Chemical and Biological Applications of Digital-Microfluidic Devices

TL;DR: This article reviews efforts to develop various LoC applications using electrowetting-based digital microfluidics, and describes these applications, their implementation, and associated design issues.

Abstract: Digital-microfluidic lab-on-a chip (LoC) technology offers a platform for developing diagnostic applications with the advantages of portability, sample and reagent volume reduction, faster analysis, increased automation, low power consumption, compatibility with mass manufacturing, and high throughput. In addition to diagnostics, digital microfluidics is finding use in airborne chemical detection, DNA sequencing by synthesis, and tissue engineering. In this article, we review efforts to develop various LoC applications using electrowetting-based digital microfluidics. We describe these applications, their implementation, and associated design issues.

Summary

Electrowetting technology

• Electrowetting is the phenomenon whereby an electric field modifies the wetting behavior of a polarizable or conductive liquid droplet in contact with a hydrophobic, insulated electrode.
• Droplets are usually sandwiched between two parallel plates; the bottom plate is the chip surface, which houses the addressable electrode array, and the top is either a continuous ground plate or a passive top plate (the chip's characteristics determine the top plate's nature).
• Thus, the top plate can be a disposable component.
• Using the electrowetting principle, the authors can transport discrete droplets in a highly controlled way over an array of electrodes.
• The LoC chip surface is coated with an insulating layer of parylene C (about 800 nm), and both the top and bottom surfaces are covered with a Teflon-AF thin.

Related work

• Much of the reported work on lab-on-a-chip (LoC) microfluidic devices has focused on miniaturization of analytical methods and protocols to improve performance and throughput.
• Most LoC devices have aimed at performing chemical or biological protocols on chip, with pre-prepared samples processed off chip.
• Digital-microfluidic systems have several advantages over continuous-flow systems; the most important are reconfigurability and architecture scalability.
• The gap is usually flooded with silicone oil that acts as a filler fluid, preventing droplet evaporation and reducing surface contamination.
• & controls many droplets independently because the electrowetting force is localized at the surface.

Applications and design issues

• In most diagnostic and chemical detection applications, a key challenge is the preparation of the analyte for presentation to the on-chip detection system.
• In diagnostics, raw physiological samples must be introduced onto the chip and then further processed by lysing blood cells and extracting DNA.
• In real-time airborneparticulate-sampling applications, the process of sample collection from an air stream must be integrated into the LoC analytical component.
• In tissue-engineering applications, the challenge is to build high-resolution (less than 10 microns) 3D tissue constructs with embedded cells and growth factors by manipulating and maintaining live cells on the chip platform.
• Here, the authors highlight these new applications, including detection of airborne sulfates obtained by air sampling, DNA pyrosequencing, and a biomimetic manufacturing process for soft-tissue engineering.

On-chip assays

• On-chip assays for determining the concentrations of target analytes is a natural application for digital microfluidics.
• Work in this area has focused on multiplexed assays, which measure multiple analytes in a single sample, as Figure 3 shows.
• Initially, scientists must determine the compatibility of each chemical substance with the electrowetting platform.

Glucose assay

• The in vitro measurement of glucose in human physiological fluids is of great importance in clinical diagnosis of metabolic disorders.
• Srinivasan, Pamula, and Fair have demonstrated an LoC for glucose assay using a colorimetric enzyme-kinetic method based on Trinder's reaction, 7 which determines glucose concentration.
• First, the authors pipette droplets of the glucose sample and the reagent onto the electrowetting chip.
• The LoC then merges and physically mixes the sample and the reagent by shuttling the coalesced droplet across three electrodes for 15 seconds, at a switching rate of 8 Hz and an actuation voltage of 50 V.
• Because absorbance measurement begins 15 seconds after the droplets merge, the measured reaction rate might not exactly equal the initial reaction rate.

Glucose assay design issues

• Any efficient or moderate-throughput microfluidic architecture inevitably requires droplets to share microfluidic resources on the chip (transport lanes, mixers, and incubators).
• 5, 10 In contrast, the transport of fluids containing proteins, such as enzyme-laden reagents and human physiological fluids, is not as straightforward, because most proteins adsorb irreversibly to hydrophobic surfaces and contaminate them.
• Therefore, to prevent contamination and enable transport, the authors must avoid contact between a liquid droplet containing proteins and the Teflon surface.
• The accuracy and repeatability of the dilution process depends on volume variations in droplet splitting and concentration variations due to incomplete mixing.
• Figure 5 shows absorbance as a function of time for nine assays.

TNT assay

• An estimated 100 million land mines buried in 65 countries throughout the world pose an enormous humanitarian problem, killing and mutilating thousands of civilians.
• In addition to land mine detection, many countries also need chemical detection of explosives to assess soil and water contamination.
• At the completion of mixing, the LoC measured absorbance with an LED.
• If a cubic meter of sampled air contains 1 mg of TNT (1 femtogram/ml of air), and all of this particulate is collected on the chip's sampling surface and concentrated in a 1-ml droplet, the droplet will contain a TNT density of 1 mg/ml, or 1,000 mg/ml, well above the assay's detection limit.
• Figure 7 shows the absorbance spectra of the color products resulting from the TNT and DNT assays.

TNT assay design issues

• Nitroaromatic compounds such as TNT and DNT react with nucleophiles such as hydroxides and alkoxides, to form colored Jackson-Meisenheimer complexes.
• Acetone, acetonitrile, and methanol have been the most popular solvent choices for TNT analysis, even though researchers have demonstrated this reaction in various organic solvents.
• All three solvents are currently incompatible with oil-medium electrowetting systems because they are miscible with silicone oil.
• DMSO is also completely miscible in all proportions with water and has a low order of toxicity.
• DMSO is also known to enhance the stability of the Jackson-Meisenheimer complex.

Sulfate assay

• Another assay developed on a digital-microfluidic platform is atmospheric sampling of sulfate particles.
• Atmospheric particulate matter contributes to adverse health effects, visibility reduction, and global climate change, all with significant socioeconomic implications. [16] [17] [18] [19].
• Here, AU is absorbance units, y is the slope of the linear curve fit, R is a measure of the deviation of the data from a linear fit, the abscissa x here represents time, and the equations represent a linear curve fit to the data.

Sulfate assay design issues

• For sulfate assay, the authors must perform droplet scanning and sample collection in air without a top plate, so as not to perturb the impactor air flow.
• To prevent evaporation, the scanning droplet can be clad in an oil encasement that travels through air with the actuated droplet.
• The authors have demonstrated oil cladding by transporting a droplet through the interface between the oil medium and the air.
• The authors diluted a solution containing 1-micron-diameter polystyrene beads and deposited it on the hydrophobic Teflon surface of an electrowetting chip.
• This small path length poses serious sensitivity issues.

DNA pyrosequencing

• The number of bases in the GenBank genetic sequence database has increased exponentially, with a doubling period of approximately 18 months, and the database currently contains about 3 3 10 10 bases, equivalent to the content of 10 human genomes (a base is a nucleotide on a DNA strand).
• In 10 years, the database will contain the equivalent of approximately 1,000 human genomes, and in 20 years, the equivalent of 100,000 human genomes.
• Achieving the productivity necessary for continued exponential growth of sequence information will require new, intrinsically scalable sequencing methods with no inherent operational limits.
• Researchers have proposed using digital-microfluidic devices in several competing technologies to reduce reagent costs, which, along with instrument cost, are the primary cost of Sanger-based sequencing (the most common sequencing method, developed by Fred Sanger).

On-chip sequencing by synthesis

• The authors are currently evaluating the digital-microfluidic platform for performing miniaturized sequencing by synthesis.
• The reaction to incorporate a nucleotide is carried out by DNA polymerase.
• The entire pyrosequencing process takes 3 to 4 seconds per nucleotide added.
• Unlike traditional pyrosequencing approaches, this approach does not limit the system to detecting light before the next nucleotide is added.
• The luciferase completes the light generation in less than 0.2 seconds.

Pyrosequencing design issues

• One of the chief design issues for performing pyrosequencing on an electrowetting chip platform is DNA immobilization through surface attachment.
• Because the platform's top plate is glass, it opens new possibilities for surface chemistry and enables convenient chip reuse.
• On-chip DNA immobilization is feasible with gold particles, although droplet transport over gold particles dislodges them.
• Additionally, biotinylated DNA is guaranteed to bind well to streptavidin because streptavidin has an extremely high affinity for biotin.
• Such scaling is critical to massively parallel on-chip DNA sequencing at high clock rates.

On-chip tissue engineering

• Tissue engineering (TE) is evolving as a potential method for the repair and reconstruction of diseased or damaged tissues.
• The scaffold should not only mimic the tissue's biological functions but also provide mechanical support of the tissue during the reconstruction process, maintain the tissue's initially fabricated 3D shape, and protect the tissue from handling during implantation and in vivo loading.
• On-chip reservoirs dispensed each solution, and the chip then combined the two droplets by actuating them into each other.
• 46 Efforts in all these areas are necessary to put the field on a solid footing and to find the unique niche that the technology can fill.
• Investigators have conducted extensive research on the basic principles and operations underlying the implementation of electrowetting-based digital-microfluidic systems.

Figures

Figure 4. Optical absorbance measurement instrumentation monitors color change due to on-chip colorimetric reactions. (cSt is centiStokes.)

Figure 10. Advanced Liquid Logic’s prototype pyrosequencing chip, with onchip reservoirs for dispensing reagents and nucleotides A, T, C, and G. The DNA is attached to sample location S.

Figure 5. Absorbance measured as a function of time for nine serial glucose assays— three different glucose concentrations (40 mg/dl, 50 mg/dl, and 120 mg/dl) assayed three times each.8 Here, AU is absorbance units, y is the slope of the linear curve fit, R is

Figure 9. Solid-phase pyrosequencing (sequencing done on a solid platform, as opposed to a liquid phase). After incorporation of a nucleotide—in this

Figure 1. Electrowetting effect on a digital-microfluidic platform. Because of the conductive top plate and the individually addressed buried

Figure 6. Calibration curve of absorbance versus TNT concentration, demonstrating a linear relation on the electrowetting chip.14

Figure 7. Absorbance spectrum of colored complexes formed in the reaction of TNT and DNT with KOH, using dimethyl sulfoxide (DMSO) as the solvent.14

Figure 3. Fully automated and integrated operation of a multiplexed assay lab on a chip (LoC), with two droplets (samples 1 and 2) undergoing pipelined glucose assays.

Figure 2. Coplanar actuation array for droplet scanning: top view (a) and side view of section A-A (b). Surface electrodes provide electrical contact to the droplet, making a top contact plate unnecessary. (This figure is reproduced, with

Figure 8. Scanning-droplet particle collection process. Arrows indicate direction of droplet scan, and frame numbers show the time sequence. The droplet picks up the deposited 1-micron beads on the surface, leaving behind a cleaned swath. The droplet does not redeposit beads once it

Full text

Chemical and Biological
Applications of Digital-
Microfluidic Devices
Richard B. Fair, Andrey Khlystov, Tina D. Tailor,
Vladislav Ivanov, and Randall D. Evans
Duke University
Vijay Srinivasan, Vamsee K. Pamula, and
Michael G. Pollack
Advanced Liquid Logic
Peter B. Griffin
Stanford University
Jack Zhou
Drexel University
&DIGITAL-MICROFL UIDIC LAB-ON-A-CHIP (LoC) tech-
nology offers a platform for developing diagnostic
applications with the advantages of portability, sample
and reagent volume reduction, faster analysis, increased
automation, low power consumption, compatibility
with mass manufacturing, and high throughput. In
addition to diagnostics, digital microfluidics is finding
use in airborne chemical detection, DNA sequencing by
synthesis, and tissue engineering.
In this article, we review efforts to develop various
LoC applications using electrowetting-based digital
microfluidics. We describe these applications, their
implementation, and associated design issues. The
‘‘Related work’’ sidebar gives a brief overview of
microfluidics technology.
Electrowetting technology
Electrowetting is the phenomenon whereby
an electric field modifies the wetting behavior of
a polarizable or conductive liquid droplet in contact
with a hydrophobic, insulated electrode.
1
Figure 1
illustrates this effect. Applying a voltage to a series of
adjacent electrodes that can be turned on or off
creates an i nterfacial tension gradient
that can manipulate droplets. Droplets
are usually sandwiched between two
parallel plates; the bottom plate is the
chip surface, which houses the address-
able electrode array, and the top is
either a continuous ground plate or
a passive top plate (the chip’s char-
acteristics determine the top plate’s nature).
In coplanar designs, both the buried activation
electrode and the exposed electrodes that ground the
droplet are located on the bottom surface.
2,3
As
Figure 2 shows, the top plate is not required for
coplanar devices, but we advise using it to contain the
oil and the droplets. Also, manufacturers or users can
customize the passive top plate with specific chemistry
or structures appropriate for each application. Thus,
the top plate can be a disposable component.
We have demonstrated electrowetting-based systems
for manipulating microliter- and nanoliter-size droplets
in LoC protocols.
4–6
Using the electrowetting principle,
we can transport discrete droplets in a highly controlled
way over an array of electrodes. We can reconfigure the
array to transport the droplets or hold the droplets as
virtual reaction chambers where mixing takes place. To
view videos of electrowetting-induced droplet motion,
visit http://www.ee.duke.edu/research/microfluidics
and http://cjmems.seas.ucla.edu/index.html.
The LoC chip surface is coated with an insulating
layer of parylene C (about 800 nm), and both the top
and bottom surfaces are covered with a Teflon-AF thin
Editor’s note:
Digital-mi crofluidic technology offers a revolutionary platform for many
chemical and biological applications. Learn how to manipulate droplets and
process chemical and biological samples on chip for clinical diagnostics,
gene sequencing, airborne chemical detection, and tissue engineering.
—Krishnendu Chakrabarty, Duke University
Biochips
0740-7475/07/$25.00
G
2007 IEEE Copublished by the IEEE CS and the IEEE CASS IEEE Design & Test of Computers
10

Related work
Much of the reported work on lab-on-a-chip (LoC)
microfluidic devices has focused on miniaturization of
analytical methods and protocols to improve perfor-
mance and throughput. Researchers have demonstrated
the benefits of miniaturization, such as smaller sample
requirements, reduced reagent consumption, de-
creased analysis time, and higher levels of throughput
and automation. Most LoC devices have aimed at
performing chemical or biological protocols on chip,
with pre-prepared samples processed off chip. Thus,
few researchers have reported work on integrating front-
end functions such as sample collection, analyte
extraction, preconcentration, and filtration, with the
required analytical operations then performed on the
chip.
1
Currently, almost all microfluidic devices are based
on continuous fluid flow in permanent microchannels in
glass, plastic, or other polymers. De Mello and Beard
present a review of sample pretreatment with continu-
ous-flow microfluidic systems.
1
However, continuous-
flow-based microfluidic devices offer very little flexibility
in scalability and reconfigurability, and they are usually
application specific.
An alternative approach to microfluidics is to
manipulate the liquid as unit-sized discrete microdro-
plets. Because of its architectural similarities to digital
microelectronic systems, we often refer to this ap-
proach as ‘‘digital’’ microfluidics. Digital-microfluidic
systems have several advantages over continuous-flow
systems; the most important are reconfigurability and
architecture scalability.
2
Electrowetting
2
and dielectro-
phoresis
3
are the two most commonly used micro-
droplet actuation techniques, although other methods
have been demonstrated, such as thermocapillary
actuation
4
and surface acoustic wave actuation.
5
Electrowetting is primarily a contact line phenomenon
consisting of electric-field-induced interfacial tension
changes between a liquid and a solid conductor.
The use of electrowetting for dispensing, transport-
ing, splitting, merging, and mixing aqueous droplets has
appeared in the literature previously.
2,6–10
Researchers
have addressed the problem of chip surface contami-
nation through biomolecular adsorption by using a sili-
cone oil medium
9
or by controlling the application of
actuation voltages.
11
Fair et al. demonstrated sample
collection and preconcentration on the same chip.
12
Chatterjee et al. demonstrated that a variety of organic
and inorganic substances can be actuated in droplet
form.
13
Their results suggest that actuation of non-
conducting liquids might not occur through electrowet-
ting but rather through some other mechanism such as
dielectrophoresis, as others have suggested.
14,15
References
1. A.J. de Mello and N. Beard, ‘‘Dealing with ‘Real’ Samples:
Sample Pre-Treatment in Microfluidic Systems,’’ Lab on
a Chip, vol. 3, no. 1, 2003, pp. 11N-19N.
2. M.G. Pollack, A.D. Shenderov, and R.B. Fair, ‘‘Electrowet-
ting-Based Actuation of Droplets for Integrated Micro-
fluidics,’’ Lab on a Chip, vol. 2, no. 1, 2002, pp. 96-101.
3. J.A. Schwartz, J.V. Vykoukal, and P.R.C. Gascoyne,
‘‘Droplet-Based Chemistry on a Programmable Micro-
Chip,’’ Lab on a Chip, vol. 4, no. 4, 2004, pp. 11-17.
4. A.A. Darhuber et al., ‘‘Thermocapillary Actuation of
Droplets on Chemically Patterned Surfaces by Program-
mable Microheater Arrays,’’ IEEE/ASME J. Microelectro-
mechanical Systems, vol. 12, no. 6, Dec. 2003, pp. 873-
879.
5. A. Renaudin et al., ‘‘Plateforme SAW De´die´e a`la
Microfluidique Discre´ te pour Applications Biologiques
[SAW Platform Dedicated to Discrete Microfluidics
for Biological Applications],’’ Proc. 2nd French Congress
Microfluidics, French Hydrotechnical Society, 2004 (in
French).
6. H. Ren, V. Srinivasan, and R.B. Fair, ‘‘Automated Electro-
wetting-Based Droplet Dispensing with Good Reproduc-
ibility,’’ Proc. 7th Int’l Conf. Micro Total Analysis Systems
(MicroTAS 03), Transducers Research Foundation, 2003,
pp. 993-996.
7. P. Paik et al., ‘‘Electrowetting-Based Droplet Mixers for
Microfluidic Systems,’’ Lab on a Chip, vol. 3, no. 1, 2003,
pp. 28-33.
8. P. Paik, V.K. Pamula, and R.B. Fair, ‘‘Rapid Droplet Mixers
for Digital Microfluidic Systems,’’ Lab on a Chip, vol. 3, no.
4, 2003, pp. 253-259.
9. V. Srinivasan, V.K. Pamula, and R.B. Fair, ‘‘A Droplet-
Based Microfluidic Lab-on-a-Chip for Glucose Detection,’’
Analytica Chimica Acta, vol. 507, no.1, 2004, pp. 145-150.
10. V. Srinivasan et al., ‘‘Clinical Diagnostics on Human Whole
Blood, Plasma, Serum, Urine, Saliva, Sweat, and Tears on
a Digital Microfluidic Platform,’’ Proc. 7th Int’l
Continued on p. 12
January–February 2007
11

film (about 50 nm) to ensure a continuous hydropho-
bic platform, which is necessary for smooth droplet
actuation. A spacer separates the top and bottom
plates, resulting in a fixed gap height. The gap is
usually flooded with silicone oil that acts as a filler
fluid, preventing droplet evaporation and reducing
surface contamination.
1
Figure 3 shows a typical LoC
platform with multiple electrodes, reservoirs, and
detection sites. The photo is a snapshot of two
pipelined glucose assays in process.
5
Unlike a continuous-flow microfluidic platform,
a digital-microfluidic platform operates under soft-
ware-driven electronic control, eliminating the need
for mechanical tubes, pumps, and valves. Digital
platform protocols work similarly to traditional
bench-top methods, except that they use more
automation and significantly smaller sample sizes.
Users can merge, split, transport, mix, and incubate
droplets by programming electrodes to carry out
specific tasks. A digital-microfluidic platform offers
many advantages for real applications. It
& has no moving parts. All operations take place
between the two plates under direct electrical
control without use of pumps or valves.
& requires no channels. The gap is simply filled with
liquid; channels exist only in a virtual sense and
can be instantly reconfigured through software.
& controls many droplets independently because the
electrowetting force is localized at the surface.
& controls or prevents evaporation with the oil
surrounding the droplets.
& uses no ohmic current. Although capacitive currents
exist, the device blocks direct current, minimizing
sample heating and electrochemic al reactions.
& works with a wide variety of liquids—that is, most
electrolyte solutions.
& makes close to 100% utilization of
the sample or reagent possible by
wasting no fluid for priming chan-
nels or filling reservoirs.
& is comp atible with microscopy.
Glass substrates and transparent
indium-tin-oxide (ITO) electrodes
make the chip compatible with
observation from a microscope.
& is extremely energy efficient—using
nanowatts to microwatts of power
per transfer.
& achieves high droplet speeds—up
to about 25 cm/s.
Figure 1. Electrowetting effect on a digital-microfluidic platform. Because
of the conductive top plate and the individually addressed buried
electrodes in the bottom plate, applying a voltage can actuate the droplet
from one electrode position to the next.
Continued from p. 11
Conf. Micro Total Analysis Systems (MicroTAS 03), Trans-
ducers Research Foundation, 2003, pp. 1287-1290.
11. J.-Y. Yoon and R.L. Garrell, ‘‘Preventing Biomolecular
Adsorption on Electrowetting-Based Biofluidic
Chips,’’ Analytical Chemistry, vol. 75, no. 19, 1 Oct.
2003, pp. 5097-5102.
12. R.B. Fair et al., ‘‘Integrated Chemical/Biochemical
Sample Collection, Pre-concentration, and Analysis on
a Digital Microfluidic Lab-on-a-Chip Platform,’’ Lab-on-
a-Chip: Platforms, Devices, and Applications, L.A.
Smith and D.
Sobek, eds., Proc. SPIE, vol. 5591, 8 Dec. 2004, pp.
113-124.
13. D. Chatterjee et al., ‘‘Droplet-Based Microfluidics
with Nonaqueous Solvents and Solutions,’’ Lab on
a Chip, vol. 6, no. 2, 2006, pp. 199-206.
14. T.B. Jones, ‘‘On the Relationship of Dielectrophoresis
and Electrowetting,’’ Langmuir, vol. 18, no. 11, 28
May 2002, pp. 4437-4443.
15. P.R.C. Gascoyne and J.V. Vykoukal, ‘‘Dielectroph oresis-
Based Sample Handling in General-Purpose Program-
mable Diagnostic Instruments,’’ Proc. IEEE,vol.92,
no.1,Jan.2004,pp.22-42.
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& uses droplet-based protocols functionally equiva-
lent to bench-scale wet chemistry. Thus, users can
simply scale down, automate, and integrate
established assays and protocols.
& permits maximum operational flexibility. Direct
computer control of each step allows conditional
execution steps.
Applications and design issues
In most diagnostic and chemical detection applica-
tions, a key challenge is the preparation of the analyte
for presentation to the on-chip detection system. In
diagnostics, raw physiological samples must be in-
troduced onto the chip and then further processed by
lysing blood cells and extracting DNA. For massively
parallel DNA sequencing, scientists can prepare
samples off chip, but they must perform synthesis steps
in a sequential on-chip format through automated
control of buffers and nucleotides to extend the read
lengths of DNA fragments. In real-time airborne-
particulate-sampling applications, the process of sam-
ple collection from an air stream must be integrated
into the LoC analytical component. One way to
accomplish this is with a collection droplet that scans
an exposed impacted surface and then is introduced
into a closed analytical section. In tissue-engineering
applications, the challenge is to build high-resolution
(less than 10 microns) 3D tissue constructs with
embedded cells and growth factors by manipulating
and maintaining live cells
on the chip platform.
Here, we highlight these
new applications, includ-
ing detection of airborne
sulfates obtained by air
sampling, DNA pyrose-
quencing, and a biomimet-
ic manufacturing process
for soft-tissue engineering.
On-chip assays
On-chip assays for de-
termining the concentra-
tions of target analytes is
a natural application for
digital microfluidics. Work
in this area has focused on
multiplexed assays, which
measure multiple analytes
in a single sample, as Figure 3 shows. Initially,
scientists must determine the compatibility of each
chemical substance with the electrowetting platform.
Compatibility issues include the following:
& Do the liquid’s viscosity and surface tension
allow droplet dispensing and transport by elec-
trowetting?
Figure 3. Fully automated and integrated operation of a multiplexed assay lab on a chip
(LoC), with two droplets (samples 1 and 2) undergoing pipelined glucose assays.
Figure 2. Coplanar actuation array for droplet scanning: top
view (a) and side view of section A-A (b). Surface electrodes
provide electrical contact to the droplet, making a top contact
plate unnecessary. (This figure is reproduced, with
permission, from R.B. Fair et al., ‘‘Integrated Chemical/
Biochemical Sample Collection, Pre-concentration, and
Analysis on a Digital Microfluidic Lab-on-a-Chip Platform,’’ Lab-
on-a-Chip: Platforms, Devices, and Applications, L.A. Smith
and D. Sobek, eds., Proc. SPIE, vol. 5591, 8 Dec. 2004, pp. 113–
124.)
January–February 2007
13

& Will th e drop let’s conten ts foul the chi p’s
hydrophobic surfaces?
& In systems with a silicone oil medium, will the che-
micals in the droplet cross the droplet-oil interface,
thus reducing the droplet’s content?
Three examples of successful on-chip assay are
glucose, TNT, and sulfate assays. These three exam-
ples use some or all of the following on-chip process
steps:
& Load prediluted sample and reagent into on-chip
reservoirs.
& Dispense droplets of analyte solutions and
reagents.
& Transport droplets.
& Mix analyte solution and reagent droplets.
& Detect reaction products.
Glucose assay
The in vitro measurement of glucose in human
physiological fluids is of great importance in clinical
diagnosis of metabolic disorders. Srinivasan, Pamula, and
Fair have demonstrated an LoC for glucose assay using
a colorimetric enzyme-kinetic method based on Trinder’s
reaction,
7
which determines glucose concentration.
5
Figure 4 shows a schematic drawing of the assay
detection system. We perform on-chip glucose assay in
three steps: dispensing, mixing, and detection. First,
we pipette droplets of the glucose sample and the
reagent onto the electrowetting chip. The LoC then
merges and physically mixes the sample and the
reagent by shuttling the coalesced droplet across three
electrodes for 15 seconds, at a switching rate of 8 Hz
and an actuation voltage of 50 V. The time for the
mixing protocol is more than is required and can be
reduced to less than five seconds.
4
At the end of the
mixing phase, the 545-nm light-emitting-diode (LED)
and photodiode setup measures the absorbance for at
least 30 seconds. Electrowetting forces hold the mixed
droplet stationary during absorbance measurement.
Because absorbance measurement begins 15 seconds
after the droplets merge, the measured reaction rate
might not exactly equal the initial reaction rate.
This device makes detection of glucose concentra-
tions possible in the range of 25 mg/dl to 300 mg/dl,
using dilution factors as low as 2 and 3, in less than 60
seconds. The results compare favorably with conven-
tional measurements on a spectrophotometer, imply-
ing no significant change in enzyme activity under
electrowetting. Reproducibility of the measurement
for the same sample concentration over multiple
measurements on the same chip was less than 2%,
indicating excellent control of droplet volumes and no
cross-contamination.
8
Glucose assay design issues
Any efficient or moderate-throughput microfluidic
architecture inevitably requires droplets to share
microfluidic resources on the chip (transport lanes,
mixers, and incubators). Cross-contamination poten-
tially can occur whenever droplets containing differ-
ent samples are manipulated in the same chip area.
Researchers have demonstrated the transport of non-
biological electrolytes using electrowetting both in air
9
and in other immiscible media such as silicone oil.
5,10
In contrast, the transport of fluids containing proteins,
such as enzyme-la den reagents and human physiolog-
ical fluids, is not as straightforward, because most
proteins adsorb irreversibly to hydrophobic surfaces
and contaminate them. In the electrowetting system,
the liquid droplet is sandwiched between two
hydrophobic (Teflon AF-coated) plates. Any contact
between the liquid droplet and the Teflon AF surface
will therefore contaminate the surface.
In addition to contaminating the surface, protein
adsorption can also render it permanently hydrophil-
ic.
11
This effect is detrimental to transport because
electrowetting works on the principle of modifying
a hydrophobic surface’s wettability. Therefore, to
prevent contamination and enable transport, we must
avoid contact between a liquid droplet containing
proteins and the Teflon surface. Thus, air is a less
desirable filler medium for assays involving proteins
because the droplet will always be in contact with the
Teflon surface.
9
Researchers have reported exceptions for the use of
air medium systems in applications in which it is
desirable to deposit proteins on surfaces, such as
matrix-assisted laser desorption/ionization mass spec-
trometry (MALDI-MS).
12
However, for glucose assays,
silicone oil, with its low surface tension and spreading
property, is an ideal alternative. From visual observa-
tions and electrical capacitance measurements during
the transport of droplets in silicone oil, we have
inferred the presence of a thin film of oil encapsulating
the droplet. This film isolates the droplet from the
Teflon surfaces , minimizing adsorption and facilitating
transport.
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Frequently asked questions

Q1. What are the contributions in "Chemical and biological applications of digital- microfluidic devices" ?

In this article, the authors review efforts to develop various LoC applications using electrowetting-based digital microfluidics. The authors describe these applications, their implementation, and associated design issues. 

Q2. What are the common methods of actuation of microdroplets?

2 Electrowetting2 and dielectrophoresis3 are the two most commonly used microdroplet actuation techniques, although other methods have been demonstrated, such as thermocapillary actuation4 and surface acoustic wave actuation. 

Q3. What is the promising method of generating a tissue scaffold?

A promising method of generating a tissue scaffold is electrowetting printing—embedding cells and growth factors in hydrogels and printing the hydrogel-containing cells onto the hydrogel-containing growth factors. 

Q4. Why do the authors need to know the sequence of the unknown strand?

Because the authors know the order in which the nucleotide addition occurs, the authors can determine the unknown strand’s sequence by formation of its complementary strand. 

Q5. What are the primary unmet needs for pyrosequencing?

The primary unmet needs are the following:& manufacturing techniques that can mimic tissueand extracellular matrix architecture with high resolution (less than 10 microns) for tissues such as myocardium (heart muscle), blood vessels, bone, and nerves; & digital automation methods for delivery of cellsand growth factors into tissue scaffolds (artificial structures that support the tissue);44 and & manufacturing techniques to place vascularstructures in engineered tissue, in which a lack of nutrient transport currently limits the size and cellular content of implants. 

Q6. What is the effect of droplet volume on the accuracy of electrowetting?

For the electrowetting system, within-run precision is mainly affected by droplet volume errors, cross-contamination between experiments, and measurement errors. 

Q7. What is the importance of glucose in clinical diagnosis?

The in vitro measurement of glucose in human physiological fluids is of great importance in clinical diagnosis of metabolic disorders. 

Q8. What are the advantages of continuousflow microfluidics?

continuousflow-based microfluidic devices offer very little flexibility in scalability and reconfigurability, and they are usually application specific. 

Q9. How does the LoC mix the sample and the reagent?

The LoC then merges and physically mixes the sample and the reagent by shuttling the coalesced droplet across three electrodes for 15 seconds, at a switching rate of 8 Hz and an actuation voltage of 50 V. 

Q10. What did the researchers do to merged the two droplets?

Applying voltages to the appropriate electrodes merged the two droplets, and transporting the merged droplet mixed it still further by enhancing the mixing flow. 

Q11. What is the first known demonstration of the electrowetting method?

The first experiments performed by us addressed whether a 2% sodium alginate solution (with a viscosity of 250 centipoise) and a calcium chloride cross-linker solution could be actuated, dispensed, and reacted on an electrowetting chip with a silicone oil medium. 

Q12. What is the main cost of Sanger-based sequencing?

Researchers have proposed using digital-microfluidic devices in several competing technologies to reduce reagent costs, which, along with instrument cost, are the primary cost of Sanger-based sequencing (the most common sequencing method, developed by Fred Sanger). 

Q13. What is the way to measure the concentration of analytes?

On-chip assaysOn-chip assays for determining the concentrations of target analytes is a natural application for digital microfluidics. 

Q14. What are the main issues that are preventing the development of digital microfluidics?

While researchers are making progress in furthering the understanding of electrowetting phenomena from a fundamental perspective, other efforts are exploring the applications, novel device structures, and CAD methods possible in digital microfluidics. 

Q15. What is the way to manipulate droplets?

Learn how to manipulate droplets and process chemical and biological samples on chip for clinical diagnostics, gene sequencing, airborne chemical detection, and tissue engineering. 

Q16. What is the effect of electrowetting on the surface of a liquid droplet?

This effect is detrimental to transport because electrowetting works on the principle of modifying a hydrophobic surface’s wettability. 

Q17. What is the main obstacle to the adoption of microfluidics?

a lack of good on-chip sample preparation methods currently is the greatest impediment to commercial acceptance of microfluidic technologies, including digital microfluidics. 

Q18. What is the role of the lysing of blood cells in the analysis of biological samples?

In diagnostics, raw physiological samples must be introduced onto the chip and then further processed by lysing blood cells and extracting DNA. 

Q19. How many microns is the optical path length in the detector shown in Figure 4?

In the detector shown in Figure 4, the optical path length is 100 to 300 microns, which is30 to 100 times smaller than in conventional systems (10 mm). 

Q20. What is the simplest method of determining the concentration of analytes?

The LoC quantitatively determines the analyte’s concentration by measuring absorption at target wavelengths using a simple LED and photodiode setup. 

Q21. What is the way to perform a miniaturized sequencing?

Design & Test of Computers18ing by hybridization,28–32 sequencing by synthesis,33 single-molecule sequencing,34,35 miniaturized electrophoresis,36–38 and miniaturized pyrosequencing (see http://www.454.com).On-chip sequencing by synthesisThe authors are currently evaluating the digital-microfluidic platform for performing miniaturized sequencing by synthesis.