Development of an Integrated Optofluidic Platform for Droplet and
Micro Particle Sensing
Microflow Analyzer for Interrogating Self Aligned Droplets and Droplet
Encapsulated Micro Objects
P. K. Shivhare
1,2
, A. Prabhakar
1
and A. K. Sen
2
1
Department of Electrical Engineering, Indian Institute of Technology Madras, Chennai, India
2
Department of Mechanical Engineering, Indian Institute of Technology Madras, Chennai, India
Keywords: Optofluidics, Microfluidics, Microflow Cytometry, Droplet Microfluidics, Microflow Analyser, Optical
Sensing, Single Cell Analysis, Emulsion, Scattered Signals, Fluorescence Detection, 3D Flow Focusing.
Abstract: Here we report the development of a micro flow analyser that integrates digital microfluidics technology with
optoelectronics for the detection of micron size droplets and particles. Digital microfluidics is employed for
the encapsulation of microparticles inside droplets that self-align at the centre of a microchannel thus
eliminates the need of complicated 3D focusing. Optoelectronics comprise a laser source and detectors for
the measurement of forward scatter (FSC), side scatter (SSC) and fluorescence (FL) signals from the
microparticles. The optoelectronics was first used with a simple 2D flow focusing channel to detect
microparticles which showed uncertainty in the data due to lack of 3D focusing. The integrated device with
digital microfluidics technology and optoelectronics was then used for the enumeration and detection of
Rhodamine droplets of different size. Rhodamine droplets of different size were characterized based on FSC,
SSC and FL. Finally, the device was used for the detection of fluorescent microbeads encapsulated inside
aqueous droplets.
1 INTRODUCTION
Integrated optical detection in a microfluidic platform
recently got an immense attention, and a new field of
study “Optofluidics” have emerged. On such
integrated platforms light and fluids are engineered
synergistically to implement a highly sensitive and
portable lab-on-chip bio-chemical sensors (Psaltis et
al. 2006; Testa et al. 2015). Innumerable integrated
optofluidic platforms were successfully demonstrated
in last few years for various applications for instance,
controlling liquid motion using light (Baigl 2012),
sunlight based fuel-production (Erickson et al. 2011),
microfabrication (Koh et al. 2015), and flow
cytometry (Godin et al. 2008). In particular
development of portable and economical microflow
cytometer is urgently required for addressing the
feeble diagnostic situations in rural areas of
developing countries. Various microflow analysers
were developed for different applications including
counting and studying biological cells (Zhang et al.),
bacteria (Verbarg et al. 2013), cellular DNA
(Ornatsky et al. 2008) droplets (Kunstmann-Olsen et
al. 2016).
Isolating and interrogating a micro-object from
bulk revealed that the differences in the micro objects
and biological cells exists due to the various natural
random processes (Xie et al. 2015). Traditional
methods, which involves measurement of the
probability distribution from a large population
ensemble of micro objects may prove to be
misleading (Carlo and Lee 2006; Huang et al. 2014)
since such study camouflages the unique behaviour
of the individual micro objects (Yang et al. 2016a).
The study and detection of this particularity may
prove to be helpful in early diagnosis of diseases like
cancer (Yang et al. 2016b), drug screening (Espulgar
et al. 2015), cellular drug metabolism (Chen et al.
2016), intracellular communication (Liu and Lin
2016), etc.
It is known from literature that Water in Oil
(W/O) or Oil in Water (O/W) droplets tends to move
toward the region with zero shear rate (Leal 1980),
this property can be exploited for eliminating the need
Shivhare P., Prabhakar A. and Sen A.
Development of an Integrated Optofluidic Platform for Droplet and Micro Particle Sensing - Microflow Analyzer for Interrogating Self Aligned Droplets and Droplet Encapsulated Micro Objects.
DOI: 10.5220/0006174801710178
In Proceedings of the 10th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2017), pages 171-178
ISBN: 978-989-758-216-5
Copyright
c
2017 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
171
of 3D flow focusing in microchannel for the
development of a microflow analyser. In addition,
ability of microfluidic platforms to manipulate small
volumes of fluids (~fL to ~nL) by efficiently
producing monodisperse droplets at very high speed
(kHz to MHz) makes it an ideal platform for single
particle analysis (Martino and Andrew 2016).
Miniaturization of traditional test tube into a
monodisperse droplet (Kintses et al. 2010) of few
micron size not only drastically decreases the volume
of sample required for analysis but also efficiently
compartmentalizes a particle for various single
particle analysis (Joensson and Andersson Svahn
2012). In addition recent developments in the field of
digital microfluidics (Jebrail and Wheeler 2010) have
equipped the researchers with a complete toolbox for
droplet manipulation which includes various
functional operations such as sorting, splitting,
mixing, incubation of droplets (Kintses et al. 2010).
Nevertheless, challenges exists in realizing a
commercially viable products. The main hindrance in
the development of a microflow analyser are
complicated techniques required for 3D flow
focusing of sample and the control of interdistance
between the micro particles in order to avoid presence
of multiple objects in the optical window. (Shivhare
et al. 2016). Similarly, several challenges are present
for single particle analysis in terms of increasing
throughput, ultra-sensitive detection of extremely
small volume (Xie et al. 2015; Yang et al. 2016b), and
dynamic control of environment (Carlo and Lee
2006) for such measurement. Techniques such as
microdissection and micromanipulators are presently
employed for single particle analysis. However, these
methods require complex manipulations (Xie et al.
2015) and in addition costly equipments further limits
availability of these techniques. Further, the optical
detection of micro particle encapsulated inside
droplet was not paid any attention. Thus, techniques
for isolation and cost-effective detection of single
micro particle from bulk are urgently required.
In this work, we report the development of an
integrated optofluidic microflow analyser for non-
invasive optical detection of 2D-focused micro
particle. The inherent property of droplets to move
toward region with zero shear rate is employed for
generating self-aligned droplets thereby eliminating
the need of complicated 3D flow focusing
mechanisms. Forward scattered (FSC), side scattered
(SSC) and fluorescent (FL) signals are collected and
mapped in a scatter plot. Further, the platform is also
used for isolating and sensing of single micro objects
by trapping the object in the discrete droplet
surrounded by an immiscible phase.
2 EXPERIMENTS
2.1 Design and Microfabrication
Figure 1 shows image of the integrated optofluidic
device used for interrogating 2D flow focused
polystyrene beads, droplets, droplet generation rate,
and measurement of FSC, SSC, and FL from a droplet
encapsulated polystyrene fluorescent beads. The
device is incorporated with an on-chip droplet
generator to generate droplets and facilitate
compartmentalization of single micro particle for
optical interrogation. In all experiments the sample
(mostly aqueous phase) was infused using sample
inlet while continuous phase (mostly oil phase) was
infused through sheath inlet. As marked, the device is
equipped with four grooves for embedding an in-situ
optical detection zone by inserting optical fiber.
Groove 1 was used to illuminate the sample using a
lensed fiber to focus the laser beam to a spot of 10 µm
in the center of microchannel, whereas fibers in
Groove 2, Groove 3, and Groove 4 was placed at 5
anticlockwise, 45
clockwise, and 135
clockwise
with respect to incident beam and used to obtain FSC,
SSC, and FL signals respectively.
Figure 1: Image of the integrated optofluidics device
employed for the measurements.
The design of the device was made using CAD
software (Autodesk® AutoCAD 2015) and clear field
photomask was printed with the resolution of 40000
dpi. Finally, Polydimethylsiloxane (PDMS) based
microfluidic device was obtained employing standard
photolithography technique followed by soft
lithography process, a detailed description of the
process is provided in the literature (Sajeesh et al.
2014). In order to insert standard available multimode
62.5/125μm optical fiber, the depth of the device
was kept at130μm. The width of the main fluid
channel and throat of droplet generator junction were
100μm and 30μ respectively.
BIODEVICES 2017 - 10th International Conference on Biomedical Electronics and Devices
172
2.2 Materials and Methods
2.2.1 Aqueous and Oil Phases
Deionized (DI) water mixed with varying
concentrations of Rhodamine Dye (Sigma Aldrich,
Bangalore, India) was used as discrete phase while
olive oil (Sigma Aldrich, Bangalore, India) was used
as continuous phase in the experiments. First, a highly
concentrated 52.19mM Rhodamine solution was
prepared by adding 100mg of Rhodamine B (Sigma
Aldrich, Bangalore) dye in 4mL of aqueous glycerol
solution (22% wt/wt). Now this concentrated solution
was further diluted to get 130μM solution. Aqueous
Rhodamine solution and olive oil were filtered with
0.2 μm PTFE and nylon filters respectively (Axiva
Sichem Biotech, Chennai, India) to avoid clogging
due to unsolicited dust in the microfluidic channel. In
order to stabilize the droplets, 5% wt/wt of Tween 80
(Sigma Aldrich Bangalore, India) was added to the
Rhodamine solution as surfactant.
2.2.2 Microbeads
Fluorescent polystyrene beads (Sigma Aldrich
Bangalore, India) of diameter 10μm and 15μm was
mixed in aqueous glycerol solution (22% wt/wt) in
order to avoid sedimentation of beads. 0.5% wt/wt of
surfactant Tween 80 (Sigma Aldrich Bangalore,
India) was added to the solution to prevent the
aggregation of the beads present in the solution. The
original bead solution was diluted with the aqueous
solution.
2.3 Optical Sensing Setup
A block diagram of optical detection system
incorporated for measurements is shown in Fig. 2.
Graded index multimode (62.5/125μm) fiber
coupled20mW, 532nm turnkey laser system
(VTEC Lasers and Sensors, The Netherlands) was
employed for illuminating the sample (Using Groove
1). The FSC data was collected using Si PIN
photodiode (DET02AFC/M, Thorlabs Inc., USA),
whereas for collecting feeble SSC and FL signal
highly sensitive Si semiconductor based avalanche
photodiode (C10508-01, Hamamatsu Photonics,
Japan) and single photon counting module
(SPCM50A/M, Thorlabs Inc., USA) are used
respectively, as compared to traditional slower and
bulky photomultiplier tubes (PMT). The use of
smaller semiconductor detectors increases portability
of the system. All these detectors were connected to
the microfluidic chip using multimode fibers (62.5/
125μm) inserted in the fiber grooves as explained in
Section 2.1 of the manuscript. An optical band pass
filter at 575/50 nm (ET575/50m, Chroma Technology
Corp., USA) was attached to single photon counting
module (SPCM) to filter out the illuminating
wavelength of 532 nm and measure fluorescence
signal.
The FSC and SSC signals were sampled at 10kHz
while FL signal was sampled at 5kHz. The data was
collected using data acquisition system (NI-USB-
6251, National Instruments, India) and in-house
software written in LabVIEW®. Data was analysed
on a computer using an in-house built Python
software to obtain width, amplitude, and area for each
pulse (each particle/droplet crossing the detection
region).
2.4 Experimental Setup
Pressure based pumping system (MFCS-EZ, Fluigent
SA, France) were used to infuse the fluids into the
microchannels. Inverted microscope (Axiovert A1,
Carl Ziess GmbH, Gemany) attached with high speed
camera (SA3, Phtoron, USA) was used to capture the
videos at 1000 frames. ImageJ (Rasband, W. S.,
ImageJ, USA) was used to analyse the videos to
obtain the droplet generation rate (
), and droplet
diameter (
).
Figure 2: Block diagram of the optical detection system.
Lasersource PINPhotodiode APD SPCM
NIDAQ
ToGroove1FromGroove2FromGroove3FromGroove4
ToComputer
Development of an Integrated Optofluidic Platform for Droplet and Micro Particle Sensing - Microflow Analyzer for Interrogating Self
Aligned Droplets and Droplet Encapsulated Micro Objects
173
3 RESULTS AND DISCUSSION
3.1 2-D focused Micro Particles
Microbeads solution and DI water were infused in the
microchannel from sample and sheath inlet
respectively. The focused streams of microbeads
were passed through detection region and FSC of
each event was obtained. Fig. 3(a) shows the scatter
plot of scattered signal

vs. residence time
(pulse-width) for large number of beads passing
through detection region. Fig. 3(b) represents the
normalized distribution of the scattered signal

for micro particles of 10μm and 15μm, data was fit
to normal distribution with
value of 0.87 and 0.79
respectively. It can be clearly observed that micro
particles of different sizes form different colonies in
the scatter plot marked with ellipses in Fig. 3(a).
Remark that residence time of beads of different
sizes are comparable, this is attributed to the fact that
the beads are focused only in horizontal plane and are
free to move in any vertical plane. Since, mobility
(Sajeesh et al. 2014) profile is parabolic along the
vertical plane (Shivhare et al. 2016), particle along
center line requires less time to traverse detection
region, as compared to the particle travelling near the
wall. The scattered signal

is proportional to size
of micro particle crossing the detection region (Cho
et al. 2010), and thus the amplitude of scattered signal
in case of bigger beads is higher than smaller beads
as can be observed from Fig. 3(a). This can also be
observed from Fig. 3(b), where mean value of signal
for normal distribution corresponding to 15μm is
higher than 10μm. However, we can clearly notice the
presence of 10μm particles in the colony of 15μm
and vice versa. This is due to the fact that since the
particles are focused in only horizontal plane multiple
beads can pass the detection region at the same instant
which introduces the error in the measurement
(Shivhare et al. 2016). Thus 2D focused micro
particles of varying sizes can be segregated with FSC
signal, however, varying mobility in the vertical plane
and presence of multiple particles in the detection
region introduce error in the measurement. This error
introduced due to the variation of mobility can be
eliminated by focusing the particle from all direction.
Thus 3D flow focusing is required for sensitive
cytometric application.
3.2 Rhodamine Droplets
Continuous stream of Rhodamine water droplets were
generated with olive oil as continuous phase and
rhodamine dye solution as discrete phase. The size of
the droplets was varied by varying the pressure of
continuous and discrete phases. FSC, SSC, and FL
were measured for these droplets. Figure 4 shows the
signal collected from a train of droplets crossing the
microchannel. It can be observed from the data that
as the droplet passes the detection zone it hinders the
path of continuous beam of laser light and scatters the
light in various direction which results in pulse in
scattered signals. Note that when droplet passes the
detection zone FSC encounters a negative pulse due
to the obstruction of continuous beam of light. This
light gets scattered in different direction due to
interface between droplet and continuous phase
which results in positive pulse in SSC. Also since
droplet is devised using fluorescent dye, a pulse in FL
signal is captured by SPCM as droplet crosses the
detection zone.
Figure 3: (a) Scatter plot of Scattered Signal (

) vs. Residence Time () (b) Normalized distribution of scattered signal

for 2D focused micro particles of sizes 10μm and 15μm.
(a)
(b)
0.000 0.003 0.006 0.009
0.4
0.6
0.8
1.0
Scattered Signal (V)
Residence Time
(
s
)
10 um
15 um
0.4 0.6 0.8
0.0
0.1
0.2
0.3
0.4
Normalized Count
Scattered Signal (V)
10 um
15 um
BIODEVICES 2017 - 10th International Conference on Biomedical Electronics and Devices
174
Figure 4: FSC, SSC, and FL data from a stream of
continuous Rhodamine droplet.
Figure 5(a) illustrates the images of the
Rhodamine droplets generated in the microchannel,
whereas Figure 5(b) represents the 3D scatter plot of
FSC-SSC-FL signal of each droplet passing the
detection zone from droplet of varying sizes. The size
(in μm) of the corresponding droplets is mentioned as
legend in the figure and scale on all images in Figure
5(a) represents 100μm. It can be observed from the
Figure 5(b) that droplets of different sizes form the
different colony in the scatter plot.
Droplets in microfluidic channel tends to move
towards the region with zero shear rate due to the
deformability induced lift forces (Leal 1980). In the
present design, due to symmetric flow conditions the
region with zero shear rate tends to be the centreline
of the microchannel which results in self 3D flow
focusing of the interrogated objects. Thus this self-
alignment of droplets eliminated the need of complex
3D flow focusing setup (Shivhare et al. 2016) and
hence need of extra bulky pumping system required.
This improves the portability of the system and also
makes it relatively less complex.
The impact of this 3D flow focusing is clearly
visible in the scattered plot (Figure 5(b)) where the
various colonies are well separated as compared to
the 2D focused micro particles as shown in Figure 3
(b). This distinction in the colonies is critical for
measurement of properties like size, type, velocity of
the object. Thus the use of droplets as the
miniaturized test tube in the cytometric applications
reduces the error due to mobility variation of micro
particles.
The other important concern in the cytometric
measurement is the control of minimum interdistance
between the particles in order to avoid the multiple
objects in the optical window (Shivhare et al. 2016).
This issue can also be addressed using droplet
microfluidics since the droplet generation rate can be
controlled using flow rate of discrete and continuous
phase (Nguyen et al. 2006).
3.3 Particle Encapsulation
The polystyrene beads were compartmentalized in the
water in oil (W/O) droplets with Olive oil as
continuous phase and microbeads solution described
in the Section 2.2.2 as discrete phase. As droplets are
generated a bead present in the solution gets trapped
inside the droplet, and thus gets completely isolated
from the rest of beads (bulk). Now using the already
reported tools for droplet manipulation (Jebrail and
Wheeler 2010) various single particle experiments
can be performed on the isolated micro particle. The
number of positive droplets (droplets containing a
micro particle) can be increased by increasing the
concentration of the micro particle. The relation
between the numbers of micro-particles in the droplet
is given by Poisson’s distribution
exp


/!, where  represents the
probability of presence of beads in a droplet with
representing mean number of beads in the volume of
each droplet (Mazutis et al. 2013). Thus, as the
concentration of bead in the solution increses,
droplets containing multiple particle, also increases.
(Chabert and Viovy 2008).
Figure 6(a) and Figure 6(b) represents the image
of a negative (droplet without micro particle) and
positive droplets (droplet with micro particle)
respectively. It can be clearly perceived from the
image that Figure 6(b) contains a micro particle.
Figure 6 (c), (d), and (e) shows the FSC, SSC, and FL
data captured from the stream of mixture of positive
and negative droplet crossing the detection zone. The
0.0 0.2 0.4 0.6 0.8 1.0
3
6
9
12
FSC (V)
Time (s)
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.5
1.0
1.5
2.0
SSC (V)
Time (s)
0.0 0.2 0.4 0.6 0.8 1.0
0
100
200
300
400
500
FL (Count)
Time
(
s
)
Development of an Integrated Optofluidic Platform for Droplet and Micro Particle Sensing - Microflow Analyzer for Interrogating Self
Aligned Droplets and Droplet Encapsulated Micro Objects
175
Figure 5: (a) Images of droplets of varying sizes flowing inside the microchannel (b) 3D scatter plot of FSC-SSC-FL signal
from Rhodamine droplets of varying sizes.
dashed rectangle (8
th
and 9
th
pulse) in the graph are
the positive droplets, while other pulses are from
negative droplets. It can be observed from the Figure
6(c) that the FSC of positive (8
th
and 9
th
pulse) and
negative droplets (except 8
th
and 9
th
pulse) are almost
similar, this is attributed to the fact that the FSC signal
is proportional to the size of droplets which remains
unaltered for positive and negative droplets.
However, SSC is proportional to the internal
granularity of the object which is being interrogated.
Since, the positive droplet contains the micro particle
the internal structure of these droplets differ from
negative ones, this change in the granularity can
clearly be observed in the SSC signal shown in Figure
6(d). Note that positive droplets (8
th
and 9
th
pulse)
possess two peaks as compared to the single peak in
negative droplets. This is further confirmed by the FL
signal shown in Figure 6(e) where peaks are present
only for the droplets containing fluorescent beads.
The developed integrated platform
simultaneously exploits the advancements in the field
of digital microfluidics and optofluidics for isolating
a particle from bulk and real time non-invasive
optical interrogation. Further, sorting mechanism can
be employed to separate and collect the positive
droplets for further analysis. This device provides a
low cost alternative to the presently employed
complex techniques for single particle analysis.
4 CONCLUSIONS
In this work, we reported the development of an
integrated optofluidic microflow analyser which
consists of a microfluidic channel and a set of optical
fiber grooves for simultaneous measurement of
various scattered signal viz. FSC, SSC, and FL signal.
An optical illumination and detection system
comprising of green laser source, Si PIN photodiode,
Si avalanche photodiode, and Single Counting
Photon Module was designed. Polystyrene beads of
10μm and 15μm were focused in horizontal plane
(two dimensional focusing) and scattered signals
were collected. The obtained scatter plot shows that
error in the measurement is introduced due to the
variation in mobility of particles and presence of
multiple objects in the optical detection window since
particles were not focused in vertical plane. Both of
these error, which are measure concern for the
development of microflow analyser, were eliminated
with the formulation of self-focused droplet inside the
microchannel. The collected scattered signals formed
the distinct colonies for droplet of varying sizes on
the scatter plot, which is critical for various
BIODEVICES 2017 - 10th International Conference on Biomedical Electronics and Devices
176
Figure 6: (a) Image of droplet without micro particle (b) Image of droplet with micro particle (c) FSC (d) SSC (e) FL signal
from a stream of mixture of positive and negative droplets in the microchannel.
cytometric measurements. Finally the proof of
concept was demonstrated for single particle analysis
by encapsulating and optically interrogating 10μm
fluorescent polystyrene beads inside the droplet.
Also, the use of droplets to compartmentalize the
micro particles drastically reduced the volume of
sample required. Thus the developed platform
incorporating droplets encapsulation of micro particle
and optical interrogation can prove to be low cost,
portable, non-invasive alternative for single particle
analysis.
ACKNOWLEDGEMENTS
The authors would like to thank SERB, DST (Project
No. MEE/15-16/340/DSTXASHS) and IIT Madras
for providing financial support for the project. We
acknowledge support of Centre of NEMS and
Nanophotonics (CNNP), IIT Madras with the
photolithography work. Also, we thank the
Interdisciplinary Research Program, IIT Madras,
which enabled this work.
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