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A Microfluidic Diode for Sorting C. elegans

Tao Honga, Lijie Yangb, Richard Muc, Guillermo Sanchezd and Deyu Lib

Queensborough Community Collegea, City University of New York, Bayside, NY 11364Department of Mechanical Engineeringb, Vanderbilt University, Nashville, TN 37235

Life and Physical Sciences Departmentc, Fisk University, TN 37208Hughes-Kellogg Biology Research Laboratoryd, Fisk University, TN 37208

Introduction

Methods

Conclusion

Acknowledgements

Future Work

Design and Result

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W (μm)a 50b 45c 40d 35e 30f 25g 20

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t=0 s t=2 s t=4 s t=6 s t=8 s t=10 s t=12 s

t=0 s t=0.1 s t=0.2 s t=0.3 s t=0.4 s t=0.5 s t=0.6 s

INLET (WORM LOADING)

OUTLET (WASTE)

• Purpose of sorting C. elegans in different developmental stages

Caenorhabditis elegans (C. elegans) has been widely used as a powerful model organism which develops from hatching to reproductive adulthood through four larval stages (L1 to L4) and exhibits distinctive stage-specific features.

• Different crawling performance of C. elegans in curved and straight channels

• State of art sorting strategy of C. elegans by microfluidic platforms

I III

II

IV

Method I&II: Based on electrotactic swimming behavior

Method III: Based on chemotactic swimming behavior

Method IV: Based on physical behavior in pressure controlled polar arrays

• Our sorting strategy: Based on crawling behavior in different sized microfluidic diodes

1 mm

L1 L2 L3 L4 Adult

Average diameter 11.7 ± 0.2 μm 17.0 ± 0.2 μm 22.2 ± 0.3 μm 29.6 ± 0.6 μm 47.9 ± 0.8 μm

• Fabrication of microfluidic devices

• Source of unsynchronized C. elegans colony

Design and print mask Mold

PDMS curing& peeling

Photolithography

PDMS replica

1. Tubing

Microfluidic Device

2. Plasma Bonding

The initial unsynchronized C. elegans colony (N2, Wild Type) was provided by Hughes-Kellogg Biology Research Laboratory, Fisk University. The sample colony used in the sorting experiment was cultured from initial colony for 48 hours in 21℃ to reach the maximum population.

Time-lapse photographs of the whole process of C. elegans (diameter = 78.0 μm) crawling through the curved channels (width = 50 μm) under + 40 μL/min for 12 seconds.

Time-lapse photographs of C. elegans (diameter = 46.9 μm) struggling in front of the straight channels (width = 65 μm) under + 40 μL/min in 0.6 seconds.

Step1: + 40 μL/min for 20 mins for worm loading and sorting

Step2: - 200 μL/min for 30 seconds for flushing eggs and dead bodies out of the channels

Pictures of different sized C.elegans trapped in each section

100 um

100 um

C. elegans (L4 and adult) have a spontaneity to compress themselves 20%-50% to penetrate the curved channels, but they cannot penetrate the straight channels with the same width under the same flow rate.

After sorting process, the C. elegans from L4 and Adult (diameter: 31-72 um) are divided in 7 sections based on their diameters. The difference in average diameter of C. elegans in each section is about 5 um.

The average critical compressive ratio increases while the width of the channel decreases, which demonstrates that larva C. elegans can compress more than adult C. elegans in the unidirectional channels.

Increasing flow rate might result in a significant enhancement of average critical compressive ratio in some specific channels.

Top view of actual device after the sorting process.

References

Widen the region of sorting by fabricating channel arrays in smaller width.

Design a collecting system for repeatable use of the device.

Study the impact of curved channels’ shape and the length of the straight channels on the diode‘s property.

Introduce chemical stimuli to the C. elegans trapped in different sections of the original device and study their difference of response.

Front view of experiment set up under the microscope

The petri dish cultured C. elegans

A B

A

BHan 2012, Lab Chip 12: 4128.Park2008, PLoS ONE 3:e2550.

Solvas 2011, Chem. Commun 47:9801.

Ai 2014, Lab Chip 14:1746. Wang 2015, Lab Chip 15:2513.Erkut 2014, figshare.com.

We thank for the financial support by the following funding, ARO: W911NF-15-1-0441;NEEC: N00174-16-C-0008 and DOD: W911SR-14-2-0001, sponsored by the EdgewoodChemical and Biological Center (ECBC). Specially thank to Qian Zhang and Lin Yang forcomments and discussions, and the help from Yin Zhang in the experiment.

microfluidic diode

400 um

Limitation: 1. Narrow sorting region (Except in method I, those devices can separate only two parts of worms, adults and the other larva stages)

2. Additive environmental stimuli (Except in method IV, attractants and electric field are applied in the device which will increase the complexity of operation)

Advantage: 1. Significantly higher accuracy of sorting in L4 to adult region (capable of dividing young adults into four different sections with ̴5 μm diameter difference)

2. Different groups of similar sized C. elegans can be separated and trapped into different sections, this property can be used to study the responsive difference to the chemicals in different stages.

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Measurement of average diameter of C. elegans in each section & average critical compressive ratio

Average diameter of C.elegans traped in each sectonThe width of channelsAverage critical compressive ratio

(Average critical compressive ratio = 1 - the width of channel / the average diameter of C.elegans trapped in the corresponding channel)

The average critical compressive rate specially increase in channel d to g under 50 μL/min while there is no noticeable difference among the value in 20, 30 and 40 μL/min.

The impact of different flow rate on the average critical compressive rate in each section

• Microfluidic diode for C. elegan sorting