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Droplet microfluidics for characterizing the neurotoxin-induced responses in
individual Caenorhabditis elegans†‡
Weiwei Shi, Hui Wen, Yao Lu, Yang Shi, Bingcheng Lin*and Jianhua Qin*
Received 29th July 2010, Accepted 15th September 2010
DOI: 10.1039/c0lc00256a
A droplet-based microfluidic device integrated with a novel floatage-based trap array and a tapered
immobilization channel array was presented for characterizing the neurotoxin-induced multiple
responses in individual Caenorhabditis elegans (C. elegans) continuously. The established device
enabled the evaluations of movement and fluorescence imaging analysis of individual C. elegans
simultaneously. The utility of this device was demonstrated by the pharmacological evaluation of
neurotoxin (6-hydroxydopamine, 6-OHDA) triggered mobility defects, neuron degeneration and
oxidative stress in individual worms. Exposure of living worms to 6-OHDA could cause obvious
mobility defects, selective degeneration of dopaminergic (DAergic) neurons, and increased oxidative
stress in a dose dependent manner. These results are important towards the understanding of
mechanisms leading to DAergic toxicity by neurotoxin and will be of benefit for the screening of new
therapeutics for neurodegenerative diseases. This device was simple, stable and easy to operate, with the
potential to facilitate whole-animal assays and drug screening in a high throughput manner at single
animal resolution.
Introduction
C. elegans, a small soil nematode, has been widely used as
a model organism for fundamental biological research due to its
availability in development, genetics and neurochemistry.
1–3
It is
the first multi-cellular organism to have its genes fully sequenced,
which is significantly suitable for the study of genetics. The basic
features of C. elegans include its small size (1 mm), short
generation time and ease of cultivation on agar or in liquid.
Especially, several aspects of human neurochemistry and phar-
macology are conserved in C. elegans, which make it a well-suited
model organism for investigating numerous diseases such as
neurodegenerative diseases, physiological processes, and drug
discovery.
4–8
In recent years, microfluidics technology has been emerging as
a powerful tool for chemical and biological research and it has
aroused more and more interests in worm research due to its
unique characteristics. Specially, the dimension of channel
microfluidics (mm to mm) matches the worm size perfectly, and
its seamless integration of active control elements may allow the
flexible operations of diverse types of worm manipulations, such
as picking, sorting, and transferring individual worms. Presently,
channel microfluidics-based worm studies have been carried out
for applications in maze exploration,
9
oxygen sensation,
10
phenotype and genetic screenings,
11–13
automatic cultivation,
14
nerve regeneration
15,16
and microvalve-based and tapered
channel-based immobilization for imaging.
17–21
Compared with continuous flow-based microfluidic platforms,
droplet microfluidics has been shown to be compatible with
many chemical and biological applications.
22,23
This platform has
dimensional scaling benefits that have enabled controlling and
rapid mixing of fluids in the droplet microreactors. This, coupled
with the precise generation and repeatability operations of
droplets, has made it a potential platform to directly synthesize
particles and encapsulate many biological entities for some
specific applications.
24–27
Due to the unique advantages of
droplet-based system, we firstly presented a droplet-based
microfluidic system to encapsulate the individual worms into
a parallel series of droplets and investigated the worm movement
behavior in response to neurotoxin (MPP+),
28
because the
neurotoxin (such as MPP+, MPTP, 6-OHDA) was found to be
able to induce mobility defects in C. elegans, which could be used
as a pharmacogenetic model to study Parkinson’s disease and
evaluation of anti-neurodegeneration drugs.
5
Strong evidence
29–34
has indicated that the neurotoxin induced mobility defects in
C. elegans might result from the specific destroying of dopami-
nergic (DAergic) neurons by neurotoxin, and the generation of
reactive oxygen species might be involved in the process of
neuritic damage and cell death. However, these events which are
targeted to dopamine neurons are not explored in our previous
work. In addition, the biocompatibility of the droplet environ-
ment for long time worm maintenance is limited due to the
property of oil phase used in the previous work.
Here, we proposed a novel droplet-based microfluidic device
for characterizing the various responses of individual C. elegans
to neurotoxin continuously. Different from our previous work,
a novel floatage-based droplet trapping method was developed in
this work, which was quite simple and stable, allowing coupling
with a tapered immobilization array easily. This device enabled
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457
Zhongshan Road, Dalian, China. E-mail: jhqin@dicp.ac.cn; bclin@dicp.
ac.cn; Fax: +86-411-84379650; Tel: +86-411-84379650
† Published as part of a LOC themed issue dedicated to Chinese
Research: Guest Editor Professor Bingcheng Lin.
‡ Electronic supplementary information (ESI) available: Movies
showing: individual adult worm encapsulation, the trapping process of
single droplets encapsulated with individual worms, the trapping
process of array droplets encapsulated with individual worms, and
individual worm immobilization. See DOI: 10.1039/c0lc00256a
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the integration of various functions including worm encapsula-
tion, droplet trapping and worm immobilization, and the eval-
uation of mobility behavior and fluorescence features of
individual C. elegans simultaneously. With this device, the
mobility defects, neuron degeneration and oxidative stress of
individual worms in response to neurotoxin (6-OHDA) could be
characterized at single animal resolution in an automatic and
high throughput manner. Neurotoxin 6-OHDA was observed to
cause obvious mobility defects, selective degeneration of DAer-
gic neurons, and increased oxidative stress in C. elegans, indi-
cating potential mechanisms leading to DAergic toxicity by
neurotoxin and the possible generation of reactive oxygen species
during neuritic damage. This will be useful for high throughput
whole animal assays and screening of new therapeutics for
neurodegenerative diseases.
Experimental
2.1 Chip design and fabrication
In this work, the microfluidic chip was composed of three func-
tional units: a droplet generator, a floatage-based trap array and
a tapered immobilization channel array, which enabled the
functions of individual worm encapsulation, droplet trapping
and worm immobilization.
The schematic illustration of the microfluidic chip is shown in
Fig. 1. The structure of the chip is composed of two layers: The
top layer consists of an 80-trap array for droplet trapping and
each trap (1 mm diameter and 1 mm height) is connected to
a tapered worm immobilization channel for imaging analysis.
The worm immobilization channel (50 mm height and 2 mm
length) has a gradually narrowed width (from 100 mmto20mm).
The joint of the trap and immobilization channel descends like
a step-stair, with the height of 500 mm, 200 mm and 50 mm
respectively. The 80 immobilization channels are all linked to the
central waste reservoir through a 20-mm high channel. The
bottom layer includes a T-junction droplet generator (the two
inlet channels are 300-mm wide and 500-mm high) and a flexural
droplet transporting channel (500-mm wide and 500-mm high).
The 80 traps in the top layer are linked in series by droplet
transportation channels in the bottom layer.
The top and bottom layers were fabricated in poly-
dimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland,
MI) using a rapid prototyping technique.
35
Briefly, SU-8 3035
photoresist (Microchem, Newton, CA) was spin-coated onto
glass wafers and patterned by photolithography. PDMS base
and curing agent with two different mixing ratios (5 : 1 and 20 : 1
by mass) were mixed thoroughly, degassed under vacuum, and
poured onto the top and bottom layer masters respectively. The
PDMS were cured at 80 C for 30 min. After cooling, the two
PDMS replicas were peeled off from masters and bonded
together immediately. Finally, the PDMS chip was baked at
100 C overnight to achieve irreversible sealing between the two
layers.
2.2 Droplet generation and trapping process
Two PEEK tubes (1/1600OD, 0.0100ID, 1531, Upchurch Scientific,
Oak Harbor, WA, USA) were used for connecting two inlet holes
of the microfluidic chip to syringe pumps (50 mL/250 mL, Kloehn
Ltd., Las Vegas, NV, USA). The syringe pumps were used for
injecting the dispersed phase and continuous phase flow and
controlling the flow rates respectively. The aqueous solution was
used as the dispersed phase and fluorinated oil FC-40 (provided
by 3M) with 2% (w/v) EA surfactant (provided by Raindance
Technologies, Inc.) was used as the continuous phase. Before
connecting the chip to syringe pumps, oil and aqueous solution
were filled into each tube respectively. Then the aqueous solution
and oil were injected into the microfluidic device under proper
flow rates. The droplets were generated by the shearing of
dispersed phase with continuous phase at the bottom layer
T-junction. The generated droplets were subsequently trapped in
the top traps.
2.3 Worm culture and 6-OHDA treatment
Wild-type N2, transgenetic strains UA57 (baIn4[Pdat-1::GFP +
Pdat-1::CAT-2]) and CL2166 (dvIs19[pAF15(gst-
4::GFP::NLS)]) C. elegans were obtained from the Caeno-
rhabditis Genetics Center at the University of Minnesota (St.
Paul) and cultivated as described in Brenner.
36
Briefly, worms
were cultivated at 20 C on nematode growth medium (NGM)
agar (0.55 g Trizma HCl, 0.24 g Tris base, 4.6 g tryptone, 2 g
NaCl and 22 g ager to 1 liter) seeded with Escherichia coli OP50
(food). Prior to seeding, bacteria were incubated overnight at
37 C and stored at 4 C.
Fig. 1 Schematic and photograph of the droplet-based microfluidic
device for individual C. elegans assay. (a) Schematic of the droplet-based
microfluidic chip. The chip consists of two layers: the bottom layer for
droplet generation and transportation, and the top layer for droplet
trapping and worm immobilization. (b) The magnified view of one
operation unit, including a droplet trap and a worm immobilization
channel. The depths of different parts of the operation unit are marked
with different colors. (c) Illustration of the structure of the device
including a top layer and a bottom layer. The inset is a photograph of the
fabricated droplet-based microfluidic chip.
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Synchronized L1 worms (UA57 and CL2166) were washed
into sterile water from agar plates at 20 C. The density of the
worm suspension was adjusted to 10–15 worms/mL. 600 mL worm
suspension was loaded to a 1.6-mL micro-centrifuge tube, and
the 6-hydroxydopamine (6-OHDA, Aldrich) stock solution (100
mM 6-OHDA/20% DMSO) was added to achieve the final
concentrations of 0, 5 mM and 10 mM. The worms were left in
the dark for 1 h at room temperature (mix gently every 10 min).
After treatment, the worms were washed with water, the super-
natant was discarded and the process was repeated.
37
Then the
worms were placed on a NGM plate and incubated at 20 C for
72 h until they reached adulthood stage. Then the worms were
analyzed on the established microfluidics platform at the 72th
hour (defined as 1st day), 120th hour (3rd day) and 168th hour
(5th day).
Detailed experimental procedures are as follows: (1) The
worms were synchronized to L1 and exposed to 6-OHDA for one
hour. (2) 6-OHDA treated L1 worms were incubated on NGM
plate to adult (defined as day 1), 1/3 of them were washed and
loaded into the chip, and the worm’s mobility behavior and
neurons/oxidative stress data were collected at day 1. (3) The rest
2/3 of worms were incubated on NGM plate to day 3, and 1/2 of
them (1/3 of the total worms) were washed and tested at day 3. (4)
The rest 1/3 of worms were incubated on NGM plate to day 5,
and then washed and tested at day 5.
2.4 Worm encapsulation and mobility data collection
Adult worms which have been treated with 6-OHDA are
collected into S mediumxat 1st day, 3rd day and 5th day, and the
density was adjusted to about 2 worms/mL. The worm suspension
was loaded into the PEEK tube connected to the water phase
syringe pump, and then the system was assembled according to
the description above (part of ‘‘Droplet generation and trap-
ping’’). After assembly, oil and worm suspension were injected at
proper flow rates respectively, and a series of droplets encapsu-
lated with individual worms were generated at the T-junction and
trapped in the traps. When 80 droplets had been generated, the
water phase syringe pump was stopped; when the entire 80
droplets were trapped, the oil phase syringe pump was stopped.
The movements of individual worms encapsulated in droplets
were recorded by a high-resolution CCD camera mounted onto
a stereozoom microscope (Leica S8APO, Germany) at fixed time
points, and each video lasted for 30 s in a format of 15fps @ 800
600. The numbers of stroke movement of worms were counted
manually.
2.5 Worm immobilization and fluorescence data collection
After worm loading and mobility data collection, the PEEK
tubes were pulled out and the two inlets were plugged up care-
fully. Waste 1 reservoir was connected with a syringe filled with
1% agarose (Agarose, Type IX-A, Sigma) solution. The plunger
was pushed gently and the agarose solution was injected into the
chip at the flow rate of 0.5–1 mLs
1
, and the worms were rushed
into the adjacent tapered channels and immobilized. The flow of
agarose solution was maintained to avoid the movements of
worms. The fluorescence images of individual worms (UA57 and
CL2166) were recorded by an inverted fluorescent microscope
(Olympus IX 71, Japan) with excitation wavelengths at 470–495
nm and detection wavelengths at 510–550 nm. The fluorescence
images were analyzed using image processing and analysis soft-
ware (IMAGE-PRO, Media Cybernetics, USA).
Results and discussion
3.1 Design of the droplet-based microfluidic device
In order to evaluate the behaviors of individual worms in
response to neurotoxin, it is important to design the microfluidic
device with the capability to realize sequential operations
including worm encapsulation, droplet trapping and worm
immobilization for mobility and neuron imaging analysis as well.
In this work, the microfluidic device was manufactured by two
PDMS layers which were mainly composed of three functional
units: T-junction droplet generator on the bottom layer, floatage-
based trap array and tapered immobilization channel array on
the top layer.
We adopted fluorocarbon oil FC-40 instead of commonly used
hydrocarbon oil (silicone oil and mineral oil) as the continuous
phase due to the following reasons: 1) The biocompatibility of
FC-40 was fully proved by previous researches, and it was widely
used in biology applications;
38
2) compared with hydrocarbon
oils, fluorocarbon oils result in less swelling of PDMS;
39
3)
fluorocarbon oils have good solubility for gases, which is
necessary for the viability of encapsulated worms;
38
4) the density
of FC-40 is larger than water, which made it possible to trap
droplets by floatage. EA fluorinated surfactant (RainDance
Technologies, MA, USA)
39
was added to the continuous phase
(2% by volume) to reduce the interfacial tension between disperse
and continuous phase and stabilize the water–oil interface, which
will make the droplets more stable and prevent the mixing of
different droplets. Droplets were generated as a result of the
shear force and interfacial tension at the water–oil interface at
the T-junction on the bottom layer.
After being generated, droplets were captured in the droplet
traps array under the effect of floatage. The floatage-based
trapping process is realized by the following steps: The generated
droplets keep on moving forward along the transportation
channel in the bottom layer; when a droplet flows just under an
empty trap on the top layer, it will move upward under the
floatage resulting from the density difference between the
aqueous and oil phase FC-40 (aqueous 1 g mL
1
< oil 1.85 g
mL
1
), and then be captured in the trap above the channel; the
following droplets will pass through along the channel without
mixing with the trapped one due to the effect of surfactant, and
then be trapped sequentially. The whole trapping process was
spontaneous and repeatable. Fig. 2a illustrates the principle of
the droplet floatage-based trapping process (ESI,‡ Movie 1).
It is noted that, the established floatage-based droplet trapping
method is quite simple and stable as compared with the flow
xS medium: 1 liter S Basal, 10 mL 1 M potassium citrate pH 6, 10 mL
trace metals solution, 3 mL 1 M CaCl
2
, 3 mL 1 M MgSO
4
. Add
components using sterile technique; do not autoclave. S Basal: 5.85 g
NaCl, 1 g K
2
HPO
4
,6gKH
2
PO
4
, 1 mL cholesterol (5 mg mL
1
in
ethanol), H
2
O to 1 liter. Sterilize by autoclaving. Trace metals solution:
1.86 g disodium EDTA, 0.69 g FeSO
4
$7H
2
O, 0.2 g MnCl
2
$4H
2
O,
0.29 g ZnSO
4
$7H
2
O, 0.025 g CuSO
4
$5H
2
O, H
2
O to 1 liter. Sterilize
by autoclaving; store in the dark.
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resistance-based way in our previous work. Moreover, the array
traps are isolated with each other in the top layer, which makes it
possible to integrate other additional operation units such as
worm immobilization channels, without destroying the
geometric connectivity of the flowing channels. Therefore, the
characterization of mobility behavior and fluorescence features
of individual C. elegans could be achieved simultaneously by
using the floatage-based trapping method.
3.2 Performance of the droplet microfluidic device for
individual worm assay
To achieve the aim of this work, the basic performances of the
droplet microfluidic device were evaluated for individual worm
assay initially. In this work, the trapping performance of the
device was demonstrated by injecting aqueous ink solution
(green) and oil (FC-40 with 2% EA surfactant) as the dispersed
and constant phase, respectively. Under the optimized conditions
for the chip geometry, the droplets were generated with a volume
of 0.5 mL at constant flow rates of the two phases (oil at 5.21 mL
min
1
, aqueous solution at 1.74 mL min
1
). As shown in Fig. 2b,
a total of 80 uniform ink droplets were subsequently trapped in
the trap array successfully, demonstrating the feasibility of this
device for droplet trapping. The entire generating and trapping
process can be accomplished within 20 min. We further investi-
gated the capability of this device for encapsulating individual
worms into droplets. Prior to the assay, the dispersed phase was
changed into an adult worm suspension in S medium at the stable
flow rates (oil at 5.21 mL min
1
and worm suspension at 1.74 mL
min
1
). The density of the adult worm suspension was adjusted to
2 worms/mL in order to ensure the individual worms be encap-
sulated into droplets. As shown in Fig. 2c, 80 aqueous droplets
with uniformity were trapped in the trap array encapsulated with
individual worms. Under the optimized conditions above, the
device enabled encapsulation of individual adult worms into
array droplets with 30–40% probability (the encapsulation
process is shown in ESI,‡ Movie 2 and 3). The movements of
individual worms encapsulated in droplets were recorded by
CCD camera mounted onto a stereozoom microscope.
Following the droplet trapping, the capability of this device to
immobilize individual C. elegans for imaging analysis was eval-
uated. After worm encapsulation and mobility data collection,
the aqueous inlet and oil inlet were plugged in. 1% agarose
solution was injected from waste reservoir 1 and flow out from
waste reservoir 2. Then, the trapped aqueous droplets would also
flow into waste reservoir 2 through immobilization channels.
Thus, the encapsulated worm would be immobilized by the size
constraints in this flowing process. In this work, 1% agarose
solution was adopted to avoid its fusion with the droplets. The
real-time movie to demonstrate the immobilization process is
shown in Fig. 3b (and ESI,‡ Movie 4). After the worms being
immobilized, the flow of agarose solution was maintained to
avoid the movements of worms. The fluorescence images of
individual worms can be recorded by an inverted fluorescent
microscope. Fig. 3c,d show the bright field and the correspond-
ing fluorescence photograph of individual worms immobilized in
the immobilization channels. Some working parameters of the
device were shown in ESI,‡ supplementary information, part 4.
Fig. 2 Demonstration of the floatage-based microfluidic device for array droplet trapping. (a) Principle of the generated droplets captured in the trap
array by floatage. (i) produced droplet 1 moves towards an empty trap at proper flow rate; (ii) when droplet 1 moves along the microchannel and locates
right below the trap, it will easily flow into the above trap due to the floatage (r
oil
>r
water
); (iii) droplet 1 occupies the trap, and the following droplet 2
passes through the below of the filled trap and keeps on moving; (iv) droplet 2 reaches the next trap; (v)droplet 2 flows upward into the next trap, andthe
following droplet 3 and 4 move on and would be trapped successively. (b) Photograph of the microfluidic device trapped with 80 uniform ink droplets. (c)
A microfluidic chip trapped with 80 aqueous droplets encapsulated with individual worms. The inset is the magnified view of five trapped droplets
encapsulated with individual worms.
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As biocompatibility of the droplet environment is important
for the worm assay, we also investigated this performance by
encapsulating L1 larvae worms (wild-type N2) into the droplets
and observing them for several days. It is noted that, we chose
FC-40 as oil phase in this work because it has been widely used in
cell-culture systems for regulating the gas supply due to its
unique advantages, like being chemically and biologically inert,
ease of sterilization and high respiratory-gas solubility, etc.
38
Here, its benefit of high solubility of oxygen was utilized to
ensure adequate oxygen supply to worms encapsulated in the
droplets. The biocompatibility of EA surfactant has been
demonstrated in previous publication.
39
Under the conditions
mentioned above, we encapsulated 18 individual L1 larvae
worms into the array droplets and recorded their survival status
every day. All of the encapsulated larvae worms could survive for
at least five days, suggesting that the droplet environment is
biocompatible for the following worm assay. (Detailed biocom-
patibility validations are shown in ESI,‡ supplementary infor-
mation, part 3.) In this experiment, the duration time of the
tested worms in the droplets was less than 2 h with good viability,
which did not produce significant stress on worms.
The obvious advantages of this system over conventional
methods in C. elegans study mainly include the following points:
a) The dimension of droplet is highly controllable and flexible
which could match worm size and restrict the motion range of the
worm, and the acquisition of behavior data of numerous indi-
vidual worms would be simply accomplished by a CCD camera
without changing the chip position to avoid the worms crawling
or swimming out of the field of view. b) The established method
enabled real-time data collection of multiple individual worms in
an array format, allowing multiple worms assay at the same time.
c) The mutiple manipulation of individual worms including
encapsulation, transportation and immobilization could be
accomplished in an automatic, continuous and fast manner
easily.
3.3 Characterizing the neurotoxin-induced responses in
individual C. elegans
The neurotoxin 6-hydroxydopamine (6-OHDA) has been
previously reported to cause Parkinson’s disease (PD)-like
symptoms in vertebrates by destroying dopamine neurons,
5,40
and it has been established as a pharmacological model to study
the Parkinson’s disease and quantitative evaluation of anti-PD
drugs.
6–8
Although rare genetic forms of PD have been identified,
the molecular determinants of dopamine neuron vulnerability
and cell death in the majority of PD cases has not been defined.
In the following work, we used the droplet-based microfluidic
device to study the neurotoxin 6-OHDA induced responses in
individual C. elegans and two transgenic strains, UA57 and
CL2166, were used in this work. In each experiment, the indi-
vidual worms were randomly selected, and their mobility
behavior, DAergic neurons degeneration and possible oxidative
stress in response to 6-OHDA were investigated.
3.3.1 Triggered mobility defects after exposure to 6-OHDA.
Parkinson’s disease is a severe movement disorder characterized
by resting tremor, spasticity and an inability to initiate move-
ment, resulting from the irreversible loss of dopamine neurons.
6
Current hypotheses of pathogenesis involve environmental toxin
exposure and the increased generation of reactive oxygen species.
In order to characterize the 6-OHDA induced mobility defects in
Fig. 3 Performance of the tapered immobilization channel array for immobilizing and imaging of C. elegans. The red frames indicated the location of
the immobilized worms. (a) Schematic of an individual worm immobilized in the tapered immobilization channel. (b) A 4-image sequence showing the
immobilization process of two individual worms. (c,d) Bright field photograph and the corresponding fluorescence photograph of individual worms
immobilized in the immobilization channels. Transgenetic strain CL2166 were used which expressed an oxidative stress-inducible and imaged by
a fluorescence microscopy at the magnification of 20using a 2objective.
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strains UA57 and CL2166, individual C. elegans were firstly
treated with different concentrations (control, 5 mM, 10 mM) of
6-OHDA. Then the treated worms of the same 6-OHDA
concentration were divided into three groups and loaded into the
chip at day 1, day 3 and day 5, correspondingly. Then the
mobility behavior of the encapsulated individual worms was
investigated. The results demonstrated that the untreated worms
exhibited free movements (e.g. higher stroke frequencies, more
sine wave-shape or C-shape) at the test days; however, the treated
worms exhibited obvious mobility defects (e.g. lower stroke
frequencies, more omega- and tetanic-shape, etc.) instead. The
average stroke frequency of the individual UA57 and CL2166
worms associated with 6-OHDA concentrations at different time
are shown in Fig. 4, respectively. From these figures, we can see
that the stroke frequency of the untreated worms was relatively
higher, without obvious decrease throughout the period of
experiment. However, the stroke frequency of worms decreased
significantly after exposure to 5 mM 6-OHDA, and the omega
and tetanic state ratio were observed to increase obviously (data
not show). In addition, after treatment of 10 mM 6-OHDA, the
worms showed a low stroke frequency at the tested days, and
a higher ratio for the appearance of omega and tetanic state (data
not shown).
3.3.2 Visualization of DAergic neurons degeneration sensitive
to 6-OHDA. To determine whether C. elegans DAergic neurons
are sensitive to 6-OHDA in vivo, worm strain UA57, which
expressed strong and specific green fluorescence in all eight
DAergic neurons was utilized in this experiment, and the six
neurons in the head were analyzed here. Briefly, after encapsula-
tion into the droplets and movement data collection, the
individual worms moved and stabilized in the immobilization
channels via positive pressure applied in waste reservoir 1. Thus,
the DAergic neurons degeneration in response to 6-OHDA with
different concentrations (5 mM, 10 mM) could be imaged and
characterized. The representative fluorescence images of GFP
expression in 12 individual UA57 worms associated with
6-OHDA concentrationsat the 3rd day was shown in Fig. 5a.A loss
of GFP expression in the specific DAergic neurons was observed in
the individual worms obviously. As shown in this figure, at the 3rd
day, the untreated worms expressed intact and strong GFP in all six
DAergic neurons in the head. However, the worms showed
a significant reduction of GFP expression after treatment with
5 mM 6-OHDA, many of the cell somas became round and blebs
appeared along the dendrites. Furthermore, after treatment with
10 mM 6-OHDA, a complete loss of GFP expression was observed
in most DAergic neurons, with occasional retention of GFP
expression in cell bodies, suggesting the obvious effects of DAergic
degeneration triggeredby neurotoxin. (For a more cleardisplay, see
ESI,‡ supplementary information, part 2.).
Based on the above studies, 6-OHDA was observed to induce
not only the mobility defects, but also the DAergic neurons
degeneration. Coupled with the data obtained from the mobility
test, it is also noted that, the degree of neuron degeneration was
closely related to the effects of mobility disorder induced by
neurotoxin (Fig. 5a). It is clear that the untreated worms
exhibited free movements with normal DAergic neurons (intact
and strong GFP expression), while, the worms treated with
6-OHDA showed obvious mobility defects and the correspond-
ing reduced GFP expression in DAergic neurons, indicating that
DAergic neurons degeneration might lead to the induced
movement disorders in C. elegans.
Fig. 4 Average stroke frequency (SEM) of UA57 and CL2166 worms in responses to 6-OHDA. The data were statistically analyzed by using the two
sample t-test (Origin 8.0, Origin Lab, MA) to evaluate the significance of the difference between different groups. (a) Average stroke frequency (SEM)
of 12 UA57 worms in responses to 6-OHDA. *p< 0.05, **p< 0.01, ***p< 0.001. (b) Average stroke frequency (SEM) of 12 CL2166 worms in
responses to 6-OHDA. *p< 0.05, **p< 0.01, ***p< 0.001. Form this figure, untreated worms (UA57 and CL2166) exhibited higher stroke frequency at
day 1, 3, 5; the 6-OHDA treated worms (UA57 and CL2166) exhibited lower stroke frequency, and the stroke frequency decreased visibly form day 1 to
day 5. The original data is shown in supplementary information, part 1.‡
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3.3.3 Triggered oxidative stress after exposure to 6-OHDA. It
was previously reported that 6-OHDA is selectively accumulated
by dopamine neuron through presynaptic dopamine
transporters, and may thereby induce neuritic damage and cell
death.
6
It has been assumed that the increase of reactive oxygen
species might be involved in the process of neuritic damage and
Fig. 5 Fluorescence images and corresponding stroke frequencies of individual UA57 and CL2166 worms in response to 6-OHDA at the 3rd day. (a)
DAergic neurons fluorescence images and corresponding stroke frequency of individual UA57 worms treated with different concentrations of 6-OHDA
at the 3rd day. The fluorescence images were taken by a fluorescence microscopy at a magnification of 400using a 40objective. The untreated worms
expressed intact and strong GFP in all six DAergic neurons in the heads, and exhibited high stroke frequencies; meanwhile, the worms treated with 5 mM
6-OHDA showed a significant reduction of GFP expression, many of the cell somas became round and blebs appeared along the dendrite, and also the
worms’ stroke frequencies decreased obviously; the worms treated with 10 mM 6-OHDA exhibited a complete loss of GFP in most DAergic neurons,
and their stroke frequencies declined to a low level (Typical fluorescence photos were shown in ESI).‡ (b) Oxidative stress fluorescence and corre-
sponding stroke frequency of individual CL2166 worms treated with different concentrations of 6-OHDA at the 3rd day. The fluorescence images were
taken by a fluorescence microscopy at a magnification of 160using a 16objective. The untreated worms exhibited high stroke frequencies without the
generation of reactive oxygen species; and the 6-OHDA treated worms showed obvious mobility defect (low stroke frequencies) and the corresponding
increased oxidative stress.
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cell death. After characterizing the mobility defects and DAergic
neurons degeneration of individual worms, we further explored
whether 6-OHDA can trigger oxidative damage during the
process of neuron degeneration. We use worm strain CL2166,
which expressed an oxidative stress-inducible GFP, for the
following experiments. The fluorescence intensity of GFP express
is relative to the degree of oxidative stress produced in C. elegans.
After being encapsulated into droplets, the real-time move-
ments of individual worms after 6-OHDA treatment were
recorded. Then, the individual worms were captured and stabi-
lized in the immobilization channels and the fluorescence image
of GFP expression were analyzed in CL2166 worms at different
times (1st, 3rd and 5th day). Fig. 5b demonstrated the repre-
sentative fluorescence images of individual worms after 6-OHDA
treatment at the 3rd day. The increase of GFP expression in the
worm body was found to be associated with the neurotoxin at
different concentrations. As shown in this figure, after treatment
with 5 mM 6-OHDA in L1 phase, the worms exhibited an
obvious enhancement in GFP expression at the 3rd day, and the
fluorescence intensity became much brighter and increased
significantly after treatment with 10 mM 6-OHDA. However, the
untreated worms expressed relatively weak GFP instead, without
the obvious generation of reactive oxygen species. Together with
the data from the mobility defects above, the results demon-
strated that 6-OHDA could induce oxidative stress, and the
appearance of mobility defects as well. Also, the level of oxida-
tive stress was closely related to the effects of mobility disorder
induced by neurotoxin (Fig. 5b). The untreated worms were
observed to exhibit free movements without the generation of
reactive oxygen species; while, the worms showed obvious
mobility defects and a corresponding increased oxidative stress
after treatment with 6-OHDA, indicating that the increased
oxidative stress might be involved in the process of neuron
degeneration, leading to mobility defects.
Fig. 6 summarizes the quantitative evaluation of the various
responses induced by 6-OHDA in strains UA57 and CL2166 (at
the 3rd day), including mobility defects, DAergic neurons
degeneration and oxidative stress, respectively. As shown in
Fig. 6, the worms exhibited an obvious dose dependant manner
for the three types of responses after neurotoxin treatment, which
were very sensitive to 6-OHDA. This will be helpful for the
identification of neuroprotective compounds of C. elegans
dopaminergic neurons against 6-OHDA.
Conclusions
We proposed a novel microfluidic device integrated with droplet
generator, floatage-based trap array and tapered immobilization
array for analyzing several responses of individual C. elegans to
neurotoxin. With this device, the mobility defects, neuron
degeneration and oxidative stress of individual worms in
response to neurotoxin 6-OHDA could be characterized
continuously, and automatically. The worms were found to
exhibit obvious mobility defects after neurotoxin treatment,
which were correlated with a specific degeneration of DAergic
neurons, and the increased oxidative stress might be involved in
the process of neuron degeneration. The established device is
quite simple, more stable and biocompatible, which has the
potential to accelerate current whole-animal assays and high-
throughput drug screening for neurodegenerative diseases at
single animal resolution.
Acknowledgements
This research was supported by the National Nature Science
Foundation of China (No.20635030 and 90713014), Key Project
of Chinese National Programs for Fundamental Research and
Development (973 program, No. 2007CB714505 and
2007CB714507), Knowledge Innovation Program of the Chinese
Academy of Sciences (KJCX2-YW-H18), and Instrument
Research and Development Program of the Chinese Academy of
Sciences (YZ200908).
We greatly thank RainDance Technologies (MA, USA) and
3M (MN, USA) for the supply of surfactant EA and FC-40.
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