Review

pubs.acs.org/CR

Advances in Microfluidic Materials, Functions, Integration, and

Applications
Pamela N. Nge, Chad I. Rogers, and Adam T. Woolley*
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States
4.7. Opportunities for Future Development 2573
5. Applications 2573
5.1. Point-of-Care 2573
5.2. Cell Analysis 2574
5.3. Nucleic Acid Assays 2575
5.4. Drug Metabolism 2575
5.5. Omics 2576
5.5.1. Proteomics 2576
5.5.2. Metabolomics 2576
CONTENTS 5.6. Environmental Analysis 2576
5.7. Space Exploration 2577
1. Introduction 2550 5.8. Opportunities for Future Development 2577
2. Materials for Microfluidics 2553 6. Conclusions and Outlook 2577
2.1. Inorganic Materials 2553 Author Information 2578
2.1.1. Silicon 2553 Corresponding Author 2578
2.1.2. Glass 2553 Notes 2578
2.1.3. Ceramics 2554 Biographies 2578
2.2. Polymers 2554 Acknowledgments 2578
2.2.1. Elastomers 2555 References 2578
2.2.2. Thermoplastics 2556
2.3. Paper 2558
2.4. Opportunities for Future Development 2559 1. INTRODUCTION
3. Functions in Lab-on-a-Chip Systems 2559 Microfluidics consist of microfabricated structures for liquid
3.1. Sample Preparation 2559 handling, with cross sections in the 1−500 μm range and small
3.1.1. Extraction and Purification 2559 volume capacity (femtoliter to nanoliter). Capillary tubes
3.1.2. Preconcentration 2561 connected with fittings,1 although utilizing small volumes, are
3.1.3. On-Chip Labeling 2562 not considered microfluidics for the purposes of this paper
3.2. Separation Methods 2562 since they are not microfabricated. Likewise, millifluidic systems
3.2.1. Chromatography 2562 made by conventional machining tools are excluded due to
3.2.2. Electrophoresis 2563 their larger feature sizes (>500 μm).
3.2.3. Fractionation 2564 Though micromachined systems for gas chromatography
3.3. Sensing and Detection 2564 were introduced in the 1970s,2 the field of microfluidics did not
3.3.1. Optical Detection 2564 gain much traction until the 1990s.3 Silicon and glass were the
3.3.2. Electrochemical Detection 2565 original materials used, but then the focus shifted to include
3.3.3. Mass Spectrometry 2566 polymer substrates, and in particular, polydimethylsiloxane
3.3.4. Biosensors 2567 (PDMS). Since then the field has grown to encompass a wide
3.4. Fluid Manipulation 2567 variety of materials and applications. The successful demon-
3.4.1. Pumps 2567 stration of electrophoresis and electroosmotic pumping in a
3.4.2. Valves 2569 microfluidic device provided a nonmechanical method for both
3.4.3. Mixing 2569 fluid control and separation.4 Laser-induced fluorescence (LIF)
3.5. Opportunities for Future Development 2570 enabled sensitive detection of fluorophores or fluorescently
4. Integration 2571 labeled molecules. The expanded availability of low-cost
4.1. Overview 2571 printing allowed for cheaper and quicker mask fabrication for
4.2. Fluidic Interconnects 2571 use in soft lithography.5 Commercial microfluidic systems are
4.3. Microdialysis 2572 now available from Abbott, Agilent, Caliper, Dolomite,
4.4. Sample Preparation: Extraction and Purifica- Micralyne, Microfluidic Chip Shop, Micrux Technologies, and
tion 2572
4.5. Droplet Microfluidics 2572
4.5.1. Closed Droplet Microfluidics 2572 Special Issue: 2013 New Frontiers in Bioanalytical Chemistry
4.5.2. Open Droplet Microfluidics 2573 Received: August 15, 2012
4.6. Digital PCR 2573 Published: February 14, 2013

© 2013 American Chemical Society 2550 dx.doi.org/10.1021/cr300337x | Chem. Rev. 2013, 113, 2550−2583
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Table 1. Summary of Properties of Materials That Have Been or Could Be Used for μCE
optical UV surface surface
material clarity transparency EOF (× 10−4 cm2 V−1 s−1) separation chemistry charge refs
glass good >280 nm 3 (pH 7.0); 4.21 ± 0.18 (pH 9.2) yes silanol negative 15, 33,
36
silicon opaque none a a silicon/silanol requires 20
oxidation
LTCC opaque none 3.00 ± 0.05 (pH 7.0) yes aluminum negative 33
oxide
Ormocer moderate >300 nm 3.3 ± 0.3 (pH 10.0, lithography); 5.3 ± 0.1 (pH 10.0, UV- yes a negative 36, 37
embossed)
PDMS good >220 nm 2.5 ± 0.5 (pH 6.0); 4.9 ± 0.9 (pH 9.0) yes silanol negative 36, 79
(native)
PDMS good >220 nm 8.2 (pH 9.0) yes silanol negative 49
(oxidized)
TPE moderate >400 nm 2.2 (10 mM SDS); ∼2.5 (pH 6.0); ∼1.7 (pH 7.0); ∼0.6 (pH yes polyester requires 49−52
9.0); ∼1.4 (pH 10.0); ∼0.10 (pH 6−10, PEG-grafted); ∼2.6 oxidation
(oxidized)
PFPE moderate >350 nm a no fluorocarbon a 55, 56
PS good >300 nm 1.54 ± 0.03 (pH 9.2) yes phenyl requires 15
oxidation
PC good >360 nm 2.22 ± 0.09 (pH 9.2) yes phenyl/ requires 15
carbonate oxidation
PMMA good >340 nm 2.07 ± 0.07 (pH 9.2) yes acrylate negative 15, 49
PEGDA good >300 nm a yes acrylate/PEG negative 76
PEGDA/ good >300 nm 0.63 ± 0.02 (pH 8.7) yes acrylate/PEG negative 75
PMMA
PDMS-co- moderate >440 nm 2.5 ± 0.7 (pH 6.0); 5.7 ± 0.8 (pH 9.0) yes silanol/PEO negative 79
PEO (5%)
PDMS-co- moderate >440 nm 1.2 ± 0.3 (pH 6.0); 2.5 ± 0.3 (pH 9.0) yes silanol/PEO negative 79
PEO
(10%)
FEP/PFA moderate a a no fluorocarbon a 81
COC good >360 nm 3.08 ± 0.28 (TBE buffer) yes cyclic olefin requires 82
oxidation
thiol−ene moderate a a no thiol/allyl a 66−68
PU (castor moderate a 2.6 (pH 7.0) yes carbamate/ negative 88
oil) ricinoleic
acid
PU moderate >300 nm 2.47 (pH 7.0) yes carbamate/ negative 89
(tecothane phenyl
TT-1095A)
zein moderate a a no zein a 91
SU-8 moderate >360 nm a yes epoxy/phenyl negative 36
paper opaque none a a cellulose a 97, 98
a
Information that was not reported in the literature.

Waters, as a few prominent examples. For a more thorough phones, video games, and medical and biological sensors.13
description of the history of microfluidics, we refer the reader to Micro-total-analysis systems, also known as laboratories-on-a-
a number of comprehensive, specialized reviews,3,6−11 as well as chip, are the chemical analogue of MEMS, as integrated
a more general 2006 review.12 microfluidic devices that are capable of automating multiple
The field of microfluidics offers many advantages compared processes relevant to laboratory sciences. For example, a typical
with carrying out processes through bulk solution chemistry, lab-on-a-chip system might selectively purify a complex mixture
the first of which relates to a lesson taught to every first-year (through filtering, antibody capture, etc.) and then separate
chemistry student. Simply stated, diffusion is slow! Thus, the target components and detect them.
smaller the distance required for interaction, the faster it will be. Microfluidic devices consist of a core of common
Smaller channel dimensions also lead to smaller sample components. Areas defined by empty space, such as reservoirs
volumes (femtoliter to nanoliter), which can reduce the (wells), chambers, and microchannels, are central to micro-
amount of sample or reagents required for testing and analysis. fluidic systems. Positive features, created by areas of solid
Reduced dimensions can also lead to portable devices to enable material, add increased functionality to a chip and can consist of
on-site testing (provided the associated hardware is similarly membranes, monoliths, pneumatic controls, beams, and pillars.
portable). Finally, integration of multiple processes (like Given the ubiquitous nature of components defined by empty
labeling, purification, separation, and detection) in a micro- space, and microchannels in particular, we focus here on a few
fluidic device can be highly enabling for many applications. of their properties. Microfluidic channels have small overall
Microelectromechanical systems (MEMS) contain integrated volumes, laminar flow, and a large surface-to-volume ratio.
electrical and mechanical parts that create a sensor or system. Dimensions of a typical separation channel in microchip
Applications of MEMS are ubiquitous, including automobiles, electrophoresis (μCE) are 50 μm width, 15 μm height, and 5
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Figure 1. Microcantilevers (a−e) and a microring resonator (f) made from silicon. (a) Schematic diagram of waveguides and microcantilever array
layout on die. (b) Optical image of two microcantilevers in a fabricated array. (c) Close up scanning electron micrograph (SEM) image of the
unclamped end of a microcantilever (left of 165 nm gap) and the differential splitter capture waveguide (right of gap). (d) Photograph of complete
integrated device showing the fluid microchannels (red) and control valves (green). (e) Cross-section of fluid microchannel at a microcantilever
array. (f) Top-view SEM image of a microring resonator and linear waveguide, visible through an annular opening in the fluoropolymer cladding
layer. Reprinted with permission from refs 18 and 19. Copyright 2009 American Chemical Society and 2011 The Royal Society of Chemistry.

cm length for a volume of 37.5 nL. Flow in these devices is magnitude of the potential decreasing as distance from the
normally nonturbulent due to low Reynolds numbers. For surface increases. The electrical double layer is the basis for
example, water flowing at 20 °C in the above channel at 1 μL/ EOF, wherein an applied voltage causes the loosely bound
min (2.22 cm/s) results in a Reynolds number of ∼0.5, where diffuse layer to move toward an electrode, dragging the bulk
<2000 is laminar flow. Since flow is nonturbulent, mixing is solution along. Charges on the exposed surface also exert a
normally diffusion-limited. Small channel sizes also have a high greater influence on the fluid in a channel as its size decreases.
surface-to-volume ratio, leading to different characteristics from Larger surface-to-volume ratios are more prone to nonspecific
those commonly found in bulk volumes. The material surface adsorption and surface fouling. In particular, noncharged and
can be used to manipulate fluid movement (such as by hydrophobic microdevice surfaces can cause proteins in
electroosmotic flow, EOF) and surface interactions. For a solution to denature and stick.
solution in contact with a charged surface, a double layer of We focus our review on advances in microfluidic systems
charge is created as oppositely charged ions are attracted to the since 2008. In doing this, we occasionally must cover
surface charges. This electrical double layer consists of an inner foundational work in microfluidics that is considerably less
rigid or Stern layer and an outer diffuse layer. An electrostatic recent. We do not focus on chemical synthesis applications of
potential known as the ζ potential is formed, with the microfluidics although it is an expanding area, nor do we delve
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into lithography, device fabrication, or production costs. Our vapor deposition) to create structures.20 Iliescu et al.20 recently
specific emphasis herein is on four areas within microfluidics: reviewed silicon and glass microfluidic systems. Silicon has a
properties and applications of commonly used materials, basic high elastic modulus (130−180 GPa) and is not easily made
functions, integration, and selected applications. For each of into active fluidic components such as valves and pumps.
these four topics, we provide a concluding section on Silicon surface chemistry based on the silanol group (−Si−OH)
opportunities for future development, and at the end of this is well developed, so modification is easily accomplished via
review, we offer general conclusions and prospective for future silanes. For example, nonspecific adsorption can be reduced or
work in the field. Due to the considerable scope of the field of cellular growth improved through chemical modification of the
microfluidics, we limit our discussion to selected examples from surface.21,22 Silicon is transparent to infrared but not visible
each area, but cite in-depth reviews for the reader to turn to for light, making typical fluorescence detection or fluid imaging
further information about specific topics. We also refer the challenging for embedded structures. This issue can be
reader to recent comprehensive reviews on advances in lab-on- overcome by having a transparent material (polymer or glass)
a-chip systems by Arora et al.10 and Kovarik et al.14 bound to silicon in a hybrid system. Such hybrid devices have
led to a renaissance in Si-based detectors for microfluidic
2. MATERIALS FOR MICROFLUIDICS systems.18,19 For example, Si microcantilevers and optical
Microfluidic device materials initially consisted of silicon and resonators have recently been interfaced with microfluidics, as
glass substrates; as the field advanced other materials were shown in Figure 1. Applications have ranged from droplet-
evaluated. These materials can be organized into three broad based polymerase chain reaction (PCR)23 and cellular culture24
categories: inorganic, polymeric, and paper. Inorganic materials to nanowires for label-free cardiac biomarker detection (Figure
use has broadened beyond glass and silicon to include 2).25
substrates such as low-temperature cofired ceramics and
vitroceramics. Polymer-based materials can be divided into
elastomers and thermoplastics. Paper microfluidics is an
emerging and substantially different technology from devices
made from either polymer or inorganic materials.
There are three main factors to consider when choosing a
design or material for a microfluidic system: required function,
degree of integration and application. Closely related, these
three factors require consideration of both material properties
and fabrication processes. Flexibility, air permeability, electrical
conductivity, nonspecific adsorption, cellular compatibility,
solvent compatibility, and optical transparency are all physical
characteristics that may be important when choosing a material.
In 2006, Shadpour et al.15 published an in-depth review on
material properties and their effects on electrophoretic
separations. Table 1 provides a current summary of character-
istics of materials that have been used or have potential to be
used in μCE. The table reports optical and surface charge
properties, as well as EOF data and whether separations have
been reported in that material. Integration of fluid movement
and control, detection instrumentation, or other aspects of chip
Figure 2. Silicon nanowire system for cardiac biomarker detection. (a)
automation can introduce a higher level of complexity in the Image of silicon nanowire (SiNW) device array chip, integrated with
fabrication process. Aqueous-based solutions can be used in a microfluidic system for fluid exchange. Fluids are deposited into the
broad range of materials, and limiting choices are often more a acrylic well through the inflow tube on the left (red arrow) and
matter of personal preference. In contrast, many organic removed from the outflow tube on the right (blue arrow). (b)
solvents will cause polymer substrates to swell, crack, or Schematic showing the layout of the SiNW device array on the chip. A
dissolve. Paper microfluidic devices are limited to capillary total of 36 clusters of 5 nanowires each were available for use. (c) SEM
action for fluid transport through a device. Active components image of a cluster of nanowires. Each nanowire is individually
made from glass or silicon can be batch fabricated but can also addressable by oxide-passivated metal contact lines running out to the
be fragile. The combination of these materials with a edge of the chip. Reprinted with permission from ref 25. Copyright
deformable component such as PDMS can enable integration 2009 American Chemical Society.
of pneumatic pumps and valves.16,17 These considerations are
only a few of those needing to be made when choosing a 2.1.2. Glass. After the initial focus on silicon, glass emerged
material for a specific application. The most important as the substrate of choice for a time. Microstructures are created
question, however, is “what are you trying to accomplish with by etching into the glass through wet or dry methods;20
your device?” formation of fluidic features requires bonding or hybrid layer
attachment to enclose. Since glass has a large, composition-
2.1. Inorganic Materials dependent elastic modulus, hybrid devices are required for
2.1.1. Silicon. Silicon was the first material used for active components such as valves and pumps (Figure 3).16,17
microfluidics2 but then was largely avoided over glass and Glass has low background fluorescence, and as with silicon,
polymers for some time.18,19 Fabrication of silicon (as well as modification chemistries are silanol based. Glass is compatible
glass) devices utilizes either subtractive methods (e.g., wet or with biological samples, has relatively low nonspecific
dry etching) or additive methods (such as metal or chemical adsorption, and is not gas permeable. Mellors et al.26,27
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Figure 3. Microfluidic valve network. Photograph of the multiplexed latching valve test device, with a four-bit demultiplexer (top box) for routing
pressure and vacuum pulses from the single “input” connection to each of 16 latching valves (bottom box). Reprinted with permission from ref 17.
Copyright 2006 The Royal Society of Chemistry.

Figure 4. μCE nanospray glass device. Schematic diagrams of (A) short- and (B) long-channel μCE−ESI-MS chips. The length of the separation
channel was 4.7 cm in panel A and 20.5 cm in panel B. The reservoirs are labeled S (sample), B (buffer), SW (sample waste), and SC (side channel).
The direction of EOF is indicated by the arrows in panel A. (C) CCD image of the electrospray plume generated from the corner of a μCE−ESI-MS
chip. Reprinted with permission from ref 26. Copyright 2008 American Chemical Society.

demonstrated all-glass devices that combined μCE and in aqueous solutions; high rates of leaching occurred with acidic
electrospray ionization-mass spectrometry (ESI-MS) by direct- and basic solutions, while solutions near physiological pH
ing the separation channel to a corner of the device to create a resulted in low leach rates. Electrodes can be deposited onto
nanospray (Figure 4). Zhao et al.28 utilized electrophoresis and LTCC using expansion matched metal pastes. In this manner,
chemiluminescence in an all-glass device to detect glutathione Almeida et al.32 used an LTCC device with potentiometric
from cellular supernatant. Wu et al.29 demonstrated a hybrid detection to analyze sulfamethoxazole and trimethoprim in fish
device containing glass/PDMS microfluidics attached to farm water. Electrophoretic separations have been demon-
electronic integrated circuits. The modular approach of this strated for phenolic compounds33,34 and inorganic ions.35
system allowed integration of different circuit layouts without Organically modified ceramics, available through Microchem
redesigning the entire device. Glass microfluidic systems are and Microresist Technology as Ormocomp, provide an
currently available commercially through Dolomite, Micralyne, optically transparent, UV-curable alternative material. 36
Agilent, and Caliper. Amino acid and protein separations have been shown within
2.1.3. Ceramics. Low-temperature cofired ceramic (LTCC) these substrates.36,37
is an aluminum oxide based material that comes in laminate
2.2. Polymers
sheets that are patterned, assembled, and then fired at elevated
temperature (Figure 5).30 Due to its laminar nature, LTCC can Polymers are organic-based, long-chain materials that have
be fabricated into complex three-dimensional devices where gained significant traction in microfluidics in the past 15 years.
each layer can be inspected for quality control before inclusion Polymers are advantageous for microfluidic device fabrication
in the stack. Fakunle and Fritsch30 demonstrated low because they are relatively inexpensive, amenable to mass
nonspecific adsorption using an enzyme-linked immunosorbent production processes (e.g., hot embossing, injection molding,
assay (ELISA) in a LTCC device. Zhang and Eitel31 etc.), and adaptable through formulation changes and chemical
investigated LTCC biostability by measuring material leaching modification.
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Figure 6. Large scale integrated PDMS microfluidic valve network.

(Top) Open and (middle) closed 8 × 8 and 6 × 6 μm2 valves.
Figure 5. Microfluidic device made from LTCC. (a) Multilayer (Bottom) demonstration of geometry dependent valving; the flow
stacking to create a screen-printed gold/LTCC microchannel device. channel on the left is closed while the one on the right remains open
(b) Photograph of a microchannel device with an edge connector. (c) when the control channel width is 4 μm. Reprinted with permission
Top down view of a device. (d) Cross-section view of the from ref 39. Copyright 2012 The Royal Society of Chemistry.
microchannel showing the four gold electrodes. The width of the
chip in panel d is not drawn to scale. Reprinted with permission from
ref 30. Copyright 2010 Springer. Thermoset polyester (TPE) is a thermally initiated material
formed by the polymerization of polyester and styrene through
UV or heat.49 TPE is hydrophobic and requires surface
2.2.1. Elastomers. Polydimethylsiloxane was first intro- modification through buffer additives or chemical reactions to
duced as a microfluidic substrate in the late 1990s.38 Now allow water to flow readily through microfluidic channels.50
PDMS is the most common microfluidic substrate in use in Although stable when exposed to many solvents, TPE can be
academic laboratories due to its reasonable cost, rapid dissolved in chlorinated solvents.51 TPE is transparent over
fabrication, and ease of implementation. Device molds are much of the visible range of the spectrum but absorbs UV
formed via conventional machining or photolithography light.52 TPE has a higher elastic modulus (1−100 MPa) than
methods, and PDMS microstructures are cast and cured on PDMS but a lower one than typical thermoset plastics (>1
these molds. Importantly, multiple layers can be stacked to GPa). Roy et al.53 demonstrated the fabrication of TPE valves
create complex fluidic designs (Figure 6).39 A low elastic similar to those possible with PDMS. Utilizing commercially
modulus (300−500 kPa) makes PDMS enabling for valves and available TPE, Brassard et al.54 demonstrated a multichannel
pumps.39 PDMS is gas permeable, which can be advantageous hybrid polymer microfluidic device for protein immobilization.
for oxygen and carbon dioxide transport in cellular studies, but The inertness of perfluorinated compounds (e.g., Teflon-
bubble formation from passage of gas through PDMS can be coated cookware) makes this class of materials attractive for
problematic. Low molecular weight oligomer chains in PDMS microfluidics; such surfaces are both oleophobic and hydro-
can leach out into solution, negatively impacting cellular phobic. Although there are several formula variations, most
studies, for example.40 A hydrophobic material, PDMS is fluoroelastomers are polyfluoropolyethers, and are sometimes
susceptible to nonspecific adsorption and permeation by described as “liquid Teflon”. Rolland et al.55 demonstrated that
hydrophobic molecules.41 Chemical modification of PDMS polyfluoropolyether diol methacrylate (PFPE-DMA) could be
can often address these issues. Plasma exposure will hydro- utilized to make valves similar to ones in PDMS. PFPE-DMA
philize the exposed PDMS surface but only lasts a short showed less swelling in the presence of organic solvents than
time.42,43 Reaction of silanes with silanols formed by plasma PDMS and had a Young’s modulus of 3.9 MPa, ∼10 times
activation or other methods slows this change in surface higher than PDMS but still acceptable for valve applications.
properties.44,45 Goral et al.46 demonstrated conditions like Rolland et al.56 further demonstrated that PFPE-DMA could be
those in vivo for hepatocytes in a perfusion-based PDMS device. molded with resolution as small as 50 nm. De Marco et al.57
Fiddes et al.47 fabricated circular channels and cultured showed that UV-cured PFPE could be bonded strongly to
endothelial cells on their surfaces to mimic conditions during PDMS; a weaker 1.5 MPa delamination pressure between
cardiovascular flow. Sun et al. 48 monitored long-term bonded PFPE layers was found. Bonding of perfluorinated
Escherichia coli culture in PDMS channels and tested bacterial materials to glass and similar substrates also tends to be very
growth inhibition using tetracycline and erythromycin to weak.58 Sharma et al.59 formed microstructured features in
demonstrate a high-throughput analysis method for antibiotics. Viton, a fluorocarbon rubber that maintains its elastomeric
Although valuable for rapid prototyping and commonly used in properties up to 200 °C, and used the large volume difference
academic microfluidic applications, PDMS is generally avoided in paraffin phase changes for membrane actuation. Dyneon
commercially. THV, a commercially available Teflon material, was utilized by
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Begolo et al. 60 for microfluidic structures for droplet Topas, Zeonex, Aline Components, and Optical Polymers Lab
manipulation with an aqueous DNA solution and fluorinated Corp.
oil (Figure 7). Polystyrene (PS) is a polymer made from repeating vinyl-
benzene subunits and is a desirable microfluidic material for cell
culture and analysis as PS is used for conventional cell culture
systems.40 The styrenic surface of PS is hydrophobic; plasma
oxidation or chemical modification is required to make it
hydrophilic.61 Young et al.61 optimized PS device fabrication
using an epoxy mold and hot embossing (Figure 8); subsequent

Figure 8. PS microfluidic devices. Images of (A) epoxy molds, (B)

Figure 7. Dyneon THV, a perfluorinated polymer used for droplet through-hole embossed PS parts, and (C) bonded devices applied in
microfluidics. (A) Droplet production in a flow focusing device neutrophil chemotaxis and nonadherent multiple myeloma cell
(typical volumes ∼10 pL) and plug production in a T-junction (typical immunostaining. Reprinted with permission from ref 61. Copyright
volumes ∼100 nL). (B) Transmission and fluorescence images of a 2011 American Chemical Society.
plug train circulating in a serpentine microchannel. Water solution
contains 0.4 ng/mL of DNA labeled with SYBR Green I. (C)
Fluorescence intensity collected over time in the center of a channel.
Each peak corresponds to the passage of a plug, and the intensity is thermal bonding resulted in strong adhesion between two PS
proportional to the DNA concentration. (D) Image of the whole chip. layers, and the formed devices sustained culture and testing of
Reprinted with permission from ref 60. Copyright 2011 The Royal
neutrophil chemotaxis for 2 days. Adaptation of “Shrinky
Society of Chemistry.
Dinks”, a child’s toy that shrinks drawn features on PS, to
microfluidic systems results in well-sealed devices and high
2.2.2. Thermoplastics. Thermoplastics are densely cross- aspect ratios.62
linked polymers that are moldable when heated to their glass Polycarbonate (PC) is a durable material created by
transition temperature but retain their shape when cooled. polymerization of bisphenol A and phosgene, resulting in
These materials are generally durable, amenable to micro- repeating carbonate groups. PC has a very high softening
machining processes, optically clear, resistant to permeation of temperature (∼145 °C), making it desirable for DNA thermal
small molecules, and stiffer than elastomers. Thermoplastics cycling applications. PC microfeatures are fabricated by hot
require latch-valve geometries16 for valves since the material is embossing with subsequent annealing of two layers using
unable to collapse on itself to form a seal. Thermoplastic raw thermal bonding.63,64 Lee and Ram65 demonstrated a method
materials are available commercially through companies such as to bond PC and PDMS to create peristaltic pumps. Chen et
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Figure 9. Poly-PEGDA microfluidics. (A) Structure of poly-PEGDA. (B) Side-view schematic of a poly-PEGDA device. (C) Bottom-view
photograph of a finished poly-PEGDA device; white bar is 0.5 cm. (D) Fluorescence comparison of PDMS and poly-PEGDA over time during flow
of a dilute, adsorptive, fluorescently labeled protein solution. (E) μCE of amino acids (top) and proteins (bottom) using a poly-PEGDA microchip.
Reprinted with permission from ref 76. Copyright 2011 American Chemical Society.

al.64 demonstrated continuous flow PCR in a 96-device plate, copolymer. Rogers et al.76 polymerized a poly(ethylene glycol)
analogous to a 96-well plate. diacrylate (PEGDA) material with resistance to permeation of
Off-stoichiometry thiol−ene is a UV curable polymer. small molecules and to nonspecific protein adsorption over
Microfluidic devices are fabricated from two different polymer time (Figure 9). Electrophoretic separations in poly-PEGDA
formations: one formula has an excess of allyl groups and the had symmetrical peaks and good resolution. Although the
other an excess of thiols.66 Carlborg et al.66 demonstrated elastic modulus of ∼0.1 GPa77 for poly-PEGDA is too high for
valves with a thiol−ene/PDMS membrane and thiol−ene use in self-collapsing valves, it has strong potential for
fluidic connectors. Good et al.67 showed that changes in application in latch-valve designs.78 Klasner et al.79 demon-
polymer formulation can be used to “tune” the Young modulus strated a PDMS-co-poly(ethylene oxide) material that had
(1−10 MPa). Lafleur et al.68 demonstrated surface attachment decreased optical clarity compared with PDMS but incorpo-
of proteins on thiol−ene microdevices. rated the nonadsorptive poly(ethylene oxide) (PEO) moiety
Poly(methyl methacrylate) (PMMA), formed through the directly within the material without requiring surface
polymerization of methyl methacrylate, is widely known under modification. Amino acid separations were demonstrated
the commercial names of Plexiglas and Lucite. PMMA patterns within devices made from this polymer.
can be formed through hot embossing or injection molding. Fluoropolymers, such as fluorinated ethylene-propylene
Several different methods for bonding to form microfluidic (FEP) and perfluoroalkoxy polymer (PFA), have many of the
networks have been demonstrated.69 PMMA has an elastic same material properties as the fluoroelastomers discussed in
modulus of 3.3 GPa and good optical clarity from the visible section 2.2.1, but they are less flexible. Grover et al.80
into the UV.70 Other advantages of this material include characterized thermally bonded, glass−FEP latch-valve devices,
biological compatibility, gas impermeability and ease of which were even resistant to highly corrosive solutions such as
micromachining at relatively low temperatures (∼100 °C). piranha. Ren et al.81 recently presented the fabrication and
Yang et al.71 demonstrated quantitation of α-fetoprotein in usage of all-Teflon chips with excellent solvent resistance
blood serum using immunoaffinity extraction coupled with (Figure 10). Although slightly opaque, the optical transparency
electrophoretic separation in an integrated PMMA microdevice. of PFA was sufficient for fluorescence and cellular imaging.81
Yang et al. 72 further showed selective extraction and The high-temperature hot embossing (∼260 °C) required to
quantification of multiple human serum proteins in integrated mold devices led to the use of a high-density, densely cross-
PMMA systems. linked PDMS mold. These all-Teflon microdevices had low
Various acrylic monomers can be polymerized on a mold, nonspecific protein adsorption compared with PDMS and PS;
analogously to PDMS, to provide micropatterned substrates moreover, cellular compatibility over 5 days and good gas
with tailored surface properties. For example, the incorporation permeability were demonstrated with HepG2 cells.
of poly(ethylene glycol) (PEG) helps to reduce nonspecific Cyclic-olefin copolymer (COC) is optically transparent82
adsorption of proteins and cells.73 Lee and co-workers74,75 and suitable for use with most solvents and aqueous
demonstrated electrophoretic separations of amino acids, solutions.83,84 Moreover, COC has good moldability and low
peptides, and proteins in a PEG-functionalized acrylic background fluorescence. Roy and Yue85 investigated the
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demonstrated using end-channel amperometric detection.

Surface modification with PEO was effective in reducing
nonspecific adsorption.89 A UV curable variant, PU acrylate,
was demonstrated to have reduced swelling and increased
biocompatibility for cell growth relative to conventional PU.90
Zein is a biodegradable polymer made from corn protein (a
readily available resource in the U.S.) and is an alternative to
petroleum-based polymers currently in use.91 Zein devices were
fabricated by pouring the protein prepolymer over a PDMS
template, drying the mold for 48 h, and subsequently removing
the dried zein layer from the PDMS. Solvent bonding was
utilized for laminating all-zein or hybrid zein−glass devices.
Although the devices were yellow in color (Figure 11),
fluorescence of rhodamine B could still be differentiated from
the background fluorescence of the polymer.
SU-8 is an epoxy-based photoresist that is often used to form
a mold to create features in another material. SU-8 offers high
aspect ratio structures and the ability to pattern multiple
Figure 10. Solvent resistance and antifouling properties of an all- layers.92 SU-8 is transparent in the visible spectrum but not in
Teflon microfluidic device. (A) A PFA chip with microchannels filled the UV, and has an orange-brown coloration. The Young’s
with acetone (colored with a red dye) and DMSO (colored with a blue modulus of 2.0 GPa (Microchem.com) enabled the creation of
dye). (B) Laminar flow of dyed organic solvents in an all-Teflon chip. cantilever-based flexible check valves (Figure 12), which were
(C) Fluorescence images of different kinds of microchannels filled tested over a series of actuation pressures and flow rates and
with a 100 μg/mL GFP aqueous solution. (D) Fluorescence images of allowed unidirectional flow.93 Heyries et al.94 demonstrated an
the channels in panel C after washing with buffer for 1 min. Reprinted SU-8/PDMS device to quantify allergen-specific antibodies.
with permission from ref 81. Copyright 2011 National Academy of
Proteins from common allergy sources (β-lactoglobulin from
Sciences.
milk, peanut lectin, and human IgG) were immobilized in an
array on a PDMS surface before aligning to the SU-8 cover
effects of oxygen, argon, and nitrogen plasma exposure on layer. Protein extracts were used to identify the blood serum of
COC bond strength and platelet adhesion; nitrogen plasma allergic and nonallergic individuals. Free-standing SU-8
treatment provided the best combination of hydrophilicity, structures were initially created through sacrificial removal of
EOF, and biocompatibility. Since COC is hydrophobic, surface an attached glass wafer.95 More recent methods can release an
modification is necessary to separate proteins in these devices; SU-8 device without sacrificing the wafer; with this approach
for example, dynamic coating with 2-hydroxylethyl cellulose integrated electrodes in an SU-8 device enabled amperometric
reduced nonspecific protein adsorption noticeably.82 Nge et detection of dopamine and epinephrine.96 SU-8/Pyrex devices
al.86 recently demonstrated on-chip labeling utilizing a reversed- with integrated electrodes for μCE electrochemical detection
phase monolith for solid-phase extraction (SPE) in a COC are sold commercially by Micrux Technologies.
device. Peng et al.87 showed that ligase detection reaction
2.3. Paper
coupled with single-pair fluorescence resonance energy transfer
could provide rapid (<20 min) detection of Staphylococcus Paper is a flexible, cellulose-based material that has recently
aureus, Staphylococcus epidermidis, and E. coli in a COC device. emerged as a promising microfluidic substrate for several
Polyurethane (PU) has been utilized in a variety of fields and reasons: (1) paper is cheap and readily available around the
has appeal in microfluidics. Piccin et al.88 demonstrated that world; (2) the material can be simply disposed by burning or
plant-derived castor oil can be reacted with a diisocyanate to natural degradation; (3) inkjet and solid wax printing enable
form PU. Fabricated devices formed irreversible bonds between easy pattern definition and functionalization; (4) the porous
layers and were stable when exposed to a range of acidic and paper structure allows for a combination of flow, filtering, and
basic aqueous solutions. μCE of neurotransmitters was also separation; (5) paper is biologically compatible; (6) paper can

Figure 11. Zein−glass microfluidic devices with complex fluidic pathways. (a) Interconnected letters composed of continuous microfluidic channels,
(b) a microfluidic network with channels and chambers, and (c) a solved microfluidic maize maze with multiple false paths. Blue food dye was used
for visualization. All scale bars are 5 mm. Reprinted with permission from ref 91. Copyright 2011 The Royal Society of Chemistry.

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Chemical Reviews Review

Figure 12. SU8 microfluidic device with check valves. (A) Cantilever-based diaphragm micropump. (B) Image of the cantilever in (i) open mode
and (ii) closed mode. Reprinted with permission from ref 93. Copyright 2011 The Royal Society of Chemistry.

Figure 13. Three-dimensional origami paper microfluidic device. (a) Chromatography paper having photolithographically patterned channels,
reservoirs, and a folding frame. (b) Top layer of the folded paper revealing four inlet reservoirs in the center of the device (four flanking circular
features are present within the 3-D structure but are visible due to the transparency of the paper). (c) Bottom layer of the folded paper. (d) The
aluminum housing used to support the 3-D paper microfluidic system. (e) An unfolded, nine-layer paper microfluidic device after injecting four
aqueous, colored solutions through the four injection ports. Reprinted with permission from ref 102. Copyright 2011 American Chemical Society.

be chemically modified through composition/formulation 2.4. Opportunities for Future Development

changes or through surface chemistry; and (7) the normally Numerous materials for microfluidics have been introduced
white background provides a contrast for color-based detection over the past 25 years, and each material comes with a set of
methods.97,98 Paper-based microfluidics rely on the passive inherent strengths and weaknesses. Many materials developed
mechanism of capillary action to pull solutions through a (e.g., PDMS) have remained firmly planted in the academic
device. Dungchai et al.99 first demonstrated electrochemical world but have failed to gain traction commercially. How can
detection in paper microfluidics using a three-electrode design the field break this barrier? The key lies in the fabrication and
to quantitate glucose, lactate, and uric acid in human serum. evaluation of materials that are not only readily mass producible
and inexpensive but also an integral part of a compelling
Nie et al.100 utilized commercially available glucometers to
application. Hybrid devices, which can reap the benefits of each
detect glucose, cholesterol, and lactose in blood serum on a material’s strengths, have shown promise in achieving this goal.
paper microfluidic device. Colorimetric detection was utilized
to quantify nitrites in saliva and ketones in urine using a paper-
3. FUNCTIONS IN LAB-ON-A-CHIP SYSTEMS
based microfluidic device.101 Liu and Crooks102 demonstrated a
3-D paper microfluidic device made from a single “origami” Microfluidic functions are the basic operations in a microchip
folded sheet of paper (Figure 13), wherein colorimetric and system that combined lead to the desired analysis capability.
Key functions include sample preparation, separation, detec-
fluorescence detection of glucose and bovine serum albumin
tion, and liquid transport. Device functions and the overall
was accomplished. Rezk et al.103 used surface acoustic waves to objective of the analysis dictate the design and hardware
improve mixing in paper microfluidic devices and to draw liquid required for each platform.
toward the channel outlet and reduce back flow into other
3.1. Sample Preparation
reservoirs that occurs with capillary filling alone. Thom et al.104
integrated galvanic cells directly into a paper microfluidic device Although the integration of sample preparation in microfluidic
to power light-emitting diodes (LEDs) to be used for devices can be challenging, significant progress has been made
in terms of filtration, extraction, purification, and enrichment.11
fluorescence detection of β-D-galactosidase once sample was
Advantages of microfluidic sample preparation include
added. Osborn et al.105 explored mixing, separation, and reduction in analysis time, automation, and improved
hydrodynamic focusing in two-dimensional paper networks. Fu throughput. Recent reviews provide deeper coverage specifically
et al.106 utilized gold nanoparticles in paper devices to improve on the topic of sample preparation.107,108
sensitivity in lateral flow measurement of human chorionic 3.1.1. Extraction and Purification. Solid-phase extraction
gonadotropin. is a useful purification and enrichment method wherein analytes
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Figure 14. (a) Schematic of the microfluidic aptasensor. (b) Cross-sectional view along line A−A in panel a, illustrating the layered structure. (c)
Photograph of a packaged device. Reprinted with permission from ref 123. Copyright 2011 Elsevier.

Figure 15. Schematic illustration of chip architecture and operation. (A) Chip is loaded with trailing and leading buffers surrounding the sample by
applying vacuum to wells 4 and 5. (B) ITP stacking is initiated by applying an electric field between wells 1 and 7. (C) When the stacked sample
reaches the junction for ITP−gel electrophoresis handoff, the cathode is switched from well 1 to well 6. (D) Gel electrophoresis separation. (E)
Typical electropherogram obtained after preconcentration; I is the fluorescence intensity, and S is the peak separation. Reprinted with permission
from ref 128. Copyright 2008 American Chemical Society.

are first retained on a solid support and then subsequently in a microfluidic platform.110 The most common SPE modes in
eluted in a concentrated form.109 SPE can be integrated with microfluidics are reversed-phase, which works for nonpolar to
other processes like thermal cycling, separation, and detection moderately polar compounds, and affinity, which provides
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greater specificity via interaction between target analyte and a In conventional isotachophoresis (ITP), a leading electrolyte,
complementary compound bound on the solid phase. sample, and trailing electrolyte are injected in that order into a
Silica-based columns (beads, particles, porous sol−gel, and column. The sample components separate according to their
bead/sol−gel hybrid) are the most common supports used for mobilities, with the fastest ion directly behind the leading
reversed-phase SPE, and they have been shown to be both electrolyte and the slowest near the trailing electrolyte; all ions
reproducible and reliable.111 The hydrophilic silanol groups at in the sample zone move at a constant speed. ITP can be
the surface of the silica are generally coupled to hydrophobic carried out similarly in microfluidic channels. For microchip
alkyl or aryl functional groups by reaction with silanes, with C8 ITP determination of bacterial urinary tract infections, 16S
and C18 being some of the most widely used coatings.112,113 rRNA from bacterial lysates obtained from patient urine
The use of monolithic columns is increasing because they can samples was monitored using molecular beacons. The
be prepared easily on-chip by UV polymerization without the combination of extraction, focusing, and detection of 16S
need to form retaining structures like frits.114,115 In addition, rRNA was realized in a single step, and clinically relevant
the porosity and surface area of monoliths can be tuned by concentrations of E. coli were detected.127 Park et al.128 used a
varying the monomer/porogen composition.116 Appropriate branched channel design (Figure 15) to preconcentrate an
hydrophobicity for reversed-phase SPE is provided by antigen and antibody mixture by ITP, followed by gel
monoliths made of butyl-, lauryl-, octadecyl-, or 2-hydroxyethyl electrophoresis to separate the immunocomplex products
methacrylates.83,109,117,118 Silica particles or the microchannel formed. Trailing, sample, and leading zones were formed in
walls in a microchip can also be coated with chitosan for the the chip, and when the stacked sample reached the injection
extraction of RNA from biological samples, because the binding junction, the system was switched to gel electrophoresis
and release of RNA from chitosan is pH-dependent.119 PMMA separation mode. The cancer marker protein α-fetoprotein
microfluidic devices with chitosan-functionalized microfabri- was separated from unreacted antibody and enriched 200-fold
cated posts have been used for the purification of human by this method. In a different study, cardiac troponin I spiked
genomic DNA from whole blood.120 into human serum was concentrated and detected in PMMA
Highly specific affinity extraction techniques based on the microchips using ITP implemented with a 10-fold reduction in
strong interaction between an analyte (target) and a receptor cross-sectional area of the microchannels.129
attached to the column have been explored in microfluidic Temperature gradient focusing is an equilibrium-gradient
devices. Anti-fluorescein isothiocyanate (FITC) was immobi- technique in which electrophoretic flow is countered by
lized on monoliths in PMMA devices and used to selectively hydrodynamic flow; a temperature gradient in a buffer having
extract FITC-tagged proteins, followed by elution and μCE temperature-dependent ionic strength results in a gradient in
separation.121 Biotinylated PCR products were selectively analyte electrophoretic velocities.130,131 The temperature
extracted on a streptavidin-modified bed fabricated in a gradient can be produced by external heating elements or by
PMMA microdevice; the purified material was subsequently Joule heating resulting from the applied electric field. A bilinear
eluted by thermal denaturation and analyzed by μCE.122 temperature gradient was produced by integrating heaters in
Aptamers, which are short nucleic acid sequences that bind to PDMS−glass microchips, with a steep gradient to preconcen-
target molecules with high specificity, are alternatives to trate samples and a shallow gradient for separation. Application
antibody-based extraction. A PDMS−glass multilayer micro- of this technique to the separation of dyes and amino acids with
fluidic device (Figure 14) packed with aptamer-functionalized similar electrophoretic mobilities showed improved peak
microbeads was used to selectivity extract and concentrate capacity and resolution compared with a linear temperature
arginine vasopressin, with its temperature-dependent binding gradient.131 In another paper, Joule heating produced by the
and release controlled by an integrated microheater and combination of AC and DC fields yielded >2500-fold
temperature sensor.123 The binding of nucleic acid sequences concentration enhancement of fluorescein.132
to their complementary immobilized probe nucleic acid The method known as “sweeping” concentrates analytes by
molecules has also been investigated in microfluidic devices, flowing a pseudostationary phase through a sample prepared
particularly in DNA microarray applications.124 To accelerate without the pseudophase; the pseudophase then accumulates
DNA hybridization in microarrays, electrokinetically controlled and concentrates the analytes into a sharp zone. Cross-channel
DNA hybridization was performed in a PDMS−glass micro- glass microchips were used for on-chip preconcentration
fluidic chip with an immobilized oligonucleotide array. The combining field amplification and bovine serum albumin
hybridization produced a measurable signal, and all processes sweeping for the detection of green fluorescent protein.133
from hybridization to detection were completed in 5 min.125 An Compared with free solution techniques, enrichment based
even shorter hybridization time of 90 s was achieved using on exclusion methods such as nanoporous filters generally
automated fluid transfer in a glass−PDMS microfluidic device offers a simpler setup. Hydrogel membranes made from acrylic
with integrated microvalves and micropumps.126 monomers can act as either neutral or charged nanoporous
3.1.2. Preconcentration. Various online sample precon- filters whose pore size and mechanical properties can be
centration techniques, utilizing analyte characteristics such as controlled by varying the polymerization conditions.134 Hatch
charge, affinity, mobility, and size, have been applied to et al.135 integrated two neutral acrylic polymer hydrogel
overcome the limited concentration detectability resulting from membranes in a glass device, one for size-based preconcentra-
the short optical pathlengths in microfluidic channels. An tion and the other for the gel electrophoresis separation matrix.
additional benefit of concentrating samples prior to analysis is Negatively charged acrylamide membranes can be formed by
improved detection of low-concentration analytes typically addition of an ionizable comonomer, such as 2-acrylamido-2-
encountered in real-world samples.107 SPE, discussed in section methylpropanesulfonate (AMPS), enabling the membrane to
3.1.1, is frequently employed for sample enrichment; here, we be ion-selective.136 Chun et al.136 showed preconcentration of
will focus on other preconcentration methods for which recent anions with an ion-conductive membrane made entirely of
advances are notable. cross-linked AMPS in glass microchips. A polymer membrane
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combining acrylamide and AMPS was fabricated in a PMMA integrated derivatization, injection, separation, and detection,
device near the injection intersection (Figure 16), and anionic enabling the study of glutathione reductase kinetics by
continuously monitoring the concentration of the generated
reduced glutathione. To improve automation and integration, a
technique was developed that combined on-chip protein SPE
enrichment in a polymer monolith followed by subsequent
fluorescent labeling and rinsing away of unreacted dye before
sample elution from the monolith. Importantly, all steps were
automated under voltage control.86
Postcolumn methods require the placement of an additional
connection after separation but before detection. A cross design
glass microchip with a postcolumn reactor positioned between
the separation channel and detection point was fabricated and
used for derivatization of separated amino acids.143 A similar
postcolumn setup was employed to continuously monitor the
Figure 16. Photograph of a microfluidic device and zoom view of a on-chip release of neurotransmitters from immobilized PC 12
preconcentration membrane. (A) Photograph of a microfluidic device cells in a multilayer PDMS−glass device incorporating a cell
used for sample preconcentration. Reservoir labels are 1, sample; 2, reactor, continuous flow sampling and μCE, with postsepara-
sample waste; and 3−6, buffer. (B) Photomicrograph of microchannel tion derivatization for fluorescent detection.144
intersection region showing position of the polymerized membrane. Other labeling methods that do not involve pre- or
Reprinted with permission from ref 134. Copyright 2011 Wiley. postcolumn channel geometries have also been described. A
T-junction microdevice with porous polymer monoliths
proteins were excluded from this membrane, providing arranged in series was used as a passive micromixer for on-
preconcentration prior to μCE separation.134 Other membranes chip labeling of lysine with fluorescamine; this serial arrange-
that have been employed include PC, titania, and Nafion. PC ment was shown to be directly responsible for enhanced mixing
nanopore membranes were integrated in PDMS microchips and increased reaction rate.145 Droplet microfluidics, further
and used to concentrate labeled serum albumin.137 Titania elaborated in section 4.5, has also been used for on-chip
membranes fabricated at the intersection of two fluidic channels labeling; droplets containing amino acids from cell lysate and
on a microfluidic device were used to electrokinetically enrich dye droplets were merged and reacted before being loaded
2,7-dichlorofluorescein.138 Proteins were also concentrated in a electrokinetically into microchannels for separation.146
PDMS−glass microfluidic device having an integrated Nafion 3.2. Separation Methods
strip.139 One additional preconcentration method, isoelectric
focusing (IEF), will be discussed under electrophoretic Common separation techniques including chromatography,
methods in section 3.2.2. electrophoresis, and fractionation have been demonstrated in
3.1.3. On-Chip Labeling. Many samples do not fluoresce microdevices. Although miniaturized electrophoretic systems
naturally and have to be derivatized to benefit from the low received more initial attention than chromatographic ones,
limits of detection of LIF. Sample labeling is often performed important progress has been made in both areas, as covered in
off-chip, but on-chip labeling has been achieved both in pre- greater detail in recent review articles on microfluidic
and postcolumn formats. Precolumn labeling was initially chromatography147,148 and electrophoretic methods.149,150
shown in a glass microchip for tagging of amino acids with 3.2.1. Chromatography. Chromatographic separation
subsequent μCE and LIF detection.140 To carry out parallel techniques are reliable, versatile, and sensitive.147 A wide
analysis of multiple unlabeled samples, a multilayer PMMA variety of separation modes are possible in liquid chromatog-
microfluidic device with integrated on-chip labeling and μCE raphy by using different supports.151 The first microfabricated
was demonstrated wherein one fluorescent tag reservoir liquid chromatography system was demonstrated by Manz et
allowed parallel labeling and analysis of up to eight samples al.,152 with an open-tubular column and a conductometric
in different channels.141 Online derivatization of reduced detector. However, in this initial paper, no separation results
glutathione with ThioGlo-1 was accomplished in a long were presented. Typical chromatography columns in commer-
serpentine reaction channel (Figure 17).142 The system cial nonmicrofabricated systems use packed particle supports,
but confining particles such as derivatized silica beads to precise
locations within microfluidic devices can be a challenge.83
Other supports like high-aspect-ratio pillars153 and monolithic
columns have also been used in microfluidic devices.114,115 A
reversed-phase butylmethacrylate monolith was fabricated in
COC microchips for high-performance liquid chromatography
(HPLC) of peptides and proteins (Figure 18). Stainless steel
needles connected the microchips to external pumps and valves
via fused silica capillaries.83 Peptide and protein mixtures were
also separated in polyimide microchips using poly(lauryl
methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-
Figure 17. Layout of a microfluidic chip used for enzyme analysis. RR, divinylbenzene) monolithic columns.151
SR, BR, and WR represent reagent reservoir, sample reservoir, buffer A pumping mechanism is required in HPLC methods for
reservoir, and waste reservoir, respectively. Reprinted with permission flow through solid supports, and several types of pumps have
from ref 142. Copyright 2010 American Chemical Society. been demonstrated. An EOF pump was created in glass
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Figure 18. Microchip liquid chromatography with a monolithic column. (A) Chip design and (B) experimental system with dynamic sample
injection. (C) Chip design and (D) experimental system for online sample cleanup and enrichment-HPLC separations. Reprinted with permission
from ref 83. Copyright 2009 American Chemical Society.

microchips by coating selected channels with a polyelectrolyte first proposed in the early 1990s, is one of the best miniaturized
multilayer; when one arm of a T-junction was coated and the separation techniques because it is fast, has good resolution,
other was not, a disparity in EOF was generated upon and does not need moving parts.157,158 Contrary to conven-
application of voltage, resulting in pressurized flow.154 EOF tional capillary electrophoresis instrumentation, which consists
micropumps, however, require high voltages and are pH of one or more discrete capillaries, many different fluidic
sensitive. In electrochemical micropumps, pressure generated channels can be fabricated on a microfluidic device for
by the build-up of electrolysis gases in a confined chamber is integration or throughput,158 as discussed below. In free
used to pump liquids in microfluidic structures.155 Electro- solution electrophoresis, analytes in a buffer migrate with
chemical micropumps integrated in glass microfluidic channels different mobilities in response to an applied electric field and
were used for the separation of fluorescently labeled amino resolve as distinct bands according to their size and charge. A
acids.155 Micropump components themselves are discussed in glass microfluidic device with a 22 cm long serpentine
greater detail in section 3.4.1. separation channel (Figure 19) was used to analyze N-glycans
Pressure-driven flow has both advantages and disadvantages. in blood serum samples from disease-free and esophageal
Good reproducibility is achieved in conventional HPLC adenocarcinoma patients, as well as from those with Barrett’s
systems because flow rates are not affected by variations in esophagus and high-grade dysplasia, who are at risk of
surface properties of the separation column. In addition, high- developing cancer. The long separation channel offered
throughput and parallel analysis can be achieved by fabricating excellent separation efficiency (700 000 plates) for these
different supports in a single device.83 The implementation of complex samples, and the disease states were successfully
liquid chromatography in microfluidic systems with pressure- differentiated by statistical analysis of the electropherograms.159
based flow using conventional pump systems requires Gel electrophoresis uses a sieving matrix to enhance
connecting to chips but addresses the fluidic resistance due fractionation. Linear polyacrylamide dissolved in the running
to small channel dimensions. Importantly, commercial micro- buffer has been used for the analysis of plasma thrombin by
fabricated HPLC systems from Waters (TRIZAIC UPLC affinity μCE in a PMMA device. Improved resolution of the
nanoTile)156 and Agilent (1260 Infinity HPLC-Chip, www. free aptamer and thrombin−aptamer complex compared with
chem.agilent.com) are available. free-solution electrophoresis was attributed to the stabilizing
3.2.2. Electrophoresis. Electrophoresis is a powerful effect of the sieving gel on the affinity complex.160
liquid-phase separation technique, which can be used to A hybrid of chromatography and electrophoresis, capillary
separate a diverse range of analytes. Microchip electrophoresis, electrochromatography (CEC), has a mobile phase moved by
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from two samples thought to be representative of potential

compounds that could be found on Mars.172
Longer channels are desirable to increase separation
efficiency, especially for complex samples that require greater
peak capacities. However, confining such channels to a small
area requires serpentine configurations, as demonstrated for the
high-resolution separation of amino acid neurotransmitters in a
20 cm long channel.173 A microfluidic device with serpentine
channels having unequal tapered turns to minimize band
broadening was fabricated and used to analyze native and
desialylated N-glycans in blood serum.174
Increased design flexibility and functionality can be achieved
in multilayer systems in which fluidic channels are fabricated in
multiple levels instead of in a single layer.175 Multilayer devices
have been shown in glass,176 PMMA,141 and combined PDMS
and glass.177 A glass multilayer device with a droplet
microfluidic module on the top layer and microchannel
network in the bottom layer was used for both serial dilution
Figure 19. Schematic of a microfluidic device with serpentine channel and enzymatic digestion.176 A multilayer PMMA microfluidic
for N-glycan analysis. Inset shows an image of an asymmetrically
tapered 180° turn. Reprinted with permission from ref 159. Copyright
device fabricated from three PMMA levels (Figure 20)
2012 American Chemical Society. integrated on-chip labeling with electrophoretic separation for
parallel analysis of multiple unlabeled protein samples.141
Magnetic bead based immunoassays were demonstrated in a
EOF, with separation based on both electrophoretic mobility multilayer PDMS−glass microfluidic device with integrated
and interaction with a stationary phase. CEC is well suited for pumps and valves for automated sample processing.177
miniaturized formats, and the designs of the microfluidic Advantages of electrophoretic methods include efficiency,
devices used are similar to those for μCE. Microchip CEC was speed, and low sample consumption. However, the concen-
first reported by Jacobson et al.161 with an open channel coated tration sensitivity, constrained by the injection of small volumes
with octadecylsilane as the stationary phase. Other stationary and the short optical pathlength, is a limitation.178 Importantly,
phases now used include coated silica beads162 or micro- the usefulness of electrophoretic techniques can be increased by
fabricated column structures163,164 and polymer monoliths.116 the integration of functionalities such as PCR, enzymatic
Charge on a polymer monolith, usually achieved by copolymer- digestion, and SPE, as described in section 4.
izing a negatively charged monomer, can be used for EOF.116 A 3.2.3. Fractionation. Other separation methods based on
monolith with anodic EOF was made by copolymerizing butyl fractionation have also been developed. Digital isoelectric
methacrylate and [2-(methacryloyloxy)ethyl] trimethylammo- fractionation, a technique that uses separate pH-specific
nium chloride and used for the capture and trypsin digestion of membranes to concentrate analytes based on their isoelectric
model proteins.114 A neutral monolith, made from the points, was integrated with immunoassay quantitation to probe
copolymerization of octadecylacrylate and pentaerythritol glycoprotein cancer biomarkers in biological samples.179 Field-
triacrylate, has been shown to exhibit cathodic EOF despite flow-fractionation, a method that separates particles through a
the absence of fixed charges.165 field perpendicular to the direction of flow, has been coupled
Isoelectric focusing is a high-resolution technique for with dielectrophoresis in H-shaped microchannels to separate
separation and analysis of amphoteric molecules such as platelets from other blood components on the basis of size.180
proteins and peptides in a pH gradient; since IEF concentrates A series of parallel nanochannels with height steps was used to
analytes, it is particularly useful for dilute samples.166 After separate inorganic and biological nanoparticles by size.181
focusing, the analytes can be mobilized by electrophoretic or 3.3. Sensing and Detection
hydrodynamic (pressure, vacuum, or gravity) means or a
combination of the two, to move ampholytes toward the end of 3.3.1. Optical Detection. Optical detection methods have
the column for detection.167 The mobilization step can be several advantages. They generally have good detection limits,
avoided by whole-column imaging of the focused sample are isolated from the fluid, and can be used to monitor a wide
bands.168 A straight IEF channel has been used for the analysis variety of compounds.182 Recent reviews specifically on optical
of FITC-labeled proteins, using a pH gradient immobilized on a detection methods in microfluidics offer more in-depth
monolithic column by the reaction of ampholines with surface coverage of this topic.183,184 Several approaches to optical
epoxide groups. Both voltage and pressure were used to detection are currently being implemented in microfluidic
mobilize focused zones for detection.169 devices; these can be classified as label-based, such as
Micellar electrokinetic chromatography (MEKC), which was fluorescence and chemiluminescence, or label-free.
first shown in microchips in 1995,170 combines the techniques Laser-induced fluorescence is the most frequently used
of electrophoresis and chromatography to separate both neutral optical method in microfluidic systems because of its low
and charged species. MEKC is based on the partitioning of detection limits.185 However, samples that do not fluoresce
analytes between a micellar pseudostationary phase and the naturally need to be derivatized, often with variants of either
surrounding fluid, with sodium dodecyl sulfate being the most fluorescein or rhodamine, which fluoresce in the green and red
common surfactant for the production of micelles.171 MEKC regions of the spectrum, respectively.185 In many instances, the
separations have been used as an orthogonal method to μCE optics for detection in microfluidics are not integrated in the
for the determination of Pacific Blue derivatized amino acids chip itself. For LIF, a laser is used for excitation and a
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light (indirect CL).191 CL detection has the advantage of not

requiring an excitation source that raises background. CL
methods require very sensitive detectors and have been
demonstrated in both off- and on-chip formats. μCE was
coupled with CL for the detection of intracellular sulfhydryl
compounds using the luminol−Na2S2O8 reaction.192 A high-
throughput immunoassay technique was developed for the
simultaneous determination of five analytes through μCE
combined with magnetic separation and CL detection.193
UV absorbance is a label-free method commonly used in
HPLC and electrophoresis systems. However, its sensitivity is
reduced by the short optical pathlengths commonly encoun-
tered in microfluidic channels.188 Both off- and on-chip UV
absorbance formats have been shown in microdevices. A cross
geometry fused-silica microchip was used for μCE of toxic
alkaloids with UV-absorbance detection.194 A silicon microchip
with integrated UV-transparent waveguides for detection that
connected to optical fibers was fabricated, and compounds
separated by CEC were detected at 254 nm.195
3.3.2. Electrochemical Detection. Miniaturization of
electrochemical detectors does not result in reduced
sensitivity.196 Conductivity, amperometry, and potentiometry
are the most commonly used electrochemical detection
methods in microfluidics. More in-depth reviews of electro-
chemical detection have been written recently by Zimmer-
man197 and Trojanowicz.198
Conductivity detection takes advantage of the conductivity
difference between background electrolyte in solution and the
analyte.199 Two modes have been implemented in microfluidic
devices. In contact conductivity mode, the detection electrodes
are in direct contact with fluid inside the channel.200 With
contactless conductivity, the electrodes are isolated from the
Figure 20. Flow process for multilayer PMMA microfluidic device solutions in the device, avoiding issues such as electrode fouling
fabrication. (a−c) Laser cutting of openings for reservoirs (larger) and and bubble formation.184,201 Electrodes for capacitively coupled
interlayer through-holes (smaller). (d−f) Hot embossing of the contactless conductivity detection were integrated in micro-
bottom layer (upper surface) and the top layer (lower surface). (g) devices by a simple microfabrication technique involving
Assembly of the middle and bottom layers through thermal bonding. patterning of electrodes on a printed circuit board followed
(h) A completed device with top layer thermally bonded to the by lamination and lithographic steps to cover the electrodes in
assembly in panel g. Reservoirs 1−8 are sample inlets for different photoresist. These devices had comparable sensitivity for
analytes. Reservoir 9 is the fluorescent label inlet. Reservoirs 10−12 inorganic cations to systems constructed via more advanced
are buffer inlet, injection waste, and waste, respectively. Reprinted with
fabrication methods.202 Coupling of μCE with both contactless
permission from ref 141. Copyright 2011 American Chemical Society.
conductivity and amperometric detection for the determination
of nitrate and nitrite ions has also been demonstrated. Copper
photomultiplier or CCD is used for detection.159,186 Integration electrodes for conductivity detection were fabricated on a
of an LED excitation source and a photodiode detector in a printed circuit board attached to the PDMS−glass device, while
microdevice has been shown, yielding relatively high limits of amperometric detection palladium electrodes were fabricated
detection of 100 nM for rhodamine 6G and 10 μM for on the glass plate before bonding with PDMS (Figure 22).199
fluorescein.187 While label-based fluorescence methods require Amperometric detection relies on electrical current produced
time-consuming sample derivatization, their detection limits are from the oxidation or reduction of a species by voltage applied
typically better than those for label-free methods. Native UV- between a reference and a working electrode. This method is
excited fluorescence is a label-free detection method that advantageous in that the electrodes can be fabricated by
typically entails excitation between 210 and 325 nm.188,189 In photolithographic processes along with the microfluidic
one format, small aromatic compounds and proteins separated device.203 μCE with amperometric detection of biological
by μCE were effectively detected using 266 nm laser excitation anions has been demonstrated, with in-channel detection
in a fluorescence microscope.190 In an integrated approach, offering improved sensitivity compared with end-channel
waveguides formed in a silicon microchip directed excitation detection.203
light to the detection region and collected transmitted light In potentiometric methods, the potential of an ion-selective
(Figure 21), allowing analytes separated by μCE and MEKC to electrode relative to a reference electrode is probed. The
be detected by both UV absorbance and native UV-excited resulting charge separation that occurs generates a potential
fluorescence.188 between the working and reference electrodes that depends on
Chemiluminescence (CL) is based on the production of light the type of ion and its concentration.204 A miniaturized
through a chemical reaction (direct CL) or when a reaction polymer system with an enzyme-functionalized ammonium-
product transfers its energy to another molecule that then emits selective electrode has been used for potentiometric determi-
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Figure 21. (A) Photograph of an on-chip UV detection microdevice. (B) SEM image of a chip showing a cross-sectional view of the detection cell to
the left and a waveguide to the right. (C) Sketch of channel and waveguide layout. (D) Schematic illustration of the setup. Reprinted with permission
from ref 188. Copyright 2009 Wiley.

Figure 22. Layout of (A) a hybrid PDMS/glass contactless conductivity detection microchip and (B) a similar chip that also offers amperometric
detection. WE, working electrode; RE, reference electrode; CE, counter electrode. Reprinted with permission from ref 199. Copyright 2010 The
Royal Society of Chemistry.

nation of creatinine and urea.205 A potentiometric ion-selective and matrix-assisted laser desorption/ionization (MALDI) are
electrode for probing generated silver ions was employed in a two ionization methods for interfacing microfluidic chips with
paper-based test for the determination of IgE.206 MS. ESI-MS114,211,212 is the more common approach for
3.3.3. Mass Spectrometry. Klepárniḱ 207 and Lee et al.208 coupling microdevices to MS. A droplet microfluidic platform
have presented insightful reviews on the use of MS in was interfaced with ESI-MS via a tip made of a fused-silica
microfluidic systems. Interfacing microfluidics with mass transfer capillary, an ESI needle, and a tapered gas nozzle
spectrometry detection produces valuable data, enabling (Figure 23), where droplets were moved into the MS inlet by
discrimination based on small differences in mass.209,210 ESI the pressure difference from the flow of N2 through the gas
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chip for the assessment of streptavidin−biotin binding.217

Optical microcavity resonator arrays operate under a similar
mechanism to SPR and have been employed in the quantitative
analysis of cancer biomarkers.218 A quartz crystal microbalance
(QCM) is an acoustical transducer, often made of a quartz layer
between two electrodes, which measures a change in resonance
frequency induced by changes on the device surface.219 An
open droplet microfluidic system integrated with a QCM
resonator was used to detect binding of lipids and proteins
from a single droplet on a functionalized sensor surface.220
Various electrochemical biosensors have also been studied.
Amperometric principles were employed in the mass transport
study of glucose and lactate in a microspike sensor. The
Figure 23. Microfluidic eductor operation. Schematic representation
of the eductor, illustrating the configuration of the transfer capillary, microspike array was affixed to a glass substrate and
ESI needle, and gas nozzle, coupled to a closed droplet microfluidic functionalized with either glucose oxidase or lactate oxidase
device. Reprinted with permission from ref 209. Copyright 2012 (Figure 24); a microfluidic setup with a glucose sensor showed
American Chemical Society. that performance was related to enzyme kinetics.221 Other
biosensors have been described as well. Microcantilever based
sensors, which convert changes in mass into a frequency shift,
nozzle.209 In the approach used by Mellors et al.27 an EOF have been optimized and used for the detection of proteins.222
pump at the junction of three channels transferred solution to Microcantilevers based on a suspended microchannel resonator
an ESI emitter (see Figure 4). Unequal pressure, resulting from have also been applied in detecting a cancer biomarker,
the coating of all channels except the EOF pump side channel activated leukocyte cell adhesion molecule, in serum.223 In this
with a positively charged polyamine, moved liquid through the study, a low-fouling carboxybetaine-derived polymer was coated
ESI tip. ESI-MS microchip systems are simpler but not suitable on the resonator surface, and adsorption of activated leukocyte
for parallel analysis, compared with MALDI-MS.210 MALDI- cell adhesion molecule led to a corresponding change in
MS, performed by placing both sample and matrix onto a target resonance frequency. Thermal biosensors measure the heat
substrate, has been interfaced with microfluidics. Parallel released or absorbed in biochemical reactions. A thermal
analysis of eight peptides from a microfluidic chip was biosensor based on two polymer membranes and heaters with a
performed using a custom built MALDI target plate. Addition- thermopile between the membranes was integrated in a PDMS
ally, a peptide mixture separated by reversed-phase liquid microchip for glucose measurements.224 Microbeads in the
chromatography on a COC device was successfully analyzed chamber had glucose oxidase and catalase immobilized to target
from the target plate.210 A PDMS−glass device has also been glucose and hydrogen peroxide, respectively, and the heat
coupled to MALDI-MS via a microflow gate and used to detect generated by the enzymatic reactions was detected by the
low concentrations of arginine vasopressin.123 thermopile. Changes to the characteristic rotational frequency
3.3.4. Biosensors. A biosensor involves a transducer that of a magnetic bead can be brought about by alterations to the
transforms a biochemical signal into an electrical signal and a bead’s physical properties or environment. This feature was
molecular recognition component on the transducer to used in a closed droplet microfluidic platform that monitored
generate a sensor response.213 Biosensors have been reviewed bacterial cell growth and measured antibiotic susceptibility.225
recently by Prakash et al.214 and Choi et al.215 A number of
biosensing methods have been applied in microfluidics. Because 3.4. Fluid Manipulation
of their simple, versatile, and straightforward application, 3.4.1. Pumps. Fluid pumping is an essential function in
optical biosensors are frequently used in the detection of microfluidic systems because it facilitates the flow of solution.
analytes.216 A common optical biosensing method is surface Integration of micropumps into microfluidic devices decreases
plasmon resonance (SPR). Surface plasmons are highly external equipment needs and reduces the dead volumes from
sensitive to changes in refractive index, which can be induced interfacing with pumps. Here we describe a number of
by target binding. SPR has been integrated in a microfluidic microfluidic pumping approaches. A summary of various

Figure 24. (A) Schematic representation of a microspike based skin patch for glucose/lactate detection. (B) Schematic representation of a
microfluidics based system to study mass transport phenomena in biosensors. Reprinted with permission from ref 221. Copyright 2012 The Royal
Society of Chemistry.

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Table 2. Summary of Various Pumping Approaches Utilized in Microfluidic Devices

category actuation mechanism approach advantages disadvantages refs
passive capillary (nonmechani- spontaneous fluid motion by capillary action simple and straightforward; no flow is not continuous over a long numerous
cal) additional fabrication steps period of time; flow cannot be
controlled easily
active electroosmotic (nonme- electroosmotic force through interaction of ease of manufacture; constant adsorption can affect flow; only works 227, 228
chanical) applied electric field with electrical double flow rate which can be with conductive solutions
layer manipulated by the applied
voltage
electrochemical (nonme- generation of gas bubbles, such as by ease of manufacture; small size; gas bubbles and electrochemical by- 204, 205
chanical) electrolysis low power products can block conduction be-
tween the electrodes and hinder
actuation
pneumatic peristaltic sequential actuation of a series of pneumatic easily integrated; fast response requires external equipment to supply 235, 237
(mechanical) microvalves with pressurized air time compressed air; pulsed fluid flow
electrohydrodynamic interaction between electrostatic forces and easily integrated; can pump a flow rate depends on material surface 239
(nonmechanical) ions in nonconducting fluids variety of liquids; requires low properties; typically low flow rates
voltages and pressures
acoustic (mechanical) force produced by the interaction of does not generate byproducts applied frequencies can denature bio- 219
longitudinal waves with the surrounding that can contaminate sample molecules and lyse cells
fluid
magnetohydrodynamic Lorentz force produced when orthogonal ease of integration; continuous possible electrolysis of water at the 242, 243
(nonmechanical) electric and magnetic fields are applied to flow electrodes; bubble formation
a conducting solution in a microchannel

pumping methods employed in microfluidic devices and their

properties is shown in Table 2. A detailed review on
micropumps and microvalves has also recently been published
by Au et al.226
Electroosmotic pumps (EOPs) that use EOF to drive liquids
within fluidic conduits have several advantages: they have a
constant flow rate, their flow direction can be reversed by
simply changing the voltage polarity, and they are easily
integrated into microfluidic devices since they have no moving
parts.227 Bubbles generated when EOPs are operated in DC
mode can be avoided by application of an AC voltage.228 An
AC microfluidic pump within a long serpentine microchannel
was used in DNA hybridization.229 An alternate way to avoid
electrolysis bubbles in EOPs is to use Ag/Ag2O electrodes that
are eventually dissolved during pumping; flow generated by
these low voltage pumps was sufficient for drug delivery.230
Low-power electrochemical pumps rely on gas bubbles
produced by the electrolysis of water.231,232 Such electro-
chemical pumps and an SU-8 tip for interfacing with MS were
integrated in a COC microchip and used to couple reversed-
phase liquid chromatography to ESI-MS (Figure 25); the
performance was tested with cytochrome c.233 Low-power
electrochemical microfluidic pumps have also been integrated
with valves in PDMS devices for delivery of reagents to a
glucose biosensor.234 Figure 25. Schematic diagram of microchip fabrication. (a) Thermal
Pneumatically actuated valves and pumps operate through and pressure bonding of the device sandwiching two silica capillaries
the displacement of a thin membrane by pressure in a layer and an SU-8 ESI emitter. (b) Hot embossing of COC against the
above a microfluidic network. Peristaltic pumping transfers fluid silicon master engraved by reactive ion etching. (c) Gold−titanium
by serial actuation of this membrane;235 peristaltic pumps electrode deposition for electrochemical pumping and ESI. Reprinted
integrated in a PDMS−glass device were used for labeling, with permission from ref 233. Copyright 2008 Springer.
dilution, and separation of amino acids in an automated
manner.236 Cole et al.235 reported a method for controlling forces and ions in nonconducting fluids. EHD micropump
multiple peristaltic pumps using fewer external connections. performance was found to be related to electrode geometry
Other designs of pneumatic micropumps involving actuation by with an asymmetric geometry yielding higher pressure with
a single control line or serpentine-shape pneumatic channels lower power than a symmetric design.239 In acoustic pumping,
are discussed by Lai and Folch237 and Huang et al.,238 the force produced by the interaction of longitudinal waves
respectively. with the surrounding fluid is exploited for liquid movement.219
Electrohydrodynamic (EHD), acoustic, and magnetohydro- An acoustic pump constructed in PMMA produced a 140 mm
dynamic (MHD) micropumps are nonmechanical because flow H2O backpressure and a 0.6 mL/min flow rate at 20−100
is achieved in the absence of valves or moving parts. EHD Hz.240 Acoustic pumping has also been used to manipulate cell
pumping is based on the interaction between electrostatic movement in PDMS−glass devices.241 MHD pumping is based
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Table 3. Summary of Common Methods Used To Make Valves in Microfluidic Devices

category actuation mechanism approach advantages disadvantages refs
passive check valve unidirectional flow; valve is opened by increase in pressure; simple; can withstand can have a large dead volume 226
(mechanical) employs fixed membranes, flaps, etc. high backpressures
capillary burst valve sudden changes in geometry or surface chemistry of simple design and efficiency is lower with low 226
(nonmechanical) microchannel fabrication surface tension liquids
active pneumatic deflection of an elastomeric membrane by application of easily integrated; large requires a control layer; needs 249,
(mechanical) external pressure actuation force external pneumatic inputs 226
phase-change employs materials with a volume difference in a phase ease of fabrication and materials can contaminate 253
(nonmechanical) change, such as a hydrogel, paraffin wax, or water/ice operation samples if in direct contact
magnetic external or integrated magnets for moving magnetic generates high force complex fabrication 248
(mechanical) materials

on the Lorentz force produced when orthogonal electric and

magnetic fields are applied to a conducting solution in a
microchannel.242 MHD pumps require lower potentials than
EOPs but become ineffective for microchannels <100 μm.243 A
micropump that combined MHD with mixing due to the force
produced by electric charges in an electric field has been
demonstrated.244
3.4.2. Valves. Valves play an important role in fluid
manipulation by controlling both movement and flow direction.
Although PDMS is the most common material used for
microfluidic valves, other thermoplastic materials,53 as well as
hybrids made from combining glass, elastomeric materials, and
thermoplastic materials, are being used increasingly.245 Table 3
gives a summary of several of the most widely used valves in
microfluidic devices, as well as some of their key characteristics.
Passive valves include check and burst valves: check valves
allow unidirectional flow, while burst valves regulate flow
through an irreversible expansion of channel size with the
application of high pressures.226 A network of check valves
actuated by finger squeezing was fabricated in PDMS and
shown to quantitatively mix liquids, enabling the colorimetric
analysis of clinically relevant concentrations of glucose and uric
acid.246 An automated PDMS−glass microfluidic system,
integrated with capillary burst valves (with a pressure limit of
4.1 kPa) that manipulated the flow of cell samples, enabled
sample preparation for laser scanning cytometry.247
Active valves rely on an external energy source for
actuation.248 Pneumatic valves that are actuated by compressed
air are widely used because of fabrication simplicity.249 A
pneumatic valve fabricated at the junction between two
channels in a PDMS microchip (Figure 26) was used to
perform reproducible hydrodynamic amino acid injections for
μCE.250 “Lifting gate” valves and pumps with a maximum
breakthrough pressure of 65 kPa were fabricated in PDMS−
glass and PDMS−plastic microdevices.249 An electrolysis
actuated valve was shown to be adequately controlled by Figure 26. Illustration of microchip design and fabrication. (A) Design
electrical signals.251 Screws embedded in the PMMA frame of a of the microchip including flow channel (solid line) and control
PDMS−PMMA valve assembly can also act as valves.252 channel (dashed line). (B) Schematic of microchip fabrication. (a)
Other active valves are phase-change or magnetic. A Flow layer containing the sample injection and separation channels.
(b) Control layer containing the valving channel on a thin PDMS film.
microvalve based on the phase change between solid and (c) Unpatterned bottom layer to enclose the control channel. (C)
liquid PEG was used in μCE of amplified nucleic acids.253 A Template for flow layer fabricated with positive photoresist. (D)
magnetically controlled valve in a device was fabricated by Template for control layer fabricated with SU-8. (E) Photograph of
placing a magnet above it and an iron plate beneath it. the final PDMS microchip. (F) Photograph of the T intersection
Attraction between the magnet and iron plate moved a spacer assembled with a pneumatic valve. Reprinted with permission from ref
into the flexible PDMS, such that actuation was controlled by 250. Copyright 2011 Wiley.
manually placing or removing the magnet.248
3.4.3. Mixing. The laminar flow regime and small channel
size in microfluidic devices favor mixing by diffusion, because in the pursuit of thorough and rapid mixing of multiple
conventional mixing mechanisms cannot be implemented samples.254 Detailed reviews of mixing systems for microfluidics
easily. Thus, several micromixer designs have been developed have been written by Jeong et al.255 and Lee et al.256 An
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Table 4. Summary of Several Mixing Techniques Employed in Microfluidic Devices

category actuation mechanism approach advantages disadvantages refs
passive T- or Y-mixers fluid streams from separate inlets combine at a channel simple design; easy fabrication slow mixing; requires long 256
intersection and mix by diffusion channels
lamination designs that split the main flow stream into multiple streams and reduces diffusion distance; fabrication is complex 264
then recombine them uniform mixing across entire
channels
chaotic advection secondary flows generated when fluid passes through twisted 2D increases contact surface; mixing is not uniform over 261
or 3D structures, zigzag microchannels, or channels with ridges mixing achieved at almost the entire channel cross
and grooves all flow rates section
active electrokinetic unstable flow caused by a force in the bulk liquid generated by ease of fabrication and inte- requires high electric fields 258
coupling of electric fields and conductivity gradients gration
acoustic acoustic waves that cause secondary flow and mixing does not generate byproducts temperature increase may 256
that applied voltages can damage biological sam-
ples
magnetohydrodynamic fluid is mixed through changing flow caused by nonsynchronous simple fabrication; fast and only works with conductive 256
magnetic and electric fields effective solutions

Figure 27. Geometry of three-dimensional serpentine mixing channel. Reprinted with permission from ref 263. Copyright 2009 Elsevier.

overview of various mixing methods used in microdevices and utilize a fluctuating electric field. A combined approach with
some of their principal properties is given in Table 4. a microfluidic T-junction with patterned regions and pulsed
Micromixers that simply rely on molecular diffusion coupled EOF showed increased mixing efficiency of rhodamine B.258 A
with chaotic advection mix fluids through designed geometries micromagnetic disk fixed in the lysing chamber of a microfluidic
that generate specific flow patterns, facilitating their integration device and driven by an external magnetic field was used to
in microdevices.257 Diffusive mixers are probably the simplest, enhance cell lysis. Cell lysis increased with mixing time and
with T-mixers being the most basic design. Electrokinetically magnet rotation speed; the chamber was integrated with sample
driven T-mixers have been used to enhance mixing in PDMS preparation and analysis to detect severe acute respiratory
devices.258 Soleymani et al.259 modified a standard T-mixer with syndrome virus.269 Rapid mixing in a PDMS microfluidic device
two additional junctions to expand the mixing zone and using acoustic streaming of single bubbles, which oscillated
increase vortex generation. An alternate Y-geometry was also under acoustic excitation producing recirculating flow, has been
explored, and the mixing performance in a wide angle Y-mixer
described.270 A focused low-power laser beam increased
was characterized.260
diffusional mixing by raising the temperature in a Y-channel
Advective flow perpendicular to the main fluid flow is
accomplished in microfluidic devices through tailored geo- micromixer for two colored water samples.271
metries designed to improve mixing.261 A 3-D vortex 3.5. Opportunities for Future Development
micromixer produced efficient mixing at low Reynolds For microfluidic devices to achieve a substantial commercial
numbers.262 Effective mixing of water and ethanol was presence, they must be better suited for real world analyses.
demonstrated in a serpentine microchannel with repeating L- One challenge lies in the area of sample preparation, which is
shaped features (Figure 27) over a range of Reynolds numbers often carried out off-chip. Automated sampling and improved
from 1 to 70 with mixing performance correlated with channel
usage of integrated sample preparation steps would lead to
geometry.263 Mixing by lamination is achieved by splitting and
more of a sample-to-answer platform. In the area of separations,
recombining a fluidic stream, which enhances the diffusion
process. Improved mixing of two fluids was achieved in a enhancements to nonelectrophoretic methods in microfluidics
laminar micromixer with asymmetric splitting of the main are needed. For instance, advances in fluid manipulation and
fluidic stream.264 Mixing in zigzag microchannels265 was shown improvements in microfluidic liquid chromatography coupled
to be comparable to results in curved ones but not as effective to MS detection should facilitate both small molecule and
as in square-wave channels.266 proteomics analyses.147 Improving detection could be done by
Active mixing systems rely on moving parts or external inexpensively miniaturizing and integrating high-sensitivity
energy inputs like pressure, electric fields, or magnetic fields methods (e.g., fluorescence and amperometry) for point-of-
and are more challenging to integrate into microfluidic care (POC) assays. Enhanced fluid manipulation methods will
systems.262,267 Voltage-induced vortices above a Nafion require simplified pumps and valves, especially ones that do not
membrane fabricated in PMMA were found to enhance require complex manifolds that interface pressurized lines with
mixing.268 Micromixers based on electrokinetic instability microdevices.
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Table 5. Summary of Common Types of Fluidic Interconnects in Microfluidic Devices

interconnect approach advantages disadvantages refs
reservoir samples/reagents placed into or removed from reservoirs using simple and straightforward not readily automated numerous
wells pipettes or syringes; wires can be inserted to electrically
address reservoirs
capillary a capillary tube is glued into the microchannel inlet reservoir can withstand high pressures difficult to properly align capillary 278, 279
connections with microchannel; increases
dead volume
“sipper chip” reagents loaded into microchip through integrated capillaries automation; high throughput; cost; air bubbles trapped during Caliper
reusable fluid delivery can be difficult to
remove
CapTite components that provide tubing-to-tubing and tubing-to-chip compact; reusable; low dead complexity from interfacing LabSmith
connections volume; can withstand high multiple components
pressures
NanoPort assembly centered over a reservoir and bonded to the reusable; low dead volume adhesive can block microchannels 278
microdevice surface with adhesive rings or epoxy

Figure 28. Microdialysis sampling integrated with microfluidics. (A) Schematic of bilayer microchip with a picture of the valving interface between
the hydrodynamic flow and electrophoretic flow. Abbreviations: B, buffer; BW, buffer waste; PB, pushback; SW, sample waste; S, sample; NO,
normally open; NC, normally closed. (B) Picture taken on gray background showing how the microdialysis tubing, access pins, and voltage leads are
inserted into the microchip. (C) Coupling of the microchip device with microdialysis sampling, associated tubing, and syringe pump. Reprinted with
permission from ref 283. Copyright 2008 American Chemical Society.

4. INTEGRATION previously, components such as pumps, valves, and mixers

that enable functions such as sample preparation, separation,
4.1. Overview etc. are key to integrated microfluidic systems. Remaining
Integration is one of the key advantages of miniaturization272 challenges in creating fully integrated microdevices are the
and can facilitate assays for POC usage.273 Indeed, the objective complexity of combining several different components in a
of lab-on-a-chip systems is to integrate all laboratory processes single platform277 and interfacing with the macroscopic
into a single device in which sample pretreatment, biochemical world.278 Here, we discuss recent successes in integrating
reactions, separation, detection, and data analysis operations, various components.
for example, can be carried out.274 Although important progress
4.2. Fluidic Interconnects
toward this goal has been made, present microfluidic systems
typically can perform only one or a few select steps on-chip.29 A key challenge in integrated microfluidic systems is in making
The integration of multiple processes on-chip can limit sample robust connections to the macroscale environment.278 Fluidic
loss, reduce analysis time, and enable new detection methods interconnects link microfluidics on a chip to an external fluid
for microfluidic analyses, as summarized in depth in recent source,279 ideally in a simple and minimal dead volume
reviews.11,275 The extent of integration can be anywhere format.280 Table 5 gives an overview of several types of fluidic
between a disposable chip in external equipment to complete interconnects employed in microfluidic devices and some of
integration of all laboratory functions.276 As discussed their main characteristics. One early approach was the “sipper
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Figure 29. Layout of an integrated affinity extraction−μCE microdevice. (a) Schematic diagram and (b) photograph of a typical microchip with
integrated affinity column. Reprinted with permission from ref 72. Copyright 2010 The Royal Society of Chemistry.

chip” technology commercialized by Caliper where reagents cells.283 Microdialysis sampling has also been interfaced with
were automatically loaded into a chip through integrated microfluidic devices via even shorter fused-silica capillaries and
capillaries via vacuum (www.caliperls.com/tech/microfluidics. used for monitoring glucose in rat brains.284
htm). LabSmith’s CapTite microfluidic interconnects (www. 4.4. Sample Preparation: Extraction and Purification
labsmith.com) provide a link between multiple ports and can be
used to interface microchips with capillaries. Additionally, Both reversed-phase and affinity SPE modules have been
NanoPort assemblies commercialized by Upchurch Scientific integrated into microdevices. C18-coated magnetic particles
(www.upchurch.com) can be affixed to microdevices for have been used for in-line SPE−μCE to analyze mixtures of
interfacing with external fluidic sources. Creation of wells parabens and fluorescent dyes. The magnetic particles were
within devices to serve as reservoirs is a simple though not trapped at the intersection of an offset-T microchip, and an
highly automated approach wherein samples or reagents can be additional side channel was connected to a syringe pump for
placed into or removed from reservoirs using pipettes or elution.285 An integrated PMMA microfluidic device with a
syringes, and wires can be inserted to electrically address monolithic affinity column coupled to μCE was used for the
channels. More sophisticated interconnects can increase device extraction and quantification of cancer biomarkers in human
functionality. Capillaries connected to microfluidic chips and blood serum (Figure 29). The chip was designed with multiple
held by glue had minimal dead volumes and could withstand reservoirs to enable uninterrupted movement of the different
pressures >800 psi.279 Multiple interconnects were embedded fluids required for loading, rinsing, eluting, and separating the
in a microfluidic device in an in-plane manner that avoided the sample.72 Immunoglobulin E and nuclear factor-κB were
need for adhesives.281 Sen et al.278 fabricated an interconnec- extracted and concentrated with an aptamer-functionalized
tion system between a COC chip and a capillary to transfer size-exclusion membrane in an integrated glass microfluidic
analyte from the chip to a desorption ESI-MS system. device that also enabled mixing, buffer exchange, and
4.3. Microdialysis
detection.286
4.5. Droplet Microfluidics
Microdialysis, a topic previously reviewed in detail by Nandi
and Lunte,282 can be used in sampling wherein analytes diffuse Droplet-based microfluidics is a technique wherein picoliter to
across a semipermeable membrane generating a continuous microliter droplets are generated and manipulated. These
flow of sample for real-time in vivo monitoring of biological droplets are advantageous for localized reactions and
events.173 Integrated microfluidic systems for microdialysis preventing diffusion of compounds beyond the droplet
sampling enable direct coupling to the sample stream, resulting confines. Approaches can be divided into “closed’ systems,
in less loss of information compared with off-line techniques.283 where flow is confined in a defined microfluidic geometry or
A major challenge in coupling microdialysis sampling with μCE “open” systems where droplets are controlled electrostatically
has been the need for a fluidic interconnect that reproducibly in an open planar region. Detailed reviews on this emerging
introduces small sample volumes while maintaining good technique have been published recently.287−289
temporal resolution.282 Coupling of microdialysis sampling with 4.5.1. Closed Droplet Microfluidics. In closed systems,
μCE via PDMS-based pneumatic valves has been achieved by droplets are generated within a fixed microfluidic design. The
connecting microdialysis tubing into the microchip sample inlet size of generated droplets is determined by microfluidic device
opening (Figure 28). This approach reduced dead volume and design, liquid properties, and the flow rates of the immiscible
lag times, facilitating studies of dopamine released from PC12 phases.287,290 Channel geometry provides a passive method for
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generating droplets; the T-junction is the simplest config- drop over immobilized FITC-labeled anti-human IgG for
uration used, wherein one liquid shears a second liquid flowing fluorescence detection. Another system has been used to detect
in the perpendicular channel arm.291,292 The formation and a nucleic acid-based pathogen signature from E. coli and a
manipulation of droplets have been reviewed by Seemann et biomarker for ovarian cancer. Droplet manipulation by
al.,287 Baroud et al.,293 and Teh et al.294 Although the droplets magnetic actuation of superparamagnetic particles enabled
are typically aqueous fluids embedded in hydrophobic carrier cell lysis, followed by DNA extraction, purification, and
solutions,295 droplets within two aqueous solutions have also amplification; surface topography facilitated some aspects of
been demonstrated.296 Single-cell secretion was analyzed using droplet manipulation.302 In a hybrid open/closed system,
closed droplet microfluidics, in which each cell was Abdelgawad et al.146 combined on-chip labeling and enzymatic
encapsulated in a droplet that had both fluorescent antibodies digestion in the open portion with MEKC separation in the
and capture antibody functionalized microspheres. The closed region.
technique was validated through the analysis of IL-10, an 4.6. Digital PCR
anti-inflammatory cytokine.297 For the study of drug−protein
binding, magnetic beads functionalized with the target human Digital PCR is a novel nucleic acid quantitation method that
serum albumin and mixed with the drug warfarin were trapped utilizes numerous separate PCR reactions of dilute samples.
into microdroplets. An asymmetric magnetic field at a T- Absolute quantitation is obtained based on binary positive/
junction generated daughter droplets of different content, from negative (one or zero template) results from an array of
which the affinity constant of warfarin could be obtained from subreactions.303,304 This method is advantageous relative to
its concentration in the daughter droplets. 298 Droplet quantitative PCR because it does not require a reference sample
generation by EOF was integrated with μCE to maintain since counting is based on reactors that either have or lack
spatial control of separated components for subsequent analysis amplicons. A PDMS microdevice with an array of microreactors
without affecting separation efficiency (Figure 30). Although that were loaded in parallel was tested and validated with
complementary DNA from embryonic stem cells.303 Moreover,
the capabilities of digital PCR in quantifying low concentrations
of different DNA types were evaluated.305 Microfluidic digital
PCR has also been used to identify viruses in single bacterial
cells obtained from their native environment.306 Multiplexed
digital PCR in droplet microfluidics was used to analyze genetic
variants of spinal muscular atrophy. Two PDMS−glass devices
were used, with droplets generated in one device and
subsequently loaded into a second device for fluorescence
readout to precisely measure copy number variants.307
4.7. Opportunities for Future Development
Considerable benefits result from integrating multiple labo-
ratory processes in a single device, such that further
development would be beneficial. Most fluidic interconnects
have a specific application rather than a general purpose. Future
work should create robust and reversible fluidic interconnects
that are applicable across a broad class of microfluidic
Figure 30. Droplet compartmentalization of the components of a
platforms. Droplet-based microfluidic systems present an
mixture separated by μCE. (a) Schematic representation of the general attractive approach to control dispersion in the manipulation
method used for the compartmentalization of the separated bands in of small discrete volumes of liquid. Higher-throughput droplet-
droplets. (b) Schematic representation of the fluidic design used to based systems for the automated analysis of real samples, such
integrate μCE with droplet compartmentalization. (c) Droplet- as large assortments of single cells, would be an important
formation region shown in detail. Reprinted with permission from advance. Highly integrated systems must be developed in a way
ref 299. Copyright 2009 Wiley. that enhances performance and reduces costs relative to
conventional methods.
μCE of amino acids showed coelution of D- and L-glutamate,
droplets having these coeluting components were transferred 5. APPLICATIONS
into a fused silica capillary for MEKC resolution.299 Due to the multitude of advantages of microfluidics, many
4.5.2. Open Droplet Microfluidics. In open systems, applications have been demonstrated, often with a biological
droplets are manipulated on the surface of a plate in the focus such as POC diagnostics, single-cell analysis, nucleic acid
absence of a confining microfluidic geometry. For electro- analysis, drug discovery and development, or biosensing.308
wetting on dielectric, an electric field increases hydrophilicity of Additional applications are found in environmental monitoring
a region so fluid can better “wet” the surface.274 Electrostatic and space exploration. Here, we give a select view of some of
forces on an array of electrodes coated with a hydrophobic the vast and diverse applications of microfluidic systems.
insulator manipulate drops with high uniformity. This approach
is well suited to doing multistep reactions compared with 5.1. Point-of-Care
conventional microfluidics, since each droplet can be set up to Point-of-care diagnostics are useful for preliminary self-
carry out a separate reaction.300,301 Miller et al.301 used such a screening and disease testing for home health assessment, as
setup for the immunoassay of human IgG in serum samples. well as in military, forensic, and space applications.308 POC
Droplets containing IgG were moved over surface-attached assays must be fast, accurate, reproducible, and readily
anti-IgG, followed by washing, incubation, and actuation of the interpreted by nonexperts.309 Various aspects of POC assays
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Figure 31. Microchip blood analysis system. (a) Scheme depicting plasma separation from a finger prick of blood by harnessing the Zweifach−Fung
effect. Multiple DNA-encoded antibody barcode arrays are patterned within the plasma-skimming channels for in situ protein measurements. (b)
Antibody arrays patterned in plasma channels for in situ protein measurement. Labels A−C indicate different DNA codes. Labels 1−5 denote DNA−
antibody conjugate, plasma protein, biotin-labeled detection antibody, streptavidin-Cy5 fluorescence probe, and cDNA−Cy3 reference probe,
respectively. The inset represents a barcode of protein biomarkers, which is read out using fluorescence detection. Reprinted with permission from
ref 313. Copyright 2008 Nature Publishing Group.

have been reviewed in considerable depth.276,310 Polymer- and microdevices. Specific applications of microfluidic systems in
paper-based microfluidic systems offer cheap and disposable cell biology11,316 and single-cell analysis317 have been reviewed
properties for POC diagnostic applications. Although a variety in greater detail previously. PDMS is the primary microfluidic
of studies have demonstrated multiple advantages of micro- device material used for cell culture315 although other materials
fluidic systems (including speed, throughput, minimal reagent such as COC, poly(ethylene terephthalate glycol) and PS have
or sample consumption, etc.), to date very few microfluidic been explored in combination with PDMS to obtain combined
POC assays have been commercialized due to challenges in elastomeric and rigid devices.318 Two-dimensional cell culture
sensitivity, quantitation, and operational complexity.310 One
(on flat surfaces) is simpler than that in a 3-D geometry, but 3-
common commercialized POC test is the lateral flow
D models are receiving increased interest because they better
immunochromatographic assay used for pregnancy testing,
the diagnosis of infectious diseases (streptococcus, influenza, reflect the natural tissue and organ structure, providing a more
and HIV), and screening for drug abuse.310 Another POC realistic model of the in vivo environment.24,319,320 A
system is the blood glucose biosensor, which relies on single- fluorescence-based system has been developed on a PDMS
use electrodes made using microfabrication or vapor deposition chip with an integrated piezoelectric actuator for cell sorting
methods.311 For detection of lower concentrations of a wider and an optical waveguide for detection.321 A magnetic-based
variety of targets, more sophisticated tests are needed including technique with immobilized biofunctionalized superparamag-
microfluidics-based systems.310 A successful microfabricated netic beads in a PDMS microchannel sorted cells from clinical
POC system is the iSTAT (Abbott, http://www. samples, yielding comparable results to those obtained with
abbottpointofcare.com/), which uses a hand-held analyzer flow cytometry.322 Affinity-based methods enable the capture of
and disposable single-use cartridges to measure various low-concentration cells, such as circulating tumor cells (CTCs)
electrolytes, metabolites, gases, and hematocrit in blood. The overexpressing the membrane protein epithelial cell adhesion
precision and reproducibility of the iSTAT were comparable to molecule (EpCAM), which is implicated in epithelial
what was obtained using clinical laboratory methods.312 Other carcinomas. A PMMA chip with immobilized anti-EpCAM
advances in the development of microfluidic systems for
selectively captured CTCs from whole blood; the cells were
potential POC usage have been demonstrated. A PDMS−glass
platform was used for rapid measurement of a panel of blood subsequently released and counted with a conductivity
plasma proteins. The chip separated plasma from whole blood, sensor.323 An affinity sorting method was also implemented
and the plasma flowed across channels patterned with a in a PDMS−glass chip with antibody-coated three-dimensional
barcode-like array of DNA-linked antibodies to detect multiple channels for efficient capture of nontarget cells that allowed
protein biomarkers (Figure 31).313 Another microfluidic POC target cells to flow through (Figure 32). The absence of a cell-
device with sample processing and detection steps similar to damaging elution step was advantageous; the method was
those in ELISA was developed for disease screening in tested on the separation of lymphocytes and mouse endothelial
resource-limited areas. Reagents, pulled into the microfluidic cells.324 A better understanding of cell-to-cell variation can be
system using a disposable syringe, interact with detection zones obtained through single-cell analysis. Human glioblastoma cells
having capture proteins followed by visual or low-cost optical cultured in a PDMS−glass microfluidic platform were imaged
detection. Tests carried out in Rwanda showed sensitivity and fluorescently to analyze multiple cell proteins simultane-
specificity comparable to commercial ELISA systems in the ously.325 The nucleic acid and protein components of lysed
simultaneous diagnosis of HIV and syphilis in whole blood.314 cells can be determined by PCR and μCE, respectively. A
5.2. Cell Analysis microfluidic device with integrated reverse transcription
The great attention focused on applications of microfluidics in quantitative PCR was used for the analysis of mRNA and
cell analysis315 has led to numerous developments, with miRNA in single cells.326 Hemoglobin from human eryth-
modules for standard procedures such as cell culture, sorting, rocytes27 and amino acid metabolites from single mouse
lysis, and separation of contents all being integrated in fibrosarcoma cells327 have been analyzed by μCE.
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glass compact disk microfluidic device. The system was tested

with a Dengue virus gene sequence and showed better
performance than flow-through hybridization under the same
conditions. 331 A PDMS−glass microfluidic device with
integrated PCR, enzymatic ssDNA generation, and electro-
chemical detection was developed (Figure 33). Determination
of genomic DNA from Salmonella showed improved detection
limits over other microchip-based PCR electrochemical
methods.332 A PC microfluidic device designed to carry out
automated lysis of bacterial cells or viruses, nucleic acid
isolation, amplification, labeling, and detection was developed
and tested with Bacillus cereus and HIV RNA spiked saliva
samples.333 A portable PDMS device was demonstrated with
integrated electrochemical detection for assaying cultured E. coli
through loop-mediated isothermal amplification.334 A COC
chip with integrated SPE monolith for nucleic acid extraction
and reverse transcription-PCR was developed for influenza
virus detection. Amplification of influenza A RNA from nasal
swabs and aspirate specimens showed comparable performance
to conventional laboratory methods.335
Figure 32. Schematic of an affinity capture microfluidic device. (a) 5.4. Drug Metabolism
Side view. Cell mixtures are loaded into the chip, where nontarget cells
are captured and target cells pass through for collection. (b) Image of High-throughput screening methods in drug discovery can
an experimental device. Red food coloring was introduced into require lengthy times with complicated and costly sample
channels to help with visualization. Reprinted with permission from ref processing.336 It is therefore desirable to develop in vitro
324. Copyright 2011 American Chemical Society. systems that better predict in vivo toxicity. Detailed reviews in
this area have been presented by Neužil et al.,337 van Midwoud
5.3. Nucleic Acid Assays et al.,338 and Esch et al.339 Microfluidics have been used to
Rapid genetic tests are usually achieved with biomolecular design cultured cell or organ system mimics on-chip to simulate
techniques such as DNA hybridization or PCR.328 Review metabolism and to test drug combinations at different
articles on microfluidic approaches for DNA hybridiza- concentrations.339 An advantage offered by integrated micro-
tion328,329 and nucleic acid amplification330 offer further details fluidic technologies is that conditions can be well regulated for
about these methods. Oscillatory flow whose direction changed better control of the cell or tissue environment.338 The
cyclically, produced by a combination of centrifugal force and microscale dimensions can be used to generate physiological-
capillary action, enabled rapid DNA hybridization in a PDMS− like conditions for fluid residence times, liquid-to-tissue ratios,

Figure 33. Assay overview. (A) Template DNA is added to a PCR reagent mixture containing phosphorylated reverse primers. (B) The template is
PCR amplified. (C, D) Lambda exonuclease is mixed with the product and digests the phosphorylated strands. (E) Prior to electrochemical analysis
in the detection chamber, MgCl2 is added to adjust the salt concentration from 1.5 to 50 mM for hybridization. (F) Before introducing sample to the
sensor, a baseline redox current is measured. Next, the ssDNA product hybridizes with the E-DNA probe modulating the redox current signal.
Finally, the E-DNA probe is regenerated to verify the hybridization event. Reprinted with permission from ref 332. Copyright 2009 American
Chemical Society.

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and cellular shear stresses. Chambers with cultured cells samples for further off-chip analysis. During simultaneous
provide organ-like metabolic function, while fluidic channels processing of samples from three yeast strains, several proteins
enable transport between these “pseudoorgans”.340 An in vitro were identified.349 A glass microfluidic immunoblotting plat-
mimic of the gastrointestinal tract was coupled to cultured liver form that integrated separation, affinity capture, and washing
cells to follow the metabolism of acetaminophen. Importantly, steps was developed for the analysis of protein isoforms. IEF,
the microfluidic system provided comparable dose-dependent followed by gel immobilization of the proteins by UV exposure
liver cell toxicity data to in vivo studies.341 Interorgan and probing via antibodies, enabled the analysis of prostate
interactions were studied in a PDMS device with two chambers specific antigen isoforms in serum with comparable results to
connected via a microfluidic channel having PC filters and slab gel IEF.350 Since proteomes can differ in a heterogeneous
PDMS membranes. The chambers contained rat intestinal and population of cells, the analysis of single cells can enhance
liver tissue, and metabolites formed in the intestinal tissue were understanding of cellular function. A PDMS−glass platform
fluidically transferred to the liver for further metabolism. The was developed for counting protein copy number in single cells
interaction between the intestine and liver was validated by transferred via an optical trap to different antibody-spotted
studying the regulation of bile acid synthesis.342 Human chambers where they were lysed with a laser pulse. The levels
hepatocytes were cultured with nonparenchymal cells to of cancer-related proteins released from the cells were then
increase their in vitro viability and function and then analyzed measured upon antibody binding within the analysis
for clearance of enzymes involved in drug metabolism and chambers.351
metabolite generation. The experiments conducted in a PS 5.5.2. Metabolomics. Metabolomics, the study of metab-
microchip showed that when fluid flow was coupled to the olite intermediates and byproducts, provides information
culture system, compounds of different clearance rates were regarding cellular function and health and can be used in
more readily resolved.343 diagnostics that identify disease states in individuals. Small
5.5. Omics reagent volumes, precise sample control, and cellular
compatibility make microfluidics ideal for metabolomics
5.5.1. Proteomics. The analysis of protein types and studies. For example, a single cell can be immobilized in a
concentrations is a key research endeavor, and microfluidics device where cellular effluent can be extracted and analyzed
offers enabling capabilities. An important challenge encoun- under different conditions.352 Contributions of microfluidics to
tered in proteomics is the analysis of small sample volumes the study of drug metabolism have been discussed previously
containing a large variety of proteins at different concen- (section 5.4). Shaw et al.353 demonstrated a microfluidic
trations.344 Miller et al.345 and Zhou et al.346 have previously laminar diffusion interface that separated proteins and
reviewed some aspects of proteomics in microfluidic devices. metabolites based on their diffusion coefficients, enabling
Most of the progress achieved in proteomics has been driven by metabolite detection using IR spectroscopy with little protein
the availability of high-resolution MS,347 so microfluidic contamination. Chen et al.354 utilized a PDMS microdevice
systems coupled to MS can play an important role in high- with a cell culture region and a reversed-phase chromatography
throughput proteomic analysis methods. A bottom-up column coupled to ESI-MS to monitor MCF-7 cell response to
proteomics approach was demonstrated using a PMMA anticancer drugs. Fluorescent labeling and imaging allowed the
microfluidic device containing functionalized pepsin−agarose; study of apoptosis in response to drug levels. Shintu et al.355
the microchip was interfaced with ESI-MS (Figure 34). made an organ-on-a-chip device that was probed via proton
Characterization with myoglobin, ubiquitin, and bovine serum nuclear magnetic resonance for metabolomic analysis of liver
albumin showed rapid and complete digestion with high and kidney cell cultures exposed to several toxic compounds.
sequence coverage.348 A multichannel COC microfluidic For further information on applications of microfluidic systems
proteomic reactor was packed with strong cation exchanger in metabolomics, the reader is referred to review articles by
beads to preconcentrate, reduce, alkylate, digest, and then elute Wurm et al.356 and Kraly et al.357
5.6. Environmental Analysis
Though the prevalence of microfluidics in environmental
analysis is less than in biological studies, some advances have
been demonstrated, as reviewed in detail by Jokerst et al.358 A
major challenge in environmental analysis is the need for
sufficient device robustness to perform unattended high-
throughput sampling in the field with complex matrices.358 A
PDMS microfluidic system with an electromechanical pump
and LED detector was developed for environmental analysis.
Flow rates for the microdevice were comparable to those for
miniaturized commercial pumps, and analysis of nitrite
solutions showed sub-ppm limits of detection.359 An aerosol
analyzer with a growth tube collector connected to a PDMS
microchip was developed for online monitoring of aerosol
composition (Figure 35). Air was pulled downward through the
Figure 34. (A) Photograph of a microfluidic chip, enhanced to
highlight the channels. (B) A microscopic view of the reactor well growth tube where the particles were enlarged and deposited
entrance with immobilized pepsin beads. Arrows indicate direction of into the microchip sample reservoir for μCE. Measurements of
flow. (C) A schematic depiction of the reactor well, side view. Single- sulfate and nitrate yielded comparable results to a conventional,
headed arrows indicate direction of flow. Reprinted with permission full-size instrument.360 A PDMS−glass device with a Y-shaped
from ref 348. Copyright 2010 Wiley. microchannel for passive mixing was constructed to measure
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Figure 35. (a) Schematic of the aerosol μCE system, showing growth tube collector, water reservoir for passive wetting of the wick, and microchip
into which the aerosols are deposited. All dimensions are in millimeters. (b) Exploded view of the microchip design. The gray lines are platinum
electrophoresis electrodes, the gold lines are gold-plated tungsten detection electrodes, the black lines are microchannels, the white circles are holes
in the PDMS, and the gray circles show the locations where the holes in the adjacent layer are aligned. The chip is assembled bottom-to-top, layers
1−4. Reprinted with permission from ref 360. Copyright 2009 American Chemical Society.

lead in water. Lead detection at ppb levels via a fluorescent drugs. Further work in effectively linking multiple separation
molecular sensor was performed by UV excitation through dimensions with MS detection should aid in the growth of both
embedded optical fibers and emission collection with different metabolomics and proteomics. In the areas of environmental
optical fibers coupled to a photomultiplier.361 A PDMS−glass and space analysis, effort should focus on creating robust and
multilayer microfluidic device with integrated heaters, temper- portable devices that can operate unattended for long periods.
ature sensors, pumps, and valves was developed for determining
microbial activity in ocean environments. Detection was based 6. CONCLUSIONS AND OUTLOOK
on total microbial ATP concentration probed by luciferin−
luciferase reaction using serpentine microchannels to facilitate The field of microfluidics has progressed substantially since its
mixing.362 introduction, with applications cutting across multiple fields
and disciplines. Biological and medical applications are a major
5.7. Space Exploration
focus of current research. Advantages provided by the unique
Automated detection and analysis systems could play a key role chemistry and physics that occurs in microscale channels and
in unmanned space missions in the search for extraplanetary life the coupling of multiple functionalities continue to drive
because the ability to analyze in situ can reduce sources of advances. We have reviewed key advances in microfluidics in
terrestrial contamination. Microfluidic systems fit this require- terms of materials and functions, their integration, and
ment nicely and are well suited for planetary probes; Mora et applications. While glass and silicon have important uses,
al.363 published a recent review on this topic. A model in situ polymeric materials have gained considerable ground, especially
extraterrestrial exploration system with automated labeling, in the area of low-cost, disposable devices. Still, as yet there is
dilution, and μCE analysis was developed and tested.236 A no “perfect material” as each comes with its own inherent
portable μCE instrument for the analysis of extraterrestrial advantages and disadvantages. Despite the great progress that
organic molecules was developed by Mathies et al.364,365 has been made in microfluidics, much remains to be done to
Baseline separation of standard polycyclic aromatic hydro- gain acceptance and broad applicability outside of academia.
carbon compounds was obtained, and the system successfully Several factors have hindered the spread of microfluidics
determined the composition of environmental and Martian beyond academics. The complexity of combining multiple
analogue samples as evidence of its potential for use in
functions into a single platform in a completely automated
planetary exploration.365
manner that circumvents the need for bulky external equipment
5.8. Opportunities for Future Development is one reason. Another issue is mass production; presently,
Microfluidic systems have found application in several different applications require different designs, limiting general
important fields; however, their commercialization is still rather fabrication of one type of device that will suit every purpose.
limited. The development of POC devices will be enhanced by Other issues include the lack of device design standards as are
further integration of processes and greater simplification of prevalent in electronics, the absence of a general standalone
their usage. For single cell analysis, methods for extracting and device reader for detection, intellectual property complications
analyzing specific cellular components need to be improved. and licensing (or nonlicensing), and a too-frequent disconnect
Future work to probe interorgan interactions using micro- between those designing devices (i.e., engineering) and the
fluidics would improve understanding of metabolic profiles of eventual end users who often have a life science focus.
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Chemical Reviews Review

The advent of alternatives to enclosed-channel microfluidics Chad I. Rogers received his B.S. in Chemistry from the University of
(such as droplet and paper microfluidics) may expand the Utah in 2006. He is currently working on his Ph.D. in Analytical
application space of the field. Indeed, the manipulation of small Chemistry at Brigham Young University under Professor Adam T.
droplets through two-dimensional grids offers a more general Woolley. His research focus is on the implementation of polymerized
platform for a range of assays; although the technique is still in poly(ethylene glycol diacrylate) as a microfluidic substrate for use in
its early years, the versatile microfluidic hardware could be pumps, valves, and sensor integration.
highly enabling. The inexpensive and very wide availability of
paper combined with its known chemistry makes paper
microfluidics a simple yet powerful option for the future, in
particular for less developed countries and in emergency
situations such as disaster zones.
The multidisciplinary nature of microfluidics demands
continued coordination between different fields, engineering
as well as physical and biological sciences, to reach its full
potential. As microfluidics enters its third decade, we expect
continued growth and significant expansion beyond simple
proof-of-concept systems and into extensive real world and
commercial applications.

AUTHOR INFORMATION
Corresponding Author
Adam T. Woolley graduated summa cum laude with a B.S. in
*Phone: 801-422-1701. E-mail: [email protected].
Chemistry from Brigham Young University in 1992. He received his
Notes Ph.D. in Chemistry in 1997 from the University of California−
The authors declare no competing financial interest. Berkeley and was a postdoctoral fellow at Harvard University. In 2000,
Biographies Woolley joined the Department of Chemistry and Biochemistry at
Brigham Young University, where he is currently a Professor and
Associate Department Chair. Professor Woolley has authored over 90
peer-reviewed papers, has given over 110 scientific presentations, and
has received 9 patents related to his work. His current research is
concentrated in three general areas: biotemplated nanofabrication, the
creation of novel and sophisticated integrated microfluidic systems for
enhanced biomarker quantitation, and the design of simple,
miniaturized biomolecular assays.

ACKNOWLEDGMENTS
We are grateful to the National Institutes of Health (Grant R01
EB006124) for support of this work.

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