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Letter
Sugar Additives Improve Signal Fidelity for ImplementingTwo-phase Resorufin-based Enzyme Immunoassays
Patrick A. Sandoz, Aram J Chung, Westbrook M. Weaver, and Dino Di CarloLangmuir, Just Accepted Manuscript • DOI: 10.1021/la5004484 • Publication Date (Web): 28 May 2014
Downloaded from http://pubs.acs.org on June 3, 2014
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Sugar Additives Improve Signal Fidelity for
Implementing Two-phase Resorufin-based
Enzyme Immunoassays
Patrick A. Sandoz†, Aram J. Chung††, Westbrook M. Weaver, Dino Di Carlo
*
Department of Bioengineering, University of California, Los Angeles, 90095, CA, USA.
KEYWORDS: Multiphase microfluidics, Water-in-oil emulsion, Enzyme immunoassay,
Resorufin, Fluorophore stability.
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ABSTRACT
Enzymatic signal amplification based on fluorogenic substrates is commonly used for
immunoassays, however, when transitioning these assays to a digital format in water-in-mineral
oil emulsions such amplification methods have been limited by the leakage of small reporting
fluorescent probes. In the present study we used a microfluidic system to study leakage from
aqueous droplets in a controlled manner and confirmed that the leakage of fluorescent resorufin
derivatives is mostly due to the presence of the lipophilic surfactant Span80, which is commonly
used to preserve emulsion stability. This leakage can be overcome by the addition of specific
sugars that most strongly interfered with the surfactants ability to form micelles in water.
Application of the microfluidic system for quantitative analysis of droplets as well as
implementation of the described sugar additives would allow for alternatives to fluorinated
surfactant-based platforms and improve the signal fidelity in enzyme immunoassays
implemented through multiphase microfluidics.
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Introduction
Over the last decade, a variety of multiphase platforms have emerged in microfluidic formats to
perform analysis of single cells or molecules (1-3). Miniaturization has allowed confinement of
small and uniform volumes and enabled the emergence of digital sensing in which the sample is
fractionated to sufficiently small volumes so that one or zero target molecules or cells is
statistically confined to each volume. The distribution of volumes with a single object
encapsulated then follows Poisson statistics and the sample concentration can be calculated (4).
The most widely demonstrated digital platforms use water-in-oil confinement (5, 6) or soda-lime
glass chambers (7) and quantify the concentration of specific nucleic acid sequences which
allows the determination of viral loads or the diagnosis of rare allele presence (8, 9). Various
other platforms demonstrate particle polymerization (10), single-cell or bacteria analysis (11) or
quantification of molecular kinetics (12).
The majority of digital platforms perform single-molecule detection using the polymerase chain
reaction (13) or enzyme immunoassays (14) within confined volumes and offer highly sensitive
quantification at the attomolar scale. During confined PCR reactions intercalating fluorescent
dyes that have increased quantum yield when associated with double stranded DNA are used for
readout. For enzyme-amplified immunoassays a secondary antibody conjugated to an enzyme is
often used to report the presence of the antigen by converting fluorogenic dyes such as resorufin
or fluorescein precursors. A common limitation of these platforms remains the ability to integrate
millions of stable, discretized and confined volumes in a restricted space for imaging and
readout. As microfluidic networks have a minimal required fabrication resolution only a
restricted number of rigid confinements can be put into a platform. Conversely, droplet based
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microfluidic systems offer continuous encapsulation of picoliter aqueous systems into a
surrounding oil phase. This approach is not limited by microfluidic fabrication and can produce
thousands of droplets per second. However, a significant challenge in creating an effective and
robust droplet system is maintaining both the physical stability of the droplet and the signal
fidelity within them. The stabilization of the phases is usually improved by the addition of a
surfactant which presents two moieties (one hydrophobic, the other hydrophilic) and reduces the
free energy of the two-phase separation. However, the presence of surfactant leads to more
complex interactions between the different phases and leakage of dyes from the droplets to the
outer phase. As a consequence previous platforms were developed on a case-by-case basis
depending on the chemistry of the system (15).
Fluorosurfactants are commonly used in these assays because of reduced dye leakage and
oxygen permeability properties; however, there are disadvantages which motivate use of non-
fluorinated systems. Isothermal droplet-based DNA amplification as well as living cell assays in
droplets make use of oxygen permeable fluorinated oil and fluorosurfactant (16, 17).
Thermocycled droplet digital PCR approaches have in some cases made use of the increased heat
transport and thermal stability of silicone-based oil and surfactants instead (5) or also employed
fluorinated oil and fluorosurfactant (13). Both combinations allowed confinement stability.
However, fluorinated surfactants have several drawbacks. First of all, their synthesis is a
complex process and the number of commercially available and affordable products remains
restricted. Even more important, these fluorinated surfactants, in particular perfluorooctanoic
acid (PFOA) and perfluorooctane sulphonate (PFOS), are harmful and due to their persistent
lifetime, they can accumulate in the blood of many species including humans. Their level of
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toxicity has even forced the U.S. Environmental Protection Agency to launch a program for
decreasing their industrial production in 2006 (18). As a consequence, this has led researchers to
turn to alternative solutions. One cost-effective non-toxic and stable two-phase system combines
for example mineral oil with a mix of sorbitan-oleate (Span80 <Sigma>) and polysorbate
(Tween80 <ThermoFisher>) as surfactant (15). This system resolves the problem mentioned
above for fluorinated oil and surfactant.
However, a general issue of stability for small molecules like fluorophores in the aqueous phase
of an emulsion remains. The low stability of amphipathic readout fluorophores in water-in-oil
emulsions has been reported several times (19-22). Recently, two groups brought new insights
characterizing the small probe leakage in water-in-oil emulsions. Chen et al. reported a model for
the different factors contributing to the leakage of small fluorescent probes out of a droplet (23).
The authors characterized the crosstalk among neighboring droplets from the droplet
permeability and the mass diffusion coefficient of the small molecular probes and described
structure effects on the leakage kinetics. Although improved, such leakage issues are not totally
resolved in fluorinated surfactants, for example, Skhiri et al. also highlighted the role of
fluorinated surfactant in the transport of small probes between neighboring droplets in fluorinert
oil (24). The authors found that the micelle concentration in the fluorinert oil phase impacts the
exchange kinetics in the emulsion.
Towards the development of a digital ELISA platform, we were able to optimize a system which
crucially decreases the fluorophore leakage from picoliter water-in-oil drops. With the addition
of sugar-derivatives in the aqueous phase, the signal is significantly conserved for the time
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required for enzymatic turnover of fluorogenic substrates. This method can address previously
mentioned fluorophore-related limitations and suggests new ways of achieving functional digital
microfluidic platforms. Moreover, we report the optimal conditions for digital sensing of
different fluorophores (resorufin, fluorescein and coumarin) where including sugar derivatives
decreases the fast fluorophore leakage from the droplets and maintains a significant signal level
over time. In addition, we demonstrate a microfluidic device that allows testing of the time-
dependent signal fidelity of various fluorophores in solution compartmentalized in mineral oil
with an appropriate surfactant.
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Experimental
Material
Amplex Ultra Red (AUR) substrate, horseradish peroxidase (HRP) and H2O2 were from the
ELISA Development Kit, Invitrogen (USA). FITC (fluorescein-based probe) was from Sigma-
Aldrich Inc. (USA). DiFMU (coumarin-based substrate) was purchased from Life Technologies
Corp. (USA). Mineral oil (paraffin heavy) and Tween80 were obtained from Thermo Fisher
Scientific Inc. (USA). Span80 (sorbitan monooleate) was obtained from Sigma-Aldrich Inc.
(USA). Sucrose, dextran (MW: 70 kDa), Fructose, Glucose, Galactose, Mannose, Arabinose,
Trehalose, Glycerol and bovine serum albumin (BSA) were purchased from Sigma Aldrich Inc.
(USA). Carboxymethyl-cellulose was from Lineco Inc. (USA). Polydimethylsiloxane (PDMS)
was purchased as Sylgard 184 from Dow Corning (USA). SU-8 was from Microchem, Newton,
MA (USA).
Chip fabrication
The microfluidic chip was fabricated by using standard photolithography and PDMS replica
molding protocols. SU-8 2050 resist was spun at 3,000 rpm on a 4-inch wafer and baked for 3
minutes at 65°C and 6 minutes at 95°C. The photoresist was exposed to UV light through a
chrome mask using a Karl Suss MA6 contact aligner (Suss, Germany) with an exposure dose of
approximately 190 mJ/cm2. The wafer was then cured at 65°C for 1 minute and 95°C for 6
minutes and developed for 5 minutes using SU-8 developer. Then the wafer was baked at 180°C
for 5 minutes. A 10:1 (base:linker) mixture of PDMS was added onto the mold and cured at
65°C for 3 hours. The final PDMS microchannel was bonded to a glass slide using an oxygen
plasma treatment and baked at 65°C for 12 hours as a final step.
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Droplet generation for emulsion stability characterization
A flow-focusing junction was designed and fabricated according to previous work (25). The
mineral oil was mixed with different concentration of surfactant combining Span80 and
Tween80 from 0 to 10% (w/w). The phases were injected at constant flow rate using glass
syringes (Hamilton Company, USA) and standard syringe pumps (Harvard apparatus, USA). The
syringes were connected to the device using peek tubing 20 (IDEX, Health & Science Corp.,
USA). 25ga (1/2”) luer stubs (Instech Laboratories Inc., USA) were used to connect the glass
syringe to the peek tube. The mineral oil and aqueous flow rates for generating droplets were
respectively 8μl/min and 2µl/min. The droplets were collected and imaged downstream in a large
chamber. The chamber thickness (50µm) was maintained by posts.
Compartmentalization array for signal fidelity assay
A two-dimensional array of parallel straight channels (30μm width) studded with lateral round-
shaped traps of 40μm width was designed. The aqueous phase with the fluorophore and the sugar
derivatives was injected using a standard syringe pump and was loaded in all lateral chambers by
dead-end filling. Then the aqueous phase injection was stopped and the mineral oil phase was
injected and maintained during the entire experiment using another syringe pump. Thus confined
droplets were produced in all lateral chambers. The two-dimensional array was time-lapse
imaged using a standard microscope (see below) and the fluorescence intensity in each droplet
was tracked following the frames in the sequence and quantified using ImageJ software. A
constant oil flow rate of 5µl/min was observed to result in quick fluorophore leakage and was
conserved for each experiment.
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In-tube emulsion
Water/Span80 phases (1:1 w/w) were vortexed in a centrifuge tube for 30-60 sec. The fractions
of remaining aqueous phase and opaque Span80 micelles were retrieved, measured and
normalized to their respective inputs.
Fluorescent signal generation
The stability of the water-in-mineral oil emulsion including surfactant was demonstrated using a
flow focusing junction (25, Fig. S1a) and the packed aqueous droplets of 40µm were imaged in a
large chamber (Fig. S1b). A concentration up to 10% of mixed surfactants in the mineral oil was
found to be sufficient to prevent the droplets to fuse over a long period of time. Amplex Ultra
Red (AUR) was chosen for its demonstrated high sensitivity as a substrate for enzyme
immunoassays (11, 14). It is a non-fluorescent substrate quickly transformed in the presence of
horseradish peroxidase (HRP) into a strong fluorescent resorufin derivative. The previously
mentioned rapid leakage through the droplets was highlighted using the fluorescent derived
product of 50µM AUR substrate transformed using HRP with 2µM H2O2. To enable optimal
HRP activity, Amplex Ultra Red was buffered at pH 6.0 which also conserves its maximum
fluorescence. FITC and DiFMU were resuspended in PBS, pH 7.4.
Imaging
Droplet generation, signal stability and signal leakage were characterized using 4x and 10x
objectives of a standard inverted fluorescence microscope (Eclipse Ti-U, Nikon Corp., JP). As
the droplets were produced at a high frequency, bright-field images were captured using a high-
speed camera at 5000 frames per second (Phantom V711, Vision Research Inc., USA) and
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processed on Phantom camera control software (Vision Research Inc., USA). The stabilized
emulsion was imaged in bright-field and fluorescence using a standard camera (DS-Qi1M, Nikon
Corp., JP) and processed on NIS-Element software (v3.22.01-2010, Nikon Corp., JP).
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Results
Using a microfluidic device integrating a flow-focusing junction prior to a large open chamber
(Fig. S1a-b), we first observed that rapid fluorophore leakage occurs in emulsions upon the
addition of stabilizing surfactant (a mix of Span80 and Tween80, 15) in the mineral oil phase
(Fig. S2a-b). The Amplex Ultra Red substrate (AUR), a widely used resorufin-based substrate
for horseradish peroxidase clearly shows this leakage behavior. We also observed that the
resorufin-derived fluorophores stayed in the oil phase and did not re-enter empty neighbouring
droplets. Reducing the concentration of surfactant decreased the fluorophore leakage as
previously described (24) but enabled the droplets to fuse.
To insure uniform and reproducible conditions, we characterized this fluorophore leakage from
aqueous drops using a microfluidic device that allowed generation of stable droplet volumes
independent of surfactant properties. The device consisted of a two-dimensional massively
parallel array of 40 µm wide semi-circular drop traps (Fig. 1a). By successively running through
the device the aqueous phase followed by the oil phase, monodisperse droplets were produced
and maintained in each trap of the array. This compartmentalization method was able to generate
more than 10’000 droplets per chip and also allowed the emulsion to be maintained independent
of the incorporation of surfactant. Moreover in this platform, droplets could be imaged over time
using epifluorescence microscopy, without significant motion and with control of the external
fluid convection and environment. Resorufin, initially encapsulated within the droplets was
observed to exit the droplets only in the presence of surfactant (Fig. 1b). Maintaining the oil flow
during the experiment convected away the exiting fluorophore at the droplet interface and
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increased the concentration gradient across the droplet, such that a higher flow rate led to an
increased droplet leakage rate (Fig. 1b-c).
Very interestingly, the addition of sucrose to the droplets mitigated the leakage of resorufin (Fig.
1b, d). After 450 seconds of imaging, signal fidelity was notably conserved up to 90% of the
initial signal intensity with the addition of sugars to the aqueous solution (Fig. 1b, bottom line;
Fig. 2a-b) compared to 25% in the absence of sugars (Fig. 1b, middle line; Fig. 2a-b). Note that
the fluorophore leakage or addition of sucrose did not induce a change of the droplet size over
time. Increasing the fluorophore solubility in the aqueous phase using dimethyl-sulfoxide
(DMSO) or saturating the oil using non-fluorescent AUR substrate which both were expected to
slow down the leakage rate did not prevent the fluorophore leakage (data not shown).
In order to further characterize the impact of sugar on the fluorophore leakage, different
concentrations and sugar-derivatives were tested. We found that inhibition of fluorophore
leakage positively correlated with the amount of sucrose included in the aqueous phase (Fig. 2a)
and that different sugar derivatives (sucrose, dextran but not cellulose) displayed similar
stabilization characteristics (Fig. 2a-b). Although dextran was tested at a higher dilution (5%
w/v) than sucrose (25% w/v), the fluorescent signal stability of the droplets was comparable in
both cases. Finally, the addition of carboxymethyl-cellulose resulting in a more viscous phase
did not prevent the fluorophore leakage and these conditions resulted in the same final leakage
rate as in the standard condition without any sugar additive (Fig. 2c).
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To assess which components of the surfactant were responsible for inducing the resorufin
fluorophore leakage, we repeated the experiments and separately added the two surfactant
fractions (Span80 and Tween80) to the mineral oil. It appeared that the Span80 fraction played a
major role in accelerating leakage (Fig. 2c) even if the combination of both fractions resulted in a
much higher leakage rate.
We also aimed to understand if sugar derivatives preferentially have an effect on either the
ability to generate surfactant micelles that would enhance leakage or on the partitioning of the
fluorophore in the aqueous phase. To address this question, we decided to generate emulsions
using water and Span80 only (without fluorophore or mineral oil). Without a fluorophore acting
as the signal reporter, the microfluidics platform could not be used for imaging and a larger
quantity of emulsion that was more easily visualized was generated by vortexing the two phases
in a centrifuge tube. The effect of an additive in the aqueous phase was reported by quantifying
the remaining aqueous phase and the dense emulsion volume fraction generated by vortexing the
aqueous and Span80 phases (Fig. S3). Note that this approach directly probes surfactant micelle
stability within the aqueous phase and separately from the stability of an aqueous emulsion in oil.
We observed different emulsion fractions using Span80 alone or Span80 combined with the
sugar derivatives in the aqueous phase, as well as saturated aqueous solutions of bovine serum
albumin (BSA) or 5% carboxymethyl-cellulose. Results confirmed a dispersive effect of the
sugar-derivatives on the emulsion as the dense emulsion volume fraction was significantly
diminished by the sugar additives (Fig. 2d and Fig. S4). Note that the Span80/water emulsion
was not generated due to a stabilized air/water interface and could be produced under vacuum
and suspended in mineral oil without observing any structural changes under the microscope.
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Finally, we found that the sugar additive impact on the fluorophore leakage was specific to the
resorufin fluorophore and different optimal additives and conditions were found to minimize the
leakage of other small fluorophores from droplets. Fluorescein isothiocyanate (FITC) and
difluoro-hydroxy-methylcoumarin (DiFMU) were tested using the microfluidic platform as
previously described. For FITC, the addition of surfactant alone already inhibited fluorophore
leakage compared to the condition in which both sugar and surfactant were absent (Fig. 3a). Yet
the highest signal fidelity was obtained by combining the sugar additives with no surfactant.
DiFMU, showed the highest maintenance within droplets in the absence of surfactant and sugar-
derivatives (Fig. 3b). When surfactant was present, the addition of sucrose did not prevent
DiFMU leakage while the addition of dextran improved signal fidelity. A summary of the effects
on the signal stability of the different fluorophores using each additive in the aqueous phase
reported as the maximum leakage rate is provided in the supplementary information (Table S1).
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Discussion
Several previous studies have highlighted the fundamental limitation imposed by the diffusion of
small fluorophores through the interface of water-in-oil emulsions (19-22). Some methods
employed to reduce fluorophore leakage include generating double emulsions (19) or separating
droplets by gas bubbles in a straight channel. Chemical modification of specific fluorophores has
also been introduced to increase stability (20) and new alternatives to synthesized surfactant are
now required to develop cost and time effective drop-based devices (26). Although effective,
these solutions greatly increased both the reagent cost and the platform complexity.
In this work, we designed a leakage quantification experiment presenting well-controlled
conditions to distinguish the role of the different emulsion components. We confirmed that the
presence of surfactant increases the leakage of certain small fluorophores such as resorufin
derivatives across droplets boundaries. The addition of a constant flow passing between the
droplets also significantly increased the leakage. Renewing continuously the mineral oil and
surfactant phase using constant injection enabled us to amplify the fluorophore leakage rate and
to perform time-lapse experiments with a limited number of secondary effects. Using such a
platform enabled testing various additives for inhibition of fluorophore leakage induced by the
surfactant.
Our results suggest that the addition of short-chained sugar derivatives inhibit the fluorophore
leakage and thus help to conserve the signal over time for digital quantification through enzyme
immunoassays. The sugar additives may act in two main ways to reduce leakage: (i) by directly
interacting with and stabilizing the fluorophore within the aqueous phase (i.e. enhancing the
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equilibrium partition coefficient), or (ii) interacting with the surfactant to either (a) prevent
formation of micelles containing fluorophore, or (b) reduce the rate of micelle shedding overall.
Since the sugar additives were able to reduce the stability of water/Span80 emulsions (i.e. lower
emulsion fraction of our in-tube experiments), they likely directly interact with the Span80
surfactant, supporting mechanism (ii) described above. As Span80 is known to generate reverse
micelles (27-29) at the droplet surface, it is possible that the sugar derivatives directly disrupt the
surfactant organization at the droplet interface reducing the formation and shedding of micelles
which contain fluorophores. Note that these potential surfactant micelles were not directly
observed and therefore would be sub-micron sized. Moreover we did not observe the formation
of a surfactant bilayer at the droplet interface.
On the other hand, kinetics of leakage has also been related to direct partitioning between the
two phases (23, 24). For example, in the model of Chen et al., reducing the fluorophore
permeability was predicted to best inhibit leakage; while reducing the diffusivity using a viscous
aqueous solution like dissolved carboxymethyl-cellulose had a less prominent effect. Using
carboxymethyl-cellulose was also demonstrated to be inefficient for inhibiting the leakage of
fluorescein-derivatives in another study (19). Our results indicate that future leakage models
should consider additional effects, in which the surfactant itself acts to facilitate fluorophore
permeation across the water-oil interface, which may especially play a role when non-
equilibrium conditions are present. In our experiments, droplets are physically separated in the
microfluidic array and the oil flow convects away exiting fluorophores / micelles and thus
modifies their partition since the system never tends to equilibrium. As a consequence, when
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linking the exposed droplet intensity decays to current published models, care should be taken to
understand the previously described boundary conditions that govern the kinetics of leakage.
The demonstrated interaction between sugars and the Span80 fraction highlights the new field of
engineering interfering molecules to solubilize small probes in the aqueous phase, reduce their
permeability and prevent transport from a drop. High concentrations of monosaccharides or
disaccharides enabled reduced generation of water/Span80 emulsions with a similar efficiency.
Thus the choice of the best non-branched or short branched sugar additive for signal
conservation might only depend on its solubility in water. Therefore sucrose or fructose that have
the highest solubility might be best suited. In addition dextran, a polysaccharide, exhibited a
particular high performance since it still reduced the resorufin leakage and the Span80 micelles
at lower concentration than other sugar derivatives. Sugar alcohols showed a lower potential at
equal concentration than sugars to reduce the Span80 emulsion. This shows that interfering with
the surfactant is related to the precise chemical structure of saccharides. The similar dispersive
effect of BSA observed on Span80 (but still smaller than sugar derivatives) might highlight
potential causes of its previously reported benefits in emulsions (19, 24).
Differences in leakage kinetics for fluorescein and coumarin derivatives show that each emulsion
system has unique characteristics depending on the chemical structure and the charge of the
fluorophore and how they specifically interact with surfactants. Ultimately, multiplexing a digital
assay using resorufin, fluorescein and coumarin-based fluorophores might require a compromise
in the addition of surfactant and sugar additives. Based on our results, a combination of these
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dyes would optimally combine surfactant and 5% dextran to best conserve the signal fidelity of
all three fluorophores.
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Conclusion
We report a new technique to overcome the poor signal stability of amphipathic readout
fluorophores in water-in-oil picoliter reaction confinement systems. The addition of sugar
derivatives in the aqueous phase enables conservation of the resorufin-based signal stability over
a time period necessary for enzymatic accumulation of fluorophore and imaging of droplets.
Improved signal stability using available cost-effective reagents unlocks new opportunities for
digital water-in-oil droplet immunoassays. Moreover this approach can be expanded to any use
of resorufin-based fluorophores in a multi-phase microfluidic format.
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Figure 1. Characterization of the microfluidic platform for quantifying fluorophore leakage in an
emulsion (a) Picture of compartmentalized aqueous droplets in the two-dimensional parallel
array of lateral traps (b) Characterization of fluorophore leakage under three conditions: no
surfactant/no sugar, surfactant/no sugar and surfactant/25% sucrose. (c) Fluorophore leakage
quantification at different oil flow rates (d) Schematic of resorufin leakage prevention from
droplets induced by sucrose in the presence of surfactant.
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Figure 2. Quantification of fluorophore leakage reduction by different sugar derivatives. (a) The
aqueous drops were loaded with the fluorophore along with different concentrations of sucrose.
(b) Same as above using dextran instead of sucrose. (c) Same as above using carboxymethyl-
cellulose additive. (d) In-tube water/Span80 emulsion (1:1 w/w).
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Figure 3. Leakage characterization of two other small fluorescent probes: (a) Fluorescein
(50µM) (b) DiFMU (50µM).
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AUTHOR INFORMATION
Corresponding Author
Dino Di Carlo: [email protected]; Department of Bioengineering, University of California,
Los Angeles, 410 Westwood Plaza, Los Angeles 90095, CA, USA.
Present Addresses
†If an author’s address is different than the one given in the affiliation line, this information may
be included here.
†Patrick A. Sandoz: Global Health Institute, School of Life Sciences, EPFL, VDG, Station 19,
Lausanne 1015, Switzerland.
††Aram J Chung: Mechanical, Aerospace and Nuclear Engineering, Rensselaer Polytechnic
Institute, Troy, NY 12180
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Funding Sources
This work was supported in part by National Science Foundation grant #1332275.
ACKNOWLEDGMENT
We thank P. Renaud and A. Ozcan for helpful discussions and guidance, G. van der Goot for
providing some reagents and S. Friebe for critical proofreading of the manuscript.
SUPPLEMENTARY INFORMATION AVAILABLE
Experimental details for water-in-oil droplets generation and emulsion stabilization, resorufin
leakage in the presence of surfactant, a comparison of other various sugar derivatives on in-tube
emulsion and a summary of the leakage characterization for resorufin, fluorescein and coumarin
derivatives. This information is available free of charge via the Internet at http://pubs.acs.org/.
ABBREVIATIONS
ELISA, enzyme-linked immunosorbent assay, HRP, horseradish peroxidase, AUR, Amplex Ultra
Red, FITC, Fluorescein isothiocyanate, DiFMU, 6,8-Difluoro-7-Hydroxy-4-Methylcoumarin,
PDMS, polydimethylsiloxane, PFOA, perfluorooctanoic acid, PFOS, perfluorooctane
sulphonate.
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Sugar Additives Improve Signal Fidelity for Implementing Two-phase
Resorufin-based Enzyme Immunoassays
Patrick A. Sandoz†, Aram J. Chung††, Westbrook M. Weaver, Dino Di Carlo
*
Department of Bioengineering, University of California, Los Angeles, 90095, CA, USA.
GRAPHIC TABLE OF CONTENT:
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