Immunology On Chip

JBA-06766; No of Pages 14
Biotechnology Advances xxx (2013) xxxxxx
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
Research review paper
Immunology on chip: Promises and opportunities

Sara Baratchi a,b,, Khashayar Khoshmanesh a, Catarina Sacristn c, David Depoil c, Donald Wlodkowic d,
Peter McIntyre b, Arnan Mitchell a,
a
School of Electrical and Computer Engineering, RMIT University, Melbourne, Australia

Health Innovations Research Institute, RMIT University, Melbourne, Australia
Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, NewYork, USA
d
BioMEMS Research Group, School of Applied Sciences, RMIT University, Melbourne, Australia
b
c
a r t i c l e
i n f o
Article history:
Received 13 June 2013
Received in revised form 4 November 2013
Accepted 17 November 2013
Available online xxxx
Keywords:
Microuidics
Lab on a chip
Immunology
Immunoassays
a b s t r a c t
Microuidics has facilitated immunological studies by enhancing speed, efciency and sensitivity of current analysis methods. It offers miniaturization of current laboratory equipment, and enables analysis of clinical samples
without the need for sophisticated infrastructure. More importantly, microuidics offers unique capabilities;
including conducting multiple serial or parallel tasks as well as providing complex and precisely controlled environmental conditions that are not achievable using conventional laboratory equipment. Microuidics is a promising technology for fundamental and applied immunological studies, allowing generation of high throughput,
robust and portable platforms, opening a new area of automation in immunology.
2013 Published by Elsevier Inc.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
The architecture of a future immuno lab-on-a-chip prototype system
On-chip manipulation of cells
. . . . . . . . . . . . . . . . . .
3.1.
Cell sorting and immobilization . . . . . . . . . . . . . .
3.1.1.
Mechanical lters . . . . . . . . . . . . . . . .
3.1.2.
Dielectrophoresis
. . . . . . . . . . . . . . . .
3.1.3.
Magnetophoresis
. . . . . . . . . . . . . . . .
3.1.4.
Acoustophoresis . . . . . . . . . . . . . . . . .
3.1.5.
Surface antigenantibody afnity . . . . . . . . .
3.1.6.
Fluorescent-activated cell sorting (FACS) . . . . . .
3.2.
Cell analysis . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Cell migration . . . . . . . . . . . . . . . . . .
3.2.2.
Flow cytometry . . . . . . . . . . . . . . . . .
3.2.3.
Immunophenotyping . . . . . . . . . . . . . . .
3.2.4.
Single molecule analysis . . . . . . . . . . . . .
3.3.
Cell lysis . . . . . . . . . . . . . . . . . . . . . . . . .
On-chip manipulation of nucleic acids . . . . . . . . . . . . . . .
4.1.
Purication of nucleic acids . . . . . . . . . . . . . . . .
4.2.
Amplication of nucleic acids . . . . . . . . . . . . . . .
4.3.
Separation of nucleic acids . . . . . . . . . . . . . . . . .
On-chip manipulation of proteins . . . . . . . . . . . . . . . . .
5.1.
Detection of proteins . . . . . . . . . . . . . . . . . . .
5.2.
Purication of proteins . . . . . . . . . . . . . . . . . .
5.3.
Separation and analysis of proteins . . . . . . . . . . . . .
5.4.
Interfacing protein separation with mass spectrometry . . . .
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Correspondence to: S. Baratchi, School of Electrical and Computer Engineering, RMIT University, Melbourne, Australia.
Corresponding author.
E-mail addresses: sara.baratchi@rmit.edu.au (S. Baratchi), arnan.mitchell@rmit.edu.au (A. Mitchell).
0734-9750/$ see front matter 2013 Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.biotechadv.2013.11.008
Please cite this article as: Baratchi S, et al, Immunology on chip: Promises and opportunities, Biotechnol Adv (2013), http://dx.doi.org/10.1016/
j.biotechadv.2013.11.008
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S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx
6.
Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Our knowledge of immunology has seen extraordinary advances
over the past three decades, driving the creation of new therapeutics
for human pathogenesis directed by the immune responses of our
bodies (Medzhitov et al., 2011). Our immune system has evolved as a
highly discriminatory defence mechanism to protect against potential
invaders. Cells in the immune system form a complex network with
other tissues and organs to defend our body. Immune cells are activated
in response to pathogens or indeed any abnormalities within the system
and begin sending signals to other cells. Many different signals have
been identied; including the expression of different cytokines or
protein markers, biochemical or mechanical interactions, cell-to-cell
contact, or cell migration, which determine the progress of immune or
inammatory responses (Male et al., 2012). To study and understand
such complex and subtle signalling mechanisms sophisticated and precise tools that can isolate each process at the microscale level are
required.
Microuidics, the science and technology of manipulating small
scale of uids (109 to 1018 l) within microscale structures, has enabled powerful platforms for fundamental and applied biomedical research (El-Ali et al., 2006). In particular, microuidics is a promising
technology for miniaturization and parallelization of immuno-assays,
while minimizing required sample volumes for precious and in some
cases unique specimens. These characteristics are especially valuable
for analysis of patient's samples with limited time, facilities and expertise. As such, microuidic systems are ideal platforms for monitoring
diseases in natural disaster affected areas or developing countries with
resource-limited settings (Sun and Morgan, 2010; Toner and Irimia,
2005; Yager et al., 2006).
The most important advantage of microuidic systems is their capability to process extremely low volumes of sample and reagents, which
signicantly reduces the cost of immunological assays and enables
studying small and/or rare cell populations from clinical patients.
More importantly, the ow remains laminar within the microuidic
systems, enabling the accurate control of ow variables such as velocity,
pressure and temperature. This facilitates the analysis of immune
responses under precisely controlled environmental conditions over
target cells (Abhyankar et al., 2006; El-Ali et al., 2006; Vickers et al.,
2012). Cells can be patterned in small clusters of a few or even single
cells to obtain deep and uncluttered insight into the heterogeneity of
the cell sample (Kim et al., 2009a). This enables studying the molecular
machinery of individual cells with a precision that cannot be matched
by conventional macroscopic counterparts (Sims and Allbritton, 2007).
Moreover, reducing the diffusion length leads to faster reaction
times in microuidic systems, enabling the dynamic analysis of immune
cell responses to different and highly controlled stimuli (Faley et al.,
2008). The increased surface-to-volume ratio of such systems also
makes possible the rapid and sensitive detection of cells, nucleic acids
or protein at very low concentrations that is essential early diagnosis
of diseases (He and Herr, 2010; Khoshmanesh et al., 2011c).
Several microuidic systems can be accommodated on a single chip
and connected in customized congurations so as to realize the desired
functionalities. They can be patterned either in parallel to increase the
ow throughput of a particular sample or to conduct the same experiment under an array of environmental conditions; (Munce et al.,
2004) or patterned in series to integrate multistep procedures such as
sorting, immobilization, lysis and chemical stimulation of cells (Easley
et al., 2006; Huang et al., 2006; Zare and Kim, 2010).
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In this review, we describe the architecture of lab-on-a-chip systems

to address a wide range of immunological studies and envision the
trajectory that such systems will create for future research in immunology. We survey different microuidic systems that have been introduced for cell based techniques, ranging from sorting, migration and
cytotoxicity assays. We also present a collection of recent and topical
microfabricated platforms for biochemical and molecular biology
studies. Our objective is to identify systems that have recently been
developed to replace the conventional bench-top infrastructures in immunology and show how, through the application of various technologies, highly integrated single chip biological assays of unprecedented
capability are emerging. We believe that the microuidic technology
will become a key player in both fundamental research and applied
immunology in the not too distant future.
2. The architecture of a future immuno lab-on-a-chip
prototype system
The envisioned immuno lab-on-a-chip can be divided into three
major parts including: cell; nucleic acid; and protein modules, as
shown in Fig. 1.
The cell module is designed for sorting, trapping, stimulation, characterization and disintegration of cells. It is comprised of three functional elements: cell sorting and immobilization; cell analysis; and cell lysis
units, which can be achieved by a variety of mechanisms and components (Fig. 1). These elements can be arranged in such a way that
the cells can be directly applied to the cell analysis module or to
the cell sorter module. Alternatively, the nucleic acid module is
dedicated for trapping and analysis of target nucleic acids. It consists
of purication, amplication and separation components. Finally, the
protein module is dedicated for detection, trapping and characterization of target proteins. It consists of detection, purication, and separation components (Fig. 1).
The immuno lab-on-a-chip can serve either as an end-point system or as an interface with various off-chip technologies. For example,
the immobilized cells can be interfaced with environmental scanning
electron microscopy (ESEM) or total internal reection uorescent
(TIRF) microscopy systems to conduct high/super resolution microscopy
of cells. Alternatively, the separated proteins can be interfaced with
electrospray ionization (ESI) or matrix assisted laser deposition ionization (MALDI) systems for mass spectroscopy of proteins and peptides.
3. On-chip manipulation of cells
3.1. Cell sorting and immobilization
The sorting of target immune cells is critical in many diagnostic,
therapeutic and basic immunological studies. Samples of interest must
often be isolated from a heterogeneous population of cells in blood or
tissue. The standard methods available for cell sorting are often labour
intensive and require multiple additional labelling steps to identify
cells. Conventional cell sorting systems generally rely on continuous
ow cytometry and are based on differential labelling of cellular populations. Flow cytometers use uidic systems to deliver the stream of
samples to the interrogation point and based on the labelling strategy
can be divided into the uorescent-activated cell sorter (FACS) or
magnetic activated cell sorter (MACS) groups. In FACS, uorescent
conjugated antibodies are used for labelling the cells while in MACS, antibody conjugated magnetic beads are employed. Despite offering high
Fig. 1. Layout of a fully integrated immuno lab-on-a-chip consisting of cell, nucleic acid and protein modules.
throughput sorting, these systems are expensive, and should be operated by expert personnel. Additionally the necessity of antibody labelling
makes them unsuitable for analysis of unknown populations of cells
for which well characterized marker antibodies are not available.
Centrifugation may also be used for separation of blood cells, based on
differential cellular density. Despite the simplicity of this method, one
important limitation is that it often requires relatively large blood sample volumes (millilitres). Additionally, for some cells, such as leukocytes,
that are sensitive to their environment, centrifugation might alter their
immunophenotypes (Gossett et al., 2010).
Microuidic platforms offer numerous advantages over conventional systems such as reduced sample volume, faster sample analysis,
high sensitivity, high temporal resolution, portability, and lower cost
(Microuidics for cell separation). The microuidic approaches can be
classied into passive/active groups according to the mode of actuation
or labelled/label-free groups according to the treatment of cells, as summarized in Table 1.

While the discussion herein is limited to mechanical; electrical (dielectrophoresis); magnetic (magnetophoresis); acoustic
(acoustophoresis); surface antigenantibody afnity; and uorescentactivated cell sorting techniques, a more comprehensive discussion
about cell separation techniques can be found in other excellent review
articles (Gossett et al., 2010; Toner and Irimia, 2005).
3.1.1. Mechanical lters
These lters are commonly used for the label free separation of cells
based on their size or deformability. The cells are driven into mechanical
constrictions that are sized such that certain phenotypes can pass
through while the others are blocked (Gossett et al., 2010). Pillar-type
lters are the most common mechanical lters formed by deposition
Table 1
A summary of techniques developed for cell sorting in microuidic platforms.
Mechanism
Description
Sorting criteria
Passive/active
Labelled/
label-free
Reference
Mechanical
Filtration of cells using mechanical barriers
Size, deformability
Passive
Label-free
Hydrodynamic
Manipulation of cells using the laminar

characteristics of the ow
Mimicking intrinsic fractionation of blood
cells in vessels due to plasma skimming,
Zweifach-Fung effect or leukocyte margination
Manipulation of cells using non-uniform electric elds
Size, shape
Passive
Label-free
Ji et al. (2008); VanDelinder

and Groisman (2007)
Davis et al. (2006)
Type of blood cells
Passive
Label-free
Shevkoplyas et al. (2005);

Yang et al. (2006)
Size, dielectric properties
Active
Label-free
Size, magnetic susceptibility
Active
Labelled
Size, density, compressibility

Surface properties
Active
Passive
Label-free
Labelled
Gascoyne et al. (2009);

Khoshmanesh et al. (2011b)
Kim et al. (2009b);
Robert et al. (2011)
Lenshof and Laurell (2011)
Nagrath et al. (2007)
Size, uorescence
Active
Labelled
Size, refractive index
Active
Label-free
Hemodynamic
Dielectrophoresis
Magnetophoresis
Acoustophoresis
Antibody/antigen afnity
Fluorescent activated
cell sorting
Optical
Manipulation of magnetically labelled cells

using magnetic elds
Manipulation of cells using standing acoustic waves
Biochemical interaction between the cell membrane
receptors and antibody functionalized surfaces
Sorting cells based on their uorescent characteristics
and light scattering
Manipulation of cells using focused laser beams
Baret et al. (2009);

Wlodkowic et al. (2011)
Perroud et al. (2008)
of microposts within the channel. These lters have been employed to

separate leukocytes from erythrocytes (Ji et al., 2008) and also to isolate
foetal nucleated erythrocytes from maternal cells (Mohamed et al.,
2007). Despite the apparent simplicity of this approach, the trapped
cells can clog the channel and complicate the separation procedure.
Membrane lters are another version of mechanical lters, which
are formed by patterning arrays of well-dened pores onto polymeric
substrates (Zheng et al., 2007). These lters have been used for capturing of circulating tumour cells (CTCs) from whole blood and further
electrolysing them (Zheng et al., 2007). In comparison, cross-ow lters
are made by patterning an array of narrow channels along the side walls
of the main channel (VanDelinder and Groisman, 2006). Smaller cells
can pass through the narrow channels while the larger cells remain
within the main channel. These lters have been used for the separation
of plasma from the whole human blood (VanDelinder and Groisman,
2006). Although this design overcomes the clogging of the main channel it can potentially lead to deformation and lysis of cells if operated
at high ow rates.
changing the frequency of the applied electric eld as well as the electrical conductivity of the medium.
Dielectrophoresis has been used for isolation of different cells based
on their cytoplasmic properties, including malaria-infected erythrocytes from blood (Gascoyne et al., 2002) and CTCs from blood samples
(Gascoyne et al., 2009). Live cells can thus be separated without
the need for labelling or modication, allowing each component to be
investigated further once sorting is complete.
Dielectrophoresis also enables the immobilization of live cells in intimate contact of each other to investigate the cellular response against
different chemical stimuli. For example, Khoshmanesh et al. investigated the cycloheximide-induced apoptosis of U937 human leukaemia
cells using an open-top dielectrophoretic platform (Khoshmanesh
et al., 2011b). The electro-thermal vortices generated within the
PDMS (polydimethylsiloxane) block enabled the rapid immobilization
of cells and efcient mixing of the specic drug with the buffer bathing
the cells (Fig. 2A). The patterned cells were later interfaced with ESEM
to analyse their morphological properties (Khoshmanesh et al., 2011a).
3.1.2. Dielectrophoresis
The induced motion of neutral particles under non-uniform electric
elds, can be used to isolate cells based on their dimensions or dielectric
properties (Pethig et al., 2010). The dielectrophoretic forces can direct
the cells towards the strong or weak electric eld regions. The nonuniform electric elds are generally created by integration of metallic
microelectrodes within microuidic platforms. However, it is possible
to generate non-uniform electric elds without the microelectrodes as
well (Shevkoplyas et al., 2005; Yang et al., 2006). Dielectrophoretic
systems are very exible as their performance can be ne-tuned by
3.1.3. Magnetophoresis
The induced motion of magnetic particles under magnetic elds has
been used for separating cells based on their dimensions or magnetic
susceptibility (Gossett et al., 2010). Magnetic elds can be readily
created by incorporation of permanent or electro-magnetic coils with
microuidic platforms (Gossett et al., 2010).
The target cells should be immune-magnetically labelled in order to
be manipulated by magnetophoresis. This is achieved by employing the
interaction of antibody-coated paramagnetic micro/nano particles
which interact with specic antigens on the surface of target cells.
Fig. 2. Microuidic platforms for sorting/immobilization of cells: (A) rapid immobilization and drug-induced death analysis of U937 human leukaemia cells using a ow-free
dielectrophoretic system, white arrows show the cell chains formed between opposite microelectrodes while red arrows show dying U937 cells stained with PI (Khoshmanesh et al.,
2011b), (B) size-based separation of platelets from white blood cells using a acoustophoretic system, (abbreviations: WBC: white blood cell, PLT: platelet), the inset shows the position
of cells with respect to acoustic waves (Dykes et al., 2011), (C) Isolation of rare circulating tumour cells from perpherial blood of cancer patients using a mechanical lter (Nagrath
et al., 2007) (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)
This method has been used to separate a variety of cells and particles,
among which are T-lymphocytes from whole blood (Kim et al.,
2009b), HIV virions from a mixture of blood cells (Chen et al., 2010b),
CD3+ T-lymphocytes from a mixture of cells (Kim et al., 2009b) and
CD34+ cells from fresh leukocytes (Jing et al., 2007). Magnetophoretic
separation based on nanoparticle uptake has also been employed to
separate macrophages from monocytes based on their endocytosis
capabilities (Robert et al., 2011). Furthermore, the intrinsic magnetic
properties of hemoglobins in erythrocytes have been exploited to achieve
the label-free separation of these cells. Specically, deoxygenated
haemoglobin is paramagnetic due to the presence of unpaired electrons,
while it becomes diamagnetic in its oxygenated form. It is this distinction
that allows the separation of red blood cells from leukocytes without
labelling them (Melville et al., 1975; Zborowski et al., 2003).
3.1.4. Acoustophoresis
The manipulation of dispersed particles by standing acoustic waves
is another separation method which has been used to separate cells
based on their size, density or compressibility (Lenshof and Laurell,
2011). This method relies on the creation of standing pressure waves
in a microchannel. These acoustic waves can be readily generated
through the use of microfabricated piezoelectric transducers within a
microuidic system (Lenshof and Laurell, 2011).
Acoustophoresis has been extensively applied to separate cells based
on their dimensions. One example is separation of plasma from the
whole blood, in which the blood cells are sequentially focused along
the channel centreline by an ultrasonic acoustic wave while the plasma
was withdrawn from the side outlets (Lenshof et al., 2009). The collected
plasma was later linked to an antibody microarray chip for prostate specic antigen detection. A similar concept has been adapted to separate
platelets from peripheral blood progenitor cells (Dykes et al., 2011)
(Fig. 2B). Another study enriched viable MCF-7 breast tumour cells
from a heterogeneous mixture of apoptotic cell, based on the fact that
the apoptotic cells are smaller than viable ones (Yang and Soh, 2012).
Acoustophoresis is currently at the state of transition to clinical laboratories and industries. It is relatively insensitive to media properties such as
pH and salt concentration, making it very compatible with a broad range
of biological assays (Lenshof and Laurell, 2011).
3.1.5. Surface antigenantibody afnity
Receptor targeting at the cell membrane is another approach for cell
separation that works by coating the microchannel surface with specic
antibodies to capture target cells (Grow et al., 2003; Ruan et al., 2002).
One example is a microchip consisting of an array of microposts functionalized with antibody (to anti-epithelial cell adhesion molecules) to
isolate CTSs in peripheral blood from patients with metastatic lung,
prostate, pancreatic, breast and colon cancers (Nagrath et al., 2007)
(Fig. 2C). Another example is the application of an antibody functionalized hydrogel for the isolation of CD34+ and Flk1+ cells (endothelial
progenitor cells) from untreated whole human blood by depleting
CD34+/Flk1 hematopoietic stem cells (Hatch et al., 2012). Alternatively, Vickers et al. developed a microuidic platform with antibodyconjugated surfaces to separate two phenotypically similar cells using
a single antibody (Vickers et al., 2012). The separation was based on variations of the binding afnity of cells as a function of shear stress, which
was modulated by changing the ow rate of the medium. The effectiveness of this method was demonstrated by separating two CD31+ cell
types: human umbilical vein endothelial cells; and human microvascular endothelial cells (Vickers et al., 2012). The capturing efciency of the
antibody-functionalized microchips can be enhanced by directing the
target cells towards the antibody coated regions via secondary forces
such as dielectrophoresis (Perroud et al., 2008). Moreover, the combination of antibodyantigen afnity with electrical impedance sensing has
been used to count the CD4+ T lymphocytes in a sample of leukocytes,
which offers a promising solution for on-chip quantitative diagnosis of
HIV (Davis et al., 2006).
3.1.6. Fluorescent-activated cell sorting (FACS)

Labelling cells uorescently and then triggering a sorting mechanism depending on the measurement outcome is commonly used for
counting, sorting and characterization of owing cells (Wlodkowic
et al., 2011). A variety of microuidic based FACS systems have been
developed for sorting cells. For example, Wolff et al. developed one of
the rst FACS systems and applied it to sort uorescent latex beads
from chicken red blood cells (Wolff et al., 2003). In this system, the
sample ow was hydrodynamically focused along the centre of the
chip, and the uorescent signal of moving particles was detected using
a photomultiplier tube, which was used to activate a ow switching
valve to divert non-uorescent cells towards the waste channel. The
device achieved a sample throughput of 12,000 cells per second. In another work, a FACS was used to sort cells according to their enzymatic
activity (Baret et al., 2009). In this system, cells were encapsulated
within droplets of a biocompatible emulsion, the droplets were stored
to enable their uorogenic substrate to be turned-over by the cellular
enzymes and subsequently sorted by dielectrophoresis according to
their uorescence intensity. The cells were then recovered from the
sorted droplets. The device achieved a sample throughput of 300 droplets per second.
One of the shortcomings of FACS platforms can be their long
switching time, which is orders of magnitude longer than that of
conventional electrostatic-droplet-based cell sorters (Wu et al., 2012).
To address this issue, Wu et al. developed a pulsed laser triggered
FACS, to induce a cavitation bubble in the laser-pulsed channel (Wu
et al., 2012). The pulsed and main channels were parallel and connected
via a narrow nozzle. The bubble expansion created a high-speed liquid
jet within the nozzle, which could deect the sample of interest towards
the collection channel in 30 sec.
3.2. Cell analysis
3.2.1. Cell migration
Cell migration is a critical phenomenon in immune recognition and
response, and is the key element for processes such as wound healing
and migration towards the site of infection or inammation. Migration
initiates with a cell's response to external signals; a change in the cell's
symmetry ensues, with the cell's front facing towards the direction of
movement. This polarization is central in cellular migration and begins
with the cellular response to a chemical gradient (i.e., chemotaxis) or
to a direct current electric eld (i.e., electrotaxis).
3.2.1.1. Chemotaxis. Boydon chamber/Transwell assay is commonly used
for studying the migration of cells toward different chemokine concentrations. The system consists of two compartments that are separated
by a microporous lter. The relevant chemoattractant solution is placed
in the lower chamber and leukocytes or endothelial cells are grown in
the upper chamber to replicate the in vivo condition. One important
limitation of this system is the inability to maintain the chemokine gradient since within few hours the chemokine will homogenously diffuse
to the upper chamber and the cells no longer migrate towards the pores.
Another limitation is that it is not possible to study the effect of shear on
migrating cells (Toetsch et al., 2009).
Microuidic platforms have advanced the traditional chemotaxis assays by providing a precise and stable gradient of chemicals as well as
incorporating uid shear stress into the assay. They have been applied
to quantitatively and qualitatively study the cell migration using
chemo-attractants gradients, which can be divided into two groups:
ow-based; or ow-free devices. Flow-based devices have been applied
to study the migration of immune cells such as neutrophils or lymphocytes (Kim and Haynes, 2012). For instance, by applying stable and dynamic gradients of chemo attractants to human neutrophils, it has been
shown that the cells can sense, analyse and prioritize multiple signals
during their chemotaxis (Kim and Haynes, 2012). Flow-free devices
minimize the effect of ow-induced shear stress on cell migration,
thus enabling the study of various readouts such as cell signalling under
dened chemical gradient conditions (Abhyankar et al., 2006). One
example is the application of a membrane-based system to generate a
chemical gradient across a microuidic channel for studying neutrophils
chemotaxis (Abhyankar et al., 2006). Another chip device has been
designed to apply a diffusion-driven gradient between two parallel channels connected via an array of straight and maze-like narrow channels to
analyse neutrophils chemotaxis (Ambravaneswaran et al., 2010).
3.2.1.2. Electrotaxis. It refers to cell migration directed by a physiologicallyrelevant direct current electric eld. The electric eld triggers cell signalling to guide cell movement, but not necessarily exerts a force on cells
(Li and Lin, 2011). Electrophoresis on the other hand can exert a force
on any charged particle and in the case of cells is associated with cell's
electrophoretic mobility. In conventional electrotaxis assays, the cell culture chamber is lled with electrolytes, and the electric eld is applied
across the chamber via agar salt bridges. Despite the simplicity of
this assay, the electric eld cannot be accurately controlled and the
throughput of the system is low (Li and Lin, 2011).
Microuidic platforms offer unique features for studying immune
cell electrotaxis as they enable the creation of uniform and controllable
electric elds within microuidic channels. Furthermore, real-time and
quantitative analysis of electrotaxis at a single cell level can be achieved.
For example, by imposing a controlled electric eld along a microuidic
channel, it has been shown that anti-CD3/CD28 activated lymphocytes
can migrate towards the cathode in the system (Li and Kolega, 2002).
It has been demonstrated that electrical stimulation can activate signalling pathways in T-lymphocytes similar to those induced by chemotaxis
stimulation, concomitantly causing the cells to migrate towards the
cathode (Lin et al., 2008).
Microuidic systems also have the potential to create simultaneously
both chemical gradients and electrical elds, to achieve deeper insight
into the migration of immune cells in electrochemical environments.
For example, studying the migration of T-cells under single and coexisting gradients of chemokine and electric eld indicated that the
combination of chemotaxis and electrotaxis can accelerate the migration
of T-cells towards the cathode (Li et al., 2012).
3.2.2. Flow cytometry
A variety of innovative ow cytometry (FCM) systems have been
reported for analysis of moving cells. One example is a FCM system
to measure relative concentrations of uorescently labelled subpopulations of lymphocytes and other leukocytes within blood samples
(Frankowski et al., 2011). The device utilized integrated optical bres
for laser excitation and detection of uorescent signals. In another
study, Skommer et al. pioneered the use of a FCM system to measure
cellular DNA content in both live and xed tumour cells, and also to
track the drug-induced activation of caspases and the dissipation of
mitochondrial inner membrane potential (m loss) with respect to
cell cycle stage of tumour cells (Skommer et al., 2013) (Fig. 3AB). Despite the essentially simple design, the system incorporated a dedicated
hardware interface comprising of a microcontroller-driven syringe
pump, spatially separated laser excitation sources, photodiodes, and
photomultiplier tubes to achieve an elegant separation. Further integration and miniaturization is certainly possible.
Alternatively, Holmes et al. (Holmes et al., 2009) developed a
microuidic impedance cytometer for the label-free differentiation of
leukocyte sub-populations (T-lymphocytes, neutrophils and monocytes). The microuidic chip took advantage of microelectrodes to measure the impedance of passing single cells at two frequencies of 503 and
1707 kHz. This enabled discrimination of leukocytes according to both
size and membrane capacitance.
3.2.3. Immunophenotyping
Phenotyping of immune cells through measurement of their functional status during different stages of the cell cycle is among the gold
standard methods of determining immune health status. The immune

system comprises a heterogeneous population of cells and the level
and proportion of different cytokines and signalling markers changes
with different conditions, as in the case of infections and malignancies
(Maecker et al., 2012). Different approaches currently exist for assessment of immune cell status. Currently, ELISA/ELISpot methods are the
gold standard methods for quantifying cellular cytokine production
(Chen et al., 2013a). However, these methods are labour intensive and
require multiple steps of washing and staining. Further, ELISpot is not
able to quantify the amount of cytokine production. Another approach
is to measure the intracellular cytokine production using ow cytometry (Chen et al., 2013a). This method has so far concurrent analysis of
up to ve cytokines, which in some cases may limit its future usefulness.
The requirements for this method might also involve using larger
amounts of the sample than what was originally desired, and could
also damage the sample in the process, making further analysis difcult
or impossible.
To address the limitations associated with conventional immunophenotyping methods different microuidic platforms have been introduced. For example, rapid detection of cell-secreted biomarker proteins
has been demonstrated by utilizing a PDMS microltration membrane
for isolation and enrichment of entering monocytes (Huang et al.,
2012). The seeded cells were stimulated with an endotoxin to secrete
tumour necrosis factor-. The cell-secreted cytokine diffused into
the immunoassay chamber to be detected and quantied in a beadbased chemiluminescence assay. The system achieved the sensitive
immunophenotyping of cells with 20-fold fewer cells than conventional
cell stimulation assays. Moreover, the total assay time was 7 times
shorter than that of an enzyme-linked immunosorbent assay (ELISA).
Mixing of antibody-conjugated polystyrene microbeads with blood
samples prior to applying the samples into the device enabled the
selective capturing, stimulation and immunophenotyping of desired
immune cells subpopulations (Chen et al., 2013b).
3.2.4. Single molecule analysis

Since its rst demonstration in 1961(Rotman, 1961), single molecule detection techniques have allowed us to visualize the dynamic behaviour of biomolecules. Specically, it has been possible to accurately
examine intra- and inter-molecular interactions and their reaction
kinetics inside living cells (Dustin and Groves, 2012). Proteins are the
key biomolecules of interest in this context, harbouring specic functions in gene expression, membrane transport and signal transduction.
One of the new objectives in cell biology is to quantitatively analyse
the molecular protein networks within cells, obtaining the dynamic
and kinetic parameters of proteinprotein interactions, protein translocations and outcomes of enzymatic reactions (Rotman, 1961).
The unique feature of microuidics is that it accelerates diffusiondominated reactions and consequently increases signal-to-noise ratio
when considering visualization and imaging of biomolecular processes
(Liu et al., 2011). Hence, microuidic chambers represent excellent platforms for single molecule analysis. TIRF is widely employed for single
molecule analysis of membrane proteins, using a laser-based excitation
depth of uorescent molecules of a few hundreds of nanometres, thus
effectively improving signal-to-noise ratios. This feature makes it an
attractive detection scheme when applied to microuidic platforms.
An important application of such a device would be in the study of immunological synapse formation, utilizing for instance, the articial
model of supported planar lipid bilayers (Kam and Boxer, 2000). In
such a system, it is possible to visualize and quantify the interaction of
immuno-receptors and other surface molecules on immune cells, to
specic antigens and ligands incorporated into lipid planar bilayers,
thus mimicking what would be an actual interaction with an antigen
presenting cell (Brian and McConnell, 1984). The Bioptechs FCS2
microuidic chamber, among others, has been widely used to study
T-cell receptor clustering and synapse formation of T-cells placed on
Fig. 3. Microuidic platforms for analysis/lysis of cells: (A) Analysis of programmed tumour cell death using a -ow cytometry chip. The chip was made of Poly(methyl methacrylate)
(PMMA), and comprised three modules: the input port with integrated cell sample and sheath uid reservoirs, a 75 m 50 m channel for 2D hydrodynamic focusing of cells, and
the output port with collection chambers (Skommer et al., 2013), (B) The optical hardware interface for (A) capable of four-colour detection using a combination of spatially separated
excitation 473 nm and 640 nm lasers, and photomultiplier tubes (PMTs) for uorescent signal collection in area, height and width parameters (Skommer et al., 2013), (C) Schematic
design of FCS2 Bioptechs chamber (Bioptechs, USA) used for studying immunological synapses, (D) T-cell receptor (TCR) clustering (red) and exclusion of CD45 molecule (green) from
microclusters. T-cells are injected into an imaging chamber containing supported planar bilayers and TCRs are visualized by TIRF. Figures show synapse formation between TCR and planar
bilayer containing major histocompatibility complex (MHC) and CD45 molecules at (iiii) 30 s and (ivvi) 30 min post contact formation (Varma et al., 2006). (E) Continuous chemical
lysis of EL-4 cells along a microuidic channel. Cells cross over from the carrier buffer to the lysis buffer as the carrier buffer is gradually washed via the bifurcation channels. The inset
shows a uorescent image of the dilution of the cell carrier buffer via the bifurcation channels (Mun et al., 2010).
glass supported planar bilayers harbouring a variety of different

antigens and adhesion molecules (Fig. 3CD) (Varma et al., 2006).
Single-molecule measurements at the immunological synapse
using high resolution microscopy can be obtained and combined with
microuidic platforms to provide fundamental insight into processes
such as the kinetics of receptorligand interactions (Dustin and
Groves, 2012; Huppa et al., 2010). Obtaining such information is
not possible from ensemble-averaged signals obtained from the
cells. Furthermore, a supported lipid bilayer system integrated into a
microuidic platform has been utilized to underpin the role of the cytoskeleton in coordinating receptor signalling in both T-cells and B-cells
(Mattila et al., 2013; Treanor et al., 2011). Indeed, recent advances in
microuidics and its unique characteristics to form multiple bilayers
in combination with super resolution imaging enable addressing key
questions in the eld of immunology.
3.3. Cell lysis
Mammalian cells are enveloped by a lipid bilayer, which forms a barrier between the cytoplasm and the extracellular environment. Cell lysis
is an important step during an immunoassay, as the efciency with
which it occurs directly affects subsequent treatments, and ultimately,
the ability to functionally analyse intracellular components of a sample

of interest. Chemical cell lysis has been the method of choice for extraction of cytoplasmic contents (Mun et al., 2010; Sasuga et al., 2008). Depending on the complexity of the sample, different treatments need to
be performed to separate cells from debris and interfering substances.
This may include ltration, centrifugation, treatment with appropriate
lysis buffers, dilution and if possible enrichment of targeted molecules.
These steps are labour intensive and time consuming. Moreover, the
volume of lysis buffer should be minimized to reduce the dilution of
target cellular analytes. Appropriate selection of lysis buffer is essential
to avoid the denaturation of target cellular analytes or their conjugated
labels. In order to reduce the complexity of macro scale cell lysis
methods, these methods have been implemented into micro scale platforms (Kim et al., 2009a).
When considering the application of cell lysis in microuidic chambers, Mun et al. have employed an effective method of continuous cell
lysis (Mun et al., 2010). In this work, the cells and lysis buffer were
injected into a microchannel through separate inlets, and the cell carrier
solution was consistently removed by means of an array of narrow bifurcation channels patterned orthogonal to the main microchannel,
fully exposing the cells to the lysis buffer (Mun et al., 2010) (Fig. 3E).
Interestingly, in another work chemical lysis at a single cell level was
demonstrated by incorporating an array of picoliter microwells for

capturing and chemical treatment of cells (Sasuga et al., 2008). Other
designs have succeeded using isolated picoliter reaction volumes within
microfabricated chambers by incorporation of microvalves (Chen et al.,
2012; Wu et al., 2004) or thermopneumatic actuators (Irimia et al.,
2004). Chemical lysis of individual cells has been also achieved by
encapsulating single cells into 500 pl droplets containing lysis buffer
(Kim et al., 2009a).
Other approaches have been used to achieve physical cell lysis in
microuidics. For instance mechanical lysis can be applied by using
sharp nanoknives fabricated within the microchannels (Di Carlo
et al., 2003). Electrical lysis by exposing cells to DC or AC electric elds
(McClain et al., 2003); while thermal lysis can occur when incorporating
microheaters into the microuidic channels; (Marshall et al., 2012)
Lastly, optical lysis can be achieved by exposing the cell to a concentrated
beam of pulsed laser light (Quinto-Su et al., 2008).
4. On-chip manipulation of nucleic acids
4.1. Purication of nucleic acids
The next critical step following the cell lysis is the purication of target molecules such as nucleic acids and proteins. Different methods
have been developed to isolate DNA or RNA. In general, these methods
involve disruption and lysis of cells followed by removal of contaminants such as protein and recovery of DNA or RNA. Removal of proteins
is usually achieved by digestion with proteinase K, organic extraction or
binding of DNA to a solid phase support. Consequently, the DNA or RNA
is usually recovered by ethanol or isopropanol precipitation (Fan et al.,
2012). These methods are usually labour intensive, time consuming
and require multiple steps to collect DNA or RNA from biological
samples such as blood or tissue. To simplify and reduce these sample
preparation challenges the column-based solid phase extraction (SPE)
has been introduced by different companies. However, these methods

still require sample handling, centrifugation steps and large volume of
sample (Kim et al., 2009a).
To reduce the challenges associated with macro scale techniques
SPE-based microuidic systems have been developed that allow for
extraction of nucleic acids using nano/micro litre size samples in an
automatic manner (Wen et al., 2008). The most common solid matrices
implemented in microuidic systems for DNA extraction are silica
micropillars (Cady et al., 2003), silica beads (Chung et al., 2004), and silica sol-gels (a form of colloidal solution that forms a coherent solid gel
upon acid or base catalysis) (Wolfe et al., 2002). Magnetic silica beads
suspended in lysis buffer have also been applied for DNA extraction.
One advantage of using magnetic beads in the chamber is the ability
to perform rapid and controllable loading, mixing and patterning of
the beads to achieve a dynamic solid matrix (Azimi et al., 2011). An
additional method for nucleic acid extraction in microuidic chambers
has been the use of photo-polymerized monoliths, as they offer several
advantages including extensive surface area, tuneable pore size,
and easy patterning. An elegant example of this application was demonstrated by Wen et al. who developed a dual stage process for DNA
purication from whole blood by using a C18 silica bead column for
capturing proteins together with a monolithic column for capturing of
DNA (Fig. 4A) (Wen et al., 2007).
While the increased surface-to-volume ratio in microuidic platforms enhances the capturing efciency it creates the further, serious
problem of non-specic binding of nucleic acids or proteins. Therefore,
it is necessary to block the non-specic binding sites by treating the surface of the device with blocking agents. A variety of blocking agents including protein blockers (e.g. bovine serum albumin (BSA), non-fat
milk, and chitosan) and polymeric blockers (e.g. polyethylene glycol
(PEG), polyethyleneimine (PEI), polyvinyl alcohol (PVA) and polyacrylic
acid (PAA)) have been reported in the literature, as comprehensively
reviewed by Kim and Herr (2013).
Fig. 4. Microuidic platforms for purication, amplication and separation of nucleic acids: (A) Dual-stage purication of DNA from whole blood, using a C18 silica bead column for
capturing proteins and a monolithic column for trapping DNA (Wen et al., 2007), (B) An integrated microchip for lysis of cells and viruses, followed by SPE extraction, PCR amplication,
labelling and immuno-chromatographic based detection of nucleic acids from the cell lysates. Reservoirs P1P6 contain lysis, inhibitor removal, wash, elution, and labelling buffers while
valves V1V4 control the ow between different components (Chen et al., 2010a), (C) Another fully integrated microchip designed for DNA-based screening for infectious pathogens from
the whole blood. Chip is composed of four modules for SPE extraction, PCR amplication, marker injection, and electrophoretic separation of DNA fragments; all connected via a network of
channels and elastomeric membrane valves (Easley et al., 2006).
The extraction of RNA is more challenging than that of DNA due to its
susceptibility to degradation by Ribonucleases (RNases) (Rogacs et al.,
2012). To overcome this issue, Sattereld et al. developed a photopolymerized monolith for on-chip purication of eukaryotic mRNA
from total RNA (Sattereld et al., 2007). The monolith was functionalized with polythymine deoxyribonucleotides (oligo-dTs) which interact
with the poly-A tail of mRNA, thus capturing the nucleic acid. In another
study, Irimia demonstrated an integrated system for RNA isolation from
human monocyte cells (Irimia et al., 2009). Following cell lysis, appropriate agents were added to inactivate RNases and degrade cellular
proteins. The nucleic acids were subsequently captured on a silica
bead column while the contaminant DNA was removed by enzymatic
digestion before releasing the captured RNA. Other work has shown
the utilization of polymer capture matrices in the purication of
human immunodeciency virus (HIV) RNA from human sera (Root
et al., 2011). Serum was electrophoresed through the polymer matrix
in the chip, which had covalently bound oligonucleotides, thus selectively trapping target nucleic acids. The chip was then heated above
the melting temperature of the captured oligonucleotides to release
the target strands.
4.2. Amplication of nucleic acids
An important step in nucleic acid analysis is the amplication of captured nucleic acids using polymerase chain reaction (PCR). This method
relies on repeated heating and cooling of nucleic acid samples through
cycles of denaturation, annealing and extension of target sequences.
Conventional PCR thermocyclers consist of a thermal block with slot
arrays for PCR tubes. The temperature of the entire block must be
changed in order to achieve temperature changes in the samples. This
prolongs processing time, due to the large mass of the block. In addition,
one important challenge for the conventional PCR methods is the identication of small changes in gene expression that are usually associated
with different disease states. Quantitative PCR currently is the most sensitive approach that can identify 1.5-fold changes (Whale et al., 2012).
Microuidic PCR platforms dramatically reduce the amount of samples, reagents and analysis time, while increasing the precision of PCR
experiments (Baker, 2010). On-chip PCR has been demonstrated by
passing samples through serpentine channels, which are heated at
their bottom surface by means of patterned heaters (Crews et al.,
2008). The heaters create three isothermal zones along each channel,
enabling the cyclic denaturation, and extension of the target nucleic
acid sequence (Crews et al., 2008). Such continuous ow PCR systems
consume far less reagents and require shorter reaction times compared
to their conventional macroscopic counterparts (Zhang and Ozdemir,
2009). However, the large surface-to-volume ratio of such systems
leads to the adhesion of chemicals onto the surface of microuidic channels, which consequently leads to cross-contamination and inhibition of
PCR reactions (Zhang and Ozdemir, 2009). These issues can be overcome by utilizing droplet-based PCR systems, in which the PCR reaction
takes place in discrete droplets dispersed within an immiscible carrier
uid such as oil. The droplets can also be driven by electrowetting
mechanisms, where the wettability of the surface is modied via an
electric eld (Heyries et al., 2011; Hua et al., 2010). The rapid advances
in microfabrication technologies has enabled the creation of highly
integrated and fully automated PCR microuidic chips equipped with
micropumps, microvalves, and micromixers (Toetsch et al., 2009).
Integration of PCR microuidic components with pre- and post-PCR
processing units, as shown in Fig. 1, can further push the boundaries
of PCR-based diagnostic assays to make us one step closer to point-ofcare diagnosis of infectious diseases (Maecker et al., 2012).
4.3. Separation of nucleic acids
Gel electrophoresis is the traditional method of purifying nucleic
acid fragments, in which a DC electric eld is applied across an agarose
gel matrix to separate the samples based on their size. Despite the popularity of this technique, several manual steps are required including
staining and separation of nucleic acids followed by visualization and
quantication of separated bands, which elongate the process.
On-chip capillary electrophoresis (CE) has been demonstrated
by etching glass or quartz substrates to form capillaries. PDMS channels
are generally avoided for CE due to their permeability, which can cause
cross-contamination of nucleic acids. Integrated PCR-CE microuidic
systems capable of performing nucleic acid amplication and separation
have been demonstrated by several groups (Chen et al., 2010a; Easley
et al., 2006; Huang et al., 2006) (Fig. 4BC).
Isotachophoresis is another variant of electrophoresis, which has
been used for purication, stacking and separation of target nucleic
acids in microuidic systems. In isotachophoresis, the target sample is
introduced between the trailing and leading electrolytes whose electrophoretic mobilities are respectively lower and higher than that of the
sample. Upon application of an electric eld, the target samples are
concentrated between the trailing and leading electrolytes. This avoids
the dispersion and diffusion of the concentrated sample, and signicantly reduces the limit of detection, which cannot be achieved by CE
microdevices (Persat et al., 2009). Isotachophoresis has been used
extensively in microuidic systems, namely for the extraction and purication of genomic DNA from malaria infected red blood cells (Marshall
et al., 2011), extraction and enrichment of ribosomal RNA from whole
blood infected with bacteria (Rogacs et al., 2012), and detection of
microRNA using photo-polymerized functionalized hydrogels (GarciaSchwarz and Santiago, 2012).
5. On-chip manipulation of proteins
The density of intracellular proteins is much higher than that of DNA
or RNA. Despite this fact, measuring the expression level of the concentration of a protein of interest, especially in small cell samples is challenging due to the limitations of protein amplication. Heterogeneous
subsets of immune cells are present during immune responses to infection. These cells harbour both phenotypic and functional differences.
Thus, capturing a broad functional spectrum of given heterogeneous
cell populations remains challenging, and requires the analysis of a
large number of effector molecules from individual cells. In this regard,
microuidic platforms have been extensively utilized for the detection,
purication, separation and analysis of proteins, as summarized below.
5.1. Detection of proteins
Immunoassays using the high afnity of antigenantibody interactions have been widely used for protein detection. A variety of immunoassays are implemented in clinical laboratories. This includes ELISA to
detect and quantify the target antigen in a sample, immuno-blotting
to detect and approximate the size of a protein, ow cytometry to measure the expression of cell surface or intracellular proteins, and immunohistochemistry to detect and localize antigens in tissue samples. The
main disadvantage of these methods is that they are labour intensive
and time consuming. Additionally, they all rely on the availability of
commercial antibody conjugates to bind to target proteins and often
require 10 to 100 l of the sample (Hauss and Mller, 2007).
Microuidic platforms enable the transportation, mixing, separation
and detection of proteins in a 11000 picoliter range of sample volumes
(Freire and Wheeler, 2006). For example, a microuidic system was
developed to detect the cytokine tumour necrosis factor (TNF-)
(Cesaro-Tadic et al., 2004). In this work, a PDMS cover precoated with
capture antibodies was placed onto an array of parallel microchannels
to capture the moving analyte molecules. The PDMS cover was
then peeled off and orthogonally placed onto an array of parallel
microchannels where uorescently labelled detection antibodies were
moving. This led to the formation of a mosaic of uorescent signals
on the surface of PDMS. Moreover, researchers have succeeded in
10
developing microuidic-based ELISA chips for the quick detection and

quantication of target proteins within a sample. One example is the
disc-based microuidic ELISA system for the detection of Hepatitis B
virus (HBV) antigen or HBV antibodies from blood samples (Lee et al.,
2009). This microchip was equipped with several liquid chambers,
laser controllable microvalves, and optical detection modules. The
system enabled the automated separation of plasma from the whole
blood, incubation of plasma with target specic antigen or antibody
conjugated polystyrene beads, several washing steps, mixing and
reacting with chemiluminescent enzyme substrates, and absorbance
detection at 450 and 630 nm within 30 min (Lee et al., 2009) (Fig. 5A).
(CRP) (Peoples and Karnes, 2008). A buffer of uorescently labelled

antibody was introduced into the chamber to bind to the captured
CRP. This was followed by the addition of an acidic elution buffer to dissociate the antibodyCRP complexes and measure the concentration of
the labelled antibody. In another work, RNA aptamer conjugated beads
were applied to purify carcinoembryonic antigen, a cancer marker, from
human serum (Koh et al., 2012). The beads were packed within a PDMS
chamber lled with an array of micropillars. The chamber was then
heated to 85 C for 5 min to unfold the aptamers, enabling the capture
of passing antigens. The chamber was washed to remove the unbound
antigens, and its temperature was increased to 85 C for 35 s to denature and release the puried antigens.
5.2. Purication of proteins

5.3. Separation and analysis of proteins
Antibody capture of target proteins is an applicable method for
purifying them. Conventional methods of purifying proteins include
chromatography, which separates a large pool of proteins in to a small
pool, based on size, charge, hydrophobicity and afnity. However, conventional chromatography is labour intensive, is not suitable for proteins in the microgram scale. Microuidic platforms have enormous
capabilities with regard to the purication of small amount of proteins
from samples. The capturing efciency of microuidic systems can be
improved in several ways. For example, Sandison et al. employed a
PDMS column lled with an array of 50 m diameter pillars, which
were functionalized with covalently bound antibodies to purify recombinant afnity-tagged proteins from a bacterial lysate (Sandison et al.,
2010) (Fig. 5B). An alternative method to increase the capturing efciency of proteins is to ll the microchannels with antibody conjugated
microbeads. For instance, a capillary column packed with antibodycoated silica beads has been applied to capture C-reactive protein
The common method to analyse the proteins is gel electrophoresis

(GE), which enables the separation of proteins in gel media based on
their electrophoretic mobility as well as molecular weight (size). As proteins electro-migrate within a gel, the smaller proteins move faster than
the larger ones, and therefore the gel acts as a size-selective molecular
sieve. Photo-polymerized sieving gels such as polyacrylamide (PA) are
quite common for patterning gel matrices within microuidic channels.
For example, Herr et al. developed a polyacrylamide gel electrophoresis
(PAGE) microuidic system for rapid detection of anti-tetanus toxin and
tetanus toxin C-fragment levels in serum samples (Herr et al., 2005).
Immuno-blotting is a powerful technique to detect and analyse
specic proteins. This method uses gel electrophoresis to separate the
proteins based on their size. The proteins are then transferred to the
membrane where they are then stained with antibodies specic for
the targeted protein. On-chip immuno-blotting of proteins has been
Fig. 5. Microuidic platforms for detection, purication and separation of proteins: (A) A fully integrated lab-on-a-disc ELISA system for detection of Hepatitis B virus antigen and antibody
from blood samples (Lee et al., 2009). The chip operation relies on spining of the disc to facilitate the transporation of liquid and laser controllable microvalves to control the ow. The
system enables the separation of plasma from the whole blood, incubation of plasma with antibody conjugated beads, several washing steps, mixing and reacting with TMB enzyme
substrates, and the absorbance detection at 450 and 630 nm within 30 min, (B) a serpentine PDMS column lled with an array of 50 m diameter pillars functionalized with covalently
bound antibodies for purication of recombinant afnity-tagged proteins from a bacterial lysate (Sandison et al., 2010), (C) the layout and operation of a microchip for automated immunoblotting of proteins realized by photopatterning of polyacrylamide gels on glass. The chip facilitates the rapid loading, stacking, PAGE separation, transfer and in gel blotting of proteins,
as indicated by blue and yellow bands. The direction of the electric current is shown by i (He and Herr, 2010) (For interpretation of the references to colour in this gure legend, the reader
is referred to the web version of this article.)
demonstrated by photo-patterning several in-line PA gel elements onto

specic regions of a glass slide to enable the loading and stacking of
samples via the large pore-size loading gel, followed by electrophoretic
separation of proteins via the small pore-size separation gel, and subsequent identication of separated proteins via antibody-functionalized
blotting gels patterned in series (He and Herr, 2009). The performance
of the above system was further improved by patterning the blotting
gel in parallel with the PAGE separation region in such a way that the
separated proteins could be laterally transferred onto the blotting
region. This enabled running several PAGE separations to optimize the
experimental conditions, prior to transferring to the blotting region
(He and Herr, 2010) (Fig. 5C).
The efciency of protein separation can be improved by
implementing multi-dimensional separation strategies. One example
is the two-dimensional (2D) separation of proteins by integrating isoelectric focusing (IEF) with PAGE on a polycarbonate microuidic chip
(Li et al., 2004). In this work, the designed system consisted of a long
horizontal channel and an array of orthogonal parallel channels. The
IEF separation was achieved by establishing a pH gradient along the
horizontal channel to discriminate the proteins based on their charge.
Alternatively, the PAGE separation was achieved by electrophoresis of
proteins along the parallel channels to discriminate them based on
their molecular weight. A similar concept has been used by Emrich
et al. (Emrich et al., 2007), in which the horizontal and orthogonal
separation channels were connected via smaller channels to minimize
the diffusion of analytes at the intersection of channels. The 2D separation of proteins was also achieved by integrating isotachophoresis
with CE on a poly(methyl methacrylate) chip (Olvecka et al., 2004).
Isotachophoresis created a sharp band of concentrated proteins
electro-migrating along the channel while CE destacked and separated
the proteins along the rest of the channel.
5.4. Interfacing protein separation with mass spectrometry
Once proteins are separated, they can be coupled to a mass
spectrometer to obtain the mass spectrum of their constituent peptide
fragments. For example, Mellors et al. reported a glass microchip capable of CE separation and ESI of proteins and peptides (Mellors et al.,
2008). The corner of the glass chip was cut using a dicing saw to serve
as the electrospray source. This enabled the direct coupling of separated
samples into a mass spectrometer without the use of external pressure
or vacuum sources or the addition of capillary spray tips.
Alternatively, a PDMS microuidic system was developed for coupling CE and matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) (Luo et al., 2009). The samples were separated
along a CE channel and compartmentalized by means of fractionation
valves before being transported into the open reservoirs through the
use of a monolithic peristaltic pump. The analyte solutions were then
mixed with a matrix solution and deposited onto a MALDI target plate
for MALDI-MS. In another work, CE separation was coupled with
MALDI-MS by means of electrowetting mechanism (Gorbatsova et al.,
2012).
6. Outlook
The continuing developments in microuidic technology enable us
to improve our understanding of biological processes, and in particular
for analytical studies of the immune system. Microuidic platforms
offer myriad advantages including the potential for fast analysis, the
incorporation of multiple processing steps for different applications,
and importantly, the ability to reduce sample volume and reagents
usage. Furthermore, microuidic systems allow the undertaking
of experiments under accurately controlled conditions, limiting the
user interaction and in some instances, improving the accuracy of the
obtained results.
11
The utility of microuidic platforms for a variety of cell-, nucleic

acid- and protein-based experiments has been demonstrated in several
studies, as summarized in Sections 3 to 5 of this review. Despite the
unique advantages offered by microuidic platforms, signicant challenges lie ahead for the successful and full integration of such systems
in routine clinical and research laboratory procedures to address different aspects of immunological problems. Some of these challenges can be
summarized as below:
First, most microuidic platforms have been operated using simple
one-step procedures (e.g. purication of nucleic acids) but no concerted effort has been made to integrate multiple steps for analysis
of clinical samples. Only a small portion of the papers in the literature have demonstrated the multistep processing of the samples.
Among those is the microchip developed by Chen et al. (Chen
et al., 2010a) to detect the presence of bacteria and viruses in saliva
samples. This microchip enabled chemical lysis of cells and viruses,
followed by SPE-based extraction, PCR amplication, labelling and
chromatographic-based detection of nucleic acids from the cell
lysates. Another example is the microchip developed by Irimia
et al. (2009) for genome wide expression analysis of samples as
few as 150 cells. The microchip facilitated the chemical lysis of
cells, followed by degrading of cellular proteins, inactivation
of RNAses, SPE-based capturing of nucleic acids, enzymatic removal
of contaminant DNA and releasing the captured RNA to be amplied
off the chip.
Second, in most cases, low complexity samples (e.g. spiked proteins)
have been used while processing of complex biological specimens
(e.g. blood) have been done in separate steps in dedicated chips.
As such, the application of highly integrated micro devices for
conducting fundamental and applied research in the eld of immunology still remains a challenge due to the complexity and heterogeneity of the samples. Integration of components relies on the
successful implementation of microvalves. A variety of microvalves
have been reported in the literature including the pneumatic-based
ones developed by Quake's group (Unger et al., 2000). However,
the fabrication and operational issues surrounding these microvalves
have limited their widespread implementation. One way to resolve
these issues is to design innovate microvalves which are less complex. One example is the disc-based ELISA microchip developed by
Lee et al. (2009) to detect viral infections in blood samples. The
microchip took advantage of laser controllable microvalves to control
the ow within the different compartments of the microchip. This enabled the serial separation of plasma from whole blood, incubation of
plasma with antibody conjugated beads followed by washing, mixing
and reaction with chemiluminescent enzyme substrates.
Third, most microuidic systems rely on bulky and expensive offchip systems such as uorescent microscopes, syringe pumps, and
signal generators. Therefore, the next challenge in the eld is to be
able to miniaturize those systems to realize fully independent
immuno lab-on-a-chip platforms, which are small, transportable,
and low-cost. For example, the mini-microscope developed by Kim
et al. (2012) enables in situ monitoring of cells. Alternatively, innovative approaches can be utilized to replace the bulky off-chip components with smaller ones. For example, the microuidic-based
cytometer developed by Holmes et al. (2009), takes advantage of
impedance measurement of passing cells to achieve a label-free differentiation of leukocytes sub-populations based on their size and
membrane capacitance. This makes the cytometer independent of
bulky optical components such as laser sources, lters, lenses and
PMTs.
12
Fourth, most studies demonstrate only the proof-of-concept capability of microuidic platforms for conducting biological experiments
while there are only a few reported works in the literature, where
the utility of microuidic chips has been demonstrated in real clinical procedures. Among these is the microchip developed by Nagrath
et al. (2007) to capture CTCs in the peripheral blood of patients with
metastatic lung, prostate, pancreatic, breast and colon cancer in a
single step without any processing steps. Another example is the
microchip developed by Rosenbach et al. (2011) for rapid RNA extraction from highly pure T lymphocytes of burn-injured patients,
which was used for downstream evaluation of the gene expression
of cytokines via quantitative real-time PCR.
Fifth, to ensure that microuidic platforms can be routinely used in
clinical laboratories, they must be highly developed and free from
technical challenges that can present obstacles to users who are
not familiar with microuidic devices. Most devices are custom
designed for each application and thus may be prone to operational
issues such as leaking of microchannels, clogging of microchannels
by entrapped bubbles, and difculty in interfacing with several
inlet/outlet tubes. There exist several excellent technological solutions to these problems and it is suggested that standards be developed to ensure that all microuidic devices benet from these
solutions.
In conclusion, immuno lab-on-a-chip systems could be equipped
with several components to address (i) sample preparation, (ii) sorting,
immobilization, stimulation, and characterization of target cells,
(iii) lysing of target cells, (iv) capture, amplication, and separation of
target nucleic acids, (v) detection, capture, and separation of target
proteins, and (vi) interfacing target immobilized samples with offchip imaging or spectroscopy technologies. Such systems require the
design and implementation of modular microuidic components,
which can be easily laid in series to form highly integrated multipurpose immuno lab-on-a-chip platforms. These integrated systems
would enable the routine laboratory techniques to be performed with
smaller sample volumes, a smaller volume but larger array of consumables, and vastly reduced infrastructure scale and cost. A positive outlook is that in the future, more effective, robust, low-cost and portable
micro devices will be available for routine clinical procedures.
Acknowledgements
K. Khoshmanesh acknowledges the Australian Research Council
(project number DE120101402), D. Wlodkowic acknowledges the
Australian Research Council and RMIT Vice Chancellor's Senior Fellowship, P. McIntyre acknowledges the National Health and Medical Research
Council (project grant APP1046860) and A. Mitchell acknowledges the
Australian Research Council for funding.
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JBA-06766; No of Pages 14

Biotechnology Advances xxx (2013) xxxxxx

Contents lists available at ScienceDirect

Research review paper

Immunology on chip: Promises and opportunities

School of Electrical and Computer Engineering, RMIT University, Melbourne, Australia

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

In this review, we describe the architecture of lab-on-a-chip systems

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

or labelled/label-free groups according to the treatment of cells, as summarized in Table 1.

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

Filtration of cells using mechanical barriers

Manipulation of cells using the laminar

Ji et al. (2008); VanDelinder

Type of blood cells

Shevkoplyas et al. (2005);

Size, dielectric properties

Size, magnetic susceptibility

Size, density, compressibility

Gascoyne et al. (2009);

Size, refractive index

Manipulation of magnetically labelled cells

Baret et al. (2009);

of microposts within the channel. These lters have been employed to

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

3.1.6. Fluorescent-activated cell sorting (FACS)

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

standard methods of determining immune health status. The immune

3.2.4. Single molecule analysis

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

glass supported planar bilayers harbouring a variety of different

the ability to functionally analyse intracellular components of a sample

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

demonstrated by incorporating an array of picoliter microwells for

has been introduced by different companies. However, these methods

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

developing microuidic-based ELISA chips for the quick detection and

(CRP) (Peoples and Karnes, 2008). A buffer of uorescently labelled

5.2. Purication of proteins

The common method to analyse the proteins is gel electrophoresis

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

demonstrated by photo-patterning several in-line PA gel elements onto

The utility of microuidic platforms for a variety of cell-, nucleic

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

Chen D, Mauk M, Qiu X, Liu C, Kim J, Ramprasad S, et al. An integrated, self-contained

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxx

Wu HK, Wheeler A, Zare RN. Chemical cytometry on a picoliter-scale integrated

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