Volume 21

Number 10
21 May 2021
Pages 1835–2060

Lab on a Chip
Devices and applications at the micro- and nanoscale
rsc.li/loc

ISSN 1473-0197

PAPER
Yaru Xing, Xianming Liu, Xing Cheng et al.
A robust and scalable active-matrix driven digital microfluidic
platform based on printed-circuit board technology
 Lab on a Chip
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A robust and scalable active-matrix driven digital

Cite this: Lab Chip, 2021, 21, 1886
microfluidic platform based on printed-circuit
board technology†
Yaru Xing,*ab Yu Liu,b Rifei Chen,b Yuyan Li,c Chengzhi Zhang,b Youwei Jiang,bd
Yao Lu,e Bingcheng Lin,e Peizhong Chen,f Ruijun Tian, f
Published on 22 March 2021. Downloaded on 6/17/2021 2:04:48 AM.

Xianming Liu*e and Xing Cheng *b

Two-dimensional digital microfluidic platforms, on which droplets are actuated by electrowetting on

Received 7th February 2021,
Accepted 20th March 2021
DOI: 10.1039/d1lc00101a
rsc.li/loc
dielectrics, have merits such as dynamic reconfigurability and ease for automation. However, concerns for
digital microfluidic platforms based on low-cost printed circuit boards, such as the scalability of the
electrode array and the reliability of the device operation, should be addressed before high throughput and
fully automatic applications can be realized. In this work we report the progress in addressing those issues
by using active-matrix circuitry to automatically drive a large electrode array with enhanced device
reliability. We describe the design and the fabrication of a robust and scalable active-matrix driven digital
microfluidic platform based on printed-circuit board technology. Reliable actuation of aqueous and organic
droplets is achieved using a free-standing double-layer hydrophobic membrane. To demonstrate the
versatility of the digital microfluidic platform, a pentapeptide is synthesized on the device within 30
minutes. With these improvements, a fully automatic, scalable, robust, reusable, and low-cost digital
microfluidic platform capable of parallel manipulation of a large number of droplets can find numerous
applications in chemical engineering, bioengineering and biomedical engineering.

1. Introduction system, a droplet is used as a fluidic carrier of samples and

Droplet microfluidic technology1–5 has gained increasing
attention in both academic and industrial communities in
recent years due to its potential of carrying out automated
laboratory tasks with high throughput and a miniaturized
volume. Many biological and clinical applications of droplet
microfluidic technology have been reported in the literature,
such as genomics6,7 and proteomics,8–10 diagnostics,11,12
immunoassays,13–15 single cell studies,16,17 data storage,18 drug
development,19,20 and more.21,22 In a droplet microfluidic
a
Harbin Institute of Technology, Harbin 150001, China
b
Department of Materials Science and Engineering, Southern University of Science
and Technology, Shenzhen 518055, China. E-mail: 11649025@mail.sustech.edu.cn,
chengx@sustech.edu.cn
c
Department of Electrical and Electronic Engineering, Southern University of
Science and Technology, Shenzhen 518055, China
d
SUSTech Academy for Advanced Interdisciplinary Studies, Southern University of
Science and Technology, Shenzhen 518055, China
e
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian
116023, China. E-mail: liuxianming@dicp.ac.cn
f
Department of Chemistry, Southern University of Science and Technology,
Shenzhen 518055, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1lc00101a
reagents, which can be actuated by various conditions and
methods, including pressure,23 thermal capillary,24 acoustic
waves,25 optical actuation,26 magnetic force,27 and electrical
actuation.28–30 Among these methods, electrowetting on
dielectrics (EWOD)31–33 with facile droplet handling and
individual addressability of droplets emerged as an attractive
actuation mechanism. On a two-dimensional plane, droplets
can be easily moved by electrodes underneath, thus greatly
reducing the clutter of channel-based fluidic systems by
circumventing valves and pumps. This simplicity in
implementation allows the EWOD-based droplet microfluidics
to be used in various bioengineering applications and is
conducive to achieving a high level of automation.34–36
However, in a conventional digital microfluidic device, a one-
on-one passive switching scheme results in a small number of
electrodes. In this situation, the number of droplets that can be
accommodated on a chip limits the scale of sample treatments
or reactions that can be conducted on the device, making the
device not very useful in practical applications.
To solve the problem above, the use of active-matrix driving
circuitry is proposed.37,38 Under this scheme, an M × N electrode
array only needs M + N electrical inputs. Therefore, the
advantage of active-matrix circuitry is that the required number
of electrical input (or contact) lines is significantly reduced. In

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the active-matrix circuitry, the switching transistors can be
individually turned on or off without interference with
neighboring sites, thus only activate a specific set of electrodes
via selected row and column lines. Thus, the active-matrix
circuitry allows for actuating individual droplets in a large array
without interfering with other droplets. In a reported digital
microfluidic chip,37 amorphous indium gallium zinc oxide (a-
IGZO)-based thin-film transistors (TFTs) were used to build the
active-matrix circuitry, and the manipulation of two droplets
was demonstrated on a 2 × 5 electrode array. To improve the
scale of droplet handling, Morgan and coworkers have
developed a 64 × 64 active-matrix digital microfluidic device
using standard TFT processing (CG-Silicon™, Sharp's
proprietary technology).38 Although the active-matrix circuitry
has over 3600 TFTs, the work demonstrated the actuation of
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only 16 droplets on the chip because multiple electrodes were
employed to actuate an individual droplet,38 thus making the
number of droplets that can be simultaneously actuated on the
chip comparable to other passive-matrix digital microfluidic
platforms. Moreover, the complex fabrication of the TFT array
Fig. 1 (a) The components and an exploded view of the active-matrix digital microfluidic system; (b) a photograph of the assembled digital
microfluidic platform; and (c) schematics of the active-matrix circuitry and the biasing scheme for droplet actuation.
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on a glass substrate, including multiple thin-film depositions,
multiple photolithography and patterning processes, and
multiple dry and wet etching steps, makes the TFT technology
unaffordable and unavailable to most end users.
In this work, we design and fabricate a digital microfluidic
platform with a 20 × 20 electrode array driven by active-
matrix circuitry on printed-circuit boards (PCBs). The
platform can manipulate up to 50 droplets simultaneously by
row and column scanning with high reliability. Compared to
the active-matrix circuitry implementation from conventional
TFTs on a glass substrate, the PCB-based active-matrix
circuitry is low cost and has a fast development cycle due to
the maturity and general availability of the PCB technology.
2. Experimental section
2.1 Design and fabrication of the active-matrix digital
microfluidic platform using PCB technology
To overcome the common issues such as the limited number
of electrodes and poor device reliability in conventional PCB-
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based digital microfluidic platforms, we design and
implement a multilayer configuration with a replaceable
polymer membrane as the dielectric layer. As shown in
Fig. 1(a), our digital microfluidic platform consists of four
components from bottom to top: (1) an active-matrix control
circuit board; (2) a 20 × 20 electrode array board; (3) a
replaceable hydrophobic polymer membrane attached on a
metal frame; and (4) an indium tin oxide (ITO) glass plate as
the top electrode.
The bottom active-matrix control circuit board is made of
a three-layered electric circuit on a PCB substrate, which
receives 20 column and 20 row control commands from a
personal computer via a data acquisition (DAQ) card (USB
6509, National Instrument, USA) and provides actuation
voltages to individual electrodes. The active-matrix control
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board has 400 high-voltage transistors (BSS131, Infineon,
Germany) and 400 pogo pin connectors (4P-2.54PH-8.0H-2R,
Shenzhen Yuansheng Electronics, China).
The electrode array (Fig. 1(a), top right corner) for droplet
actuation is made on a standard double-layer PCB substrate.
The size of each electrode is 2.54 mm × 2.54 mm with a gap
of around 100 μm between neighboring electrodes. The
electrode array is easily connected to the active-matrix control
circuit board by pogo pin connections when the two PCB
substrates are stacked together.
To make the electrode surface hydrophobic for droplet
actuation, we use a free-standing parylene-C/Teflon-AF double
layer membrane affixed to a metal frame for easy handling
(Fig. 1(a), top left corner). The fabrication of this membrane
and the critical role it played in enhancing device reliability
are described later. Finally, a counter electrode required for
droplet actuation by EWOD is made of an ITO-coated glass
plate. The ITO-glass plate is coated with a 50 nm Teflon-AF
layer by spin-coating the solution (1 wt% in FC-40, 3M, USA)
at 1000 rpm for 60 s. A spacer of around 50 microns
encircling the electrode array is formed using double-sided
tape. The spacer between the electrode array board and the
top cover glass plate provides the space for droplets to move
around and ensures a partially sealed environment to avoid
excessive droplet evaporation for a prolonged period of
experiments.
The four components are assembled with the help of a
pair of plastic fixtures and four metal screws at the corners,
as shown in the center of Fig. 1(a). Our assembled device
(Fig. 1(b)) is designed and constructed to produce an active-
matrix digital microfluidic platform that is capable of
handling multiple microliter droplets in parallel. The
modular design allows the individual components in the
digital microfluidic platform to be optimized and upgraded
in future development based on the needs of actual
applications.
The voltage for droplet actuation is applied to the
electrode array through the active-matrix control circuit. The
active-matrix circuitry consists of row lines, column lines,
and switching transistors, as shown in Fig. 1(c). The row and
column bus lines are addressed by the digital outputs of the
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DAQ electronics. As shown in the magnified view of four unit
cells in the active-matrix circuitry on the right-hand side of
Fig. 1(c), the gate terminal of a transistor is connected to the
row line and the column line is connected to the source
terminal of the transistor. The drain terminal of the
transistor is connected to an actuation electrode in the
electrode array via a pogo pin connector. The actuation
electrode and the drain terminal of the transistor are both
connected to the output of a high voltage power source
(QFC1020H, Dalian Quantum Fluid Control Technology
Company, China), which provides a high DC voltage via a
shunt resistor (1 MΩ, PINGCON, China). The driving software
for the active-matrix circuitry is scripted with LabView
(National Instrument, USA). The interface program also
controls the DAQ card to send row and column addressing
signals to the transistors.
2.2 Fabrication of the free-standing parylene-C/Teflon-AF
membrane
The fabrication process involves six steps as shown in
Fig. 2(a). A silicon wafer is first primed by spin-coating a
polyvinyl alcohol (PVA) solution of 3 wt% at 1000 rpm for 60
s. The PVA film is baked at 95 °C on a hot plate for 15 min.
The wafer is then deposited with a 3.5 μm thick parylene-C
film using low-pressure chemical vapor deposition (CVD)
equipment (LH300, LaChi Enterprise, Republic of China).
Then, an aluminum frame is adhered onto the parylene-C
layer with double-sided adhesive tapes to ensure that the
polymer membrane remains flat and self-standing after being
released from the silicon substrate. The silicon wafer with
the parylene-C layer is immersed in deionized (DI) water for
8 hours to dissolve the PVA film, which is a water-soluble
polymer. The polymer membrane together with the
aluminum frame is then gently peeled off from the substrate
and dried at 165 °C in a hot oven for 20 min. Finally, the
parylene-C film with the aluminum frame is affixed on
another clean silicon wafer using adhesive tapes. A Teflon-AF
solution (1 wt% in FC-40, 3M, USA) is spin-coated on the
parylene-C film at 1000 rpm for 60 s, and the Teflon-AF film
is baked at 165 °C on a hotplate for 15 min. The completed
free-standing parylene-C/Teflon-AF bilayer film of 65 mm ×
65 mm in size is shown in Fig. 2(b). Fig. 2(c) shows the
attachment and replacement of the free-standing membrane
on the actuation electrode array.
2.3 Droplet preparation and manipulation on the digital
microfluidic platform
Droplets of various contents are prepared and placed on the
digital microfluidic platform for device reliability testing and
functionality demonstration. Acidic solutions of HCl (0.5%
and 1%) are prepared by mixing a concentrated HCl solution
with DI water. Basic solutions of NaOH (0.1 M, 0.5 M, and 1
M) are prepared by dissolving sodium hydroxide in DI water.
Salt solutions of KCl (0.1 M, 0.5 M, and 1 M) are prepared by
dissolving potassium chloride in DI water. Dye droplets with
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Fig. 2 (a) The fabrication procedure for the free-standing hydrophobic parylene-C/Teflon-AF bilayer membrane; (b) a photograph of the free-
standing polymer membrane with a metal frame; (c) a schematic showing the free-standing hydrophobic membrane attachment on the electrode
array before use and replacement after use.
a concentration of 0.01% are prepared by dissolving
commercial dyes of amaranth (red color, CAS no: 915-67-3),
erioglaucine disodium salt (blue color, CAS no: 3844-45-9),
sunset yellow FCF (yellow color, CAS no: 2783-94-0), and fast
green FCF (CAS no: 2353-45-9) in DI water. All dyes are
purchased from Aladdin, USA. Droplets are pipetted onto the
hydrophobic membrane and actuated using the electrode
array.
2.4 Reagent preparation for peptide synthesis
We synthesize a pentapeptide on the digital microfluidic
platform to demonstrate its application. Reagents for peptide
synthesis are prepared off chip. A magnetic bead dispersion
(Dynabead M-270 Amine, ThermoFisher, USA) of 50 μL is
pipetted into a 2 mL Eppendorf tube. The magnetic beads
are washed with 500 μL N,N-dimethylformamide (DMF,
General Reagent, China) twice using a strong magnet to hold
the beads. To attach the rink ligand to the magnetic beads,
16.2 mg of rink ligand (2-(4-(((((9H-fluoren-9-yl)methoxy)
carbonyl)amino)(2,4-dimethoxyphenyl)methyl)phenoxy)acetic
acid, Ark Pharm, USA), 13.7 mg of 2-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU,
GL Biochem, China), 6 μL of N,N-diisopropylethylamine
(DIPEA, Energy Chemical, China), and 300 μL of DMF are
mixed in a disposable vessel for 3 min, and the activated rink
ligand solution is added into the rinsed magnetic bead
dispersion. The mixture in the tube is placed on a
mechanical shaker at room temperature for 2 h. Then the
liquid from the reaction Eppendorf tube is expelled and the
magnet beads are rinsed with 500 μL of DMF twice. The
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magnetic beads with the rink ligand are then dispersed in
200 μL DMF for later use.
Fmoc-protected amino acids are purchased from
Chinapeptides, China. Four amino acid solutions are
activated by mixing each Fmoc-protected amino acid (Fmoc-
Gly-OH, 2.5 mg; Fmoc-Glu(OtBu)-OH, 3.5 mg; Fmoc-His(Trt)-
OH, 4.8 mg; Fmoc-Pro-OH, 1.4 mg) with 1.4 mg of HATU, 1.3
μL of DIPEA, and 100 μL of DMF in a 1 mL Eppendorf tube.
The solutions are freshly prepared before use. A 20 v/v percent
piperidine solution is prepared by pipetting 20 μL piperidine
into 80 μL DMF in a tube. The solution is agitated for 1 min.
All reagents (a dispersion of magnetic beads with the rink
ligand, activated Fmoc-protected amino-acids, and solvents)
prepared from the aforementioned steps are used for peptide
synthesis on the digital microfluidic platform. The detailed
reaction steps performed on chip are described in section 3.4.
2.5 Post processing for peptide analysis using a mass
spectrometer
After pentapeptide synthesis, the magnetic beads are rinsed
with dichloromethane (DCM, Changtai Technology, China)
twice to remove DMF. The pentapeptide is detached from the
magnetic beads after mixing the beads with 50 μL
trifluoroacetic acid (TFA, Energy Chemical, China) for 30 min
with agitation. The pentapeptide is dried by nitrogen gas
blowing and then resolubilized in a 10 mL mixture of
acetonitrile and water in a 50 : 50 ratio. After centrifuging and
filtering, the pentapeptide solution is mixed with the matrix
in a 1 : 1 ratio. A 0.5 μL mixture is fed into a MALDI TOF
mass spectrometer (Autoflex speed LRF, Bruker, USA) for
peptide analysis.
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2.6 Peptide yield analysis
To evaluate the yield of the peptide synthesis, we perform
both off-chip and on-chip synthesis and the peptide product
is quantified using a microvolume UV-vis spectrophotometer
(NanoDrop One, Thermo Fisher Scientific, USA). To
accumulate enough product for NanoDrop analysis, the
peptide products from 9 droplets (∼18 μL) of magnetic
bead dispersion as prepared in section 2.5 are collected.
The peptide product from the magnetic beads are cleaved
by TFA and redissolved in a mixture of methyl cyanide and
water in a 1 : 1 ratio for NanoDrop analysis. The absorptions
of the amino acids and the pentapeptide standard of known
concentration are acquired at the wavelength of 205 nm.
The concentration of the peptide product is then
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determined by absorption measurement. The pentapeptide
standard is synthesized off-chip following a standard
procedure.
3. Results and discussion
3.1 Droplet handling on the digital microfluidic platform
With active-matrix control circuitry and reliable operation,
the digital microfluidic platform developed in this work
Fig. 3 An example of droplet manipulations on the active-matrix digital microfluidic device. (a and b) Fifty scattered droplets spell out a pattern of
“SUSTech” through EWOD actuation; (c–f) concurrently mixing and splitting multiple droplets.
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can handle a large number of droplets in parallel using an
array of 400 electrodes. The concurrent manipulation of
multiple dye droplets with red, yellow, green, and blue
colors is demonstrated on our device. A total of 50
aqueous droplets in different colors are pipetted onto the
device (Fig. 3(a)). The red, yellow, and green droplets are
simultaneously actuated using the Labview program to
compose the letters “SUSTech” (Fig. 3(b)). Similar to the
droplets with high ionic content, highly reliable actuation
of dye droplets is observed. A blue droplet is moved back
and forth on the electrode array at a speed of 50 mm s−1
for a duration of up to 5 hours. The durability of droplet
actuation is comparable with those devices with a thick
dielectric layer in oil medium.39 It is worth mentioning
that the actuation of the droplets in our experiments is
carried out in air medium to avoid potential
contamination from the oil medium, which can be
problematic for subsequent off-chip analysis such as mass
spectroscopy. The mixing and splitting of multiple droplets
are easily accomplished on our device as shown in
Fig. 3(c–f). A video showing various types of droplet
handling on the digital microfluidic platform is included
in the ESI.
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3.2 Quality and reliability of the free-standing parylene-C/
Teflon-AF bilayer polymer membrane
Using the fabrication process described in Fig. 2(a), the
hydrophobic polymer membrane produced here is free of
scratches and wrinkles, and water droplets can move easily
under gravity at a 15.5° tilting angle (Digital inclinometer,
China). Prior to placing the parylene-C side of the polymer
membrane on the electrode array board, a small amount of
silicone oil is applied on the bottom electrode array. The
silicone oil can form a thin and smooth oil film by filling in
the gaps of 45 μm deep and 100 μm wide between
neighboring electrodes and help affix the polymer membrane
on the electrode array board by capillary force.
The hydrophobic polymer membrane serves multiple
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In the standard PCB technology, the trenches between
adjacent electrodes on a typical PCB substrate are fabricated by
wet etching. Due to the thickness of the top copper layer in the
standard PCB fabrication, the trench can be tens of microns
deep. The deep trenches make the electrode array surface
uneven and they impede the movement of the droplets. To
lower the surface unevenness of the PCB substrates, tedious
chemical mechanical polishing (CMP) and physical vapor
deposition (PVD) technologies were adopted by Gong and
Kim.40 However, in practice, these planarization methods have
difficulty achieving good results due to the roughness of the
copper surface and the warping of the PCB substrates.
Compared to the conventional method of directly coating
parylene on the PCB electrode array, the free-standing film
yields an outstandingly flat and smooth surface, as shown in

purposes. First, it provides a smooth hydrophobic surface
with low contact angle hysteresis for easy actuation of
droplets through underlying electrodes. Second, the polymer
membrane can be easily peeled off and replaced by a freshly
prepared film to avoid cross contamination between
experiments. This allows all other PCB components to be
reused to lower the cost of operation for the digital
microfluidic platform. Finally, in some applications where
solid-phase separation is needed, the analyte or synthesis
product can be left on the membrane and the membrane can
be removed and transported to other analytical equipment
such as mass spectrometers for off-chip detection and
analysis.
Fig. 4(a) and (b). Because direct parylene coating by chemical
vapor deposition (CVD) produces a conformal morphology
which follows the surface of the metal electrodes, there is still a
45 μm deep trench between neighboring electrodes with a
roughness of 90 nm on the top surface of the electrodes on the
PCB substrate. The surface morphology of the electrode array
with the direct parylene coating method is examined using a
helium ion microscope (HIM, ORION NanoFab, Zeiss, Germany)
(Fig. 4(e)). A section of the electrode after parylene coating is cut
by a focused gallium beam (Fig. 4(f)). Although the bisected
electrode is evenly covered with parylene, there is a deep trench
with a rugged surface on the bottom of the trench. As a result,
when a droplet is actuated from one electrode to its adjacent

Fig. 4 (a) Optical 3D image of the PCB electrode in the conformal parylene-C and Teflon-AF coated digital microfluidic chip; (b) optical 3D image
of the PCB electrode covered by the free-standing bilayer membrane; (c) a schematic diagram of droplet actuation on conformal parylene-C and
Teflon-AF coated electrodes. The actuation voltage is 350 volts; (d) a schematic diagram of droplet actuation on free-standing membrane covered
electrodes. The actuation voltage is 85 volts; (e) the HIM image of an electrode and a trench with conformal parylene-C and Teflon-AF coating
shows the electrode gap; and (f) the HIM image of a cut-through of part (e) shows the copper in grey and parylene-C/Teflon-AF in dark grey.

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one, it fills into the 45 μm deep and 100 μm wide trench

between the electrodes and then has to be pulled out by the
EWOD force in the course of actuation (Fig. 4(c)). In our
experiment, the threshold voltage required to actuate the
droplets on the metal electrodes with a 3.5 μm thick parylene
and 50 nm Teflon-AF layer is 350 volts. When the PCB
electrodes are covered by the free-standing polymer membrane,
the trench is filled with silicone oil and the droplets experience
a flat and uniform surface with a surface roughness of only 3
nm during actuation. In this case, the droplet slides onto the
adjacent electrode easily (Fig. 4(d)) and consequently the driving
voltage required for droplet actuation is reduced to 85 volts.
A comparison of the electric field distributions at critical
regions of the digital microfluidic platforms is provided with
COMSOL simulations using the geometrical parameters of the
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digital microfluidic platforms and the actuation voltages used

in the experiments. The overall electric field distributions and
the magnified views at several critical regions for the device with
conformal parylene-C and Teflon-AF coating and an actuation
voltage of 350 volts are shown in Fig. 5(a), (c), (e), and (g), while
those for the device with our replaceable free-standing polymer
membrane and an actuation voltage of 85 volts are shown in
Fig. 5(b), (d), (f), and (h). The greatest distinctions in the electric
field intensities are found at the corresponding regions of the
two devices. In Fig. 5(c) and (e), the strongest electric fields at
the tip of the droplet inside the electrode gap and at the lower
advancing edge of the droplet are 176 volts per μm and 217 volts
per μm, respectively. Both values significantly exceed the
theoretical dielectric strength or breakdown electric field of the
Teflon-AF film, which is 59 volts per μm. It can be inferred that
the Teflon-AF layer deposited with the conventional method
might easily be damaged during operation and cause a
reliability issue for droplet actuation. Contrarily, the electric
field strength inside the electrode gap regions filled with
silicone oil and covered with the free-standing polymer
membrane is just 2 volts per μm (Fig. 5(d)). The electric field at
the lower advancing edge of the droplet is 55 volts per μm
(Fig. 5(f)), which is still lower than the dielectric strength of the
Teflon-AF film. Furthermore, the electric fields at the upper
advancing edges of the droplets in contact with the Teflon-AF Fig. 5 COMSOL simulations of the electric field distributions in the
films on the top plates are also calculated. In the conventional digital microfluidic platforms with the (a) conformal parylene-C/Teflon-
AF coating and (b) replaceable free-standing polymer membrane. The
method, the Teflon-AF film experiences an electric field of 82
magnified views of the electric fields at the tips of the droplets inside
volts per μm and can experience electric breakdown during the electrode gap (red rectangles in (a) and (b)) are shown in (c) and (d),
droplet actuation. Such catastrophic damage is observed in respectively. The magnified views of the electric fields at the advancing
control experiments, in which a hole in the Teflon-AF film is edges of the droplets touching the Teflon-AF films on the bottom
formed due to electric breakdown (Fig. 5(i)). For the digital electrodes (blue rectangles in (a) and (b)) are shown in (e) and (f),
respectively. The magnified views of the electric fields at the advancing
microfluidic platform with the free-standing polymer
edges of the droplets touching the Teflon-AF films on the top
membrane, the electric field at the corresponding location is electrodes (yellow rectangles in (a) and (b)) are shown in (g) and (h),
just 18 volts per μm under the required actuation voltage, respectively. The optical microscopic images of the Teflon-AF coated
leaving the surface untouched for a prolonged period of top electrodes show (i) damages in the insulation layer with the
operation (Fig. 5(j)). conformal parylene-C/Teflon-AF coating and (j) intact insulation layer
with the replaceable free-standing polymer membrane coating.

3.3 Robust operation of the digital microfluidic platform

The driving voltage needed to effectively actuate droplets in dielectric film (e.g., thickness, dielectric constant, and surface
digital microfluidics is mainly related to the properties of the roughness) and the droplet (e.g., polarity and viscosity). By

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Table 1 Reliability of droplet actuation on the digital microfluidic actuation of droplets with different ionic contents on our
platform with a free-standing polymer bilayer membrane
digital microfluidic platform. All droplets can be actuated in
Droplet type Actuation duration Number of actuations a partially sealed ambient environment by a voltage below
100 volts. The DI water droplet can be actuated back and
DI water >6 h >21 600
1 M NaOH 200 min 12 000 forth on ten electrodes in a line for more than 6 hours with a
0.5 M NaOH >6 h >21 600 driving voltage applied every second, which indicates the
0.1 M NaOH >6 h >21 600 droplet can be actuated for more than 21 600 times on the
1 M KCl 230 min 13 800
digital microfluidic platform without error. After a long
0.5 M KCl >6 h >21 600
0.1 M KCl >6 h >21 600 actuation time, we observed a slight reduction of droplet size
1% HCl 100 min 6000 due to evaporation within the space sealed with the top glass
0.5% HCl >6 h >21 600 plate, the bottom electrode PCB, and the spacer in-between.
DMF 20 min 1200
For solutions with a considerable ionic concentration,
such as 1 M NaOH, 1 M KCl, and 1% HCl, the actuation
lifetime is reduced to 100–200 min due to residue
reducing the electric field during droplet actuation, the contamination left behind by the moving droplets. After
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digital microfluidic platform with the free-standing parylene- repetitively passing by the ionic droplets through the same
C/Teflon-AF bilayer membrane shows excellent device area, the surface of the polymer membrane loses its
stability and reliability. Table 1 summarizes the reliable hydrophobicity and rendered the device inoperable. However,

Fig. 6 (a) A schematic of the solid-phase peptide synthesis procedures; (b–e) sequence of the pictures illustrating synthesis of peptides in droplets
on the digital microfluidic platform. Droplets (∼2 μL) including magnet beads and piperidine are mixed for 1 min for deprotection (b and c) and
then washed with DMF three times (d). Magnet bead droplets are mixed with one of the five Fmoc-protected amino acids (Pro, His, Gly, Glu and
Gly) for 1 min for the coupling reaction (e).

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we are still able to actuate the droplets with considerable
ionic concentrations by more than 6000 times before failure
as shown in Table 1, which is more than enough to complete
any analytical or synthetic task on the device. Furthermore,
the contamination issue can be alleviated by placing clean DI
water droplets next to the target droplet. The residue
contamination left behind by the target droplet can be picked
up by the cleaning droplets when they are actuated in
tandem.
Unlike a conventional PCB-based EWOD device in which
catastrophic electric breakdown is often the main failure
mechanism, no electric breakdown is observed in our device
with the free-standing polymer bilayer membrane. The
remarkable electrical robustness overcomes the primary
obstacle for digital microfluidic platforms and paves the way
Published on 22 March 2021. Downloaded on 6/17/2021 2:04:48 AM.
for numerous practical applications in chemical engineering,
bioengineering and biomedical engineering.
3.4 Peptide synthesis in droplets on the digital microfluidic
platform
The schematic of the Fmoc solid-phase synthesis of a penta-
peptide is shown in Fig. 6(a). The magnetic beads with a
protecting group on the alpha amine as carriers are
deprotected with piperidine and then coupled with an Fmoc-
protected amino acid. The deprotection and coupling cycles
are repeated several times with different amino acids to
produce a target polypeptide. The synthesis of a pentapeptide
(Pro, His, Gly, Glu and Gly) is automatically implemented on
our active-matrix digital microfluidic system, as shown in
Fig. 6(b). Reagents are first fed onto the electrodes through
Fig. 7 MALDI-TOF mass spectrum of the pentapeptide synthesized on the digital microfluidic platform.
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Lab on a Chip
fluid input ports driven by programmable precision micro-
pumps (Healtell, China). Electric signals are applied to the
actuation electrodes in the 20 × 20 array to dispense, move,
and mix droplets of reactants. The pentapeptide synthesis
has 20 droplet manipulation steps (four steps per amino acid
cycle and five cycles in total). The droplet arrangements and
their manipulation sequences in one amino acid cycle are
shown in Fig. 6(b–e). Because the electrode array is large,
four reaction sites can be arranged on the digital microfluidic
platform to allow for parallel synthesis of peptides. First,
twenty-four 2 μL droplets including four magnetic bead
droplets, four piperidine droplets, and sixteen DMF droplets
are introduced onto the electrode array for deprotection and
washing (Fig. 6(b)). Second, the piperidine droplets and the
magnetic bead droplets are mixed for 1 min to remove Fmoc
to expose the amine group (Fig. 6(c)). Third, the magnetic
beads are fixed with a strong magnet on the top plate and
the remaining solvent droplet is actuated away to separate
the magnetic beads from the waste liquid, and the DMF
droplets are moved to the magnetic bead site to wash the
beads three times (Fig. 6(d)). When washing the magnetic
beads, the magnet is removed to release the magnetic beads
into the DMF droplet. Then the magnet is placed on the top
plate again to separate the beads from the DMF rinsing
droplet. Finally, the magnetic bead droplets are mixed with
droplets containing one of the five activated amino acids
(Pro, His, Gly, Glu and Gly) for 1 min for the coupling
reaction (Fig. 6(e)). After each amino acid coupling, the beads
are washed with DMF droplets three times.
Fig. 7 shows a MALDI-TOF mass spectrum (MS) for the
pentapeptide synthesized on our digital microfluidic
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Lab on a Chip
platform. The theoretical mass of Fmoc-PHGEG (Fmoc-Pro-
His-Gly-Glu-Gly) is 717.736, which is consistent with the
strongest peak of 717.431 in the MS spectrum. For single
amino acid (Gly) attachment, the ratio between on-chip and
off-chip synthesis is 65%. The reduced yield for on-chip
synthesis is believed to be due to magnetic bead loss in
solid–liquid separation during reaction and rinsing steps. For
the pentapeptide, the product from on-chip and off-chip
synthesis is 0.68 nmol and 1.79 nmol, respectively. The yield
is calculated to be in the range of 3.25–6.5% for on-chip
synthesis, depending on the concentration of active chemical
sites on the magnetic beads (0.75–1.5 nmol μL−1). The results
confirm the feasibility of automatic peptide synthesis on the
digital microfluidic platform. Due to the small droplet size,
mass transport is quick, so the reaction time in each step is
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reduced to about 1 min, which is significantly shorter than
the 1 h time needed in a conventional peptide synthesizer.
The total synthesis time for the pentapeptide is 30 min on
the digital microfluidic platform, while the same process can
take up to 8 h in a commercial peptide synthesizer.
The ability to configure multiple reaction sites on the
digital microfluidic platform allows for simultaneous
synthesis of different peptides for library construction, or
parallel synthesis of the same peptide at multiple sites for
enhanced throughput. Thus, the digital microfluidic platform
with a large electrode array has the versatility to adapt for
different synthesis goals. The much-reduced time for peptide
synthesis in a small droplet due to faster chemical transport
is significant for throughput. It may also open the possibility
to synthesize longer peptides (e.g., tens or even hundreds of
amino acids) in a reasonable time frame using solid-phase
synthesis.
4. Conclusion
We develop a scalable and robust digital microfluidic
platform with active-matrix driving circuitry implemented
with PCB technology. The digital microfluidic platform is
assembled from four layers of components which can be
individually swapped or upgraded for ease-of-use, low-cost
operation, and possibility of future improvement. The active-
matrix circuitry allows simultaneous actuation of up to 50
droplets on the digital microfluidic platform. The size of the
electrode array can be easily extended into tens of thousands
because of the scalability of the active-matrix circuitry, so the
digital microfluidic platform has the potential to drive
thousands of droplets in parallel. To achieve highly reliable
manipulation of droplets, we develop a free-standing
parylene-C/Teflon-AF bilayer polymer membrane with a
surface roughness of a few nanometers for the electrode
array. Droplets with high ionic contents can be actuated by
tens of thousands of times without dielectric breakdown due
to a lower driving voltage required for droplets on a smooth
hydrophobic surface. The free-standing hydrophobic
membrane can also be easily replaced to avoid cross-
contamination among different experiments. The digital
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microfluidic platform developed in this work overcomes the
common issues in previously reported droplet devices, such
as poor operation reliability and a very limited number of
concurrent droplets. The digital microfluidic platform
reported here may offer a robust, highly scalable, low cost,
easy to use, and contamination-insensitive device for
automatic and parallel droplet handling, and hence
unleashes the full potential of droplet-based digital
microfluidics. A pentapeptide is successfully synthesized on
the digital microfluidic platform, demonstrating its potential
in practical applications. By varying the chemical and
biological content in the droplets, the device may automate
high-throughput analytical and synthetic tasks in chemical
engineering, bioengineering and biomedical engineering
applications.
Author contributions
Funding acquisition: X. Cheng, X. Liu and Y. Lu;
experimental design: X. Cheng, Y. Liu, R. Chen, Y. Xing, Y.
Jiang, B. Lin, R. Tian, and X. Liu; experimental procedure: Y.
Xing, Y. Liu, R. Chen, Y. Li, C. Zhang, P. Chen, R. Tian, X.
Liu, and Y. Jiang; writing, Y. Xing, X. Liu, and X. Cheng;
review and editing: Y. Xing and X. Cheng.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We are grateful to use SUSTech Core Facilities for the
fabrication of the free-standing polymer membranes. The
financial support from the National Natural Science
Foundation of China (grant number: 31927802) is gratefully
acknowledged.
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