Plug-in tubes allow tunable oil removal, droplet packing, and reaction incubation for time-controlled droplet-based assays
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Plug-in tubes allow tunable oil removal, droplet packing, and reaction incubation for time-controlled droplet-based assays Cite as: Biomicrofluidics 15, 024108 (2021); https://doi.org/10.1063/5.0047924 Submitted: 17 February 2021 . Accepted: 20 March 2021 . Published Online: 05 April 2021 Meng Sun, Gembu Maryu, Shiyuan Wang, Qiong Yang, and Ryan C. Bailey ARTICLES YOU MAY BE INTERESTED IN A low-cost 3D printed microfluidic bioreactor and imaging chamber for live-organoid imaging Biomicrofluidics 15, 024105 (2021); https://doi.org/10.1063/5.0041027 Accomplishment of one-step specific PCR and evaluated SELEX process by a dual- microfluidic amplified system Biomicrofluidics 15, 024107 (2021); https://doi.org/10.1063/5.0045965 Automated calibration of 3D-printed microfluidic devices based on computer vision Biomicrofluidics 15, 024102 (2021); https://doi.org/10.1063/5.0037274 Biomicrofluidics 15, 024108 (2021); https://doi.org/10.1063/5.0047924 15, 024108 © 2021 Author(s).
Biomicrofluidics ARTICLE scitation.org/journal/bmf
Plug-in tubes allow tunable oil removal,
droplet packing, and reaction incubation
for time-controlled droplet-based assays
Cite as: Biomicrofluidics 15, 024108 (2021); doi: 10.1063/5.0047924
Submitted: 17 February 2021 · Accepted: 20 March 2021 · View Online Export Citation CrossMark
Published Online: 5 April 2021
Meng Sun,1,2 Gembu Maryu,1 Shiyuan Wang,1 Qiong Yang,1,a) and Ryan C. Bailey2,a)
AFFILIATIONS
1
Department of Biophysics, University of Michigan, Ann Arbor, Michigan 48109, USA
2
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA
a)
Authors to whom correspondence should be addressed: qiongy@umich.edu and ryancb@umich.edu
ABSTRACT
Here, we report a unique microfluidic technique that utilizes a membrane filter and plug-in tubes to remove oil and pack water-in-oil drop-
lets for controlled incubation of droplet-based assays. This technique could be modularly incorporated into most droplet-generation devices
without a need to alter the original designs. Our results show that removing excess oil to form tightly packed droplets allows for extended
and controllable incubation for droplets traveling in microchannels. The efficiency of this technique was evaluated and confirmed using a
time-dependent enzyme assay with a fluorometric readout. The system is also readily generalizable to control inter-droplet distance, crucial
for studying droplet communication and pattern formation.
Published under license by AIP Publishing. https://doi.org/10.1063/5.0047924
I. INTRODUCTION the on-chip and off-chip incubation techniques aforementioned.
The key component is a thin polytetrafluoroethylene (PTFE)
Despite the increasing popularity of high-throughput droplet
microfluidics in analyzing low-input targets such as single mole- membrane (Fig. 1) embedded underneath a microchannel, allow-
ing fluorinated oil to be filtered and drained through a plug-in
cules, cells, or microorganisms in isolated pL–nL volumes,1–4 the
tube while leaving aqueous droplets flowing over on the top,
limited channel length and high flow rates of droplet-generation
forming packed droplets for subsequent incubation and detection.
devices5,6 make it hard to achieve the extended incubation times
The technique is unique in construction and offers distinct supe-
required for most biological assays. To extend the droplet-dwelling/
riorities over other droplet-based oil-removal designs: (i) the
incubation time, techniques for on-chip7,8 and off-chip9,10 droplet plug-in tube can be inserted at any downstream location with no
incubation have been developed. Off-chip incubation techniques modification to the original device required; (ii) it does not rely
often require droplets to be manually collected into a microtube on internal channel networks to extract oil8,10,11 or external reser-
and then reinjected into a second microfluidic device; they are thus voir to store droplets,12,13 reducing the complexity of fluid manip-
suited for reactions requiring relatively long incubation time but ulation; (iii) the filter membrane separates the main channel and
not sensitive to the time taken for transferring samples and leading draining tube, preventing droplets from being lost into the oil
to a broad randomization of droplets upon reinjection into the extraction channel;10,11 and (iv) the incubation time can be con-
second device. On-chip methods of incubation offer a streamlined trolled using plug-in tubes of different lengths or diameters with
and order-preserving method of time delay, but the period is often no need to modify channel length or flow rates. We apply this
limited by constraints on device size since time is volume in a con- technique to dye-labeled droplets and a droplet-based enzyme
tinuously flowing microfluidic device to less than 15 min. assay to measure enzymatic activities at variable incubation times
Here, we report a method incorporating plug-in tubes on micro- from 3 to 20 min. The factors that affect oil-removal efficiency/
fluidic devices for efficient oil extraction and time-controlled droplet droplet-packing density and droplet incubation performance were
incubation, which compensates the advantages and disadvantages of also investigated.
Biomicrofluidics 15, 024108 (2021); doi: 10.1063/5.0047924 15, 024108-1
Published under license by AIP Publishing.
Biomicrofluidics ARTICLE scitation.org/journal/bmf
FIG. 1. Concept demonstration of the oil-removal and droplet-packing device. (a) Schematic showing oil removed through a membrane filter in a droplet-generation device.
(b) Top- and (c) side-view of the oil-removal module. (d) An experimental demonstration of oil removal and droplet packing in a microfluidic device. The flow rates of oil
and black dye are 2 and 1.5 μl/min; the outlet of the tube is left open (the oil is drained by gravity).
II. MATERIAL AND METHODS pump was used to drain the oil (see a representative experiment in
The key component to fabricate the oil-removal module is a Movie S1 of the supplementary material), enabling a quantitative
hydrophobic Fluoropore PTFE membrane filter with a 0.22-μm tuning of the droplet-packing density.
pore size (FGLP02500, EMD Millipore, Billerica, MA, USA).
Microbore PTFE tubings are used for device and fluidic connec- III. RESULT AND DISCUSSION
tions (SK-06417-11/21/31, Cole Parmer, Vernon Hills, IL, USA). A. Effect of flow rates on oil extraction efficiency
Other regular materials, in addition to all chemicals and instru-
ment, can be found in the supplementary material. The effect of the oil-withdrawal rate on oil-removal efficiency
was first tested at a fixed droplet-generation rate (2 and 1 μl/min for
oil and water infusion). The oil-withdrawal rate was then increased
A. Device fabrication
(indicated as D) from 0.0 to 2.1 μl/min in steps of 0.3 μl/min. When
A thick polydimethylsiloxane (PDMS) slab (3–5 mm) with D is low, the droplets with the remaining oil exit from the oil-
microfluidic channels (40 μm in depth) was first fabricated via a removal module into a narrower channel, resulting in irregular
soft photolithography method and then bonded to a glass slide spaces between unpacked droplets [Figs. 2(a) and 2(b)]. Droplets
spin-coated with PDMS. The access ports were punched by 18- packing increased as D increased [Fig. 2(c)] until it approached
and 20-gauge needles (Jensen Global, Inc., Santa Barbara, CA, the oil infusion rate (indicated as O), where a fully packed
USA), including the one for the oil draining tube. As illustrated in droplet population was generated and observed in the chamber
Figs. 1(a)–1(d), a 3 × 3 mm2 piece of a filter membrane was placed [Fig. 2(d)]. The interval length between droplets was measured
flat on top of the draining hole and pushed to the bottom of the before (I1) and after (I2) the oil removing process, and it was
channel by a blunt PTFE tube (1.5-cm long), forming a circular mem- found that I2 linearly decreases as the oil-withdrawal rate D
brane that separates the channel from the tube and allows oil to be fil- increases [I2 = −114.3D + 221.1 with r2 = 0.9858, Fig. 2(e), blue
tered through the 0.22-μm pores into the tube outlet while keeping line]. The oil-removal efficiency, defined as E% = (1 − I2/I1) × 100%,
aqueous droplets (typically 100 μm in diameter) remained flowing in was inversely proportional to I2 and increased with the oil-
the channel. The diameter of the filtering chamber was defined by the withdrawal rate [Fig. 2(e), black line]. Both experimental measure-
outer diameter of the inserted tube, which was 1.07 mm, and the ments of I2 (filled blue squares) and E% (filled black circles) match
depth was made to be the same as the channel height. their respective theoretical values (empty triangles) calculated based
The oil-removal device was initially tested by leaving the outlet on the ratios of the oil withdraw rate D and the infusion rate O,
of the draining tube open. Flipping the device over, the heavier oil which give: I2 = I1 × (1 − D/O) and E% = D/O × 100%. It should be
(ρ = 1.614 g/ml) could be efficiently removed by gravity under the noted that the oil-removal efficiency of 100% (when the neighboring
tested flow rate [Fig. 1(d)]. To further investigate the effect of the oil- droplets are closely packed) does not mean that the oil is removed
withdrawal rate on oil-removal efficiency, an additional syringe completely. A minimal amount of oil remains outside of the
Biomicrofluidics 15, 024108 (2021); doi: 10.1063/5.0047924 15, 024108-2
Published under license by AIP Publishing.
Biomicrofluidics ARTICLE scitation.org/journal/bmf
FIG. 3. (a) Droplets tagged with a black dye using a K-junction device. (b)
Stitched picture showing black droplets traveling in a connecting tubing within a
confined zone after oil removal. (c) Contrast adjusted image of (b) for grayscale
evaluation of the droplet distribution in the tubing. The flow rate of the droplets
in the tubing: 1.6 μl/min. The arrow indicates the flow direction. Scale bars:
500 μm.
removed from the droplet train, results in highly packed droplets
that can be controllably incubated, as described below.
B. Importance of removing oil and forming packed
droplets in time-controllable droplet-based assays
Droplet incubation in tubing has been reported previously,6,14
FIG. 2. The effect of the oil-withdrawal rate on oil-removal efficiency at a fixed but it has not been shown to allow precise incubation on account of
droplet-generation rate (O = 2.0 and W = 1.0). A series of images at (a) D = 0.0, loosely packed droplets within the tubing, which can then have differ-
(b) D = 0.6, (c) D = 1.2, and (d) D = 1.8. O, W, and D represent the flow rate of ential transport rates within the tubing. To demonstrate improved
oil infusion, water infusion, and oil withdrawal, unit: μl/min. (e) The plot of oil- droplet incubation afforded by droplet packing, a K-junction device15
removal efficiency E (black circles) and droplet space I2 (blue squares, averaged was used to tag ∼1000 droplets with a black dye by applying an
from ∼60 drops) vs oil withdrawing rate D. The hollow triangles represent theo-
electrical droplet injection potential for 10 s [Fig. 3(a)]. The sub-
retical values.
sequent plug of packed droplets is then incubated within the
tubing [Fig. 3(b)]. Although there is still some droplet diffusion, a
large majority of the dyed droplets (>99%) are confined within a
droplets to maintain droplet separation. Further increasing the oil- 20-s zone [Fig. 3(c)]. This occurs because the droplets are tightly
withdrawal rate causes droplets to deform, break up, and merge packed, which limits dispersion within the external incubation
when they transition between channels in different dimensions. tubing (Movie S2 in the supplementary material), with the observed
The water infusion rate was then altered to change the volume dispersion likely due to droplets rearrangement at the intersection of
fraction of water to investigate the effect on the oil-removal effi- two channels having different dimensions or at curved features
ciency under fixed oil infusion and withdrawal rates. When the within the channels.16 In contrast, droplets that have not been tightly
volume fraction of aqueous and oil phases decreased from 1.0 to packed via controlled oil removal show a high degree of dispersion,
0.25, there was no significant difference in the oil-removal efficiency, with large observed gaps of dyed droplets within the undyed back-
but slightly less packed droplets were observed at lower volume frac- ground droplets (Movie S3 in the supplementary material). This
tion (Fig. S1 in the supplementary material). The same trend was homogenous droplet incubation time is important for steps in which
discovered upon increasing the flow rates (Fig. S2 in the supplementary precise timing of in-droplet chemistries needs to be maintained,
material). It follows that lower packing efficiencies may be observed including application such as reagent injection,17 droplet fusion,
under extreme flow conditions. or synchronization.18,19 The described oil extraction module is
These findings indicate that the oil-removal efficiency is valuable for restoring tight droplet dispersion (Fig. S3 in the
largely dictated by the oil-withdrawal rate. Near matching of the supplementary material), making this approach versatile for many
oil-withdrawal and infusion rates, more than 95% of the oil can be different potential applications.
Biomicrofluidics 15, 024108 (2021); doi: 10.1063/5.0047924 15, 024108-3
Published under license by AIP Publishing.
Biomicrofluidics ARTICLE scitation.org/journal/bmf
C. Demonstration of a homogenous enzymatic assay β-D-galactopyranoside to fluorescent resorufin via a reaction with a
through packed droplets β-galactosidase enzyme. The devices were assembled on a confocal
To demonstrate the flexibility of altering incubation time with microscope with 561 nm excitation and a detection bandpass filter
packed droplets, we bridged a droplet-packing device [Fig. 4(a) (i)] with a 40 nm transmission window centered at 593 nm (additional
information can be found in the supplementary material). In this
to a detection device [Fig. 4(a) (iii)] with another piece of exter-
way, the fluorescent resorufin generated by the enzymatic conver-
nal plugged-in tubing. Two aqueous streams containing enzyme
sion of the substrate was detected in individual droplets after
and substrate solutions, respectively, were combined into drop- respacing them downstream of Device II [Fig. 4(a)]. Figure 4(b)
lets, with subsequent oil removal to form packed droplets (Movie shows four independent trace lines of droplet intensity after
S4 in the supplementary material). The enzyme and substrate- droplets were incubated for various times from 3 to 20 min (a full
containing droplets were then incubated for varying amounts of 10-s acquisition was recorded and presented in Fig. S4 of the
time flowing through tubing that bridged the packing and detec- supplementary material). Each trace represents the fluorescence
tion devices [Fig. 4(a) (ii)]. Reaction incubation time was varied intensity of droplets after incubation for the specified times. As
by using bridging tubing with different diameters and lengths expected, the average fluorescent intensity increases with increas-
(see Table S1 in the supplementary material) to achieve reactions ing reaction time owing to more complete substrate conversion
ranging from 3 to 20 min. [Fig. 4(c)]. Importantly, the width of the plugs of fluorescent
The enzymatic assay performed to show the homogeneity of droplets retains their temporal homogeneity even up to the
reactions within packed droplets even during long incubation times 20-min reaction time. This is due to the tightly packed droplets
was the conversion of the non-fluorescent substrate resorufin that travel through the incubation tubing that are not able to
FIG. 4. The connection of two devices via a bridging tubing for a droplet-based enzymatic assay. (a) Pictures showing packed droplets (i) formed after oil removal, (ii)
incubated in a 5-cm long tubing (above the focus plane), and (iii) reinjected and respaced in a droplet detection device for the measurement of enzymatic activities.
Flow rate: O = 2.0, W = 1.0, and D = 1.8 μl/min. (b) Enzymatic activities measured in photon counts after a different incubation time: blue, 3 min; red, 6 min; dark green,
10 min; and light green, 20 min. Flow rate: O = 2.0, W1 (substrate) = 0.5, W2 (enzyme) = 0.5, and D = 1.8 μl/min. (c) Enzymatic activity (averaged from ∼100 droplets)
vs incubation time.
Biomicrofluidics 15, 024108 (2021); doi: 10.1063/5.0047924 15, 024108-4
Published under license by AIP Publishing.Biomicrofluidics ARTICLE scitation.org/journal/bmf
spread out during transit. In fact, the relative standard deviations NIGMS-R35GM119688), the National Science Foundation
of the height of all of these traces is less than 4.5%, implying that (Early Career Grant No. 1553031; MCB No. 1817909), and the
the droplets have kept their generation order in a packed zone Alfred P. Sloan Foundation. The authors also wish to thank the
while traveling in the incubation tubing. By comparing droplets Single Molecule Analysis in Real-Time (SMART) Center of the
spanning with and without oil removal shown in Fig. 3 and University of Michigan, seeded by NSF MRI-R2-ID Award No.
Movie S3 of the supplementary material, it is expected that a DBI-0959823 to Nils G. Walter, as well as J. Damon Hoff for training
nearly fivefold expansion of the droplets zone without oil being and use of Alba v5 confocal spectroscopy.
first removed. This expansion could induce a larger variation for
droplet traveling/incubation time, which, for a time-sensitive DATA AVAILABILITY
kinetic assay shown in Fig. 4(c), could, in theory, result in a wide
distribution of fluorescence intensity. In this way, we demonstrate The data that support the findings of this study are available
the value of oil removal and tight droplet packing as a way to within the article.
achieve highly homogenous droplet reaction times even during
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Biomicrofluidics 15, 024108 (2021); doi: 10.1063/5.0047924 15, 024108-5
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