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From microfluidic application to

nanofluidic phenomena issue

Reviewing the latest advances in microfluidic and nanofluidic

research

Guest Editors Professors Albert van den Berg, Harold Craighead and Peidong Yang

Please take a look at the issue 3 table of contents to access

other reviews in this themed issue
 CRITICAL REVIEW www.rsc.org/csr | Chemical Society Reviews

Microfluidic lab-on-a-chip platforms: requirements, characteristics

and applicationsw
Daniel Mark,zb Stefan Haeberle,zab Günter Roth,zab Felix von Stettenzab and
Roland Zengerlez*abc
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Received 16th September 2009

First published as an Advance Article on the web 25th January 2010
DOI: 10.1039/b820557b

This critical review summarizes developments in microfluidic platforms that enable the miniaturization, integration,
automation and parallelization of (bio-)chemical assays (see S. Haeberle and R. Zengerle, Lab Chip, 2007, 7,
1094–1110, for an earlier review). In contrast to isolated application-specific solutions, a microfluidic platform provides
a set of fluidic unit operations, which are designed for easy combination within a well-defined fabrication technology.
This allows the easy, fast, and cost-efficient implementation of different application-specific (bio-)chemical processes.
In our review we focus on recent developments from the last decade (2000s). We start with a brief introduction into
technical advances, major market segments and promising applications. We continue with a detailed characterization
of different microfluidic platforms, comprising a short definition, the functional principle, microfluidic unit operations,
application examples as well as strengths and limitations of every platform. The microfluidic platforms in focus are
lateral flow tests, linear actuated devices, pressure driven laminar flow, microfluidic large scale integration, segmented
flow microfluidics, centrifugal microfluidics, electrokinetics, electrowetting, surface acoustic waves, and dedicated
systems for massively parallel analysis. This review concludes with the attempt to provide a selection scheme for
microfluidic platforms which is based on their characteristics according to key requirements of different applications
and market segments. Applied selection criteria comprise portability, costs of instrument and disposability, sample
throughput, number of parameters per sample, reagent consumption, precision, diversity of microfluidic unit
operations and the flexibility in programming different liquid handling protocols (295 references).
a
Laboratory for MEMS Applications, Department of Microsystems
Engineering (IMTEK), University of Freiburg,
Introduction
Georges-Koehler-Allee 106, 79110 Freiburg, Germany.
E-mail: [email protected]; Fax: +49 761 203 7539;
Almost 10 000 papers have been published over the last
Tel: +49 761 203 7477 10 years on the topic of microfluidics1 and the annual numbers
b
HSG-IMIT—Institut für Mikro- und Informationstechnik, of new publications are still increasing continuously. According
Wilhelm-Schickard-Straße 10, 78052 Villingen-Schwenningen,
Germany to the ISI Web of Science they currently receive around 40 000
c
Centre for Biological Signalling Studies (bioss), citations per year (see Fig. 1). Additionally, over 1000 patents
Albert-Ludwigs-University of Freiburg, Germany referring to microfluidics have been issued in the USA alone.2
w Part of the themed issue: From microfluidic application to nano-
fluidic phenomena.
Consequently, microfluidics is established very well in
z All authors contributed equally to this paper. academia and industry as a toolbox for the development of

Mr Daniel Mark studied Dr Stefan Haeberle received his

physics at the University of PhD at the Laboratory for
Ulm, Germany and the Uni- MEMS Applications at the
versity of Oregon, USA, re- Department of Microsystems
ceiving an MSc degree and Engineering (IMTEK) at the
German diploma in 2006/2007. University of Freiburg, Germany
In 2007, he started his work as in 2009. He received his diploma
an R&D engineer and PhD degree in microsystem engineer-
candidate at the Institute of ing in 2004 from the University
Microsystems Technology of Freiburg. His research con-
(IMTEK) of the University centrates on the development of
of Freiburg, focussing on lab-on-a-chip systems based on
lab-on-a-chip applications for the pressure driven and centri-
medical diagnostics. In 2008, fugal microfluidic platform. He
Daniel Mark he became group leader of the Stefan Haeberle recently accepted a position at a
centrifugal microfluidics team of global consulting firm.
the joint lab-on-a-chip research division of IMTEK and the Hahn
Schickard Society. His research experience includes microfluidic
design, prototyping, and validation of biomedical applications.

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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1153–1182 | 1153
 new methods and products in life sciences. However, the
number of commercial products based on microfluidics is,
with few exceptions, still quite low. The question is: will
microfluidics remain a toy for academic and industrial re-
search or will it finally make the transition to an end-user
product?
Looking into the past, the first microfluidic technology was
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developed in the early 1950s when efforts to dispense small

amounts of liquids in the nanolitre and picolitre range were
made, providing the basis for today’s ink-jet technology.3 In
terms of fluid propulsion within microchannels with
sub-millimetre cross sections, the year 1979 set a milestone
when a miniaturized gas chromatograph (GC) was realized by
Terry et al. on a silicon (Si) wafer.4 The first high-pressure
liquid chromatography (HPLC) column microfluidic device,
fabricated using Si-Pyrex technology, was published in 1990
by Manz et al.5 By the end of the 1980s and the beginning of

Dr Günter Roth studied inter-

disciplinary physics and bio-
chemistry in parallel at the
Eberhard-Karls-University in
Tübingen, Germany. He
received the German diploma
in physics 2001 for a micro-
structure to separate cell lysate
and in biochemistry 2002 for Fig. 1 Growth of publications (a) and citations (b) of articles related
establishing an micro-ELISA to microfluidics.1 The data from 2009 are incomplete due to the
with one micron spatial resolu- editorial deadline of this review (November, 24, 2009) but already
tion. At the EMC micro- show a further increase in publications and citations.
collections GmbH, Tübingen,
Germany he developed two
the 1990s, several microfluidic structures, such as microvalves6
Günter Roth different high-throughput
screening platforms within his and micropumps7,8 had been realized by silicon micromachining,
PhD thesis. In 2007, he was post-doc in the Institute for Cell providing the basis for automation of complex liquid handling
Biology, Tübingen, Germany and finally joined the Laboratory for protocols by microfluidic integration.9,10 This was the advent of
MEMS Applications at IMTEK, University of Freiburg, as group the newly emerging field of ‘‘micro total analysis systems’’
leader for lab-on-a-chip assay development in July 2008. (mTAS11), also called ‘‘lab-on-a-chip’’.12

Dr Felix von Stetten studied Prof. Dr Roland Zengerle

Agricultural Engineering and received his diploma in physics
Dairy Sciences at the Technical from the Technical University
University of Munich, Germany. of Munich in 1990, and a PhD
After additional studies in Bio- from the ‘‘Universität der
technology and a research Bundeswehr München’’ based
period in food microbiology on the development of micro-
he received his PhD in micro- pumps in 1994. Since 1999 he
biology, also from the Techni- has been full professor at the
cal University of Munich in Department of Microsystems
1999. Then he spent three Engineering (IMTEK) at the
years in the diagnostic indus- University of Freiburg,
try and was involved in the Germany. Today Dr Zengerle
development of methods for in addition is a director at the
Felix von Stetten sample preparation, real-time Roland Zengerle Institut für Mikro- und
PCR and DNA-arrays. After- Informationstechnik of the
wards he joined the Laboratory for MEMS Applications at Hahn-Schickard-Gesellschaft (HSG-IMIT) and vice director
IMTEK, University of Freiburg, where he became involved in of the Centre for Biological Signalling Studies (bioss). The
biofuel cell- and lab-on-a-chip-research. Today Felix von Stetten research of Dr Zengerle is focused on microfluidics and
heads the joint research division for lab-on-a-chip of IMTEK nanofluidics. He acts also as European editor of the journal
and HSG-IMIT. ‘‘Microfluidics and Nanofluidics’’.

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c The Royal Society of Chemistry 2010
 At the same time, much simpler yet very successful available in the field of microfluidics into solutions that are
microfluidic analysis systems based on capillary liquid compatible to each other and therefore can be combined
transport in wettable fleeces emerged: First very simple within a given microfluidic platform.
‘‘dipsticks’’ for e.g. pH measurement based on a single fleece According to their dominating main liquid propulsion
paved the way for more complex ‘‘test strips’’ that have principle, we subdivide microfluidic platforms into 5 groups,
been sold as ‘‘lateral-flow tests’’ since the late 80s.13 Examples namely: capillary, pressure driven, centrifugal, electrokinetic
that are still on the market today are test strips for and acoustic systems, as depicted in Fig. 2. Each listed
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pregnancy,14 drug abuse,15–17 cardiac markers18 and also platform within these groups will be discussed. As a guide,
upcoming bio-warfare protection.19 Among the devices that we provide a characterization of the respective platforms in
completely automated a biochemical analysis by microfluidic Table 1. After providing a short general introduction to the
integration into one miniature piece of hardware, the test unique properties, requirements, and applications for micro-
strips became the first devices that obtained a remarkable fluidic platforms, this review focuses on a detailed discussion
market share with billions of units sold per year. Yet they of the microfluidic platforms listed in Fig. 2. For each
remain one of the few microfluidic systems which are sold in platform, the characterization and the general principle is
high numbers. presented first. After that the microfluidic unit operations as
Until today, in many cases, the revenue in the field of well as application examples are briefly discussed. Finally,
lab-on-a-chip is created on a business-to-business, rather each platform is characterized by providing an overview of its
than a business-to-consumer basis,20 as the vast majority of strengths and limitations. We conclude by an attempt to
research in the field only approaches the stage of demonstrations provide a selection scheme for microfluidic platforms
and is not followed up by the development of products for which is based on platform characteristics and application
end-users. Among the hurdles for market entry are high initial requirements.
investments and running fabrication costs.21 Regardless of the This review does not claim completeness. It contains
10 000 available publications, offering solutions for almost examples of microfluidic platforms which were selected as
every problem that might occur, the development of a fitting to our platform definition. The review should, however,
lab-on-a-chip product is still a risky adventure. Quite often provide the reader with some orientation in the field and the
the existing microfluidic building blocks are not compatible to ability to select platforms with appropriate characteristics on
or combinable with each other. In addition, in some cases the the basis of application-specific requirements.
fabrication technologies do not match or are too expensive.
Therefore implementing an application specific assay on a chip
is still a very complex and cumbersome task bearing technical The framework for microfluidic platforms: unique
risks and with it also financial risks. properties, requirements and applications
Instead of the development of individual and isolated
Microfluidics as an enabling technology: from classical liquid
lab-on-a-chip solutions, the constraint of using building blocks
handling to single-cell handling
to form well-defined microfluidic platforms enables the
implementation of biochemical assays in a much better, A number of classical, macroscopic liquid handling systems
foreseeable and less risky manner. A microfluidic platform for performing analytical and diagnostic assays have been in
comprises an easily combinable set of microfluidic unit-operations use for many decades. Examples are petri dishes, culture bottles
that allows assay miniaturization within a consistent fabrica- and microtitre plates (also called microplates). Petri dishes
tion technology. Hence, the intention of this review is to were first described in 188722 and culture bottles23 have been
provide an overview and classification of existing micro- in use since around 1850. Since roughly 60 years ago, they
fluidic platforms that enable the miniaturization, integration, have been manufactured as plastic disposables. In comparison,
automation and parallelization of (bio-)chemical assays in an microtiter plates are quite ‘‘modern,’’ having first been de-
easy, consistent and therefore less risky manner. This classification scribed in 1951.24 Based on these standards, highly automated
also enables us to categorize the huge amount of literature liquid handling solutions have been developed within the last

Fig. 2 Microfluidic platforms classified according to main liquid propulsion principle.

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 Table 1 The table provides a definition of a microfluidic platform in general, followed by a short characterization of every microfluidic platform
presented in the following chapters of this review

Microfluidic platform Characterization

Definition of a microfluidic platform A microfluidic platform provides a set of fluidic unit operations, which are designed for easy combination
within a well-defined fabrication technology. A microfluidic platform paves a generic and consistent way for
miniaturization, integration, automation and parallelization of (bio-)chemical processes.
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Lateral flow tests In lateral flow tests, also known as test strips (e.g. pregnancy test strip), the liquids are driven by capillary
forces. Liquid movement is controlled by the wettability and feature size of the porous or microstructured
substrate. All required chemicals are pre-stored within the strip. The readout of a test is typically
done optically and is quite often implemented as color change of the detection area that can be seen by the
naked eye.

Linear actuated devices Linear actuated devices control liquid movement by mechanical displacement of liquid e.g. by a plunger.
Liquid control is mostly limited to a one-dimensional liquid flow in a linear fashion without branches or
alternative liquid pathways. Typically liquid calibrants and reaction buffers are pre-stored in pouches.

Pressure driven laminar flow A pressure driven laminar flow platform is characterized by liquid transport mechanisms based on pressure
gradients. Typically this leads to hydrodynamically stable laminar flow profiles in microchannels. There is a
broad range of different implementations in terms of using external or internal pressure sources such as using
syringes, pumps or micropumps, gas expansion principles, pneumatic displacement of membranes, etc. The
samples and reagents are processed by injecting them into the chip inlets either batch-wise or in a continuous
mode.

Microfluidic large scale integration Microfluidic large scale integration describes a microfluidic channel circuitry with chip-integrated microvalves
based on flexible membranes between a liquid-guiding layer and a pneumatic control-channel layer. The
microvalves are closed or open corresponding to the pneumatic pressure applied to the control-channels. Just
by combining several microvalves more complex units like micropumps, mixers, multiplexers, etc. can be built
up with hundreds of units on one single chip.

Segmented flow microfluidics Segmented flow microfluidics describes the principle of using small liquid plugs and/or droplets immersed in a
second immiscible continuous phase (gas or liquid) as stable micro-confinements within closed microfluidic
channels. Those micro-confinements are in the picolitre to microlitre volume range. They can be transported by
pressure gradients and can be merged, split, sorted, and processed without any dispersion in microfluidic channels.

Centrifugal microfluidics In centrifugal microfluidics all processes are controlled by the frequency protocol of a rotating microstructured
substrate. The relevant forces for liquid transport are centrifugal force, Euler force, Coriolis force and capillary
force. Assays are implemented as a sequence of liquid operations arranged from radially inward positions to
radially outward positions. Microfluidic unit operations include metering, switching, aliquoting, etc.

Electrokinetics In electrokinetics platforms microfluidic unit operations are controlled by electric fields acting on electric
charges, or electric field gradients acting on electric dipoles. Depending on buffers and/or sample, several
electrokinetic effects such as electroosmosis, electrophoresis, dielectrophoresis, and polarization superimpose
each other. Electroosmosis can be used to transport the whole liquid bulk while the other effects can be used
to separate different types of molecules or particles within the bulk liquid.

Electrowetting Electrowetting platforms use droplets immersed in a second immiscible continuous phase (gas or liquid) as
stable micro-confinements. The droplets reside on a hydrophobic surface that contains a one- or two-
dimensional array of individually addressable electrodes. The voltage between a droplet and the electrode
underneath the droplet defines its wetting behavior. By changing voltages between neighboring electrodes,
droplets can be generated, transported, split, merged, and processed. These unit operations are freely pro-
grammable for each individual droplet by the end-user enabling online control of an assay.

Surface acoustic waves The surface acoustic waves platform uses droplets residing on a hydrophobic surface in a gaseous environ-
ment (air). The microfluidic unit operations are mainly controlled by acoustic shock waves travelling on the
surface of the solid support. The shock waves are generated by an arrangement of surrounding sonotrodes,
defining the droplet manipulation area. Most of the unit operations such as droplet generation, transport,
mixing, etc. are freely programmable.

Dedicated systems for massively Within the category of dedicated systems for massively parallel analysis we discuss specific platforms that do
parallel analysis not comply with our definition of a generic microfluidic platform. The characteristics of those platforms are
not given by the implementation of the fluidic functions but by the specific way to process up to millions of
assays in parallel. Prominent examples are platforms used for gene expression and sequencing such as mi-
croarrays, bead-based assays and pyro-sequencing in picowell-plates.

few decades (‘‘pipetting robots’’) and are the current ‘‘gold systems by offering new opportunities. Expectations often
standard’’ for automated sample processing in pharma and quoted in this context are:25
diagnostics. They offer a huge potential for many applications  Portability/wearability
since they are very flexible as well as freely programmable.  Higher sensitivity
Microfluidic platforms have to compete against these established  Lower cost per test

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c The Royal Society of Chemistry 2010
  Shorter time-to-result
 Less laboratory space consumption
Additionally, scaling effects lead to new phenomena and
permit entirely new applications that are not accessible to
classical liquid handling platforms, such as:
 Well-defined, laminar flow
 Controllable diffusion enabling defined concentration
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gradients on the length scales of single-cells

 Surface forces dominate over gravitational forces
 Liquid compartments of the size of a single cell or smaller
 High-speed serial processing (at single cell level)
 High degree of parallelization (up to around 106)
In the following, the effects and phenomena leading to Fig. 3 Concept of differential manipulation in a single bovine
the above-mentioned expectations and the potential for new capillary endothelial cell using multiple laminar flows. (a, b), Chip
layout: 300 mm  50 mm channels are used to create laminar interfaces
applications will be outlined briefly.
between liquids from different inlets. (c) Fluorescence image of a cell
It is obvious that the amount of reagent consumption can be
locally exposed to red and green fluorophores in a laminar flow.
decreased significantly by scaling down the assay volume. (d) Migration of fluorophores over time (scale bars, 25 mm). This
Additionally, by reducing the footprint of each individual test, shows the high potential for accurate spatial control and separation of
a higher degree of parallelization can be achieved in a limited liquids achievable in microfluidic laminar flows. Adapted by permission
laboratory space. A prime example for microfluidic tests with from Macmillan Publishers Ltd: Nature,28 copyright 2001.
minimal reagent consumption are parallel reactions in hundreds
of thousands of individual wells with picolitre-volumes,26 which market share have emerged from this field so far. In the
took genome sequencing to a new level27 hardly achievable by next chapter, we will outline hurdles and present emerging
classical liquid handling platforms. paradigm changes that will influence future research in
With decreasing length scales, surface phenomena (e.g. capillary microfluidics.
forces, surface charges, etc.) become increasingly dominant over
The need for the microfluidic platform approach
volume phenomena. This permits purely passive liquid actuation
based on capillary forces used in the popular lateral flow assays Definition of a microfluidic platform: A microfluidic platform
also know as capillary test strips. Another effect is the onset of provides a set of fluidic unit operations, which are designed for
laminar flow at low Reynolds numbers in small channels. This easy combination within a well-defined fabrication techno-
enables the creation of well-defined and stable liquid–liquid logy. A microfluidic platform paves a generic and con-
interfaces down to cellular dimensions. Therefore, large con- sistent way for miniaturization, integration, automation and
centration gradients can be applied and the effects monitored at parallelization of (bio-)chemical processes.
the single cell level28 (Fig. 3). In summary, laminar flow con-
ditions and controlled diffusion enable temporally and spatially In the last two decades, thousands of researchers spent a
highly resolved reactions with little reagent consumption. huge amount of time to develop micropumps,33–36 micro-
A different paradigm using the possibility of controlling valves,37 micromixers,38,39 and microfluidic liquid handling
interfaces in microfluidic applications is the concept of droplet- devices in general. However, a consistent fabrication and
based microfluidics, also called ‘‘digital microfluidics’’.29 The interfacing technology as one prerequisite for the efficient
on-demand generation of liquid micro-cavities either in air or a development of lab-on-a-chip systems is very often still
second immiscible liquid enables the manipulation of missing. This missing link can only be closed by establishing
small quantities of reagents down to single cells with high a microfluidic platform approach which allows the fast and
throughput.30 Control and manipulation of such droplets easy implementation of (bio-)chemical protocols based on
can be achieved by another favorable aspect of the high common building blocks. The idea follows the tremendous
surface-to-volume ratio in microfluidics: the possibility impact of platforms in the application-specific integrated
to control the liquid flow by electrically induced forces or circuit (ASIC) industry in microelectronics, where validated
electrowetting.31 Having the huge background of theoretical elements and processes enabled faster design and cheaper
and practical knowledge in electronics, this is obviously a fabrication of electronic circuitries.
desirable property. Additional helpful properties of small Conveying this to the microfluidic platform approach, a set
assay volumes are fast thermal relaxation and low power of validated microfluidic elements is required, each able to
consumption for liquid manipulation and thermal control. This perform a certain basic fluid handling step or unit operation.
can speed up assays that require thermocycling, such as PCR, Such basic unit operations are building blocks of laboratory
which was realized in numerous microfluidic applications.32 protocols and comprise fluid transport, fluid metering, fluid
This short summary shows that there is the potential for mixing, valving, separation or concentration of molecules or
many novel applications and improvements over the state- particles (see Table 2) and others. Every microfluidic plat-
of-the-art within the above-mentioned criteria of sensitivity, form should offer an adequate number of microfluidic unit
cost, time, and size. However, despite a myriad of publications operations that can be easily combined and thereby enable
about microfluidic components, principles and applications, easy implementation of application-specific assays within that
only a limited number of successful products with a relevant given platform.

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 Table 2 Common features of microfluidic platforms from portable systems with preferably multi-parameter
capabilities.
Microfluidic unit operations Fabrication technology
These diverse fields of applications are associated with a
 Fluid transport  Validated manufacturing number of analytical and diagnostic tasks. This outlines the
 Fluid metering technology for the whole set of field for the microfluidic technology, which has to measure
 Fluid valving fluidic unit operations (prototyping
 Fluid mixing and mass fabrication) itself against the state-of-the-art in performance and costs.
 Separation Table 3 gives an overview on some important requirements of
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 Accumulation/amplification  Seamless integration of different the different market segments and application examples, with
elements
 Reagent storage & release . . . preferable in a monolithic way respect to the following selection criteria:
 Incubation . . . or by a well defined easy  Portability/wearability: miniaturized, hand-held device
 ... packaging technique with low energy consumption
 Throughput: number of samples/assays per day
 Cost of instrument: investment costs of the instrument
This concept, however, does not imply that every micro- (‘‘reader’’)
fluidic platform needs to provide a complete set of all the unit  Cost of disposables: defining the costs per assay (together
operations listed in Table 2. It is much more important that with reagent consumption)
the different elements are connectable, ideally in a monolithically  Number of parameters per sample: number of different
integrated way or at least by a well defined, ready-to-use parameters to be analyzed per sample
interconnection and packaging process. Therefore at least  Low reagent consumption: amount of sample and/or
one validated fabrication technology is required to realize reagents required per assay
complete microfluidic solutions from the individual elements  Diversity of unit operations: the variety/completeness of
within a microfluidic platform. laboratory operations that can be realized
 Precision: the volume and time resolution that is possible
 Programmability: the flexibility to adapt liquid handling
Market requirements and platform selection criteria
protocols without fabricating a new chip
The requirements on microfluidic platforms differ greatly between These criteria will be discussed for each of the platforms
different market segments. Following a roadmap on microfluidics described in this review.
for life sciences,40 the four key market segments for microfluidic
lab-on-a-chip applications are, according to their market size:
Biochemical applications for microfluidic platforms
in vitro diagnostics, drug discovery, biotechnology, and ecology.
The largest market segment, in vitro diagnostics, can be Here, a short overview of the fields of applications that are
subdivided into point-of-care testing (e.g. for self-testing in typically addressed by microfluidic platforms is presented.
diabetes monitoring or cardiac marker testing in emergency A first field of application is biotransformation, the break-
medicine) and central laboratory-based testing (e.g. core down and generation of molecules and products by the help of
laboratory in a hospital). Especially the self- and point-of-care enzymes, bacteria, or eukaryotic cell cultures. This comprises
testing segments offer huge potential for microfluidics, since fermentation, the break down and re-assembly of molecules
portability and/or wearability is an important requirement. (e.g. fermentation of sugar to alcohol), and (bio)synthesis the
Drug discovery in the pharmaceutical industry is the second build-up of complex molecules (e.g. antibiotics, insulin, inter-
largest segment. Here, enormous effort is undertaken to feron, steroids). Especially in the field of process development
identify new promising drug candidates in so called high- challenges are to handle a large number of different liquids
throughput screening (HTS) or massively parallel analysis.41 under controlled conditions such as temperature or pH, in
After screening promising candidates, so-called hits have to be combination with precise liquid control down to nL or even
validated and characterized (hit characterization). In this pL volumes. Some examples of microfluidic liquid handling
context cell-based assays have received increasing interest over platforms are given for fermentation in micro bioreactors,44–51
recent years.42,43 These assays often require the handling of the biosynthesis of radiopharmaceuticals,52 and antibody
single cells, which becomes possible using microfluidic approaches. screening, phage- and ribosome-display technologies.53,54
This market segment requires high sample throughput and low Another major field of application is analytics. The analysed
costs per test. molecule (analyte) can be from a variety of biomolecules,
The third segment is the biotech market with fermentation- including proteins and nucleic acids. Here, the main require-
based production (e.g. for biopharmaceuticals or food). This ments are effective mixing strategies and highly precise liquid
industry shows a great demand for on-line process monitoring metering and liquid handling which are needed to get accurate
and analyses in the field of process development. Here, quantitative results. Also, automation and portability/
low sample volumes and flexibility (programmability) are wearability combined with a large set of unit operations for the
important factors. implementation of complex analytical protocols are required.
Ecology is another market segment, comprising the field As an emerging field, cellular assays are the most challenging
of agricultural- and water-analysis, either as on-site spot format, since the cells have to be constantly kept in an
tests or as continuous monitoring. Included are also applica- adequate surrounding to maintain their viability and activity
tions related to homeland security, e.g. the detection of (control of pH, O2, CO2, nutrition, etc.). Cellular tests are
agents that pose biological threats. This market benefits useful to assess the effect of new pharmaceutical entities at

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 Table 3 Market segments for microfluidic lab-on-a-chip applications and their requirements*
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different dosing concentrations on toxicity, mutagenicity, In general, microfluidic substrates should be inert against
bioavailability and unwanted side effects. The most exciting the expected sample and assay reagents which might comprise
prospect is the establishment of assays with single-cell organic or inorganic solvents or extreme pH values.57 Like-
analyses.55,56 Requirements on cellular assays include high- wise, the sample must not be affected by the microfluidic
throughput solutions as well as a low reagent consumption substrate in any way that could influence the analytical result.
per test. For example, nucleic acids are critical molecules because of
After this short overview, the next chapter will summarize their negative charge and tendency to adhere to charged
the liquid handling challenges that arise from the different surfaces such as metal oxides. Similar problems occur with
liquids associated with these fields of applications. proteins or peptides which exist in a variety of electrical
charges, molecular sizes, and physical properties. In addition
to possible adsorption onto the surfaces, the catalytic activity
Requirements on microfluidic platforms related to liquids with
of enzymatic proteins can be reduced by interaction with the
biochemical content
substrate.58–61 A general counter-measure against the inter-
Performing microfluidics with pure water cannot be compared action of biomolecules and microfluidic substrates is to block
to the challenge of developing a microfluidic platform for the substrates with another suitable biomolecule which is
handling of liquids with biochemical content. Here, a large added in excess. For instance, bovine serum albumin (BSA)
variety of changing liquid properties needs to be considered, adsorbs to nearly any surface thus passivating it.62,63 Another
ranging from surface tension, non-Newtonian viscosities and significant challenge in microfluidic production technology is
the contact angle on a certain surface. In addition, when to maintain the activity of proteins during processes such as
handling biological samples, such as blood, an inter-sample thermal bonding64,65 or UV curing steps. In addition, the long-
variation, e.g. due to physiological differences between term stability of pre-stored dry reagents is required, hence
patients, has to be managed by the microfluidic system. In materials with low vapor transition rates have to be selected.
the following, a short summary of typical sample materials Experience shows that this set of challenges needs to be
and their interactions with the microfluidic substrate is considered at the very beginning of a fluidic design, since the
provided. Also, strategies to prevent unfavorable interactions listed problems can jeopardize the functionality of the whole
are outlined. system if addressed too late.

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 Lateral flow tests
Characterization of lateral flow tests
In lateral flow tests, also known as test strips (e.g. pregnancy
test strip), the liquids are driven by capillary forces. Liquid
movement is controlled by the wettability and feature size of
the porous or microstructured substrate. All required chemicals
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are pre-stored within the strip. The readout of a test is typically

done optically and is quite often implemented as a color change
in the detection area that can be seen by the naked eye.

General principle
The first immunoassay performed in a capillary driven system
was reported in 1978.66 Based on this technique, the commonly
known ‘‘over-the-counter pregnancy test’’ was introduced into
the market in the middle of the ‘80s. Today, this microfluidic
platform is commonly designated as a ‘‘lateral flow test
(LAT)’’.13 Other terms are ‘‘test strip’’, ‘‘immunochromato-
Fig. 4 Schematic design of a lateral flow test (according to ref. 68),
graphic strip’’, ‘‘immunocapillary tests’’ or ‘‘sol particle immuno-
(a) Sample pad (sample inlet and filtering), conjugate pad (reactive
assay (SPIA)’’.67 Astonishingly, hardly any publications from
agents and detection molecules), incubation and detection zone with test
a microfluidic point of view or in terms of material classifi- and control lines (analyte detection and functionality test) and final
cation exist, and apparently many ‘‘company secrets’’ are kept absorbent pad (liquid actuation). (b) Start of assay by adding liquid
unpublished.68 sample. (c) Antibodies conjugated to colored nanoparticles bind the
The ‘‘standard LAT’’ consists of an inlet port and a detec- antigen. (d) Particles with antigens bind to test line (positive result),
tion window (Fig. 4(a)). The core comprises several wettable particles w/o antigens bind to the control line (proof of validity).
materials providing all biochemicals for the test and enough
capillary capacity to wick the sample through the whole strip.
the pad.70 The conjugation pad is made of cross-linked silica
The sample is introduced into the device through the inlet into
and is used as dry-reagent storage for antibodies specific to the
a sample pad (Fig. 4(b)), which holds back contaminations
antigen conjugated to the signal generating particle. The
and dust. Through capillary action, the sample is transported
conjugates are typically colored or fluorescent nanoparticles
into the conjugate pad, where antibodies conjugated onto a
with sizes up to 800 nm, which flow without obstruction
signal-generating particle are rehydrated and bind to the
through the fleeces together with the sample. Most often
antigens in the sample (Fig. 4(c)). This binding reaction
colloidal gold19 or latex71 and more rarely carbon, selenium,
continues as the sample flows in the incubation and detection
quantum dots, or liposomes72 are the choice of nanoparticles.
pad. On the test line a second type of antibody catches the
The length, material (mainly nitro-cellulose) and pore-size
particles coated with antigens, while a third type of antibody
(50 nm to 12 mm, depending on the applied nanoparticles) of
catches particles which did not bind to an analyte on the
the detection and incubation pad define the incubation time.68
control line. The control line shows a successfully processed
The detection and enrichment of the conjugates is achieved
test while the detection line shows the presence or absence of a
on the antibody-bearing lines. Analyte detection is performed
specific analyte (Fig. 4(d)). Typically the result becomes visible
on the test line and proof of assay validity on the control
after 2 to 15 min.
line. The readout is typically done by naked eye for absence
Over the last decades, LAT transformed from a simply
(1 colored line) or presence (2 colored lines) of a minimum
constructed device into a more and more sophisticated
analyte amount. A readout with a reader enables quantitative
high-tech platform with internal calibrations and quantitative
analyte detection.69,73 For multi-analyte detection68 or semi-
readout by a hand-held reader (Fig. 5).69
quantitative setups74 several test lines are applied.
Within the last few years, new LAT designs have been
Unit operations
developed in combination with the device-based readout in
The different pads in the test strip represent different functions hand-held systems. Here a complex capillary channel network
such as loading, reagent pre-storage, reaction, detection, provides the liquid actuation (Fig. 5). Antibodies conjugated
absorption and liquid actuation. The characteristic unit operation to nanoparticles or special enzymes are pre-stored at the inlet.
of LATs is the passive liquid transport via capillary forces, The incubation time is defined by the filling time of the
acting in the capillaries of a fleece, a microstructured surface, capillary network. Typically, readout is done quantitatively
or a single capillary. The absorption volume of an absorption by fluorescence or electrochemical detection. The time-to-
pad defines how much sample is wicked through the strip and result is usually several seconds. Blood glucose or coagulation
provides metering of the sample.68 The sample pad usually monitoring are the most common applications for such quan-
consists of cellulose or cross-linked silica and is used for titative readouts.69 To accommodate aging, batch-to-batch
filtering of particles and cells as well as separating the analyte variations and sample differences, and also to achieve higher
from undesired or interfering molecules, which is absorbed in precision and yield of the assay, several internal controls and

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Fig. 5 LAT for blood coagulation with hand-held readout according to Cosmi et al.69,73 (image (a) courtesy of Roche Diagnostics). (a) Loading
of blood. (b) The blood flows from the inlet into the fluidic network rehydrating the coagulation chemistry. The ‘‘drop detect’’ electrodes detect
whether blood is applied and measure the incubation times. Several capillaries are filled and the filling is monitored with according electrodes.
A Ag/AgCl electrode is used as standard electrode for calibration and analysis. Finally the analyte gets quantified by optical or electrochemical
detection.

calibrations are automatically performed during analysis by hand-held).69,73 High-throughput or screening applications
the readout device. are possible, but quite difficult to implement.
In total, the lateral flow test is a well established platform
Application examples with a large but limited field of applications and consequently
Lateral flow tests were among the first successfully com- a benchmark for the home-care and in vitro diagnostics (IVD)
mercialized microfluidic products. A huge amount of assays sector in terms of cost per assay and simplicity.
have been developed on the capillary test strip platform during
the past 30 years.75 Today, they serve a wide field of applications, Linear actuated devices
including health biomarkers (pregnancy,13,76 heart attack,70
blood glucose,77 metabolic disorders78), small molecules (drug Characterization of linear actuated devices
abuse,16 toxins,79 antibiotics80), infectious agents (anthrax,81 Linear actuated devices control liquid movement by mechanical
salmonella,82 viruses83), immunodiagnostics,84 RNA applications,81 displacement of liquid e.g. by a plunger. Liquid control is
and even whole bacteria.85 Some of the more recent designs mostly limited to a one-dimensional liquid flow in a linear
and publications even show the detection of DNA83 without fashion without branches or alternative liquid pathways.
the need of amplification by PCR, which would open yet Typically liquid calibrants and reaction buffers are pre-stored
another vast field of new applications. The first trials for in pouches.
massively parallel screening in combination with microarrays
were made in lateral flow tests.70,81 General principle
One of the first examples of a linear actuated device was the
Strengths and limitations
i-STATs for quantitative bedside testing, introduced in the
The fact that 6 billion glucose test strips were sold in 200786 early 1990s by Abbott Point of Care Inc., NJ, USA. It relied
already indicates that the LAT may be seen as a gold-standard on active liquid actuation by displacement.87 Compared to
microfluidic platform in terms of cost, handling simplicity, lateral flow tests, this principle was one step ahead in result
robustness, market presence and the number of implemented quantification and possible applications, but also in complexity
lab-on-a-chip applications.68 The amount of sample and of the processing device and disposable test carrier.
reagent consumption are moderate, and the concept is mainly The characteristic actuation principle of the linear actuated
used for qualitative or semi-quantitative assays. Especially the platform is the mechanical linear propulsion of liquids with no
complete disposable test carriers with direct visual readout, branching. Normally, the liquid actuation is performed by a
easy handling, and a time-to-result between seconds and plunger which presses on a flexible pouch, displacing its
several minutes are predestined for untrained users. content. Another common attribute is the pre-storage of all
The simplicity of the test strip is also its major drawback. required reagents (liquid and dry) on the disposable test
Assay protocols within capillary driven systems follow a fixed carrier (cartridge). Systems based on this platform thus offer
process scheme with a limited number of unit operations, fully integrated sample-to-result processing in a relatively
imprinted in the microfluidic channel design itself. Highly short time.
precise liquid handling and metering is also extremely
Unit operations
challenging.68 The dependency of the purely capillary liquid
actuation on the sample properties can also be a major Basically, the linear actuated platform relies on only two unit
problem, leading to false positive or negative results14 or operations: liquid transport and reagent storage. Liquid
decreased precision. New designs allow applications with transport is achieved by mechanical displacement (e.g. with
quantitative analysis, but require a readout device (mainly a plunger). By pressing on flexible compartments of the

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 disposable, the liquid can be transported between reservoirs.87 A second example is the lab-in-a-tube (Liatt) analyzer from
Alternatively, a weakly bonded connection to an adjacent IQuum.92 This bench-top device with disposable test tubes
reservoir can be disrupted, or the connection to a neighbouring contains all necessary reagents for amplification-based nucleic
cavity selectively blocked.88 Liquid reagent storage can easily acid tests. It integrates sample preparation, amplification and
be implemented by integrating pouches into the cartridge. detection and is a fully integrated sample-to-result platform
Mixing can also be realized on the linear actuated platform with response times between 30 and 60 min. Handling of the
by moving liquids between neighbouring reservoirs.88 platform requires only a few steps: The sample (e.g. 10 mL of
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whole blood) is collected in the collection tube that is integrated

Application examples into the disposable, the barcode on the disposable is scanned,
and the tube is then inserted into the analyzer. The disposable
One example of a linear actuated device is of course
features compartmentalized chambers in a tube which contain
the previously mentioned i-STATs analyzer from Abbott
different reagents and can be connected via peelable seals
Point-of-Care.89 Using different disposable cartridges, several
(Fig. 7). Liquid control is performed by actuators that com-
blood parameters (blood gases, electrolytes, coagulation,
press the compartments, displacing the liquid into adjacent
cardiac markers, and hematology) can be determined with
chambers.88 Sample preparation includes a nucleic acid puri-
the same portable hand-held analyzer for automated sample
fication step: magnetic beads serve as solid nucleic acid
processing and readout (Fig. 6(a)). Since only the disposable
binding phase and are controlled by a built-in magnet. For
polymer cartridge is contaminated with the blood sample and
nucleic acid amplification, compartments can be heated and
thus has to be disposed after performing the diagnostic assay,
the liquid is transferred between two different temperature
the analyzer device itself is reusable. Typical response times of
zones thus cycling the sample. The system is capable of
the system are in the order of a few minutes.
real-time fluorescence readout.
The system features an integrated calibration solution that
is pre-stored in the disposable. The analysis process takes only
a few steps: As depicted in Fig. 6, the blood sample (a few Strengths and limitations
drops) is filled into the cartridge by capillary forces (b), and The presented commercially available examples show that
placed into the analyzer (c). First, the calibrant solution is automation and time-reduction by microfluidic systems with
released and provides the baseline for an array of thin-film active processing devices can indeed be achieved in a market-
electrodes integrated in the disposable. Then the sample is relevant context. The potential of the linear actuated device
pushed into the measuring chamber and displaces the calibrant. platform certainly lies in its simplicity and the ability for long-
Thereby, the blood parameters which can be determined by term liquid reagent storage. The presented application exam-
the sensor array of the specific disposable are measured and ples are portable and show a high degree of assay integration,
presented at the integrated display of the hand-held analyzer. requiring no external sample pre- or post-processing steps.
Several studies showed good agreement between laboratory Typical liquid (sample) volumes handled on the platform are
results and this POC-system.87,90,91 in the range of 10–100 mL, which is adequate for point-of-care
diagnostic applications (capillary blood from finger tip).
While disposables can generally be mass-produced, these
can become somewhat expensive due to the integration of
sensors (i-STATs) and liquid reagents (i-STATs and Liatt).

Fig. 7 Functional principle and processing steps in a nucleic acid test

in the lab-in-a-tube analyzer according to Chen et al.88 The disposable
contains pouches with reagents (light blue) which are actuated by
plungers while clamps open and close fluidic connections to adjacent
Fig. 6 Images and handling procedure of the i-STATs analyzer. pouches. (a) Sample is inserted (red). (b) Sample is mixed with pre-
(a) Photograph depicting the portable i-STATs analyzer for clinical stored chemicals containing magnetic capture-beads. (c) Unwanted
blood tests.89 (b) Depending on the blood parameters to be measured, sample components are moved to a waste reservoir while the capture-
a certain disposable cartridge is filled with blood by capillary forces beads are held in place by a magnet. (d, e) Further processing steps
from the finger tip and (c) afterwards loaded into the analyzer for allow sequential release of additional (washing) buffers and heating
assay processing and readout (images courtesy of Abbott Point of steps (red block) for lysis and thermocycling demands. The system
Care Inc., NJ, USA). allows optical readout by a photometer (PM).

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 Time-to-result varies between minutes and approximately one
hour, depending on the assay.
The advantage of full integration with pre-stored reagents
comes at the price of an imprinted protocol that cannot be
changed for a specific test carrier. The number of unit opera-
tions is somewhat limited, in particular separation, switching,
and aliquoting as well as precise metering are difficult to
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realize. This hinders the implementation of more complex

assays and laboratory protocols in linear actuated systems, Fig. 8 Contacting on the laminar flow platform. Three different
liquid streams are symmetrically contacted at an intersection point.
such as integrated genotyping with a plurality of genetic
This microfluidic structure is also referred to as a ‘‘flow focusing
markers or multiparameter assays.
structure’’.93

Pressure driven laminar flow

By varying the ratio of the flow rates, the lateral width of the
Characterization of pressure driven laminar flow central streamline within the common outlet channel can be
A pressure driven laminar flow platform is characterized by adjusted very accurately. Consequently, micro-objects
liquid transport mechanisms based on pressure gradients. suspended in the liquid flowing through the central channel
Typically this leads to hydrodynamically stable laminar flow are focused and aligned to this well-defined streamline position.
profiles in microchannels. There are a broad range of different If the available duration for a (bio-)chemical reaction needs to
implementations in terms of using external or internal pressure be limited, the contacted liquid streams can again be separated
sources such as using syringes, pumps or micropumps, gas further downstream as shown in ref. 103.
expansion principles, pneumatic displacement of membranes, For the separation of micro-objects like living cells or micro-
etc. The samples and reagents are processed by injecting beads from a liquid stream, several technologies have been
them into the chip inlets either batch-wise or in a continuous presented relying either on geometrical barriers,103 or magnetic
mode. forces.104,105 Sorting of micro-objects, i.e. the selective separation
based on size or any other feature, was implemented using
General principle magnetic forces,106,107 acoustic principles,108 dielectrophoresis,109
or hydrodynamic principles97–99,110 on the pressure driven lami-
As mentioned earlier, liquid flow in microchannels is typically
nar flow platform. The common principle of all these techno-
strictly laminar over a wide range of flow rates and channel
logies is a force acting selectively on the suspended micro-objects
dimensions. Pressure driven laminar flow offers several
(particles or cells), while the liquid stream stays more or less
opportunities for assay implementation:
unaffected.
 Predictable velocity profiles
A great number of valving principles exist on the pressure
 Controllable diffusion mixing
driven laminar flow platform, summarized in a review by Oh
 Stable phase arrangements, e.g. in co-flowing streams
and Ahn.37 Active as well as passive solutions have been
These advantages have been utilized for several lab-on-a-
presented. However, no standards have emerged so far, so
chip applications in the past. Probably the oldest example is
the choice and implementation of valves remains a difficulty
the so-called ‘‘hydrodynamic focusing’’ technology,93 used to
on this platform. A possible approach is to transfer the valving
align cells in continuous flow for analysis and sorting in flow
functionality off-chip,111 thus decreasing the complexity and
cytometry.94,95 Today, many technologies still use laminar
cost of the disposable.
flow effects for particle counting96 or separation.97–101 However,
pressure driven laminar flow can also be utilized to implement
other (bio-)chemical assays for lab-on-a-chip applications Application examples
as described within this section. In particular, nucleic acid- One recently established technology on the pressure driven
based diagnostic systems received a great deal of interest in laminar flow platform is so called ‘‘phase transfer magneto-
the last decade, since the first introduction of a combined phoresis (PTM)’’.104 Magnetic microparticles flowing through
microfluidic PCR and capillary electrophoresis in 1996 by a microfluidic channel network are attracted by a rotating off-
Woolley et al.102 chip permanent magnet, and can consequently be transferred
between different co-flowing liquid streams. As a first applica-
Unit operations
tion, DNA purification with magnetic beads was success-
The basic unit operation on the pressure driven laminar flow fully demonstrated with a yield of approximately 25%104
platform is the contacting of at least two liquid streams at a (first prototype). Thus, this system provides continuous
microfluidic channel junction (see Fig. 8). This leads to con- DNA-extraction capability which could serve as an automated
trolled diffusional mixing at the phase interface, e.g. for sample preparation step for flow-through PCR, in e.g. bio-
initiation of a (bio-)chemical reaction.103 It can also be applied process monitoring (of fermentation) applications.
for the lateral focusing of micro-objects like particles or cells in Other microfluidic applications based on the manipulation of
the channel.93 The required ‘‘flow focusing’’ channel network magnetic microparticles with external permanent magnets have
consists of one central and two symmetric side channels, been shown. One example is the free-flow magnetophoresis,106,107
connected at a junction to form a common outlet channel. which can be utilized to sort magnetic microparticles by size.

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 A large number of microfluidically automated components A difficulty of the platform is the necessity to connect the
for batch-wise nucleic acid diagnostics based on pressure pressure source to the (disposable) chip, which decreases the
driven laminar flow chips have been published and summed portability and requires additional manual steps. Another
up in several reviews.32,112,113 However, a totally integrated challenge is the Taylor dispersion115 of streamwise dispersed
system remains a challenge, since the integration of sample samples which can make it hard to accurately track analyte
preparation proved difficult,113 although it seems to be in concentrations. Unit operations on the platform are optimized
reach, as the next two examples show. for mixing and separation processes and somewhat limited in
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Easley et al. showed integrated DNA purification, PCR, other aspects such as aliquoting.
electrophoretic separation and detection of pathogens in less
than 30 min.114 The assay was performed on a pressure
driven four layer glass/PDMS chip with elastomeric valves. Microfluidic large scale integration
Temperature cycling for PCR was achieved by IR radiation. Characterization of microfluidic large scale integration
Only the sample lysis step was not integrated in the micro-
fluidic chip. Detection of Bacillus anthracis from infected mice Microfluidic large scale integration describes a microfluidic
and Bordetella pertussis from a clinical sample was successfully channel circuitry with chip-integrated microvalves based on
demonstrated. flexible membranes between a liquid-guiding layer and a
An integrated mTAS system for the detection of bacteria pneumatic control-channel layer. The microvalves are closed
including lysis, DNA purification, PCR and fluorescence or open corresponding to the pneumatic pressure applied to
readout has also been published recently.111 A microfluidic the control-channels. Just by combining several microvalves
plastic chip with integrated porous polymer monoliths more complex units like micropumps, mixers, multiplexers,
and silica particles for lysis and nucleic acid isolation etc. can be built up with hundreds of units on one single
was used for detection (Fig. 9). A custom-made base device chip.
provided liquid actuation and off-chip valving by stopping
General principle
liquid flow from the exits of the chip, utilizing the incompressi-
bility of liquids. Detection of 1.25  106 cells of Bacillus The microfluidic large scale integration (LSI) platform arose in
subtilis was demonstrated with all assay steps performed 1993.116 At the same time, a novel fabrication technology for
on-chip. microfluidic channels, called soft lithography made its appearance.
Soft lithography is based on the use of elastomeric stamps,
Strengths and limitations
molds and conformable photomasks to fabricate and replicate
One strength of the platform lies in its potential for continuous microstructures.117 Using this technology, the monolithic
processing of samples. Continuous sample processing is of fabrication of all necessary fluidic components within one
utmost importance for online monitoring of clinical para- single elastomer material (polydimethylsiloxane, PDMS)
meters, process control in fermentation, water quality control became possible, similar to the silicon-based technology in
or cell sorting. Typically one or a few parameters are microelectronics. PDMS, also known as silicone elastomer, is
monitored. The application examples showed one system an inexpensive material offering several advantages compared
capable of continuous DNA extraction as well as other to silicon or glass. It is a cheap, rubber-like elastomer with
implementations that integrated complex batch-wise protocols good optical transparency and biocompatibility. A detailed
such as nucleic acid analysis. The platform is in principle review on the use of PDMS for different fields of applications
compatible with polymer mass-production technologies can be found in ref. 118.
such as injection molding, enabling inexpensive disposable The strength of the technology became obvious, when
microfluidic chips. Stephen Quake’s group expanded the technology towards

Fig. 9 Chip for integrated detection of bacteria including lysis, DNA isolation and PCR published by Sauer-Budge et al.111

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 the multilayer soft-lithography process, MSL.119 With this increase of reaction kinetics by nearly two orders of magnitude
technology, several layers of PDMS can be hermetically has been demonstrated in surface binding assays.123
bonded on top of each other resulting in a monolithic, However, the key feature to tap the full potential of the
multilayer PDMS structure. This enables the fabrication of large scale integration approach is the multiplexing technology
microfluidic chips with densely integrated microvalves, pumps allowing for the control of N fluid channels with only 2 log2 N
and other functional elements. Today, this technology is control channels. Based on this principle, a microfluidic
pushed forward by the company Fluidigm Corp., CA, USA. storage device with 1000 independent compartments of
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approximately 250 pL volume and 3574 microvalves has been

demonstrated.120
Unit operations
Based on the high elasticity of PDMS, the elementary micro- Application examples
fluidic unit operation is a valve which is typically made of a One application example on the microfluidic LSI platform is
planar glass substrate and two layers of PDMS on top of each the extraction of nucleic acids (NA) from a small amount of
other. One of the two elastomer layers contains the fluidic cells124,125 for cell-based assays. For the extraction of NA from
ducts while the other elastomer layer features pneumatic a cell suspension, the cell membrane has to be destroyed first
control channels. To realize a microfluidic valve, a pneumatic (chemical lysis of the cell). Afterwards, the NA are specifically
control channel crosses a fluidic duct as depicted in Fig. 10(a). separated from the residual cell components using a solid
A pressure p applied to the control channel squeezes the phase extraction method based on a NA affinity column
elastomer into the lower layer, where it blocks the liquid (paramagnetic beads). This extraction protocol is completely
flow. Because of the small size of this valve, on the order implemented on the microfluidic platform using the basic unit
of 100  100 mm2, a single integrated fluidic circuit can operations for valving, metering, mixing and switching of
accommodate thousands of valves. Comparable to developments liquids. Measurable amounts of mRNA were extracted in an
in microelectronics, this approach is called ‘‘microfluidic large automated fashion from as little as a single mammalian cell
scale integration’’ (LSI).120 and recovered from the chip.124 Based on this technology, the
The valve technology called NanoFlext (Fluidigm) is the development of a nucleic acid processor for complete single
core technology of the complete platform. For example, by cell analysis is under way.126–128
placing two such valves at the two arms of a T-shaped channel Also many other applications have been implemented on the
a fluidic switch for the routing of liquid flows between several LSI platform over the last few years: protein crystallization,129
adjacent channels can be realized. Liquid transport within the immunoassays,130 automated culturing of cells131 or multi-
fluid channels can be accomplished by external pumps cellular organisms132 and DNA synthesizing.133
while the PDMS multilayer device merely works passively as From a commercial perspective, Fluidigm Corp. has
integrated valves, or an integrated pumping mechanism can be launched three different products based on the large scale
achieved by combining several micro-valves and actuating integration platform within the last years: the BioMarkTM
them in a peristaltic sequence (Fig. 10(d)). technology for molecular biology (e.g. TaqMans assay), the
Metering of liquid volumes can be achieved by crossed fluid TOPAZs system for protein crystallography, and the
channels and a set of microvalves. Therefore, the liquid is Fluidigms EP1 system for genetic analysis. The EP1 system
initially loaded into a certain fluid channel and afterwards in particular, bears great potential for high-throughput screening
segmented into separated liquid compartments by pressurizing applications such as sequencing.134 multiparallel PCR,135
the control channel. single-cell analysis,136 siRNA-137 or antibody-screening,138
Also mixing can be realized using the above described kinase-139 or expression-profiling.140
pumping mechanism by the subsequent injection of the liquids
into a fluidic loop (Fig. 10(e)) through the left inlet (right outlet
Strengths and limitations
valve is closed). Afterwards, the inlet and outlet valves are
closed and the three control channels on the orbit of the mixing The microfluidic LSI platform certainly has the potential to
loop are displaced with a peristaltic actuation scheme leading to become one of the most versatile microfluidic platforms
a circulation of the mixture within the loop.122 Thereby the especially for high-throughput applications. It is a flexible
liquids are mixed and can be flushed out of the mixer by a and configurable technology which stands out by its suitability
washing liquid afterwards. Using this mixing scheme, the for large scale integration. The PDMS fabrication technology
is comparably cheap and robust, and thus suitable to fabricate
disposables. Reconfigured layouts can be assembled from a
small set of validated unit operations and design iteration
periods for new chips are in the order of days. Some of the
system functions are hardware defined by the fluidic circuitry
but others like process sequences can easily be programmed
Fig. 10 Realization of the main unit operations on the multilayer externally.
PDMS-based LSI platform.121 The NanoFlext valve (a) can be closed Limitations of the platform are related to the material
(b) by applying a pressure p to the control channel. Therewith, properties of PDMS: for example, chemicals which the
microfluidic valves (c), peristaltic pumps (d) and mixing structures elastomer is not inert to cannot be processed, and elevated
(e) can be designed. temperatures such as in micro-reaction technology are not

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 feasible. Also for the implementation of applications in the Table 4 Overview and examples of unit operations and applications
field of point-of-care diagnostics, where a hand-held device is on the segmented flow microfluidic platform
often required, the LSI platform seems not to be beneficial at Microfluidic unit operations Reference
the moment. Thereto external pressure sources and valves
would have to be downsized to a smaller footprint, which is Droplet generation 29; 142; 144–147; 166; 167
Droplet merging 29
of course technically feasible, but the costs would be higher in Droplet splitting 149
comparison to other platform concepts. However, as a first Droplet sorting 29
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step towards downsizing the liquid control equipment, the use Droplet internal mixing 29; 159; 160
Droplet sorting 168
of a Braille system was successfully demonstrated.141
Applications Reference

Segmented flow microfluidics (Single) cell analysis 30; 143; 166; 169
Single organism analysis 168; 170
Characterization of segmented flow microfluidics DNA assays 171–173
Drug screening 167
Segmented flow microfluidics describes the principle of using Protein crystallization 174–179
Chemical synthesis 144; 152; 155
small liquid plugs and/or droplets immersed in a second
immiscible continuous phase (gas or liquid) as stable micro-
confinements within closed microfluidic channels. Those To use droplets inside channels as reaction confinements,
micro-confinements are in the picolitre to microlitre volume the different reactants have to be loaded into the droplet.
range. They can be transported by pressure gradients and can Therefore, a method to combine 3 different sample liquid
be merged, split, sorted, and processed without any dispersion streams by a sheath flow arrangement with subsequent injection
in microfluidic channels. as a common droplet into the carrier fluid has been shown by
the group of Rustem F. Ismagilov at the University of Chicago,
General principle IL, USA149 (see Fig. 11). Different concentrations and ratios of
The segmented flow microfluidic platform relies on a multiphase two reagent sub-streams plus a dilution buffer merge into one
fluid flow through microchannels. Generally, the applied droplet and perform a so called on-chip dilution.150 The mixing
technologies can be divided into the following categories: ratios can be adjusted by the volume flow ratio of the three
 2-phase gas–liquid streams.
 2-phase liquid–liquid Using a combination of two opposing T-junctions
 3-phase liquid–liquid connected to the same channel, the formation of droplets of
In principal, droplets of a dispersed liquid phase are immersed alternating composition has been demonstrated.151 Using a
in a second continuous gas (2-phase gas–liquid) or liquid similar technique, the injection of an additional reactant into a
(2-phase liquid–liquid) phase within a microchannel. Thereby, liquid plug moving through the channel at an additional
the inner liquid droplets are separated by the continuous downstream T-junction has been demonstrated.152 Not only
carrier liquid along the channel. If the size of the inner phase liquid chemical reagents but also other components like cells
exceeds the cross sectional dimensions of the channel, the have been loaded into droplets.153
droplets are squeezed to form non-spherical segments, also The merging of different sized droplets showing different
called ‘‘plugs’’. Following this flow scheme, the platform is velocities to single droplets has been demonstrated successfully.149
called segmented flow microfluidics. In the same work, the controlled splitting of droplets at a channel
In some applications, the stability of the phase-arrangement branching point has been shown. Using a similar method, the
is increased by additional surfactants as the third phase, formation of droplet emulsions with controlled volume fractions
stabilizing the plug interface (3-phase liquid–liquid).142 An and drop sizes has been realized.154
external pressure is applied for the transport of the plugs. A Mixing inside the droplets can be accelerated by a recirculating
comprehensive general discussion of the platform can also be flow due to shear forces induced by the motion along the
found in recent review papers.29,143,144 stationary channel wall.155 This effect is even more pro-
nounced if two liquids of differing viscosities are mixed within
Unit operations the droplet.156 Based on the recirculating flow, a mixing
The most elementary unit operation on the segmented flow scheme for the segmented flow platform has been proposed
platform is the initial generation of the droplets (see Table 4).
This step can also be considered a metering, since the liquid
volumes involved in the subsequent reaction within the droplet
are defined during the droplet formation process. Generally,
two different microfluidic structures have been reported for a
controlled and continuous generation of droplets: the flow
focusing structure as depicted in Fig. 8145,146 and the T-shaped
junction,147,148 respectively. The size of the droplet is influenced Fig. 11 Droplet-based drug screening. The plugs containing the
by the strength of the shear forces at the channel junction drugs (D1 to D4) get mixed with a bacterial solution and a viability
(higher shear forces lead to smaller droplets) for both droplet dye. In the case of potent drugs the bacteria die and the droplet shows
formation mechanisms. no staining. Image adapted from Boedicker et al.167

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c The Royal Society of Chemistry 2010
 using serpentine microchannels.157 Within each channel namely the protein solution, a buffer and the precipitant
curvature the orientation between the phase pattern in the within oil as the carrier phase.174,180 The precipitant con-
droplet and the direction of motion is changed so that centration inside the droplet is adjusted via the buffer and
the inner recirculation leads to stretching and folding of the precipitant flow rates, respectively. Therewith, different
phases. Under favorable conditions, sub-millisecond mixing concentrations are generated and transferred into a glass
can be achieved and has been employed for multi-step syn- capillary for later X-ray analysis.175 The effect of mixing on
thesis of nanoparticles.152 A detailed and theoretical description the nucleation of protein crystallization has been investigated
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of this mixing effect is given in ref. 158. by combining the described crystallization structure with a
Besides the mixing within liquid droplets dispersed into serpentine mixing channel.179 Fast mixing has been found to
another liquid carrier phase, mixing within the carrier phase be favorable for the formation of well-crystallized proteins
can also be accelerated by a segmented flow. The injection of within the droplets.178
gas-bubbles into a continuous liquid stream forming a Recently, a chip for rapid detection and drug susceptibility
segmented gas–liquid flow has been described by Klavs Jensen screening of bacteria has also been presented167 as one example
and his group at MIT.159,160 The gas bubbles are introduced of a high-throughput screening application. The channel
into the liquid flow and initiate recirculation flows within the design is depicted in Fig. 11. Plugs of the bacterial solution,
liquid segments in between due to the motion along the a fluorescent viability indicator, and the drugs to be screened
channel wall. The gas bubbles can be completely separated are injected into the carrier fluid. The different drug solutions
from the liquid stream using a planar capillary separator after (antibiotics: vancomycin (VCM), levofloxixin (LVF), ampicillin
the reaction is finished. Using that technology, the synthesis of (AMP), cefoxitin (CFX), oxicillin (OXA), and erythromycin
colloidal silica particles has been demonstrated.161 Another (ERT)) are separated by an air spacer plug within the drug
microfluidic mixing scheme based on a gas–liquid segmented trial channel. Plugs containing VCM were used as baseline,
flow uses an additional repeated separation and re-combining because VCM inhibited this Staphylococcus aureus strain in
of the channel.162 macro-scale experiments. No plugs containing VCM or LVF
The incubation time of the reagents combined inside a droplet had a fluorescence increase greater than three times the base-
at the injection position can easily be calculated at a certain point line, indicating that MRSA was sensitive to these antibiotics.
of observation from the travelling distance of the droplet divided
by the droplet velocity. Thus, the incubation time can be Strengths and limitations
temporally monitored by simply scanning along the channel The main advantages of the segmented flow microfluidic
from the injection point to positions farther downstream. This platform are the small volume liquid segments (controllable
is a unique feature of the platform and enables the investigation with high precision in the nanolitre range), acting as reaction
of chemical reaction kinetics on the order of only a few milli- confinements. This leads to little reagent consumption as well as
seconds.150 On the other hand, also stable incubation times on a high number of different experiments that can be performed
the order of a week have been demonstrated.163 This is enabled by within a short period of time, which makes the platform a
separating the droplet compartments with a carrier fluid that promising candidate for high-throughput screening applications,
prevents evaporation and diffusion. Using this approach, several e.g. in the pharmaceutical industry. The quasi-batch-mode
60 nL liquid droplets containing one or a few cells were generated operation scheme within nanolitre to microlitre-sized droplets
within a microfluidic chip and afterwards flushed into a Teflon is beneficial since it represents a consistent further development
capillary tube for cultivation. The cell densities were still as high as of classic assay protocols in e.g. well plates. The large number of
in conventional systems after 144 h of growth within the droplets. existing unit operations enables the effective manipulation of the
Additional unit operations based on charged droplets and liquid segments. Furthermore, the completely enclosed liquid
electric fields have been added to the segmented flow platform droplets allow the incubation and storage of liquid assay results
by David A. Weitz and co-workers.164 Using dielectro- over a long period of time without evaporation.
phoresis, the sorting of single droplets out of a droplet train However, a limitation of the platform is that handling of
(switching) at rates up to 4 kHz has been shown.165 The small overall sample volumes is not possible due to the volume
segmented flow technology augmented with electric field-based consumption during the run-in phase of the flow within the
unit operations is currently commercialized by the company microchannels. This and the manual connection to external
Raindance Technologies, MA, USA. pumps renders the platform less suitable for point-of-care
applications. Another drawback is the need for surfactants
Application examples that are required for high stability of the plugs. They some-
times interfere with the (bio-)chemical reaction within the
Table 4 gives an overview of the microfluidic unit operations plugs and thus can limit the number of possible applications
and applications that have been already implemented on the on the platform.
segmented flow platform. They all take advantage of the
enclosed reaction confinement within the droplets, either for
analytical applications (cell analysis, single organism analysis, Centrifugal microfluidics
DNA assays, drug screening, protein crystallization) or
Characterization of centrifugal microfluidics
chemical synthesis.
Protein crystallization, for example, is realized on the In centrifugal microfluidics all processes are controlled
segmented flow platform by forming droplets out of three liquids, by the frequency protocol of a rotating microstructured

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 substrate. Relevant forces for liquid transport are centrifugal small flow rates in the order of nL s1 as well as high
force, Euler force, Coriolis force and capillary force. Assays throughput continuous flows up to 1 mL s1 192 can be
are implemented as a sequence of liquid operations arranged generated. Therefore, scaling of flow rates over 6 orders of
from radially inward positions to radially outward positions. magnitude independent of the chemical composition, ionic
Microfluidic unit operations include metering, switching, strength, conductivity or pH value of the liquid can be
aliquoting, etc. accomplished, opening a wide field of possible applications.
Also, liquid transport at rest can be achieved by capillary
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General principle forces, depending on the channel geometry and the wetting
properties of the liquid.
The approach of using centrifugal forces to automate sample
Liquid valves can be realized by several different micro-
processing dates back to the end of the 1960s.181 At that time,
fluidic structures on the centrifugal platform. In general, they
centrifugal analyzers were first used to transfer and mix a
can be purely passive, as depicted in Fig. 12, or require an
series of samples and reagents in the volume range from 1 mL
active component outside the microfluidic substrate. First, the
to 110 mL into several cuvettes, followed by spectrometric
passive valves will be summarized: A very simple valve arises at
monitoring of reactions and real-time data processing.
the sudden expansion of a microfluidic channel, e.g. into a
Controlling microfluidic networks by just one rotary axis has
bigger reservoir: the geometric capillary valve (Fig. 12(a)). The
an obvious charm to it, since no connections to the macro-
valving mechanism of this capillary valve is based on the
world, such as pumps, are required. Moreover, the required
energy barrier for the proceeding of the meniscus, which is
centrifugal base devices can be simple and therefore robust.
pinned at the sharp corner. This barrier can be overcome
Rotational frequencies can be controlled very well and a
under rotation due to the centrifugal pressure load of the
radially constant centrifugal pseudo-force guarantees pulse-
overlying liquid plug.189,193,194 For a given liquid plug
free liquid flow. Scientific work and applications based on
position, length, liquid surface tension and contact angle, the
centrifugal microfluidics have continuously been published
valve is influenced by only the frequency of rotation, and a
since these early beginnings, although most attention to the
critical burst frequency oc can be attributed to every valve
topic arose again in the last two decades, as summarized in
structure. Another possibility to stop the liquid flow within a
several reviews.121,182–184 However, the concept is still some-
channel is the local hydrophobic coating of the channel walls
what exotic compared to the large number of pressure driven
(hydrophobic valve) (Fig. 12(b)).183,195–197 This valve is opened
systems existing today, possibly attributed to the difficulty of
as soon as the rotational frequency exceeds the critical burst
monitoring liquid flow under rotation and the dependency of
frequency oc for this geometry and surface properties. A third
liquid flow on microchannel surface quality.185 This results in
method (Fig. 12(c)) utilizes the stopping effect of compressed
high initial investment in monitoring equipment and proto-
air in an unvented receiving chamber. This centrifugo-
typing lines. Nevertheless, considerable advances towards
pneumatic valve stops liquid up to much higher pressures
integrated systems have been made in the last few decades.
than capillary valves for small receiving chamber volumes
In the beginning of the 1990s, the company Abaxis186
(r40 mL). The air counter-pressure in the unvented receiving
developed the portable clinical chemistry analyzer.187 This
chamber can be overcome at high centrifugal frequencies, at
system consists of a plastic disposable rotating cartridge for
which the liquid–air interface becomes unstable and enables a
processing of the specimen, preloading of dried reagents on
phase exchange, permitting liquid flow.198,199 Another method
the cartridge, and an analyzer instrument for actuation and
is based on a hydrophilic S-shaped siphon channel (hydrophilic
readout.
siphon valve), wherein the two liquid–gas interfaces are lever-
A next generation of centrifugal devices emerged from the
aged at high frequencies of rotation183 (Fig. 12(d)). Below
technical capabilities offered by microfabrication and micro-
a critical frequency oc however, the right-hand meniscus
fluidic technologies.188–191 Length scales of the fluidic structures
proceeds beyond the bend, thus allowing the centrifugal force
in the range of a few hundred micrometres allow parallel
to drain the complete liquid from the siphon.
processing of up to a hundred units assembled on a single
disk. This enables high throughput by highly parallel and
automated liquid handling. In addition, assay volumes can
be reduced to less than 1 mL. Particular fields such as drug
screening,189 where precious samples are analyzed, benefit
from these low assay volumes.
Today, many basic unit operations for liquid control on the
centrifugal microfluidic platform are known and new ones are
continuously being developed, enabling a number of applications
in the fields of point-of-care testing, research, and security.

Unit operations
Liquid transport is initiated by the centrifugal force, fx, directed Fig. 12 Passive centrifugal microfluidic valves. (a) Positioning of
outwards in the radial direction. The centrifugal force can be valves relative to center of rotation and centrifugal force, (b) geometric
scaled over a wide range by the frequency of rotation o. capillary valve,189 (c) hydrophobic valve,195 (d) centrifugo-pneumatic
Together with a tunable flow resistance of the fluidic channels, valve198 and (e) hydrophilic siphon valve.183

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 One example of an active valve is an irradiation-triggered second using magnetic microparticles, located in the mixing
‘‘sacrificial’’ valve published by Samsung Advanced Institute of chamber, has also been demonstrated.205 Accelerated mixing
Technology (Laser Irradiated Ferrowax Microvalve, LIFM).200 A can also be achieved by an interplay of capillary and intermittent
ferrowax plug is used to close channels off during the fabrication centrifugal forces.206
of the microfluidic network. A laser source in the processing For routing (switching) of liquids, a switch utilizing the
device can be utilized to melt the ferrowax plug and thus allow transversal Coriolis force to guide liquid flows between two
liquid passage (normally-closed valve). A modification of this outlets at the bifurcation of an inverse Y-shaped channel207 or
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technique also allows closing channels off by illuminating a at a nozzle leading into a chamber208 has been presented.
ferrowax reservoir that expands into a channel and seals it Depending on the sense of rotation, the Coriolis force is either
(normally-open valve). An advantage of this valve is that it directed to the left or to the right, guiding the liquid stream
allows liquid control depending solely on the moment of the into one of two downstream reservoirs at the bifurcation.
laser actuation, so it does not depend on the rotational speed or Another method for liquid routing based on different wetting
liquid properties. This comes at the cost of a more complex properties of the connected channels has been reported by
production process and base device. An alternative approach for Gyros AB, Sweden.209 The liquid stream is initially guided
the active control of liquid flows on the centrifugal platform is towards a radial channel, exhibiting a hydrophobic patch at
followed by the company Spin-X technologies, Switzerland. A the beginning. Therefore, the liquid is deflected into a branching
laser beam individually opens fluidic interconnects between non-hydrophobic channel next to the radial one. For high
different channel layers on a plastic substrate (Virtual Laser frequencies of rotation, the approaching liquid possesses
Valve, VLV). This enables online control of the liquid handling enough energy to overcome the hydrophobic patch and is
process on the rotating module for adjusting metered volumes therefore routed into the radial channel.210 A further possibility
and incubation times within a wide range. Due to this, the to switch liquid flows is to utilize an ‘‘air cushion’’ between an
Spin-X platform works with a standardized fluidic cartridge that initial first liquid entering a downstream chamber and a
is not custom made for each specific application, but can be subsequent liquid. The centrifugally generated pressure of
programmed online during a running process. the first liquid is transmitted via the air cushion to the
Combining one of the above-mentioned valve principles at subsequent liquid and forces it via an alternative route into a
the radially outward end of a chamber with an overflow chamber placed to the side of the main channel.211
channel at the radially inward end results in a metering The separation of plasma from a whole blood sample is the
structure.201 The metered liquid portion is directly set by prevalent first step within a complete analytical protocol for
the volume capacity of the chamber. With highly precise the analysis of whole blood. Since blood plasma has lower
micro-fabrication technologies, small coefficients of variations density compared to the white and red blood cells it can be
(CV, standard deviation divided by mean value), e.g. a found in the upper phase after sedimentation in the artificial
CV o 5% for a volume of 300 nL202 and also metered volumes gravity field under rotation. The spatial separation of the
of as little as 5 nL have been achieved.196 By arranging obtained plasma from the cellular pellet can be achieved via
several metering structures interconnected via an appropriate a capillary channel that branches from the sedimentation
distribution channel, simple aliquoting structures can be chamber at a radial position where only plasma is expected.187
realized.198,203 These structures split a sample into several Another method uses pre-separation of the cellular and
defined volumes, enabling the conduction of several assays plasma phase during the sample flow through an azimuthally
from the same sample in parallel. aligned channel of 300 mm radial width.197 The obtained
Different mixing schemes have been proposed on the plasma fraction is thereafter split from the cellular com-
centrifugal platform. Considering mixing of continuous ponents by a decanting process. Another concept enables
liquid flows within a radially directed rotating channel, the plasma separation of varying blood sample volumes in a
perpendicular Coriolis force automatically generates a transverse continuous process. The sedimentation occurs in an azimuthally
liquid flow.192 A continuous centrifugal micromixer, utilizing curved channel due to centrifugal and Coriolis forces, enabling
the Coriolis stirring effect, showed an increasing mixing up to 99% separation efficiency between two outlets for
quality towards very high volume throughputs of up to a diluted sample with 6% hematocrit.212 An overview of
1 mL s1 per channel192 (Coriolis mixer). Besides the mixing centrifugal microfluidic unit operations and related applications
of continuous liquid flows, also the homogenization of discrete can be found in Table 5.
and small liquid volumes located in chambers is of importance
especially when analyzing small sample volumes (batch-mode
Application examples
mixing), since homogenous mixing obviously speeds up diffusion-
limited chemical and biological reactions due to the close Table 5 shows some applications that have been realized
proximity between analytes. One possibility to enhance the mixing on the centrifugal microfluidic platform. At the top of the
is the active agitation of the liquid within a mixing chamber by applications section, sample preparation modules (plasma
inertia related shear forces (Euler force), induced by a fast change separation, DNA extraction) are shown. This is followed by
of the sense of rotation (shake-mode-mixing)201 or change of assays based on the detection of proteins, nucleic acids
rotational frequency (unidirectional shake-mode-mixing).204 and small molecules (clinical chemistry). Two additional
Shake-mode mixing leads to reduced mixing times on the order applications are presented at the end of the table, demonstrating
of several seconds compared to several minutes for pure diffusion- chromatography and protein crystallization. Some instructive
based mixing. A further downscaling of mixing times below one examples are discussed in more detail below.

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 Table 5 Overview and examples of unit operations and applications port of the microstructure. By mixing the blood sample with
for the centrifugal microfluidic platform the reagents, an enzymatic reaction is initiated, changing the
Microfluidic unit operations Reference color of the mixture depending on the alcohol concentration.
After sedimentation of the residual blood cells, the absorbance
Capillary valving 183; 189; 191; 193; 194; 213–220 is monitored in a real-time manner via a laser beam that
Hydrophobic valving 183; 195–197
Siphon valving 183; 186; 187; 204; 221; 222 is reflected into the disk plane on integrated V-grooves.229
Laser-triggered valving 200; 223–225 Using this automated assay and readout protocol the
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Centrifugo-pneumatic 198; 211 concentration of alcohol in human whole blood was

valving
Metering 183; 187; 191; 195–197; 200–202; determined within only 150 s. The results were comparable
221; 222; 224 to common point-of-care tests and required a minute blood
Aliquoting 181; 183; 186; 187; 195; 198; 226 volume of just 500 nL.
Mixing 181; 183; 186; 187; 191; 192; 200–202; Also a protein crystallization assay has been demonstrated
204; 205; 217; 221; 222; 224; 226–229
Coriolis switching 183; 201; 207; 211; 212; 230 on the centrifugal microfluidic platform.196 First, a defined
Reagent storage 217; 231 volume of the protein solution is dispensed into the protein
Applications Reference
inlet and transported into the crystallization chamber. After-
wards, the pre-loaded precipitant is metered under rotation
Integrated plasma separation 183; 197; 201; 212; 221–224; 232 and transferred into the crystallization chamber as soon as a
Cell lysis and/or DNA 224; 230; 233
extraction hydrophobic valve breaks. In the last step, the pre-loaded oil is
Protein-based assays 181; 189; 195; 201; 213; 217; 219; released at yet a higher frequency and placed on top of the
221–223; 226; 234 liquid stack within the crystallization chamber, to prevent
Nucleic acid-based assays 213; 218; 235
evaporation. The successful crystallization of proteinase K
Clinical chemistry assays 186; 187; 201; 202; 214–216; 222;
229; 236 and catalase was demonstrated.
Chromatography 237 Samsung Advanced Institute of Technology showed a fully
Protein crystallization 196 integrated immunoassay for Hepatitis B and other antibodies,
starting from 150 mL whole blood on a centrifugal base
device including a laser for controlling ferrowax valves and a
Madou et al. from the University of California, Irvine readout-unit.223 A limit of detection comparable to a con-
showed a series of capillary valves to perform enzyme-linked ventional ELISA and an assay time of 30 min were reported.
immunosorbent assays (ELISAs) on the centrifugal platform.219 On the same platform, enrichment of pathogens and subsequent
The different assay liquids are held back in reservoirs connected DNA extraction was also shown (Fig. 13).224 The microfluidic
to the reaction chamber via valves of different burst frequency. structure features an integrated magnet that controls the
The capillary valves are opened subsequently by increasing position of coated magnetic particles which are used to capture
the frequency of rotation. It was shown that in terms of target pathogens and lyse them by laser irradiation. With a
detection range the centrifugally conducted assay has the same total extraction time of 12 min, down to 10 copies/mL DNA
performance as the conventional method on a 96-well plate, but concentration in a spiked blood sample of 100 mL could be
with less reagent consumption and shorter assay time. specifically extracted and detected in a subsequent external
Gyros AB, Sweden209 use a flow-through sandwich immuno- PCR. Reagents are loaded by the operator prior to the
assay at the nanolitre scale to quantify proteins within process.
their Gyrolabt Workstation. A column of pre-packed and
streptavidin-coated microparticles is integrated into each one
of 112 identical assay units on the microfluidic disk. Each
unit has an individual sample inlet and a volume definition
chamber that leads to an overflow channel. Defined volumes
(200 nL) of samples and reagents can be applied to the
pre-packed particle column. The laser induced fluorescent
(LIF) detector is incorporated into the Gyrolabt Workstation.
Using this technology, multiple immunoassays have been
carried out to determine the imprecision of the assay result.
The day-to-day (total) imprecisions (CV) of the immunoassays
on the microfluidic disk are below 20%.195 The assays are
carried out within 50 min with sample volumes of 200 nL. In
comparison, the traditional ELISA performed in a 96-well
plate typically takes several hours and requires sample volumes
of several hundred microlitres.
A fully integrated colorimetric assay for determination of Fig. 13 Centrifugal microfluidic structure for pathogen-specific cell
alcohol concentrations in human whole blood has been shown capture, lysis and DNA purification published by Cho et al.224 The
on the centrifugal Bio-Disk platform.202 After loading the microfluidic network comprises structures for plasma separation,
reagents into the reagents reservoir, a droplet of untreated mixing, and laser-triggered valves. For manipulation of the magnetic
human blood taken from a finger tip is loaded into the inlet capture-beads, a movable magnet is integrated into the cartridge.

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 Strengths and limitations General principle
Two major advantages of the centrifugal microfluidic platform One of the first applications for electrokinetics was the analysis
are the modular setup of the system with disposable and easily of chemical compounds via electrophoretic separation within
exchangeable plastic cartridges and the many existing unit capillaries in 1967,238 long before the term ‘‘microfluidics’’
operations, which allow highly precise liquid handling. The emerged. In the beginning, glass capillaries made from drawn
fabrication costs of the disposables are governed by the glass tubes were used, whereas today well defined micro-
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specific implementation of unit operations. Necessary global channels are established and commonly used. The actuation
or local surface modification or the integration of active principle of the electrokinetic platform relies on the movement
(ferrowax) valves, post-replication treatment, assembly and of liquid in an induced electric double layer and charged
reagent pre-storage steps can increase the cost of the dis- particles (ions) in an electric field applied along a microfluidic
posables. Mostly, they are made out of plastic and thus channel. The simple setup of electrokinetic systems consisting
suitable for mass-production. The presented unit operations of microfluidic channels and electrodes without moving
allow the automation of complex assay protocols. The cost for parts explains the early advent of electrokinetic platforms
the base instrument depends heavily on readout and temperature for microfluidic lab-on-a-chip applications.
control modules. The motor required for liquid control is
generally required to be able to achieve very stable and defined
Unit operations
rotational speed and acceleration, also adding to the costs.
However, compared to (several) high-precision syringe pumps, In a microfluidic channel, a charged solid surface induces an
this solution is generally cheaper and allows a higher degree of opposite net charge in the adjacent liquid layer (electric double
integration. Due to the rotational symmetry of the disks, layer). As soon as an electric potential is applied along the
optionally some degree of parallelization can be achieved. channel, the positively charged liquid molecules are attracted
Also, the rotational symmetry is beneficial for fast readout by electrostatic forces and thus move towards a corresponding
and temperature uniformity between cavities at the same electrode (Fig. 14(a)). Due to viscous coupling, the bulk liquid
radial position. is dragged along by the moving layer and liquid actuation with
However, as soon as any additional actuation or sensing a planar velocity profile is generated (electroosmotic flow
function is required on the module during rotation and if a (EOF)239). The velocity profile is constant and dispersion only
contact free interfacing is not applicable, things become occurs by molecular diffusion. This motion is superimposed by
challenging from a technical point of view. Especially inter- the movement of ions and charged molecules, which are
facing to electric readout modules on the disk is difficult, since attracted or repelled by the electrodes depending on their
the rotating setup does not allow for wire connections between charge (Fig. 14(b)). The velocity of the molecule depends on
the disposable and the base instrument. The platform its charge and hydrodynamic radius and enables the distinction
also lacks flexibility compared to others that allow online between different molecular entities. This effect is used for
programming of fluidic networks within one piece of hardware separation of charged molecules and is called electrophoresis.
that fits all, since most of the logic functions as well as Based on the electroosmotic flow, metering of volumes down
their critical frequencies are permanently imprinted into to the picolitre range can be achieved. While the sample liquid
the channel network. However, the Virtual Laser Valve is injected and crosses an intersection point of two perpendi-
technology is an exception in this respect and allows online cular channels, the electrodes and therefore the flow along the
programming in a centrifugal system. Space restrictions are main channel is switched off. Then, the electrodes in the side
also an issue, since the required footprint (disk surface) channel are activated. This displaces a small plug at the
increases quadratically with the number of connected unit intersection into the side channel, resulting in metering of a
operations (radial length). The low centrifugal forces near sample volume depending on the geometry of the intersection
the center of rotation and the difficulty of transporting liquids area. The mixing of two co-flowing streams was shown on the
radially inward are other challenges in the fluidic design electrokinetic platform by applying an AC voltage.238 A
process. Also, completely portable solutions are currently still 20-fold reduction in mixing time compared to molecular
only a vision. diffusion has been reported. Also complete biological assays
comprising cell lysis, mixing, and DNA amplification have
been presented.240
A modification to electrophoresis is free-flow electro-
Electrokinetics phoresis, which enables the continuous separation of a mixture
Characterization of electrokinetics
In electrokinetics platforms microfluidic unit operations are con-
trolled by electric fields acting on electric charges, or electric field
gradients acting on electric dipoles. Depending on buffers and/or
sample, several electrokinetic effects such as electroosmosis,
electrophoresis, dielectrophoresis, and polarization superimpose
each other. Electroosmosis can be used to transport the whole Fig. 14 Basic electrokinetic effects (according to Atkins239).
liquid bulk while the other effects can be used to separate different (a): electroosmotic flow (EOF), (b): electrophoresis (EP), (c) dielectro-
types of molecules or particles within the bulk liquid. phoresis (DEP).

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 according to charge with subsequent collection of the sample microarray. This was the first step in the direction of a
band of interest.241 For this, an transverse electric field platform for massively parallel analysis.
is applied in pressure driven flow within a broad and flat
microchamber. While passing this extraction chamber, the Strengths and limitations
species contained in the sample flow are deflected depending
Electroosmotic actuation of liquids enables pulse-free pumping
on their charge and thus exit the chamber through one of
without any moving parts. Liquid manipulation at high
several outlets.
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precision can be achieved by the existing unit operations.

Another electrokinetic effect is based on polarization of
In addition, electroosmotic flow does not lead to Taylor
particles within an oscillating electrical field or field gradient
dispersion115 as in pressure driven systems and thus enables
(dielectrophoresis), as depicted in Fig. 14(c). Dielectrophoresis is
high efficiency separations. The seamless integration with
applied in many fields, e.g. for the controlled separation and
electrophoresis, an established technology in use for 100 years,255
trapping of submicron bioparticles,242 for the fusion and transport
is another obvious strength. In microfluidic systems, applications
of cells,243 gene transfection244 or the separation of metallic from
can benefit from faster heat dissipation, better resolution,
semiconducting carbon nanotubes.12,245,246 Other applications are
and faster separation. Miniaturization of electrophoretic
cell sorting247,248 and apoptosis of cells.249,250
analysis enables the automation and parallelization of tests with
small dead volumes, thus reducing the required amount of
Application examples sample.
A technical problem in capillary electrophoresis systems is
Capillary electrophoresis systems were the first micro total the changing pH-gradient due to electrolysis or electrophoresis
analysis systems and emerged as single chip solutions from the itself. Also streaming currents which counteract the external
analytical chemistry field in the 1990s.251 Several companies electric field or gas bubbles as a result of electrolysis at the
utilize microfluidic capillary electrophoretic chips for chemical electrodes are problematic. Also a massively parallel setup is
analysis, with capillaries of typically 10 to 100 mm diameter.252 problematic due to the heat generated by the electrophoresis
Today, Caliper Life Sciences, MA, USA252 and Agilent itself. In addition, the realization of hand-held devices is
Technologies, CA, USA253 offer microfluidic chips for DNA challenging due to the necessity of high voltages in com-
and protein analysis. Liquid propulsion is provided via bination with high energy consumption. Overall, miniaturized
electroosmosis and combined with capillary electrophoretic electrophoresis is established as a fast and efficient method for
separation. The sample is electroosmotically transported and the separation and analysis of bio-molecules.
metered inside the chip, then separated via capillary electro-
phoresis and analysed by fluorescence detection. (Fig. 15).
The whole assay is performed within minutes, instead of hours
Electrowetting
or days.
The first combinations of microfluidic integrated electro- Characterization of electrowetting
phoresis with microarrays were published in 1998 by Nanogen
Electrowetting platforms use droplets immersed in a second
Inc., CA, USA.254 This approach resulted in a 20-fold faster
immiscible continuous phase (gas or liquid) as stable micro-
hybridization and more specific binding of DNA onto the
confinements. The droplets reside on a hydrophobic surface
that contains a one- or two-dimensional array of individually
addressable electrodes. The voltage between a droplet and the
electrode underneath the droplet defines its wetting behavior.
By changing voltages between neighboring electrodes, droplets
can be generated, transported, split, merged, and processed.
These unit operations are freely programmable for each
individual droplet by the end-user enabling online control of
an assay.

General principle
The electrowetting effect was first described by Lippmann in
1875.256 Interest in this effect was spurred again in the 1990s,
when researchers started placing thin insulating layers on the
metallic electrodes to separate it from the often conductive
Fig. 15 Microfluidic realization of capillary electrophoresis analysis liquids in order to eliminate electrolysis.257 The basic electro-
on the electrokinetic platform (adapted from ref. 121) (r Agilent
wetting effect is depicted in Fig. 16(a). The wettability of a
Technologies, Inc. 2007. Reproduced with permission, courtesy of
solid surface increases due to polarization and electric fields as
Agilent Technologies, Inc.). After the sample has been transported to
the junction area (a) it is metered by the activated horizontal flow and soon as a voltage is applied between the electrode and the
injected into the separation channel (b). Therein, the sample components liquid droplet above (separated by the dielectric insulating
are electrophoretically separated (c) and readout by their fluorescence layer).257 This so-called ‘‘electrowetting-on-dielectric’’
signal (d). The complete microfluidic CE-chip is depicted in the (EWOD)258 effect is therefore a tool to control the contact
center. angle of liquids on surfaces.

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c The Royal Society of Chemistry 2010
 the use of three electrodes. Two droplets are individually
guided to electrodes separated from each other by a third
one. Deactivating these two electrodes and activating the third
separation electrode pulls the droplets together.265 The most
basic type of mixing within droplets on the EWOD platform is
Fig. 16 The electrowetting effect (according to Mugele and Baret257). an oscillation, forwards and backwards, between at least two
(a) If a voltage V is applied between a liquid and an electrode electrodes. Another mixing scheme is the repetitive movement
Open Access Article. Published on 25 January 2010. Downloaded on 10/11/2024 7:20:44 PM.

separated by an insulating layer, the contact angle of the liquid–solid of the droplet on a rectangular path. The shortest mixing time
interface is decreased and the droplet ‘‘flattens’’. (b) Hydrophobic for two 1.3 mL droplets in linear oscillation on 4 electrodes was
surfaces enhance the effect of electrowetting. For ‘‘electrowetting- about 4.6 s.266 In another work, the mixing times of 1.4 mL
on-dielectrics’’ (EWOD) several individual addressable control droplets could be further reduced to less than 3 s using
electrodes (here on the bottom) and a large counter-electrode are two-dimensional arrays.267
used. The droplet is pulled to the charged electrodes.
Application examples
This invention paved the way for the application of the
Applications based on EWOD are in the development phase
electrowetting effect as a liquid propulsion principle for
and quite close to market products. For example, an enzymatic
lab-on-a-chip systems.259,260 To utilize the EWOD technology
colorimetric assay for (point-of-care) diagnostic applications
for programmable liquid actuation, a liquid droplet is placed
has been successfully implemented, and glucose concentration
between two electrodes covered with insulating, preferably
in several biological liquids (serum, plasma, urine, and saliva)
hydrophobic, dielectric layers (Fig. 16(b)). The liquid droplet
was determined with comparable results to standard methods.262
is steered by the electrode array on one side and by a large
The microfluidic chip layout for the colorimetric glucose assay
planar ground electrode on the opposite side. Activating
is depicted in Fig. 17. It features reservoirs, injection structures
selected electrodes allows programming of a path which the
(metering) and a network of electrodes for droplet transport,
droplet follows. The droplet needs to be large enough to cover
splitting and detection.
parts of at least four addressable electrodes at all times,
Also the use of an EWOD system for the automated sample
allowing two-dimensional movement. If a voltage is applied
preparation of peptides and proteins for matrix-assisted laser
to one of the control electrodes covered by the droplet, it
desorption-ionization mass spectrometry (MALDI-MS) was
moves onto the activated electrode pad. Successive activation
reported. In that work, standard MALDI-MS reagents,
of one electrode after the other will drag the droplet along a
analytes, concentrations, and recipes have been demonstrated
defined path. This freedom to program the liquid movement
to be compatible with the EWOD technology, and mass
enables the implementation of different assays on the
spectra comparable to those collected by conventional
same chip.
methods were obtained.268 Also a PCR assay has been realized
The universal applicability of moving droplets by EWOD
on the platform by temperature cycling of a droplet at rest.269
was shown with several media such as ionic liquids, aqueous
Additional information about the EWOD platform can be
surfactant solutions,261 and also biological fluids like whole
found in a comprehensive review.270
blood, serum, plasma, urine, saliva, sweat, and tear fluid.262
Strengths and limitations
Unit operations
The strengths of the platform are the very small liquid volumes
The droplet formation, i.e. initial metering, is the elementary in the nanolitre range that can be handled with high precision,
unit operation of the platform. Metered droplets can be and the freedom to program the droplet movement. This cuts
produced from an on-chip reservoir in three steps.262 First, a down sample and reagent consumption and allows a maximum
liquid column is extruded from the reservoir by activating a of flexibility for the implementation of different assay protocols.
series of adjacent electrodes. Second, once the column over-
laps the electrode on which the droplet is to be formed, all the
remaining electrodes are turned off, forming a neck in the
column. The reservoir electrode is then activated during
the third and last step, pulling back the liquid and breaking
the neck, leaving a droplet behind on the metering electrode.
Using this droplet metering structure, droplets down to 20 nL
volume can be generated with a standard deviation of less than
2%.262 A similar technology can be used for the splitting of a
droplet into several smaller droplets.31 Since the droplet
volume is of great importance for the accuracy of all assays,
additional volume control mechanisms such as on-chip
capacitance volume control263 or the use of numerical methods Fig. 17 Electrowetting platform (EWOD). Implementation of a
for the design of EWOD metering structures264 have been colorimetric glucose assay in a single chip. Four reservoirs with
proposed. Once the droplets are formed, their actuation is injection elements are connected to an electrode circuitry, where the
accomplished by the EWOD effect as described above. droplets are mixed, split and transported to detection sites for readout
Also the merging of droplets can be achieved easily with (adapted from Srinivasan et al.262).

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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1153–1182 | 1173
 The simple setup without any moving parts can be fabricated
using standard lithographic processes. The programmable con-
trol of small droplets has its particular potential in assay
optimization, since it allows the protocol to be varied over a
certain range on the same chip.
However, although the sample and reagent consumption is
low, portable systems for e.g. point-of-care applications
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have not yet been demonstrated due to the bulky electronic

instrumentation required to operate the platform. Another
drawback is the influence of the liquid properties on the Fig. 18 Surface acoustic wave (SAW) (according to Tan et al.274).
The shock waves induce a stream on the solid–liquid interface and lead
droplet transport behaviour, i.e. different patient materials
finally to a movement of the droplet (amplitude of acoustic wave not
will show different wetting abilities and thus lead to differences
to scale).
in volume or movement speed. Also the long-term stability of
the hydrophobic surface coatings and the contamination risk
is problematic, since every droplet can potentially contaminate leaving behind a small metered liquid portion due to
the surface and thus lead to false results and also change the the interplay between the surface tension force (keeping the
contact angle for the successor droplets. Another issue is the droplet on the spot) and the acoustic force (pushing the
possible electrolysis caused by the electric fields themselves. droplet forward). Since those two forces scale differently over
Strategies for high-throughput applications have not been the droplet size, the splitting of the initial droplet into two
demonstrated to date. droplets (one sitting on the metering spot and the other
In summary, the EWOD technique bears great potential propagating forward) occurs. The smaller droplet is not
to manipulate many single droplets in parallel. While first transported since it stays unaffected by the acoustic wave.
applications have been shown, the EWOD concept is still at a Also aliquoting has been shown by moving the initial droplet
stage of development, shortly before entering the IVD markets.270 over a hydrophobic/hydrophilic checkerboard pattern.271
Mixing is an intrinsic unit operation of the SAW platform.
A droplet which is placed on the substrate and is influenced by
Surface acoustic waves a SAW shows internal liquid circulation due to the vibrating
Characterization of surface acoustic waves forces of the wave. This internal circulation leads to mixing.271

The surface acoustic waves platform uses droplets residing

on a hydrophobic surface in a gaseous environment (air). Application examples
The microfluidic unit operations are mainly controlled by A PCR protocol has been implemented on the SAW platform,
acoustic shock waves travelling on the surface of the solid based on 200 nL droplets and an additional heating element
support. The shock waves are generated by an arrangement of placed underneath the substrate surface for temperature
surrounding sonotrodes, defining the droplet manipulation cycling while the droplet is at rest.275 However, since the
area. Most of the unit operations such as droplet generation, nanolitre-sized droplet possesses a high surface-to-volume
transport, mixing, etc. are freely programmable. ratio, the liquid volume would decrease rapidly due to
General principle evaporation at the elevated temperatures required for the
PCR reaction. Therefore, the aqueous liquid droplet is covered
An alternative to the electrowetting-based transportation of with a droplet of immiscible mineral oil with a smaller contact
droplets on a plane surface has been proposed by the group of angle. This droplet-in-droplet configuration can still be
Achim Wixforth at the University of Augsburg, Germany.271 moved via surface acoustic waves on the substrate surface.
The approach is based on surface acoustic waves (SAW), The concentration of DNA could be monitored by online
which are mechanical waves with amplitudes of typically only fluorescent measurement providing a sensitivity of 0.1 ng.275
a few nanometres. The surface acoustic waves are generated by
a piezoelectric transducer chip (e.g. quartz) fabricated by
placing interdigital electrodes (interdigital transducer, IDT) Strengths and limitations
on top of a piezoelectric layer. Liquid droplets situated on the As in the EWOD platform, the SAW platform also allows the
hydrophobic surface of the chip can be moved by the SAWs if handling of small nanolitre-sized liquid volumes in droplets on
the acoustic pressure exerted on the liquid droplet is high planar surfaces. The transport mechanism using surface
enough (Fig. 18).272 The actuation of small amounts of liquids acoustic waves though is more flexible since it depends only
with viscosities extending over a large range (from 1 to 1000 mPa s) on the viscosity and surface tension of the liquid.
has been shown.273 This approach is also sometimes referred to as However, the programmability is in turn limited since
‘‘flat fluidics’’, because no cover or slit is required as in the EWOD the position of the interdigital electrodes and especially
approach. the hydrophobic/hydrophilic areas determine the possible
liquid handling processes. Another disadvantage is the long-
Unit operations
term stability and the complexity of these hydrophobic and
Metering is accomplished by moving a liquid droplet over a hydrophilic surface coatings, and thus costs of the disposable
small hydrophilic ‘‘metering spot’’ via surface acoustic waves, chip as well as the instrument.

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c The Royal Society of Chemistry 2010
 Dedicated systems for massively parallel analysis sequencing requires new and massively parallel testing in
numbers of hundred thousands to billions. These tests con-
Characterization of massively parallel analysis sume a lot of time, material, effort, and money, but could lead
Within the category of dedicated systems for massively parallel to precious results (e.g. in case of a new blockbuster drug).283
analysis we discuss specific platforms that do not comply The challenging task to monitor millions of different binding
with our definition of a generic microfluidic platform. reactions is partially solved by microarrays284 (mainly in the
The characteristics of those platforms is not given by the case of DNA and RNA) or bead-based assays in combination
Open Access Article. Published on 25 January 2010. Downloaded on 10/11/2024 7:20:44 PM.

implementation of the fluidic functions but by the specific with picowell plates.
way to process up to millions of assays in parallel. Prominent Microarrays284 are matrices with spots of different chemical
examples are platforms used for gene expression and sequencing compounds on a surface (Fig. 19(a)). The number of spots ranges
such as microarrays, bead-based assays and pyro-sequencing from a few dozen to up to several millions. The microarray is
in picowell-plates. incubated with the sample and each spot interacts with the
sample in parallel, leading to as many parallel assays as there
are spots on the microarray. Typically a microarray is read out
General principle
by fluorescence and used for nucleic acid or protein analysis.
In this chapter, solutions for highly parallel assay processing Picowell plates285,286 consist of millions of small wells (o50 mm
are presented. These are not per se microfluidic platforms by in diameter) (Fig. 19(c)). In each well, either one chemical
our definition, since they do not offer a set of easily combined compound or one single cell is deposited. After the deposition,
unit operations and are quite inflexible in terms of assay the picowell plate acts as a ‘‘microarray’’ with each position
layout. They are nevertheless presented here, since the small bearing a unique chemical compound or cell. Afterwards, all
reaction volumes per assay and partly the liquid control assays are performed similar to a microarray.
systems are based on microfluidic platforms. The significant In bead-based assays278,287 small solid phase spheres
market for repetitive analyses, which allows high development (Fig. 19(b)) or particles are used. Each bead bears one unique
costs for proprietary, optimized systems, does not necessarily chemical compound. Such a bead library can consist of
require a platform approach, but can benefit from microfluidic billions of different beads. For screening, the beads are mixed
production technologies and liquid handling systems. and incubated with the sample and consecutively with the assay
The massively parallel assay systems are a result of the buffers, performing one assay on each bead in parallel. The
increasing demand of the pharmaceutical industry for repetitive readout is commonly fluorescence based and the positive beads
assays276,277 to cover the following objectives: are sorted out and analysed one by one in series. Typically this
 Screening of chemical libraries with millions of technique is used for binding assays or DNA analysis.
compounds278 The pioneers of each field who introduced this system to
 Screening of known drugs against new targets, different the market are: Microarrays by Affymetrix, CA, USA,288
cell lines or patient material279,280 bead-based arrays by Luminex Corp., TX, USA289,290 and
 Multiparameter analysis of cell signaling and single cell Illumina Inc., CA, USA,291,292 and picowell plates by 454 Life
analysis281 Sciences, CT, USA.286
 All -omic analyses such as genomics, transcriptomics,
proteomics, glucomics, metabolomics. . .282
Microfluidic components and applications
With every newly discovered receptor or protein, all known
drugs, pre-drugs, and chemical compounds should be tested Here, the microfluidic actuation principles that are utilized in
for interaction by means of binding, activity change, or massively parallel analysis are outlined briefly. This is followed
enzymatic activity. Also the analysis of gene activity or gene by some commercial application examples. Due to the similar

Fig. 19 Images of the different systems for massively parallel screening. (a) Microarray284 after binding, providing two different fluorophores in
red and green. Unchanged genes remain yellow. Up- or down-regulated genes appear in red or green. (b) 3 mm silica spheres, as an example for
bead-based assays,278,287 deposited on the front end of glass fibers. (c) Empty wells of a picowell plate.285,286 In each well single cells or beads are
deposited, incubated and analyzed.

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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1153–1182 | 1175
 principle, microarrays and picowell plates are presented deposited on one end of a glass fiber connected to a detector.
together, followed by bead-based assays. The spheres are incubated with a DNA sample, and in the case
of a binding event, the according sphere emits a light signal
Micorarrays/picowell plates into the glass fiber. The current system allows handling of
millions of unique compounds.294
For micorarrays/picowell plates, liquid actuation and metering
can be achieved by different actuation principles. Mainly Strengths and limitations
capillary filling of a cartridge,288 or pressure driven systems
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Today, many manual steps and skilled personnel are required

are used.283,284 In other cases, the liquid actuation is achieved
for the described systems and a ‘‘real’’ microfluidic platform is
by centrifugal systems, electrophoresis, surface acoustic waves,
still not reached. However, microarrays, picowell plates and
electrowetting, and several other principles. Incubation and
bead-based assays are a very useful combination of solid phase
mixing is realized by diffusion and in some cases enhanced by
and liquid handling for massively parallel assays in the number
sonication, surface acoustic waves, or electric fields. Washing
of millions. The material consumption per assay is quite low and
is achieved by displacing the sample with the consecutive
the reaction time quite fast. The time-to-result is longer com-
liquid. The classical (parallel) readout of binding or inter-
pared to a single assay, but several orders of magnitude faster
action between the molecules is performed by fluorescence
compared to serially performing the same number of assays.
(Fig. 19(a) and (c)).288 An interesting feature is that some of
A significant limitation of these systems is the reliability,
the picowell plates are made from glass fiber bundles and thus
reproducibility, and identification of artefacts. Therefore a
present a perfect interface between the light generating bead
positive binding event in these systems is always counter-
and the detector, often a CCD camera.286,291,292
checked in a microtiter plate experiment to verify the binding
Today, the company Affymetrix offers microarrays with
event. The whole system itself cannot be designed as hand-held
>2 000 000 unique compounds. The fluidic system is quite
and is quite expensive (several 10 000 h per run for sequencing),
simple. The sample is manually loaded with a pipette into the
but is inexpensive in terms of cost-per-assay and material
chip, and capillary forces transfer the sample to the incubation
consumption (less than a cent per sequenced base).295
chamber. Incubation and mixing is enhanced by a moving air
bubble actuated by slow rotation.
The company 454 Life Sciences offers picowell plate systems Criteria for the selection of a microfluidic platform
for the performance of massively parallel gene sequencing.286
After the previous discussion of the platform approach and
Beads containing roughly 10 million identical DNA copies are
the presentation of some prominent examples for microfluidic
loaded into the picowell plate with a pressure driven system,
platforms, this section will attempt to summarize the strengths
where each bead sediments into one cavity. Different bio-
and limitations of each platform presented in Fig. 2. This
molecules are washed over the wells, interacting with the beads
should provide the reader with some guidance to select
inside. In the case of a positive reaction, a quantitative
platforms based on the selection criteria presented in
enzymatic reaction, the pyro-sequencing,293 results in the
Table 3. The given platform characteristics are based on the
emission of light. This system allows for parallel sequencing
reviewed literature and the experience of the authors, taking
of 106 beads in a single run.
into consideration properties such as the material of the
disposable, necessary processing equipment, production
Bead-based assays
technologies, published variety of unit operations, published data
For bead-based assays, liquid actuation and metering is most concerning precision, throughput, or multiparameter testing.
often pressure driven or performed with a pipetting robot in a Beneficial platforms can be selected by identifying imperative
microtiter plate. Mixing can be performed by any kind of requirements of a certain application, e.g. portability, low
mixing process according to the different actuation principles reagent consumption and high precision for point-of-care
(diffusion, sonication, SAW, shaking, electrokinetic, electro- diagnostics, which are then compared to the characteristics
phoretic, pressure driven pumping through microchannels, of the available platforms. The platform characteristics are
etc.). The beads are separated from the liquid by centrifugation compiled to the best of our knowledge in Table 6. Nevertheless
or with the help of magnetic fields and can then be transferred the performance of certain platforms according to some of the
into another liquid. Typically, detection and readout are en- criteria is still debatable and could easily change in the
abled with a fluorescent marker. The beads are then analyzed future with upcoming innovations in this fast developing field.
either sequentially or in parallel. For sequential analysis the Nevertheless the strong position of classical liquid handling
beads are transferred into a capillary and cross several laser technologies using pipetting robots can clearly be seen.
beams and detectors one after the other. In that case, the beads It is obvious that some of the microfluidic platform
bear a coding to identify them.289,290 For the massively parallel approaches are dedicated to certain fields of application. For
analysis the beads are transferred onto a planar surface or into a example, the classical liquid handling technology enables high
picowell plate (Fig. 19(b) and (c)). sample throughput and is programmable with high flexibility,
Bead-based assays have been commercialized by Luminex but the main drawback is the lack of portability/wearability
since 1997.289 A microtiter plate is used for incubation and a and the high equipment costs for complex automated work-
capillary for bead transfer into the reader. Illumina291,292 stations. These properties limit its use to large laboratories.
expanded this concept radically by the use of 3 mm silica The lateral flow test platform fulfils the requirements for
spheres, each bearing a unique DNA strand. The spheres are point-of-care diagnostic applications quite well (moderate

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c The Royal Society of Chemistry 2010
 Table 6 Characteristics of microfluidic platforms with respect to certain selection criteria
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reagent consumption, good portability, and additionally low acceptable period of time at a minimum consumption of
costs). However, as soon as the diagnostic assay requires reagents per test. Consequently flexibility is less important,
higher precision or exceeds a certain level of complexity and throughput and costs are the main issues. Thus,
(e.g. if an exact metering of the sample volume or sample approaches like segmented flow and dedicated systems for
aliquoting is required), new approaches like linear actuated massively parallel analysis are interesting candidates for these
devices and centrifugal microfluidics become advantageous for applications.
point-of-care applications. They enable more sophisticated An increasing number of application examples benefits from
liquid handling functions, which is for instance required for the transfer of unit operations and fabrication technologies
nucleic acid-based tests. between research groups by literature, collaboration or com-
The pressure driven laminar flow platform is especially mercial supply (e.g. foundries). This shows the advance of the
interesting for online monitoring applications, since it enables platform approach in the research community. We strongly
continuous flows compared to the merely ‘‘batch-wise’’ operation believe that this trend of platform-based development will
of most of the other microfluidic platforms (i.e. handling discrete continue and speed up the variety of assay implementations
liquid volumes). in the field of microfluidics. If research time and development
Some of the platforms can also be considered as ‘‘multi- costs of microfluidic applications can be reduced significantly
application’’ platforms, which is of special interest in the by this approach, and the spectrum of applications increases
field of research instrumentation. Here, portability is of less correspondingly, this could finally lead to the commercial
importance, and the number of multiple parameters per breakthrough of microfluidic products.
sample as well as programmability (potentially also during
an assay run) gains impact. The microfluidic large scale
Acknowledgements
integration and the droplet-based electrowetting and surface
acoustic waves platforms are such versatile examples. We would like to thank our colleagues Peter Koltay, Junichi
For high-throughput screening applications, on the contrary, Miwa and Sven Kerzenmacher for their helpful suggestions
a high number of assays need to be performed within an and assistance during the preparation of this manuscript. We

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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1153–1182 | 1177
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