Group Title: 7th International Conference on Multiphase Flow - ICMF 2010 Proceedings
Title: 17.2.1 - Micro-Scale Liquid-Liquid Segmented Flows for Enzyme Activity Monitoring
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Permanent Link: http://ufdc.ufl.edu/UF00102023/00415
 Material Information
Title: 17.2.1 - Micro-Scale Liquid-Liquid Segmented Flows for Enzyme Activity Monitoring Micro and Nano-Scale Multiphase Flows
Series Title: 7th International Conference on Multiphase Flow - ICMF 2010 Proceedings
Physical Description: Conference Papers
Creator: Kim, N.
Lee, W.
Balamurugan, S.
Murphy, M.C.
Soper, S.A.
Nikitopoulos, D.E.
Publisher: International Conference on Multiphase Flow (ICMF)
Publication Date: June 4, 2010
 Subjects
Subject: microfluidics
hydrophobic
fluorescence
spectroscopy
 Notes
Abstract: Liquid-liquid segmented flows in microchannels fabricated on polymer test chips were investigated experimentally and combined with fluorescence correlation spectroscopy (FCS) as an optical detection method for high throughput screening. Three different microchannel configurations were used to examine effects of the injection channel geometry on the characteristics and stability of the resulting segmented two-phase flows. The injection channel cross-section size and the area expansion ratio (ER) with respect to the test channel were significant influencing parameters. The topological characteristics of the dispersed segmented flows (droplet and plug size and pitch) as functions of carrier fluid volumetric flow ratio (βC) were determined by image processing of CCD camera frames obtained using bright field illumination on a microscope. Velocities of the dispersed phase for segmented flows were measured using double-pulsed laser illumination to be 1.12 – 1.32 times faster than the flow superficial velocity. As a set of experiments was repeated, we noticed that wetting of aqueous droplets on the microchannel caused unstable and unpredictable segmented flow regimes. In order to prevent the aqueous dispersed fluid from wetting on the channel surface, a two-step procedure for the fluorination of polymer surface was developed to make microchannel surfaces more fluorophilic and hydrophobic. The fluorescence signals from each droplet were detected through an avalanche photodiode and fed into a counter/timer board and subsequently analyzed with custom-built software.
General Note: The International Conference on Multiphase Flow (ICMF) first was held in Tsukuba, Japan in 1991 and the second ICMF took place in Kyoto, Japan in 1995. During this conference, it was decided to establish an International Governing Board which oversees the major aspects of the conference and makes decisions about future conference locations. Due to the great importance of the field, it was furthermore decided to hold the conference every three years successively in Asia including Australia, Europe including Africa, Russia and the Near East and America. Hence, ICMF 1998 was held in Lyon, France, ICMF 2001 in New Orleans, USA, ICMF 2004 in Yokohama, Japan, and ICMF 2007 in Leipzig, Germany. ICMF-2010 is devoted to all aspects of Multiphase Flow. Researchers from all over the world gathered in order to introduce their recent advances in the field and thereby promote the exchange of new ideas, results and techniques. The conference is a key event in Multiphase Flow and supports the advancement of science in this very important field. The major research topics relevant for the conference are as follows: Bio-Fluid Dynamics; Boiling; Bubbly Flows; Cavitation; Colloidal and Suspension Dynamics; Collision, Agglomeration and Breakup; Computational Techniques for Multiphase Flows; Droplet Flows; Environmental and Geophysical Flows; Experimental Methods for Multiphase Flows; Fluidized and Circulating Fluidized Beds; Fluid Structure Interactions; Granular Media; Industrial Applications; Instabilities; Interfacial Flows; Micro and Nano-Scale Multiphase Flows; Microgravity in Two-Phase Flow; Multiphase Flows with Heat and Mass Transfer; Non-Newtonian Multiphase Flows; Particle-Laden Flows; Particle, Bubble and Drop Dynamics; Reactive Multiphase Flows
 Record Information
Bibliographic ID: UF00102023
Volume ID: VID00415
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: 1721-Kim-ICMF2010.pdf

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7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010



Micro-Scale Liquid-Liquid Segmented Flows for Enzyme Activity Monitoring


N. Kim*, W. Leet, S. Balamurugant, M.C. Murphy*, S. A. Sopert and D. E. Nikitopoulos*

*Department of Mechanical Engineering, tDepartment of Chemistry
Center for BioModular Multi-Scale Systems (CBM2)
Louisiana State University, Baton Rouge, LA 70803, USA
meniki@me.lsu.edu


Keywords: Microfluidics, hydrophobic, fluorescence, spectroscopy




Abstract

Liquid-liquid segmented flows in microchannels fabricated on polymer test chips were investigated experimentally and
combined with fluorescence correlation spectroscopy (FCS) as an optical detection method for high throughput screening.
Three different microchannel configurations were used to examine effects of the injection channel geometry on the
characteristics and stability of the resulting segmented two-phase flows. The injection channel cross-section size and the area
expansion ratio (ER) with respect to the test channel were significant influencing parameters. The topological characteristics of
the dispersed segmented flows (droplet and plug size and pitch) as functions of carrier fluid volumetric flow ratio (tc) were
determined by image processing of CCD camera frames obtained using bright field illumination on a microscope. Velocities of
the dispersed phase for segmented flows were measured using double-pulsed laser illumination to be 1.12 1.32 times faster
than the flow superficial velocity. As a set of experiments was repeated, we noticed that wetting of aqueous droplets on the
microchannel caused unstable and unpredictable segmented flow regimes. In order to prevent the aqueous dispersed fluid from
wetting on the channel surface, a two-step procedure for the fluorination of polymer surface was developed to make
microchannel surfaces more fluorophilic and hydrophobic. The fluorescence signals from each droplet were detected through
an avalanche photodiode and fed into a counter/timer board and subsequently analyzed with custom-built software.


Introduction

Liquid-liquid segmented flows in microchannels have drawn
attention to the application of the fluid handling in
biochemical analytical devices because of their potential
advantages over single phase flows. While heat and mass
transfer (Burs and Ramshaw 2001) in biochemical reactions
are improved with the higher surface to volume ratios of
miniaturized fluidic systems, the efficiency of the reactions
are limited by the slow diffusive mixing due to the laminar,
low Reynolds number flow in microchannels. The parabolic
velocity profile of single-phase flow driven by pressure
gradients under no-slip conditions (i.e., Poiseuille flow)
produces broad residence time distributions (RTD). Broadly
dispersed molecules also tend to adsorb on the channel walls,
non-specifically. The adsorption of target molecules on the
walls reduces the efficiency of biochemical reactions due to
the loss of working molecules for reactions (Prakash et al.
2008).
However, separating a flow with another immiscible fluid
induces the recirculation of streamlines in the separated fluid
plugs, which enhances mixing of the molecules and prevents
the molecules of interest from dispersing along the channels.
In gas-liquid two-phase flow, target molecules distribute
within the liquid plug and mixing is intensified by the
circulating streamline. However, dispersion and adsorption


of molecules across neighboring liquid plugs, although less
than in single phase flow, still occurs through liquid films
and corer flows between the gas bubble and channel walls
(Muradoglu et al. 2007). For liquid-liquid segmented flow,
encapsulated dispersed fluids in the shape of spherical
droplets or elongated plugs serve as independent
biochemical reactors by confining the dispersion of target
molecules (Gunther et al. 2004, Song et al. 2003), which are
normally widely dispersed in pressure-driven single phase
flow. Interaction of shear and interfacial forces on the
interface between the dispersed and carrier fluids induces
recirculation of the streamlines inside both the dispersed
fluid and the carrier fluid plugs, intensifying mixing (Song et
al. 2003), which is normally reduced by the laminar
characteristics of microchannel flows. If there is no
coalescence of the neighboring dispersed fluid, the contents
of the encapsulated dispersed fluids are preserved without
cross-contamination. This is the one of the reasons that
liquid-liquid segmented flow is a potential candidate for use
in high throughput screening. Another important
characteristic of liquid-liquid segmented flow is the presence
of the continuous thin film of carrier fluid separating the
dispersed fluid from the channel wall, which prevents target
molecules from being adsorbed on the channel wall. This is
the only potential source of cross-contamination between
successive dispersed plugs containing different reagent









mixtures.
In virtue of those advantages, liquid-liquid segmented flows
have been demonstrated in many practical applications
including, continuous segmented flow polymerase chain
reaction (PCR) (Beer et al. 2008, Curcio and Roeraade 2003,
Mohr et al. 2007), encapsulation of enzymes in lipid vesicles
(Jeanne et al. 2008) using microfluidic jetting, protein
crystallization (Li et al. 2006), and fabrication of magnetic
hydrogel microparticles (Hwang et al. 2008). The
manipulation of the dispersed droplets and plugs (Adamson
et al. 2006, Song et al. 2003, Yamada et al. 2008) were
studied in order to replace conventional micro titer plates
with a series of continuous separated droplets in
microchannels for high throughput screening in drug
discovery (Chen and Ismagilov 2006). Each encapsulated
droplet served in the designed function as an independent
microreactor with increased mass and heat transfer and
maintains the distinct identity of contents without cross
communication between neighboring dispersed fluid
volumes.
As an ultra sensitive detection method for enzyme activity in
single-molecule level, fluorescence cross-correlation
spectroscopy (FCCS) has been developed. FCCS monitors
coincident signals of two spectrally distinct fluorophores
from a small number of molecules to generate information
on enzyme activity appropriate for drug screening (Kettling
et al. 1998). Droplets loaded with dual labeled substrates and
enzymes as well as enzyme inhibitors can be interrogated
using FCCS to determine the effects of the inhibitor on
enzyme activity. When this is coupled to a microfluidic
platform, using the parallelization and automation
capabilities associated with microfluidics can significantly
enhance the throughput so as to generate ultra-high
throughput screening systems.
In order to design microfluidic bio-analytical systems using
liquid-liquid segmented flows effectively and predictably, it
is necessary to understand their fundamental behavior. To
this effect, an experimental investigation of liquid-liquid
segmented flows in properly modified polymer microfluidic
channels was carried out. Details of segmented flow regimes
and maps and velocity measurements of dispersed fluid
parcels were performed. As an ultra sensitive readout method
for fluorescent signals from each segmented droplet,
fluorescence correlation spectroscopy was demonstrated.

Experimental

A. Test chip configurations
Configurations of the microchannels fabricated on the
polycarbonate (PC) chips are shown in Fig. 1. Three
different cross-sectional expansion ratios for the injection to
the test channel transition were used to observe the effect of
different cross-sectional expansion ratios on the segmented
flow regimes in the test channel. The nominal dimensions of
the injection channels are shown in Table I. This enabled
examination of the influence of the configuration of the
injection in the microfluidic incompressible two-phase
system on the evolution and characteristics of the two-phase
flow generated in the microfluidic test channel. Carrier and
dispersed fluids were introduced into each inlet and met at a
cross-junction channel. Carrier fluid from the sides
periodically pinched the elongated cylindrical dispersed fluid
thread from the central branch. Shear and interfacial forces


7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

Injection channel Test channel

Carrier Dispersed Outlet
fluid inlet fluid inlet




5mm


Injection channel


Injection channel


Test channel


Test channel


Carrier Dispersed
fluid inlet fluid inlet




5mm
- <-- Channel to outlet


Figure 1: Schematics of the hot embossed polycarbonate test
chip configurations. (a) Type I with an expansion ratio from the
injection to the test channel of 1:16 (b) Type II with an
expansion ratio of 1:4 and (c) Type III with an expansion ratio of
1:2.


at the interface between the dispersed and carrier fluids
generated a continuous string of mono-dispersed droplets in
the central branch after the cross-junction (Thorsen et al.
2001, Utada et al. 2005). This two-phase flow entered the
test channel, which was deeper (Type I) and wider (Type I, II
and III) in a serpentine configuration along the test channel.
The serpentine configuration was chosen due to its compact
footprint and because it allowed simultaneous observation of
flow over almost the full test section channel length. The U-
and 90- bends in the path of the test channel had a nominal
centreline radius of 105 pm ( 6%) for all chips. The


Carrier Dispersed Upstream pressure port Outlet
fluid Inlet fluid Inlet o





5mm Downstream pressure port






7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


cross-junction of the injection section and expansion area
from the injection to the test channels had rounded corners
as shown in Fig. l(b). This was a result of the 100 /pm
cutting tool radius (r) used in micromachining of the mold
insert, which produced the chip.

B. Microfabrication
All polycarbonate chips were replicated by hot embossing
with micro-milled brass mold inserts. This thermoforming of
polymer microfluidic chips enabled rapid production of
multiple copies with stable production quality. A mold insert
with the fluidic features was micro-machined on a 4.75"
diameter brass substrate (Alloy 353, McMaster-Carr, Atlanta,
GA) using a KERN MMP 2522 (KERN Micro- und
Feinwerktechnik GmbH, Eschenlohe, Germany)
micro-milling machine. The milling bit radii (PMT,
Janesville, WI) ranged from 25 pum to 100 pum. The brass
mold insert was used to thermoform multiple chips in
polycarbonate sheets (GE LEXAN9 9034, 2.36 mm thick,
Modem Plastics, Bridgeport, CT) using a hot embossing
machine (HEX02, JENOPTIK Mikrotechnik, Jena,
Germany). After post-processing, including cutting and
drilling holes at the inlet and outlet, chips were cleaned using
the following sequence: soaked in a weak (1%) mild
detergent solution (5 min); ultrasonic agitation (2 min) to
remove the organic lubricant used during demolding; and
rinsed in deionized water (>120 min). After machining and
cleaning, the chips were dried at 950C (20 min residence) in
a temperature-controlled oven and thermally bonded to 250
mm thick PC cover slips (CT301325, Goodfellow, Oakdale,
PA) through a controlled process. A bonding jig designed to
apply controlled, uniformly distributed clamping pressure
was used. Thermal bonding was carried out at 160 OC (20
min residence) with two cooling steps, to 950C over 30 min
and then to ambient over 150 min. This thermal bonding
process induced changes in channel geometries from the
nominal values because the temperature of the bonding
process reached slightly above the glass transition
temperature (Tg) of polycarbonate with the clamped pressure.
The resulting channel width and depth after the thermal
bonding were measured using a microscope equipped with a
digital height gage (ME-50 IA DigiMicro, Nikon
Instruments Inc. Melville, NY) and a digital readout
(Quadra-Chek 2000, Metronics Inc. Bedford, NH). The
measured channel dimensions are listed in Table I.
All microchips were made from the same PC stock and batch
and processed identically using the same parameters. The
unavoidable use of a detergent during the cleaning of the


figure z: Hot embossed polycarbonate (Fr) test clips (lype
II)


microchannel could introduce a surfactant contamination of
the microchannel walls so, prior to running experiments, the
channels were soaked and flushed for at least 1-2 days using
deionised water after the thermal bonding was completed. A
photograph of fabricated PC Type II chips is shown in Fig. 2.
Capillary tubes with a 175 pm inner diameter (1577 PEEKTM
tube, Upchurch Scientific Inc., Oak Harbor, WA) were
connected to the drilled holes at the inlet and outlet and
sealed with epoxy (5minute Epoxy, Devcon, Danvers, MA).
Previous work (Kim et al. 2007) showed that the root mean
square (RMS) surface roughness of the fabricated channel
walls ranged from -200 to -400 nm giving a relative
roughness on the order of 0.0019 with respect to the
hydraulic diameter.

C. Test fluids and channel surface treatment
Deionized water (18.2 MQ-cm at 250C, TOC of 5 ppb,
Millipore, Billerica, MA) with 0.1% (v/v) blue food-coloring
(McCormick, Sparks, MD) was used as the dispersed phase
test fluid. Perfluorocarbon (perfluorotripropylamine, FC
3283, 3M, St. Paul, MN) with 10% (v/v) of a nonionic
fluoro-soluble surfactant, 1H, 1H, 2H, 2H-Perfluorooctanol,
97% (PFO, Alfa Aesar, Ward Hill, MA), was used as the
carrier phase test fluid. Measurements of the physical
properties of the test fluids like density, interfacial tension
and viscosity were essential for the analysis of the
experimental results. Density was measured using an
electronic scale (B120S, Sartorius Corp, Edgewood, NY),
interfacial tension was measured from a pendant water drop
suspended in carrier fluid (Hansen and Rodsrud 1991) using


Table I: Measured dimensions of the injection and test channels, nominal dimensions are shown in parenthesis


Injection channel (pm) Test channel (pm)
Expansion ratio
Width Depth Width Depth

Type I 43.6 (50) 41.1 (50) 188.2 (200) 189.8 (200) 1:16

Type II 42.8 (50) 184.6 (200) 193.7 (200) 184.6 (200) 1:4

Type III 97.6 (100) 196.2 (200) 196.9 (200) 196.2 (200) 1:2






7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

Table II: Properties of the dispersed and carrier test fluids

Dynamic
Density Interfacial force vis
p (kg/m3) 7 (mN/m) p (mPais)

DI-Water 997.83 0.72 -0.9509 0.006

FC 3283 1,808.58 9.46 54.15 0.13 1.4796 0.005

FC3283 + PFO (10% v/v) 1,777.54 8.06 13.49 0.33 1.7816 0.012

- All properties were measured at room temperature (220C 1)


a contact angle and surface tension measurement instrument
(FTA125, First Ten Angstroms, Inc., Portsmouth, VA), and
viscosity was measured using a calibrated U-shaped glass
tube viscometer (9721-B50, Cannon Instrument, State
College, PA). Each property was measured more than 10
times at room temperature (220C 1) and shown in Table II
with less than 3% coefficient of variation.
In order to improve the wetting of the fluorocarbon carrier
fluid on the channel walls and prevent the dispersed fluid
from wetting the microchannel wall, the microchannel walls
were coated using the following process. Vaporized 1H, 1H,
2H, 2H-perfluorodecyltrichlorosilane (ABCR GmbH & Co.
KG, Germany) was driven by compressed argon (Ar) gas
into the microchannels for 1 hour and then the microchannel
was filled with FC 3283 + PFO (10% v/v) solution and
baked in a convection oven (HAFO 1602, VWR, West
Chester, PA) at 60 OC for Ihr.

D. Experimental apparatus
The experimental apparatus is shown in Fig. 3(a). Carrier



Neutral Density
Beam expander ND (50% & 70% Plate beam splitte
Fiber optic 3=> 12 mm transmittance) 50/50
illuminaton Mirror -
Pressure transducers S
Syringe pump 2
outlet --I .. DIsprId.ld
Polymer chip Syringe pump 1
Microscope er
e I er unitu
SMercury lamp L 2
SMerc Lauer r Plate beam splitte
Computer for CCD camera 10/90
image record 30 mJ Nd:YAG laser
532 nm
(a)


Figure 3: Schematic diagram of (a) experimental apparatus
and (b) instrumentation setup for two-color fluorescence
cross-correlation spectroscopy


and dispersed phase fluids were introduced into test chips
through capillary tubes from glass syringes (Gastight 1000
Series, Hamilton, Reno, NV) driven by syringe pumps
(NE500, New Era Pump Systems, Inc., Wantagh, NY). The
optical observations and measurements were conducted on
inverted fluorescence microscopes (IX70 and IX81,
Olympus, Center Valley, PA) using 2X, 4X and 10X
objectives equipped with a digital CCD camera
(SharpVISION 1400-DE, IDT, Inc., Tallahassee, FL).
Different illumination sources were used for specific
observations. Continuous bright field back-illumination from
a fiber-optic light-guide with a diode array and diffuser was
used for the acquisition of clear flow images for observations
of segmented flow topology and extraction of dimensions
using image processing. A double pulsed 532nm Nd:YAG
laser was used for the measurement of dispersed fluid
droplet and plug velocities. All experimental apparatus were
installed on a vibration-isolated optical table
(VH3660W-OPT, Newport Corp., Irvine, CA) to remove
external vibrations. Quantitative information was extracted
from the acquired images using image processing routines
developed in-house based on the OPTIMASTM (ver. 6.51,
Media Cybernetics, Inc., Bethesda, MD) image processing
software.
Figure 3(b) shows the experimental setup for the
microfluidic system combined with FCCS in this study. The


TABLE III: Uncertainties of experimental parameters


Parameter Uncertainty, (%)

Flow rate, Q 1

Channel width, w 0.5

Channel depth, d 1.5

Hydraulic Diameter, Dh 0.74

Density, p 0.3

Viscosity, p 0.5

Interfacial tension, y 1.7

Reynolds number, Re 2.09






7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


:. "'* -' "


(b)


' ./ ; " ('. ) .. "




(d)







4 Q /-;.


Figure 4: Liquid-liquid segmented flow regimes of the 50 ,um
x 50 pm injection channel chip (Type I chip with an expansion
ratio of 16) under white field illumination (a) Droplet flow in
the expansion area (4X objective) with homogeneous carrier
fluid volumetric flow ratio, Pc = 0.93 under white field
illumination (b) Droplet flow in the test channel (10X
objective) with Pc : 0.95 under laser illumination (c) Droplet
flow, Pc = 0.74 (d) Irregular Segmented flow, fc = 0.75 (e)
scattered Droplet flow, tc = 0.5 (f) Irregular Segmented flow,
; 0.5 (g) Plug flow,;: 0.37 (h) Plug flow,;: 0.37.


fluorescence bursts from each droplet were fed into a
counter/timer computer board (PCI-6602, National
Instruments) having a temporal resolution of 12.5 ns and
subsequently analyzed with custom-built software written
using LabVIEW 7.0.
Each measurement of the experimental variables included
some level of uncertainty due to bias and precision error.
These uncertainties propagate through mathematical
operations and create an uncertainty in the result (Beckwith
et al. 1993, Weilin et al. 2000). Those uncertainties of the
experimental variables were determined based on the
product data specification and the propagation of the
measured uncertainties [Table III].

Flow Regimes

Three distinct liquid-liquid segmented flow regimes, Droplet,
Plug, and transient Irregular Segmented flows were observed


Figure 5: Liquid-liquid segmented flow regimes of the 50 um x
200 fm injection channel chip (Type II chip with an expansion
ratio of 4) under white field illumination (a) Droplet flow in the
expansion area (4X objective) with homogeneous carrier fluid
volumetric flow ratio, Pc = 0.87 (b) Droplet flow in the test
channel (2X objective) with Pc = 0.87 (c) Irregular Segmented
flow, Pc = 0.69 (d) Irregular Segmented flow, ic = 0.67 (e) Plug
flow, tc = 0.4 (f) Plug flow, tc = 0.4 (g) Plug flow, tc = 0.11 (h)
Plug flow,;: 0.11.


in the test channels. Distinct flow regimes in the expansion
area between the injection and the test channel and in the test
channel sections were specified based on their topology in
terms of the carrier fluid volumetric flow ratio. This is the
fraction of the volumetric flow rate of the carrier fluid over
the total volumetric flow rates of the carrier and dispersed
fluids, pc = Qc/(Qc + QD), where Qc and QD are the
volumetric flow rates of the carrier and dispersed fluids,
respectively.
The images in the first column in Figs. 4(a), 4(c), 4(e) and
4(g) were acquired from the expansion area of the Type I
chip with an expansion ratio of 16 using a 4X objective
under white field illumination for quantitative extraction of
geometrical data. The second column in Figs. 4(b), 4(d), 4(f)
and 4(h) shows images acquired from the test channel
through a 10X objective with 532 nm Nd:YAG
double-pulsed laser illumination for the measurement of the
velocities of the dispersed droplets and plugs. Figures 4(a)
and 4(b) show regular water droplets flowing along the
centerline of the channels. With increasing of the dispersed
fluid flow rate and a constant carrier fluid flow rate, the









_I L I I







(a) (b)





ili


(c) (d)

Figure 6: Liquid-liquid segmented flow regimes of the 100 X
200 injection channel chip (Type II chip with an expansion ratio
of 2) under white field illumination (a) Plug flow at the
cross-junction area (4X objective) at carrier fluid volumetric
flow ratio,;? 0.83, (b) Plug flow at the test channel, tc = 0.83,
(c) Plug flow,;: 0.5, and (d) Plug flow,;: 0.2.


distance between the neighboring droplets was reduced,
increasing frequency of droplet injection [Fig. 4(c)]. Some of
droplets in the test channels coalesced producing an Irregular
Segmented flow regime [Fig. 4(d)] due to disturbances like
the variation of the surface roughness and the curvature
effects resulting from the serpentine test channel bends.
Scattered Droplet flow was observed in the expansion area
with increased dispersed fluid flow rate only in the Type I
chip, which had the highest expansion ratio [Fig. 4(e)].
Intimate contact between the scattered droplets resulted in
coalesced larger irregular droplets and plugs in the test
channels subject to roughness and bend effects. Unlike
instantaneous coalescence of the contacting gas bubbles
observed in gas-liquid, two-phase flow in microchannels
(Kim et al. 2007), dispersed aqueous droplets in the
perfluorocarbon carrier fluid maintained their own interfaces
for a while even as they adhered closely to each other. Below
Pc z 0.32, Figures 4(g) and 4(h) show regular Plug flows
with sufficient distance between the plugs to prevent the
dispersed liquid plugs from merging.
Images in the first and second columns in Fig. 5 display
segmented flow in the expansion area and test channel of the
Type II chip. Droplet, Plug, and transient Irregular
Segmented flows were also observed. Unlike irregular flow
regimes developed by coalescence of neighboring droplets
[Fig. 4(c)] and scattered droplets [Fig. 4(e)] on the Type I
chip, irregular flow regimes on the Type II chip were
contributed by both coalescence of neighboring droplets and
irregular injection in the expansion area. Figure 6 shows
segmented flow regimes from the Type III chip. Most of the
segmented flow regimes observed on this chip were regular
Plug flows without a transient Irregular Segmented flow
regime for the carrier and dispersed fluids flow rates used in
this observation window. Due to the relatively larger
injection channels with the lower expansion ratio, more
stable and longer plugs were generated in the test channel.
Figure 6 shows the dependence of the length of dispersed


7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

plug, the distance between dispersed plugs, and the residence
time (i.e., frequency) of the dispersed plugs on the different
combinations of carrier and dispersed flow rates.
For the cross-junction injector, the injection channel's
cross-sectional area had a strong influence on the two-phase
flow regime generated in the test channel. Smaller injection
channel cross-sectional areas favored the formation of
droplet flow regimes, and when these regimes were created
at higher volumetric flow ratios they were unstable because
of the closer packing of the larger numbers of generated
droplets. Larger injector cross-sectional area channels tended
to primarily produce plugs. These trends were expected,
considering that when the injection channels had a smaller
cross-sectional area, plugs of the dispersed fluid with a
smaller volume were periodically generated at the
cross-junction through pinch-off. As the injection channel
expanded into the larger test channel the smaller volume
plugs formed droplets. At higher volumetric flow ratios the
frequency of the plugs increased and so does the injection of
droplets into the larger test channel. The flow regime


0.002

0.001
0.0008
0.0001
0.0004


0.0001


0.001


JD (mis)

Figure 7: Liquid-liquid segmented flow regime map and
transition lines between regimes observed from the test channel
of Type I chip with an expansion ratio (ER) of 16. (*: Droplet
flow, V: Irregular Segmented flow, and : Plug flow).


0.002

0.001
0.0008
0.0006
0.0004


0.0001


0.001


JD (mis)

Figure 8: Liquid-liquid segmented flow regime map and
transition lines between regimes observed from the test channel
of Type II chip with an expansion ratio (ER) of 4. (*: Droplet
flow, V: Irregular Segmented flow, and : Plug flow).


.. .' I'e lgar
Droplet / ~sprn nted
S /* "V VV
..' v v '.

* /* y' v rV
S/ / Plug

/./ ,4./ v ,/o ooo
-0 / 0
Drophlt&rmgulr segmented Type I chip with ER 16
---- Irregular segmentedPlug -Type I chip with ER 16
-------- Drople/timgular segmened Type II chip with ER 4
-..-..- Irregular segmented/Pkug Type II chip with ER 4


Droplet segrhe a
y, .", v o oo .'n
oT .4 0 0 0 /.
> 3 v/ o yno
i / /' Plug
S/ /'/ o o/' 0 0 0 0o
o / o 0./ ca o oo
- e 0 0 00
SDropletkregular segmenwed Type II chip with ER 4
--- Iregular segmentedlPlug Type II chip with ER 4
.- -.-.-. ropIep regular signed Type I chip with ER 1
.. Regular segmentedlPlug Type I chip with ER 16





7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


generated in the injector channel dictated the nature of the
regime in the test channel.

Flow Maps

Based on the observed topological segmented flow regimes,
Figures 7 and 8 show the flow maps and transition lines
between regimes observed with the Type I and II chips,
respectively. The data points in the figures represent each
liquid-liquid segmented flow state with respect to carrier
fluid superficial velocity (Jc) and the dispersed fluid
superficial velocity (JD). The superficial velocity is defined
as the volumetric flow rate of the dispersed or carrier fluid
divided by the cross-sectional area of the test channel. The
Droplet and Plug flow regimes were shifted towards the
higher dispersed and lower carrier fluid superficial velocities
with increasing expansion ratio. The transient Irregular
Segmented flow was favored in the higher expansion ratio
channel and the interval of transient Irregular Segmented
flow between the Droplet and Plug flow regimes was shorter
for the low expansion ratio channels. In the lowest expansion
ratio chip, the Type III chip with an expansion ratio of 2,
most of the flow patterns were regular Plug flows without
transient Irregular Segmented flow patterns.


Droplet Velocity Measurement


(a)


* Measured velocity vs c Type I chip with ER 1



-
Piug I Irregular
S Segmented
1 [




II"
Ii Droplet


0.2 0.4 0.6 0.8 1.0
Carrier fluid volumetric flow ratio, PC


Measurement of the velocity of the dispersed droplets and
plugs was accomplished by recording two consecutive image
frames separated by a known time interval (Atp) and
measuring the distance traveled (Al). A camera in double
exposure mode and a 532 nm Nd:YAG double pulsed laser
with an external timing controller were used for the velocity
measurements. Figures 9(a) and 9(b) show the overlap of
two consecutive images acquired by the double pulsed laser
with a 2.5 msec pulse separation time in the Plug and
Droplet flow regimes, respectively. The velocity of the
dispersed fluid (VD) was calculated by dividing the transit
distance (Al) of a dispersed droplet or plug by the fixed pulse
separation time (Atp). Image processing was used to extract
the centroids of the droplets and plugs in each
two-dimensional image frame and the distance traveled by
the centroids during the pulse separation time was calculated.
Measured velocities were scaled by the sum of the
superficial velocities of the dispersed and carrier fluids:


VDJ-=


VD
JD +J


The measured velocities were greater than the corresponding
superficial velocities. In the Droplet flow regime, which had
no contact between the droplets and the channel walls, the
scale factor was 1.32 with a coefficient of variation of 7.6%.
In the Plug flow regime, the scale factor was smaller at 1.12
with a coefficient of variation of 4.5%.
There are two possible reasons for the decrease in the scale
factor from the Droplet to the Plug flow regimes. Smaller
droplets centered within the channel are convected with a
velocity closer to the maximum velocity of the parabolic
velocity profile in the neighborhood of the centerline in the
microchannel. Another factor is that the relatively larger


(c)


Figure 9: Overlap of the consecutive images taken with the
double pulsed laser with a 2.5 msec pulse separation for the (a)
Plug flow and (b) Droplet flow regimes (c) measured velocity
(VD) on the Type I chip was scaled by sum of superficial
velocities of the disperses and carrier fluids (J Jc +JD).


volume of the plugs filled more of the cross-section of the
microchannel and the higher drag force due to friction in the
thin films between the plugs and channel walls slowed them
down.

Wetting of the Dispersed Fluid

One advantage of liquid-liquid segmented flow for
bio-analytical applications is that each dispersed droplet or
plug works as an independent biochemical reactor. Wetting
of the channel surfaces with the dispersed fluid is not
desirable since the adsorption of molecules of the preceding
plug on the channel walls would result in
cross-contamination in the following plug. Wetting of the
channel walls with dispersed segmented flow were observed
in these experiments. Once wetting nucleated on any
microchannel surface, the flow regimes could no longer be
predicted based on the carrier fluid volumetric flow ratio and
the wetted patch on the surface caused unpredictable flows.
This wetting commonly occurred after long plug flow
experiments at low carrier fluid volumetric flow ratios,
because under these conditions the longer aqueous plugs led
to breakdown of the carrier fluid liquid films between the
long plug and channel wall. Furthermore, the surface
modification used to make the channel walls more
hydrophobic can deteriorate after extended use exposing
wetting patches on the surface. Figure 10 shows examples of






7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


O-plasma

PMMA PMMA-O
65 2 63+30


(c) (d)


Figure 10: Wetting of dispersed fluid on the channel surface (a)
in Droplet flow, and (b) Plug flow. (c) and (d) different
segmented flow regimes observed under the same flow
conditions in the same chip due to wetted patches in the
injection channel, Qc 20 pm/min, QD 3 pm/min and fc
0.87.


the dispersed fluid wetting the test channel walls for the
Droplet [Fig. 10(a)] and Plug flow regimes [Fig. 10(b)].
Wetting of the injection channel wall with the dispersed fluid
induced different flow patterns even under the same flow
conditions in terms of the carrier fluid volumetric flow ratio
[Figs. 10(c) and 10(d)]. Water deposited on the injection
channel walls restrained following droplets until the drag
force was high enough to cause release of the larger attached
droplet. For this reason, the resulting segmented flow regime
was Plug flow rather than the Droplet flow generated in the
non-wetted injection channel.
In order to obtain a consistent and predictable flow regime
with a fixed volumetric flow ratio, we have developed a
two-step procedure to fluorinate the surfaces of polymer
materials including poly-methylmethacrylate (PMMA) and
polycarbonate (PC) used for microfluidic devices. For these
fluorination reactions, we used planar polymer surfaces with
the reaction requiring two basic steps: The first step involved
treating the polymer surface with oxygen plasma for 30 s.
This treatment introduced polar oxygen functional groups
including hydroxyl groups onto the surface as shown by XPS.
We refer to this functional material as PMMA-O or PC-O
depending on the polymer material used. In the second step,
the activated polymer surface was treated with a 2 mM
solution of heptadecafluoro- 1,1,2,2-tetrahydrodecyl)
trichlorosilane (CF3(CF2)7CH2CH2SiCl3), HFTTCS, in
FC-3283 for 2 h. This treatment step did not affect the
transparency of the polymer substrate and the resultant
surface showed a water contact angle of 116 30, which is
an expected value for a monolayer of HFTTCS (Schweikl et
al. 2007). The modification process is shown in Fig. 11. A
control PMMA substrate (i.e., no oxygen plasma treatment)
was also treated with a 1 mM solution of HFTTCS in
FC-3283 for 2 h and showed a water contact angle of 65 +
2. These results indicated that the increase in water contact
angle of the PMMA-O treated material following reaction


CF,(CF),CH2CH2SCI,

FC-3283 PMMA-F
116+3


Figure 11: Reaction scheme used for the fluorination of various
polymer surfaces using PMMA as an example along with the
water contact angles of the surfaces after each step of the
reaction.



with HFTTCS was due to the formation of a covalently
tethered HFTTCS monolayer with plasma-generated surface
hydroxyl groups. This was further confirmed by XPS.
Figure 12 shows an X-ray photoelectron spectroscopy (XPS)
spectrum of PC, PC after oxygen plasma treatment (PC-O)
and PC-O after HFTTCS treatment (PC-F). The PC and
PC-O spectra showed the expected Cl and Ols peaks
however, the intensity of Ols peak for PC-O was increased
compared to the Ols peak in the PC spectrum. The Ols/Cls
ratio showed that this ratio increased 1.68 times upon a 30 s
oxygen plasma treatment; 0.1520 for the PC surface to
0.2552 for the PC-O surface. This confirmed the
incorporation of oxygen-containing polar hydrophilic groups
on the PC surface following oxygen plasma treatment. The
survey spectrum of PC-F surface showed the Fls, Si2p and
Si2s peaks. The Fls/Cls ratios of PC-F surface obtained
from the high-resolution spectrum taken at 30 and 90 were
0.6196 and 0.3477 respectively. The high Fls/Cls ratio
obtained from first 15 A from the surface compared to 35 A
shows that, the surface contain high concentration of the
perfluoro silane. This showed that, the HFTTCS reaction
occurred surface selectively.
Surface roughness is an important factor, which can affect
the water contact angle. It is known that increased surface
roughness can cause increases in the water contact angle of
the surface (Yeh et al. 2008). It has also been reported that,




80000.
Fls









PC
Soss


S4000- 400 01




ancing Energ (eV)


Figure 12: XPS spectrum of unmodified polycarbonate (PC),
30 s oxygen plasma treated polycarbonate (PC-O), and PC-O
film after 2 h HFTTCS treatment (PC-F)






7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


Figure 13: The topography of the PMMA surfaces obtained
from tapping mode AFM. (a) Native PMMA, (b) PMMA-O,
and (c) PMMA-F surfaces. The scan range for all the images is
10 pm by 10 pm, and the Z-range is 115 nm.


gas plasma treatment may also increase the surface
roughness due to etching of the polymer surface during
plasma treatment (Ward et al. 2005). Therefore, we
examined the surface topography of native PMMA and
modified PMMA surfaces. Figure 13 shows the surface
topographies of PMMA, PMMA-O and PMMA-F surfaces
obtained by tapping mode AFM. The PMMA-O surface
showed some sharp features due to etching of PMMA during
oxygen plasma treatment. The PMMA-F surface also
showed features similar to that of PMMA-O, but these
features appeared to be less defined and sharp. The root
mean square roughness (RMS roughness) of the PMMA
surface (2.23 nm) did not change considerably after
HFTTCS reaction (4.46 nm). Since the surface roughness of
PMMA does not increase significantly during the
fluorination reaction, the increase in water contact angle
(WCA) was mainly due to the introduction of surface
perfluoro groups. This along with the water contact angle
value of 116, which is the expected value for the good
HFTTCS monolayer, showed that the increase in the water
contact angle was due to the reaction and not due to change
in surface roughness.

Fluorescence correlation spectroscopy

As a reliable detection method using droplet flow,
single-color fluorescence correlation spectroscopy (FCS) has
been developed and demonstrated to detect photonic signals
from fluorescent microspheres suspended in each
continuously segmented droplet. FCS is an ultrasensitive
method for monitoring low concentration molecules through
a correlation analysis of fluctuations in photonic intensity.
Figure 14 shows a preliminary result for the detection of
intensity signals from fluorescent polystyrene microspheres
suspended in each droplet at different fluid flow rates using
Type I chip. Microscopic images of the droplet flow and
corresponding plots of fluorescence signal peaks show the
increased droplet frequency with increased fluid flow rate.
The method is sensitive and fast enough to read most of
photonic signals from the single microsphere in a droplet.


(a)









(c)


(b)


Figure 14: Microscopic images of droplet flows in
micro-channels and fluorescent signals at different flow rates
(a), (b) dispersed fluid flow rate, QD = 1 pL/min and carrier
fluid flow rate, Qc = 20 pL/min and (c), (d) dispersed fluid
flow rate, QD = 3 pL/min and carrier fluid flow rate, Qc = 20
p/L/min.



Conclusions

Liquid-liquid segmented flows in polymer microchannels
were studied experimentally using polymer chips with three
different cross-sectional expansion ratios (1:16, 1:4, and 1:2)
from the injection to the test channels. Flow regimes and
maps were determined based on the flow topology from each
chip. Effects of the different expansion ratios on the regimes
and irregularity of the segmented flows were examined. The
droplet and plug regimes were shifted to the higher carrier
and lower dispersed fluid superficial velocities and the plug
flow regime was broader with the lower expansion ratio
channels. The transient Irregular Segmented flow was
favored in the higher expansion ratio channel and the
transition interval between the Droplet and Plug flow
regimes was shorter for the low expansion ratio channels.
Measured velocities of dispersed fluid droplets and plugs
were faster than the sum of the dispersed and liquid
superficial velocities. The wetting issue of dispersed fluid on
the channel wall surface was discussed and a procedure for
the polymer surface fluorination was developed. We also
demonstrated an optical system that can provide sensitive,
fast readout modality to monitor regularly spaced droplets
for high throughput studies. The counting electronics were
configured so as to process photon bursts with a time
resolution of 12.5 ns (80 MHz) suitable for high-throughput
screenings.

Acknowledgements

This work was supported by the National Science
Foundation under grant EPS-0346411 and MRI grant
NSF-9977576 (CTS) as well as the State of Louisiana Board
of Regents under grant LEQSF(2005-06)-ENH-TR-20. The









authors gratefully acknowledge the contributions of Jason
Guy at the Center for BioModular Multi-Scale Systems
(CBMM) and Dr. Proyag Datta at Center for Advanced
Microstructures and Devices (CAMD) for their technical
support in the fabrication of polymer microfluidic chips.

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