanced bonding of polydimethylsiloxane with parylene and its optimization

Polydimethylsiloxane (PDMS) and parylene are among the most widely used polymers in biomedical and microfluidic applications due to their favorable properties. Due to differences in their chemical structure and fabrication methods, it is difficult to integrate them together on a single microfluidic device. In this paper, we have demonstrated a method to bond patterned PDMS with parylene without the use of high temperature or pressure in a two-step process. The steps include (1) the attachment of cured PDMS surface to parylene using microcontact printing to form a weak bond followed by (2) a plasma exposure of sealed assembly to SF6, N2 and O2 gases, which enhanced the quality of bond by approximately fourfold to 1.4 MPa. We systematically investigated the effect of gas flow rates, chamber pressure, plasma time and power using Taguchis design of experiment method. Composition of the bond formed in this process was evaluated to understand the mechanism of bond formation. Microfluidic channels fabricated from a PDMS replica and a flat parylene-coated surface, bonded using this method, have been able to withstand burst pressures of up to 146 kPa compared to 35 kPa for PDMS prepolymer microcontact printed assembly.

Effect of pulse direct current signals on electrotactic movement of nematodes Caenorhabditis elegans and Caenorhabditis briggsae

The nematodes (worms) Caenorhabditiselegans and Caenorhabditisbriggsae are well-known model organisms to study the basis of animal development and behaviour. Their sinusoidal pattern of movement is highly stereotypic and serves as a tool to monitor defects in neurons and muscles that control movement. Until recently, a simple yet robust method to initiate movement response on-demand did not exist. We have found that the electrical stimulation in a microfluidic channel, using constant DC electric field, induces movement (termed electrotaxis) that is instantaneous, precise, sensitive, and fully penetrant. We have further characterized this behaviour and, in this paper, demonstrate that electrotaxis can also be induced using a pulse DC electric signal. Worms responded to pulse DC signals with as low as 30% duty cycle by moving towards the negative electrode at the same speed as constant DC fields (average speed of C. elegans = 296 ± 43 μm/s and C. briggsae = 356 ± 20 μm/s, for both constant and pulse DC electric fields with various frequencies). C. briggsae was found to be more sensitive to electric signals compared to C. elegans. We also investigated the turning response of worms to a change in the direction of constant and pulse DC signals. The response for constant DC signal was found to be instantaneous and similar for most worms. However, in the case of pulse DC signal, alterations in duty cycle affected the turning response time as well as the number of responding worms. Our findings show that pulse DC method allows quantitative measurement of response behaviour of worms and suggest that it could be used as a tool to study the neuronal basis of such a behaviour that is not observed under constant DC conditions.

A microfluidic phenotype analysis system reveals function of sensory and dopaminergic neuron signaling in C. elegans electrotactic swimming behavior

The nematode (worm) C. elegans is a leading multicellular animal model to study neuronal-basis of behavior. Worms respond to a wide range of stimuli and exhibit characteristic movement patterns. Here we describe the use of a microfluidics setup to probe neuronal activity that relies on the innate response of C. elegans to swim toward the cathode in the presence of a DC electric field (termed “electrotaxis”). Using this setup, we examined mutants affecting sensory and dopaminergic neurons and found that their electrotactic responses were defective. Such animals moved with reduced speed (35–80% slower than controls) with intermittent pauses, abnormal turning and slower body bends. A similar phenotype was observed in worms treated with neurotoxins 6-OHDA (6- hydroxy dopamine), MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine) and rotenone (20–60% slower). We also found that neurotoxin effects could be suppressed by pre-exposing worms to a known neuroprotective compound acetaminophen. Collectively, these results show that microfluidic electrotaxis can identify alterations in dopamine and amphid neuronal signaling based on swimming responses of C. elegans. Further characterization has revealed that the electrotactic swimming response is highly sensitive and reliable in detecting neuronal abnormalities. Thus, our microfluidics setup could be used to dissect neuronal function and toxin-induced neurodegeneration. Among other applications, the setup promises to facilitate genetic and chemical screenings to identify factors that mediate neuronal signaling and neuroprotection.

Agar-polydimethylsiloxane devices for quantitative investigation of oviposition behaviour of adult Drosophila melanogaster

Drosophila melanogaster (fruit fly) is a model organism and its behaviours including oviposition (egg-laying) on agar substrates have been widely used for assessment of a variety of biological processes in flies. Physical and chemical properties of the substrate are the dominant factors affecting Drosophilas oviposition, but they have not been investigated precisely and parametrically with the existing manual approaches. As a result, many behavioral questions about Drosophila oviposition, such as the combined effects of the aforementioned substrate properties (e.g., exposure area, sugar content, and stiffness) on oviposition and viability, and their threshold values, are yet to be answered. In this paper, we have devised a simple, easily implementable, and novel methodology that allows for modification of physical and chemical composition of agar substrates in order to quantitatively study survival and oviposition of adult fruit flies in an accurate and repeatable manner. Agar substrates have been modified by surface patterning using single and hexagonally arrayed through-hole polydimethylsiloxane (PDMS) membranes with various diameters and interspacing, as well as by substrate stiffness and sugar content modification via alteration of chemical components. While pure PDMS substrates showed a significant lethal effect on flies, a 0.5 mm diameter through-hole access to agar was found to abruptly increase the survival of adult flies to more than 93%. Flies avoided ovipositing on pure PDMS and on top of substrates with 0.5 mm diameter agar exposure areas. At a hole diameter of 2 mm (i.e., 0.25% exposure area) or larger, eggs were observed to be laid predominately inside the through-holes and along the edges of the PDMS-agar interface, showing a trending increase in site selection with 4 mm (i.e., 1% exposure area threshold) demonstrating natural oviposition rates similar to pure agar. The surface-modified agar-PDMS hybrid devices and the threshold values reported for the substrate physical and chemical conditions affecting oviposition are novel; therefore, we advocate their use for future in-depth studies of oviposition behaviour in Drosophila melanogaster with accuracy and repeatability. The technique is also useful for development of novel assays for learning and decision-making studies as well as miniaturized devices for self-assembly of eggs and embryonic developmental investigations.

Materials and methods for the microfabrication of microfluidic biomedical devices

Abstract: The materials that have been employed for the construction of microfluidic devices have been diverse, ranging from traditional materials, such as silicon and glass, to newer polymeric materials. Similarly, the methods for microfabrication have included lithography, casting, injection molding and hot embossing, to name a few. In this chapter, we provide an overview of the various materials and methods that have been used in a diverse range of microfluidic applications. Details on the physical and chemical properties of the materials, as well as the performance characteristics of the microfabrication methods, are provided.

Plasma enhanced bonding of polydimethylsiloxane (PDMS) with parylene

PDMS and parylene are among the most widely used polymers in biomedical and microfluidic applications. However, integration of both these materials on a single device has not been possible due to low bond strength achieved using conventional methods. In this paper, we demonstrate plasma-enhanced bonding of PDMS slabs with parylene thin films (from 0.4MPa for PDMS glued assembly to >1.4MPa tensile strength enhancement) without the usage of high temperature or pressure. This was achieved by systematic investigation of bonding parameters using Taguchis design of experiment method. The application of this bond has also been demonstrated in PDMS-parylene microfluidic channels.

Semi-Empirical Estimation of Dean Flow Velocity in Curved Microchannels

Curved and spiral microfluidic channels are widely used in particle and cell sorting applications. However, the average Dean velocity of secondary vortices which is an important design parameter in these devices cannot be estimated precisely with the current knowledge in the field. In this paper, we used co-flows of dyed liquids in curved microchannels with different radii of curvatures and monitored the lateral displacement of fluids using optical microscopy. A quantitative Switching Index parameter was then introduced to calculate the average Dean velocity in these channels. Additionally, we developed a validated numerical model to expand our investigations to elucidating the effects of channel hydraulic diameter, width, and height as well as fluid kinematic viscosity on Dean velocity. Accordingly, a non-dimensional comprehensive correlation was developed based on our numerical model and validated against experimental results. The proposed correlation can be used extensively for the design of curved microchannels for manipulation of fluids, particles, and biological substances in spiral microfluidic devices.

A hybrid microfluidic device for on-demand orientation and multidirectional imaging of C. elegans organs and neurons

C. elegans is a well-known model organism in biology and neuroscience with a simple cellular (959 cells) and nervous (302 neurons) system and a relatively homologous (40%) genome to humans. Lateral and longitudinal manipulation of C. elegans to a favorable orientation is important in many applications such as neural and cellular imaging, laser ablation, microinjection, and electrophysiology. In this paper, we describe a micro-electro-fluidic device for on-demand manipulation of C. elegans and demonstrate its application in imaging of organs and neurons that cannot be visualized efficiently under natural orientation. To achieve this, we have used the electrotaxis technique to longitudinally orient the worm in a microchannel and then insert it into an orientation and imaging channel in which we integrated a rotatable glass capillary for orientation of the worm in any desired direction. The success rates of longitudinal and lateral orientations were 76% and 100%, respectively. We have demonstrated the application of our device in optical and fluorescent imaging of vulva, uterine-vulval cell (uv1), vulB1\2 (adult vulval toroid cells), and ventral nerve cord of wild-type and mutant worms. In comparison to existing methods, the developed technique is capable of orienting the worm at any desired angle and maintaining the orientation while providing access to the worm for potential post-manipulation assays. This versatile tool can be potentially used in various applications such as neurobehavioral imaging, neuronal ablation, microinjection, and electrophysiology.