immunology on chip

. . . .no lab-. . . .tion .

. . .

. . .

3.1.5. Surface antigenantibody afnity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

. . . . . . . . . . 0

. . . . . . . . . . 0

. . . . . . . . . . 0

Biotechnology Advances xxx (2013) xxxxxx

JBA-06766; No of Pages 14

Contents lists available at ScienceDirect

Biotechnology Advances5. On-chip manipulation of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.1. Detection of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.1. Purication of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2. Amplication of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3. Separation of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.6. Fluorescent-activated cell sorting (FACS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Cell analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.2.1. Cell migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2.2. Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2.3. Immunophenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2.4. Single molecule analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.3. Cell lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. On-chip manipulation of nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05.2. Purication of proteins . . . . .5.3. Separation and analysis of proteins5.4. Interfacing protein separation with

Correspondence to: S. Baratchi, School of Electrical an Corresponding author.

E-mail addresses: [email protected] (S. Baratc

0734-9750/$ see front matter 2013 Published by Elsehttp://dx.doi.org/10.1016/j.biotechadv.2013.11.008

Please cite this article as: Baratchi S, et al, Imj.biotechadv.2013.11.008. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1.3. Magnetophoresis . . .3.1.4. Acoustophoresis . . . .Contents

1. Introduction . . . . . . . . .2. The architecture of a future immu3. On-chip manipulation of cells .

3.1. Cell sorting and immobiliza3.1.1. Mechanical lters3.1.2. Dielectrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0on-a-chip prototype system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Immunoassays

ImmunologyMicrouidicsLab on a chip

robust and portable platformResearch 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, Australiab Health Innovations Research Institute, RMIT University, Melbourne, Australiac Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, NewYork, USAd BioMEMS Research Group, School of Applied Sciences, RMIT University, Melbourne, Australia

a b s t r a c ta r t i c l e i n f o

Article history:Received 13 June 2013Received in revised form 4 November 2013Accepted 17 November 2013Available online xxxx

Keywords:

Microuidics has facilitated immunological studies by enhancing speed, efciency and sensitivity of current anal-ysis methods. It offers miniaturization of current laboratory equipment, and enables analysis of clinical sampleswithout the need for sophisticated infrastructure. More importantly, microuidics offers unique capabilities;including conductingmultiple serial or parallel tasks as well as providing complex and precisely controlled envi-ronmental conditions that are not achievable using conventional laboratory equipment. Microuidics is a prom-ising technology for fundamental and applied immunological studies, allowing generation of high throughput,

s, opening a new area of automation in immunology. 2013 Published by Elsevier Inc.

j ourna l homepage: www.e lsev ie r .com/ locate /b iotechadv. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

d Computer Engineering, RMIT University, Melbourne, Australia.

hi), [email protected] (A. Mitchell).

vier Inc.

munology on chip: Promises and opportunities, Biotechnol Adv (2013), http://dx.doi.org/10.1016/

..

.

Several microuidic systems can be accommodated on a single chipand connected in customized congurations so as to realize the desired

cells. Conventional cell sorting systems generally rely on continuousow cytometry and are based on differential labelling of cellular popu-

2 S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxfunctionalities. They can be patterned either in parallel to increase theow throughput of a particular sample or to conduct the same experi-ment under an array of environmental conditions; (Munce et al.,2004) or patterned in series to integrate multistep procedures such assorting, immobilization, lysis and chemical stimulation of cells (Easley

lations. Flow cytometers use uidic systems to deliver the stream ofsamples to the interrogation point and based on the labelling strategycan be divided into the uorescent-activated cell sorter (FACS) ormagnetic activated cell sorter (MACS) groups. In FACS, uorescentconjugated antibodies are used for labelling the cells while inMACS, an-6. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

Our knowledge of immunology has seen extraordinary advancesover the past three decades, driving the creation of new therapeuticsfor human pathogenesis directed by the immune responses of ourbodies (Medzhitov et al., 2011). Our immune system has evolved as ahighly discriminatory defence mechanism to protect against potentialinvaders. Cells in the immune system form a complex network withother tissues and organs to defend our body. Immune cells are activatedin response to pathogens or indeed any abnormalitieswithin the systemand begin sending signals to other cells. Many different signals havebeen identied; including the expression of different cytokines orprotein markers, biochemical or mechanical interactions, cell-to-cellcontact, or cell migration, which determine the progress of immune orinammatory responses (Male et al., 2012). To study and understandsuch complex and subtle signalling mechanisms sophisticated and pre-cise tools that can isolate each process at the microscale level arerequired.

Microuidics, the science and technology of manipulating smallscale of uids (109 to 1018 l) within microscale structures, has en-abled powerful platforms for fundamental and applied biomedical re-search (El-Ali et al., 2006). In particular, microuidics is a promisingtechnology for miniaturization and parallelization of immuno-assays,while minimizing required sample volumes for precious and in somecases unique specimens. These characteristics are especially valuablefor analysis of patient's samples with limited time, facilities and exper-tise. As such, microuidic systems are ideal platforms for monitoringdiseases in natural disaster affected areas or developing countries withresource-limited settings (Sun and Morgan, 2010; Toner and Irimia,2005; Yager et al., 2006).

The most important advantage of microuidic systems is their capa-bility to process extremely low volumes of sample and reagents, whichsignicantly reduces the cost of immunological assays and enablesstudying small and/or rare cell populations from clinical patients.More importantly, the ow remains laminar within the microuidicsystems, enabling the accurate control of ow variables such as velocity,pressure and temperature. This facilitates the analysis of immuneresponses under precisely controlled environmental conditions overtarget 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 singlecells to obtain deep and uncluttered insight into the heterogeneity ofthe cell sample (Kim et al., 2009a). This enables studying the molecularmachinery of individual cells with a precision that cannot be matchedby conventional macroscopic counterparts (Sims and Allbritton, 2007).

Moreover, reducing the diffusion length leads to faster reactiontimes inmicrouidic systems, enabling the dynamic analysis of immunecell responses to different and highly controlled stimuli (Faley et al.,2008). The increased surface-to-volume ratio of such systems alsomakes possible the rapid and sensitive detection of cells, nucleic acidsor protein at very low concentrations that is essential early diagnosisof diseases (He and Herr, 2010; Khoshmanesh et al., 2011c).et al., 2006; Huang et al., 2006; Zare and Kim, 2010).

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

In this review, we describe the architecture of lab-on-a-chip systemsto address a wide range of immunological studies and envision thetrajectory that such systemswill create for future research in immunol-ogy. We survey different microuidic systems that have been intro-duced for cell based techniques, ranging from sorting, migration andcytotoxicity assays. We also present a collection of recent and topicalmicrofabricated platforms for biochemical and molecular biologystudies. Our objective is to identify systems that have recently beendeveloped to replace the conventional bench-top infrastructures in im-munology and show how, through the application of various technolo-gies, highly integrated single chip biological assays of unprecedentedcapability are emerging. We believe that the microuidic technologywill become a key player in both fundamental research and appliedimmunology in the not too distant future.

2. The architecture of a future immuno lab-on-a-chipprototype system

The envisioned immuno lab-on-a-chip can be divided into threemajor parts including: cell; nucleic acid; and protein modules, asshown in Fig. 1.

The cell module is designed for sorting, trapping, stimulation, char-acterization and disintegration of cells. It is comprised of three function-al elements: cell sorting and immobilization; cell analysis; and cell lysisunits, which can be achieved by a variety of mechanisms and compo-nents (Fig. 1). These elements can be arranged in such a way thatthe cells can be directly applied to the cell analysis module or tothe cell sorter module. Alternatively, the nucleic acid module isdedicated for trapping and analysis of target nucleic acids. It consistsof purication, amplication and separation components. Finally, theprotein module is dedicated for detection, trapping and characteriza-tion of target proteins. It consists of detection, purication, and separa-tion components (Fig. 1).

The immuno lab-on-a-chip can serve either as an end-point sys-tem or as an interface with various off-chip technologies. For example,the immobilized cells can be interfaced with environmental scanningelectron microscopy (ESEM) or total internal reection uorescent(TIRF)microscopy systems to conduct high/super resolutionmicroscopyof cells. Alternatively, the separated proteins can be interfaced withelectrospray ionization (ESI) or matrix assisted laser deposition ioniza-tion (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 mustoften be isolated from a heterogeneous population of cells in blood ortissue. The standard methods available for cell sorting are often labourintensive and require multiple additional labelling steps to identifytibody conjugated magnetic beads are employed. Despite offering high

and opportunities, Biotechnol Adv (2013), http://dx.doi.org/10.1016/

3S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxthroughput sorting, these systems are expensive, and should be operat-ed by expert personnel. Additionally the necessity of antibody labellingmakes them unsuitable for analysis of unknown populations of cellsfor which well characterized marker antibodies are not available.Centrifugation may also be used for separation of blood cells, based ondifferential cellular density. Despite the simplicity of this method, oneimportant limitation is that it often requires relatively large blood sam-ple volumes (millilitres). Additionally, for some cells, such as leukocytes,that are sensitive to their environment, centrifugation might alter theirimmunophenotypes (Gossett et al., 2010).

Microuidic platforms offer numerous advantages over convention-al 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 beclassied into passive/active groups according to the mode of actuation

Fig. 1. Layout of a fully integrated immuno lab-on-a-chip

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008or labelled/label-free groups according to the treatment of cells, as sum-marized in Table 1.

While the discussion herein is limited to mechanical; elec-trical (dielectrophoresis); magnetic (magnetophoresis); acoustic(acoustophoresis); surface antigenantibody afnity; and uorescent-activated cell sorting techniques, a more comprehensive discussionabout cell separation techniques can be found in other excellent reviewarticles (Gossett et al., 2010; Toner and Irimia, 2005).

3.1.1. Mechanical ltersThese lters are commonly used for the label free separation of cells

based on their size or deformability. The cells are driven intomechanicalconstrictions that are sized such that certain phenotypes can passthrough while the others are blocked (Gossett et al., 2010). Pillar-typelters are the most common mechanical lters formed by deposition

consisting of cell, nucleic acid and protein modules.


Table 1A 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 Ji et al. (2008); VanDelinderand Groisman (2007)

Hydrodynamic Manipulation of cells using the laminarcharacteristics of the ow

Size, shape Passive Label-free Davis et al. (2006)

Hemodynamic Mimicking intrinsic fractionation of bloodcells in vessels due to plasma skimming,Zweifach-Fung effect or leukocyte margination

Type of blood cells Passive Label-free Shevkoplyas et al. (2005);Yang et al. (2006)

Dielectrophoresis Manipulation of cells using non-uniform electric elds Size, dielectric properties Active Label-free Gascoyne et al. (2009);Khoshmanesh et al. (2011b)

Magnetophoresis Manipulation of magnetically labelled cellsusing magnetic elds

Size, magnetic susceptibility Active Labelled Kim et al. (2009b);Robert et al. (2011)

Acoustophoresis Manipulation of cells using standing acoustic waves Size, density, compressibility Active Label-free Lenshof and Laurell (2011)Antibody/antigen afnity Biochemical interaction between the cell membrane

receptors and antibody functionalized surfacesSurface properties Passive Labelled Nagrath et al. (2007)

ize,

ize,

4 S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxof microposts within the channel. These lters have been employed toseparate leukocytes from erythrocytes (Ji et al., 2008) and also to isolatefoetal nucleated erythrocytes from maternal cells (Mohamed et al.,2007). Despite the apparent simplicity of this approach, the trappedcells can clog the channel and complicate the separation procedure.

Membrane lters are another version of mechanical lters, whichare formed by patterning arrays of well-dened pores onto polymericsubstrates (Zheng et al., 2007). These lters have been used for captur-ing of circulating tumour cells (CTCs) from whole blood and furtherelectrolysing them (Zheng et al., 2007). In comparison, cross-ow ltersaremade by patterning an array of narrow channels along the sidewallsof the main channel (VanDelinder and Groisman, 2006). Smaller cellscan pass through the narrow channels while the larger cells remainwithin themain channel. These lters have been used for the separationof plasma from the whole human blood (VanDelinder and Groisman,2006). Although this design overcomes the clogging of the main chan-nel it can potentially lead to deformation and lysis of cells if operatedat high ow rates.

3.1.2. DielectrophoresisThe induced motion of neutral particles under non-uniform electric

elds, can be used to isolate cells based on their dimensions or dielectricproperties (Pethig et al., 2010). The dielectrophoretic forces can directthe cells towards the strong or weak electric eld regions. The non-uniform electric elds are generally created by integration of metallic

Fluorescent activatedcell sorting

Sorting cells based on their uorescent characteristicsand light scattering

S

Optical Manipulation of cells using focused laser beams Smicroelectrodes within microuidic platforms. However, it is possibleto generate non-uniform electric elds without the microelectrodes aswell (Shevkoplyas et al., 2005; Yang et al., 2006). Dielectrophoreticsystems are very exible as their performance can be ne-tuned by

Fig. 2. Microuidic platforms for sorting/immobilization of cells: (A) rapid immobilizatiodielectrophoretic system, white arrows show the cell chains formed between opposite micro2011b), (B) size-based separation of platelets from white blood cells using a acoustophoretic sof cells with respect to acoustic waves (Dykes et al., 2011), (C) Isolation of rare circulating tet al., 2007) (For interpretation of the references to colour in this gure legend, the reader is r

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008changing the frequency of the applied electric eld aswell as the electri-cal conductivity of the medium.

Dielectrophoresis has been used for isolation of different cells basedon their cytoplasmic properties, including malaria-infected erythro-cytes from blood (Gascoyne et al., 2002) and CTCs from blood samples(Gascoyne et al., 2009). Live cells can thus be separated withoutthe need for labelling or modication, allowing each component to beinvestigated further once sorting is complete.

Dielectrophoresis also enables the immobilization of live cells in in-timate contact of each other to investigate the cellular response againstdifferent chemical stimuli. For example, Khoshmanesh et al. investigat-ed the cycloheximide-induced apoptosis of U937 human leukaemiacells using an open-top dielectrophoretic platform (Khoshmaneshet al., 2011b). The electro-thermal vortices generated within thePDMS (polydimethylsiloxane) block enabled the rapid immobilizationof cells and efcient mixing of the specic drug with the buffer bathingthe cells (Fig. 2A). The patterned cells were later interfaced with ESEMto analyse their morphological properties (Khoshmanesh et al., 2011a).

3.1.3. MagnetophoresisThe induced motion of magnetic particles under magnetic elds has

been used for separating cells based on their dimensions or magneticsusceptibility (Gossett et al., 2010). Magnetic elds can be readilycreated by incorporation of permanent or electro-magnetic coils withmicrouidic platforms (Gossett et al., 2010).

uorescence Active Labelled Baret et al. (2009);Wlodkowic et al. (2011)

refractive index Active Label-free Perroud et al. (2008)The target cells should be immune-magnetically labelled in order tobe manipulated by magnetophoresis. This is achieved by employing theinteraction of antibody-coated paramagnetic micro/nano particleswhich interact with specic antigens on the surface of target cells.

n and drug-induced death analysis of U937 human leukaemia cells using a ow-freeelectrodes while red arrows show dying U937 cells stained with PI (Khoshmanesh et al.,ystem, (abbreviations: WBC: white blood cell, PLT: platelet), the inset shows the positionumour cells from perpherial blood of cancer patients using a mechanical lter (Nagratheferred to the web version of this article.)


5S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxThis 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) andCD34+ cells from fresh leukocytes (Jing et al., 2007). Magnetophoreticseparation based on nanoparticle uptake has also been employed toseparate macrophages from monocytes based on their endocytosiscapabilities (Robert et al., 2011). Furthermore, the intrinsic magneticproperties of hemoglobins in erythrocytes have been exploited to achievethe label-free separation of these cells. Specically, deoxygenatedhaemoglobin is paramagnetic due to the presence of unpaired electrons,while it becomes diamagnetic in its oxygenated form. It is this distinctionthat allows the separation of red blood cells from leukocytes withoutlabelling them (Melville et al., 1975; Zborowski et al., 2003).

3.1.4. AcoustophoresisThe manipulation of dispersed particles by standing acoustic waves

is another separation method which has been used to separate cellsbased on their size, density or compressibility (Lenshof and Laurell,2011). This method relies on the creation of standing pressure wavesin a microchannel. These acoustic waves can be readily generatedthrough the use of microfabricated piezoelectric transducers within amicrouidic system (Lenshof and Laurell, 2011).

Acoustophoresis has been extensively applied to separate cells basedon their dimensions. One example is separation of plasma from thewhole blood, in which the blood cells are sequentially focused alongthe channel centreline by an ultrasonic acoustic wave while the plasmawaswithdrawn from the side outlets (Lenshof et al., 2009). The collectedplasma was later linked to an antibody microarray chip for prostate spe-cic antigen detection. A similar concept has been adapted to separateplatelets from peripheral blood progenitor cells (Dykes et al., 2011)(Fig. 2B). Another study enriched viable MCF-7 breast tumour cellsfrom a heterogeneous mixture of apoptotic cell, based on the fact thatthe apoptotic cells are smaller than viable ones (Yang and Soh, 2012).Acoustophoresis is currently at the state of transition to clinical laborato-ries and industries. It is relatively insensitive to media properties such aspH and salt concentration, making it very compatible with a broad rangeof biological assays (Lenshof and Laurell, 2011).

3.1.5. Surface antigenantibody afnityReceptor targeting at the cell membrane is another approach for cell

separation that works by coating themicrochannel surfacewith specicantibodies to capture target cells (Grow et al., 2003; Ruan et al., 2002).One example is a microchip consisting of an array of microposts func-tionalized with antibody (to anti-epithelial cell adhesion molecules) toisolate 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 functional-ized hydrogel for the isolation of CD34+ and Flk1+ cells (endothelialprogenitor cells) from untreated whole human blood by depletingCD34+/Flk1 hematopoietic stem cells (Hatch et al., 2012). Alterna-tively, Vickers et al. developed a microuidic platform with antibody-conjugated surfaces to separate two phenotypically similar cells usinga single antibody (Vickers et al., 2012). The separationwasbased on var-iations of the binding afnity of cells as a function of shear stress, whichwasmodulated by changing the ow rate of themedium. The effective-ness of this method was demonstrated by separating two CD31+ celltypes: human umbilical vein endothelial cells; and human microvascu-lar endothelial cells (Vickers et al., 2012). The capturing efciency of theantibody-functionalized microchips can be enhanced by directing thetarget cells towards the antibody coated regions via secondary forcessuch as dielectrophoresis (Perroud et al., 2008).Moreover, the combina-tion of antibodyantigen afnity with electrical impedance sensing hasbeen 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).

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.0083.1.6. Fluorescent-activated cell sorting (FACS)Labelling cells uorescently and then triggering a sorting mecha-

nism depending on the measurement outcome is commonly used forcounting, sorting and characterization of owing cells (Wlodkowicet al., 2011). A variety of microuidic based FACS systems have beendeveloped for sorting cells. For example, Wolff et al. developed one ofthe rst FACS systems and applied it to sort uorescent latex beadsfrom chicken red blood cells (Wolff et al., 2003). In this system, thesample ow was hydrodynamically focused along the centre of thechip, and the uorescent signal of moving particles was detected usinga photomultiplier tube, which was used to activate a ow switchingvalve to divert non-uorescent cells towards the waste channel. Thedevice achieved a sample throughput of 12,000 cells per second. In an-other work, a FACS was used to sort cells according to their enzymaticactivity (Baret et al., 2009). In this system, cells were encapsulatedwithin droplets of a biocompatible emulsion, the droplets were storedto enable their uorogenic substrate to be turned-over by the cellularenzymes and subsequently sorted by dielectrophoresis according totheir uorescence intensity. The cells were then recovered from thesorted droplets. The device achieved a sample throughput of 300 drop-lets per second.

One of the shortcomings of FACS platforms can be their longswitching time, which is orders of magnitude longer than that ofconventional electrostatic-droplet-based cell sorters (Wu et al., 2012).To address this issue, Wu et al. developed a pulsed laser triggeredFACS, to induce a cavitation bubble in the laser-pulsed channel (Wuet al., 2012). The pulsed andmain channels were parallel and connectedvia a narrow nozzle. The bubble expansion created a high-speed liquidjetwithin the nozzle, which could deect the sample of interest towardsthe collection channel in 30 sec.

3.2. Cell analysis

3.2.1. Cell migrationCell migration is a critical phenomenon in immune recognition and

response, and is the key element for processes such as wound healingand migration towards the site of infection or inammation. Migrationinitiates with a cell's response to external signals; a change in the cell'ssymmetry ensues, with the cell's front facing towards the direction ofmovement. This polarization is central in cellular migration and beginswith the cellular response to a chemical gradient (i.e., chemotaxis) orto a direct current electric eld (i.e., electrotaxis).

3.2.1.1. Chemotaxis. Boydon chamber/Transwell assay is commonly usedfor studying the migration of cells toward different chemokine concen-trations. The system consists of two compartments that are separatedby a microporous lter. The relevant chemoattractant solution is placedin the lower chamber and leukocytes or endothelial cells are grown inthe upper chamber to replicate the in vivo condition. One importantlimitation of this system is the inability to maintain the chemokine gra-dient since within few hours the chemokine will homogenously diffuseto the upper chamber and the cells no longermigrate towards the pores.Another limitation is that it is not possible to study the effect of shear onmigrating cells (Toetsch et al., 2009).

Microuidic platforms have advanced the traditional chemotaxis as-says by providing a precise and stable gradient of chemicals as well asincorporating uid shear stress into the assay. They have been appliedto quantitatively and qualitatively study the cell migration usingchemo-attractants gradients, which can be divided into two groups:ow-based; or ow-free devices. Flow-based devices have been appliedto study the migration of immune cells such as neutrophils or lympho-cytes (Kim and Haynes, 2012). For instance, by applying stable and dy-namic gradients of chemo attractants to human neutrophils, it has beenshown that the cells can sense, analyse and prioritize multiple signalsduring their chemotaxis (Kim and Haynes, 2012). Flow-free devices

minimize the effect of ow-induced shear stress on cell migration,


samples prior to applying the samples into the device enabled theselective capturing, stimulation and immunophenotyping of desired

6 S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxthus enabling the study of various readouts such as cell signalling underdened chemical gradient conditions (Abhyankar et al., 2006). Oneexample is the application of a membrane-based system to generate achemical gradient across a microuidic channel for studying neutrophilschemotaxis (Abhyankar et al., 2006). Another chip device has beendesigned to apply a diffusion-driven gradient between twoparallel chan-nels connected via an array of straight andmaze-like narrow channels toanalyse neutrophils chemotaxis (Ambravaneswaran et al., 2010).

3.2.1.2. Electrotaxis. It refers to cellmigration directed by aphysiologically-relevant direct current electric eld. The electric eld triggers cell signal-ling to guide cell movement, but not necessarily exerts a force on cells(Li and Lin, 2011). Electrophoresis on the other hand can exert a forceon any charged particle and in the case of cells is associated with cell'selectrophoretic mobility. In conventional electrotaxis assays, the cell cul-ture chamber is lled with electrolytes, and the electric eld is appliedacross the chamber via agar salt bridges. Despite the simplicity ofthis assay, the electric eld cannot be accurately controlled and thethroughput of the system is low (Li and Lin, 2011).

Microuidic platforms offer unique features for studying immunecell electrotaxis as they enable the creation of uniform and controllableelectric elds within microuidic channels. Furthermore, real-time andquantitative analysis of electrotaxis at a single cell level can be achieved.For example, by imposing a controlled electric eld along a microuidicchannel, it has been shown that anti-CD3/CD28 activated lymphocytescan migrate towards the cathode in the system (Li and Kolega, 2002).It has been demonstrated that electrical stimulation can activate signal-ling pathways in T-lymphocytes similar to those induced by chemotaxisstimulation, concomitantly causing the cells to migrate towards thecathode (Lin et al., 2008).

Microuidic systems also have the potential to create simultaneouslyboth chemical gradients and electrical elds, to achieve deeper insightinto the migration of immune cells in electrochemical environments.For example, studying the migration of T-cells under single and co-existing gradients of chemokine and electric eld indicated that thecombination of chemotaxis and electrotaxis can accelerate themigrationof T-cells towards the cathode (Li et al., 2012).

3.2.2. Flow cytometryA variety of innovative ow cytometry (FCM) systems have been

reported for analysis of moving cells. One example is a FCM systemto measure relative concentrations of uorescently labelled subpopula-tions of lymphocytes and other leukocytes within blood samples(Frankowski et al., 2011). The device utilized integrated optical bresfor laser excitation and detection of uorescent signals. In anotherstudy, Skommer et al. pioneered the use of a FCM system to measurecellular DNA content in both live and xed tumour cells, and also totrack the drug-induced activation of caspases and the dissipation ofmitochondrial inner membrane potential (m loss) with respect tocell cycle stage of tumour cells (Skommer et al., 2013) (Fig. 3AB). De-spite the essentially simple design, the system incorporated a dedicatedhardware interface comprising of a microcontroller-driven syringepump, spatially separated laser excitation sources, photodiodes, andphotomultiplier tubes to achieve an elegant separation. Further integra-tion and miniaturization is certainly possible.

Alternatively, Holmes et al. (Holmes et al., 2009) developed amicrouidic impedance cytometer for the label-free differentiation ofleukocyte sub-populations (T-lymphocytes, neutrophils and mono-cytes). Themicrouidic chip took advantage of microelectrodes tomea-sure the impedance of passing single cells at two frequencies of 503 and1707 kHz. This enabled discrimination of leukocytes according to bothsize and membrane capacitance.

3.2.3. ImmunophenotypingPhenotyping of immune cells through measurement of their func-tional status during different stages of the cell cycle is among the gold

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008immune cells subpopulations (Chen et al., 2013b).

3.2.4. Single molecule analysisSince its rst demonstration in 1961(Rotman, 1961), single mole-

cule detection techniques have allowed us to visualize the dynamic be-haviour of biomolecules. Specically, it has been possible to accuratelyexamine intra- and inter-molecular interactions and their reactionkinetics inside living cells (Dustin and Groves, 2012). Proteins are thekey biomolecules of interest in this context, harbouring specic func-tions in gene expression, membrane transport and signal transduction.One of the new objectives in cell biology is to quantitatively analysethe molecular protein networks within cells, obtaining the dynamicand kinetic parameters of proteinprotein interactions, protein translo-cations and outcomes of enzymatic reactions (Rotman, 1961).

The unique feature of microuidics is that it accelerates diffusion-dominated reactions and consequently increases signal-to-noise ratiowhen considering visualization and imaging of biomolecular processes(Liu et al., 2011). Hence,microuidic chambers represent excellent plat-forms for single molecule analysis. TIRF is widely employed for singlemolecule analysis of membrane proteins, using a laser-based excitationdepth of uorescent molecules of a few hundreds of nanometres, thuseffectively improving signal-to-noise ratios. This feature makes it anattractive detection scheme when applied to microuidic platforms.An important application of such a device would be in the study of im-munological synapse formation, utilizing for instance, the articialmodel of supported planar lipid bilayers (Kam and Boxer, 2000). Insuch a system, it is possible to visualize and quantify the interaction ofimmuno-receptors and other surface molecules on immune cells, tospecic antigens and ligands incorporated into lipid planar bilayers,thus mimicking what would be an actual interaction with an antigenpresenting cell (Brian and McConnell, 1984). The Bioptechs FCS2microuidic chamber, among others, has been widely used to studystandard methods of determining immune health status. The immunesystem comprises a heterogeneous population of cells and the leveland proportion of different cytokines and signalling markers changeswith different conditions, as in the case of infections and malignancies(Maecker et al., 2012). Different approaches currently exist for assess-ment of immune cell status. Currently, ELISA/ELISpot methods are thegold standard methods for quantifying cellular cytokine production(Chen et al., 2013a). However, these methods are labour intensive andrequire multiple steps of washing and staining. Further, ELISpot is notable to quantify the amount of cytokine production. Another approachis to measure the intracellular cytokine production using ow cytome-try (Chen et al., 2013a). This method has so far concurrent analysis ofup tove cytokines,which in some casesmay limit its future usefulness.The requirements for this method might also involve using largeramounts of the sample than what was originally desired, and couldalso damage the sample in the process, making further analysis difcultor impossible.

To address the limitations associated with conventional immuno-phenotypingmethods different microuidic platforms have been intro-duced. For example, rapid detection of cell-secreted biomarker proteinshas been demonstrated by utilizing a PDMS microltration membranefor isolation and enrichment of entering monocytes (Huang et al.,2012). The seeded cells were stimulated with an endotoxin to secretetumour necrosis factor-. The cell-secreted cytokine diffused intothe immunoassay chamber to be detected and quantied in a bead-based chemiluminescence assay. The system achieved the sensitiveimmunophenotyping of cells with 20-fold fewer cells than conventionalcell stimulation assays. Moreover, the total assay time was 7 timesshorter than that of an enzyme-linked immunosorbent assay (ELISA).Mixing of antibody-conjugated polystyrene microbeads with bloodT-cell receptor clustering and synapse formation of T-cells placed on


7S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxglass supported planar bilayers harbouring a variety of differentantigens and adhesion molecules (Fig. 3CD) (Varma et al., 2006).

Single-molecule measurements at the immunological synapseusing high resolution microscopy can be obtained and combined withmicrouidic platforms to provide fundamental insight into processessuch as the kinetics of receptorligand interactions (Dustin andGroves, 2012; Huppa et al., 2010). Obtaining such information isnot possible from ensemble-averaged signals obtained from thecells. Furthermore, a supported lipid bilayer system integrated into amicrouidic platform has been utilized to underpin the role of the cyto-skeleton in coordinating receptor signalling in both T-cells and B-cells(Mattila et al., 2013; Treanor et al., 2011). Indeed, recent advances inmicrouidics and its unique characteristics to form multiple bilayersin combination with super resolution imaging enable addressing keyquestions in the eld of immunology.

3.3. Cell lysis

Mammalian cells are enveloped by a lipid bilayer, which forms a bar-rier between the cytoplasm and the extracellular environment. Cell lysisis an important step during an immunoassay, as the efciency withwhich it occurs directly affects subsequent treatments, and ultimately,

Fig. 3.Microuidic platforms for analysis/lysis of cells: (A) Analysis of programmed tumour ce(PMMA), and comprised three modules: the input port with integrated cell sample and sheathe output port with collection chambers (Skommer et al., 2013), (B) The optical hardware intexcitation 473 nm and 640 nm lasers, and photomultiplier tubes (PMTs) for uorescent signadesign of FCS2 Bioptechs chamber (Bioptechs, USA) used for studying immunological synapsesmicroclusters. T-cells are injected into an imaging chamber containing supported planar bilayerbilayer containing major histocompatibility complex (MHC) and CD45 molecules at (iiii) 30 slysis of EL-4 cells along a microuidic channel. Cells cross over from the carrier buffer to the lyshows a uorescent image of the dilution of the cell carrier buffer via the bifurcation channels

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008the ability to functionally analyse intracellular components of a sampleof interest. Chemical cell lysis has been themethod of choice for extrac-tion of cytoplasmic contents (Mun et al., 2010; Sasuga et al., 2008). De-pending on the complexity of the sample, different treatments need tobe performed to separate cells from debris and interfering substances.This may include ltration, centrifugation, treatment with appropriatelysis buffers, dilution and if possible enrichment of targeted molecules.These steps are labour intensive and time consuming. Moreover, thevolume of lysis buffer should be minimized to reduce the dilution oftarget cellular analytes. Appropriate selection of lysis buffer is essentialto avoid the denaturation of target cellular analytes or their conjugatedlabels. In order to reduce the complexity of macro scale cell lysismethods, these methods have been implemented into micro scale plat-forms (Kim et al., 2009a).

When considering the application of cell lysis in microuidic cham-bers, Mun et al. have employed an effective method of continuous celllysis (Mun et al., 2010). In this work, the cells and lysis buffer wereinjected into amicrochannel through separate inlets, and the cell carriersolution was consistently removed by means of an array of narrow bi-furcation 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

ll death using a -ow cytometry chip. The chip was made of Poly(methyl methacrylate)th uid reservoirs, a 75 m 50 m channel for 2D hydrodynamic focusing of cells, anderface for (A) capable of four-colour detection using a combination of spatially separatedl collection in area, height and width parameters (Skommer et al., 2013), (C) Schematic, (D) T-cell receptor (TCR) clustering (red) and exclusion of CD45 molecule (green) froms and TCRs are visualized by TIRF. Figures show synapse formation between TCR and planarand (ivvi) 30 min post contact formation (Varma et al., 2006). (E) Continuous chemicalsis buffer as the carrier buffer is gradually washed via the bifurcation channels. The inset(Mun et al., 2010).


demonstrated by incorporating an array of picoliter microwells forcapturing and chemical treatment of cells (Sasuga et al., 2008). Otherdesigns have succeeded using isolated picoliter reaction volumeswithinmicrofabricated 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 byencapsulating single cells into 500 pl droplets containing lysis buffer(Kim et al., 2009a).

Other approaches have been used to achieve physical cell lysis inmicrouidics. For instance mechanical lysis can be applied by usingsharp nanoknives fabricated within the microchannels (Di Carloet al., 2003). Electrical lysis by exposing cells to DC or AC electric elds(McClain et al., 2003);while thermal lysis can occurwhen incorporatingmicroheaters into the microuidic channels; (Marshall et al., 2012)Lastly, optical lysis can be achieved by exposing the cell to a concentratedbeam 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 tar-get molecules such as nucleic acids and proteins. Different methodshave been developed to isolate DNA or RNA. In general, these methodsinvolve disruption and lysis of cells followed by removal of contami-nants such as protein and recovery of DNA or RNA. Removal of proteinsis usually achieved by digestionwith proteinase K, organic extraction orbinding of DNA to a solid phase support. Consequently, the DNA or RNAis usually recovered by ethanol or isopropanol precipitation (Fan et al.,2012). These methods are usually labour intensive, time consumingand require multiple steps to collect DNA or RNA from biologicalsamples such as blood or tissue. To simplify and reduce these sample

has been introduced by different companies. However, these methodsstill require sample handling, centrifugation steps and large volume ofsample (Kim et al., 2009a).

To reduce the challenges associated with macro scale techniquesSPE-based microuidic systems have been developed that allow forextraction of nucleic acids using nano/micro litre size samples in anautomatic manner (Wen et al., 2008). The most common solid matricesimplemented in microuidic systems for DNA extraction are silicamicropillars (Cady et al., 2003), silica beads (Chung et al., 2004), and sil-ica sol-gels (a form of colloidal solution that forms a coherent solid gelupon acid or base catalysis) (Wolfe et al., 2002). Magnetic silica beadssuspended in lysis buffer have also been applied for DNA extraction.One advantage of using magnetic beads in the chamber is the abilityto perform rapid and controllable loading, mixing and patterning ofthe beads to achieve a dynamic solid matrix (Azimi et al., 2011). Anadditional method for nucleic acid extraction in microuidic chambershas been the use of photo-polymerized monoliths, as they offer severaladvantages including extensive surface area, tuneable pore size,and easy patterning. An elegant example of this applicationwas demon-strated by Wen et al. who developed a dual stage process for DNApurication from whole blood by using a C18 silica bead column forcapturing proteins together with a monolithic column for capturing ofDNA (Fig. 4A) (Wen et al., 2007).

While the increased surface-to-volume ratio in microuidic plat-forms enhances the capturing efciency it creates the further, seriousproblem of non-specic binding of nucleic acids or proteins. Therefore,it is necessary to block the non-specic binding sites by treating the sur-face of the device with blocking agents. A variety of blocking agents in-cluding protein blockers (e.g. bovine serum albumin (BSA), non-fatmilk, and chitosan) and polymeric blockers (e.g. polyethylene glycol(PEG), polyethyleneimine (PEI), polyvinyl alcohol (PVA) andpolyacrylicacid (PAA)) have been reported in the literature, as comprehensively

s: (tegres. Rer frker

8 S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxpreparation challenges the column-based solid phase extraction (SPE)

Fig. 4.Microuidic platforms for purication, amplication and separation of nucleic acidcapturing proteins and a monolithic column for trapping DNA (Wen et al., 2007), (B) An inlabelling and immuno-chromatographic based detection of nucleic acids from the cell lysatvalves V1V4 control theowbetween different components (Chen et al., 2010a), (C) Anoththewhole blood. Chip is composedof fourmodules for SPE extraction, PCR amplication,ma

channels and elastomeric membrane valves (Easley et al., 2006).

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008reviewed by Kim and Herr (2013).

A) Dual-stage purication of DNA from whole blood, using a C18 silica bead column forated microchip for lysis of cells and viruses, followed by SPE extraction, PCR amplication,eservoirs P1P6 contain lysis, inhibitor removal, wash, elution, and labelling buffers whileully integratedmicrochip designed for DNA-based screening for infectious pathogens frominjection, and electrophoretic separation of DNA fragments; all connected via a network ofand opportunities, Biotechnol Adv (2013), http://dx.doi.org/10.1016/

9S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxThe extraction of RNA ismore challenging than that of DNAdue to itssusceptibility to degradation by Ribonucleases (RNases) (Rogacs et al.,2012). To overcome this issue, Sattereld et al. developed a photo-polymerized monolith for on-chip purication of eukaryotic mRNAfrom total RNA (Sattereld et al., 2007). The monolith was functional-izedwith polythymine deoxyribonucleotides (oligo-dTs)which interactwith the poly-A tail of mRNA, thus capturing the nucleic acid. In anotherstudy, Irimia demonstrated an integrated system for RNA isolation fromhuman monocyte cells (Irimia et al., 2009). Following cell lysis, appro-priate agents were added to inactivate RNases and degrade cellularproteins. The nucleic acids were subsequently captured on a silicabead column while the contaminant DNA was removed by enzymaticdigestion before releasing the captured RNA. Other work has shownthe utilization of polymer capture matrices in the purication ofhuman immunodeciency virus (HIV) RNA from human sera (Rootet al., 2011). Serum was electrophoresed through the polymer matrixin the chip, which had covalently bound oligonucleotides, thus selec-tively trapping target nucleic acids. The chip was then heated abovethe melting temperature of the captured oligonucleotides to releasethe target strands.

4.2. Amplication of nucleic acids

An important step in nucleic acid analysis is the amplication of cap-tured nucleic acids using polymerase chain reaction (PCR). This methodrelies on repeated heating and cooling of nucleic acid samples throughcycles of denaturation, annealing and extension of target sequences.Conventional PCR thermocyclers consist of a thermal block with slotarrays for PCR tubes. The temperature of the entire block must bechanged in order to achieve temperature changes in the samples. Thisprolongs processing time, due to the largemass of the block. In addition,one important challenge for the conventional PCR methods is the iden-tication of small changes in gene expression that are usually associatedwith different disease states. Quantitative PCR currently is themost sen-sitive approach that can identify 1.5-fold changes (Whale et al., 2012).

Microuidic PCR platforms dramatically reduce the amount of sam-ples, reagents and analysis time, while increasing the precision of PCRexperiments (Baker, 2010). On-chip PCR has been demonstrated bypassing samples through serpentine channels, which are heated attheir 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 nucleicacid sequence (Crews et al., 2008). Such continuous ow PCR systemsconsume far less reagents and require shorter reaction times comparedto their conventional macroscopic counterparts (Zhang and Ozdemir,2009). However, the large surface-to-volume ratio of such systemsleads to the adhesion of chemicals onto the surface ofmicrouidic chan-nels, which consequently leads to cross-contamination and inhibition ofPCR reactions (Zhang and Ozdemir, 2009). These issues can be over-come by utilizing droplet-based PCR systems, in which the PCR reactiontakes place in discrete droplets dispersed within an immiscible carrieruid such as oil. The droplets can also be driven by electrowettingmechanisms, where the wettability of the surface is modied via anelectric eld (Heyries et al., 2011; Hua et al., 2010). The rapid advancesin microfabrication technologies has enabled the creation of highlyintegrated and fully automated PCR microuidic chips equipped withmicropumps, microvalves, and micromixers (Toetsch et al., 2009).Integration of PCR microuidic components with pre- and post-PCRprocessing units, as shown in Fig. 1, can further push the boundariesof PCR-based diagnostic assays to make us one step closer to point-of-care 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

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008gel matrix to separate the samples based on their size. Despite the pop-ularity of this technique, several manual steps are required includingstaining and separation of nucleic acids followed by visualization andquantication of separated bands, which elongate the process.

On-chip capillary electrophoresis (CE) has been demonstratedby etching glass or quartz substrates to form capillaries. PDMS channelsare generally avoided for CE due to their permeability, which can causecross-contamination of nucleic acids. Integrated PCR-CE microuidicsystems capable of performingnucleic acid amplication and separationhave been demonstrated by several groups (Chen et al., 2010a; Easleyet al., 2006; Huang et al., 2006) (Fig. 4BC).

Isotachophoresis is another variant of electrophoresis, which hasbeen used for purication, stacking and separation of target nucleicacids in microuidic systems. In isotachophoresis, the target sample isintroduced between the trailing and leading electrolytes whose electro-phoretic mobilities are respectively lower and higher than that of thesample. Upon application of an electric eld, the target samples areconcentrated between the trailing and leading electrolytes. This avoidsthe dispersion and diffusion of the concentrated sample, and signi-cantly reduces the limit of detection, which cannot be achieved by CEmicrodevices (Persat et al., 2009). Isotachophoresis has been usedextensively in microuidic systems, namely for the extraction and puri-cation of genomic DNA frommalaria infected red blood cells (Marshallet al., 2011), extraction and enrichment of ribosomal RNA from wholeblood infected with bacteria (Rogacs et al., 2012), and detection ofmicroRNA using photo-polymerized functionalized hydrogels (Garcia-Schwarz and Santiago, 2012).

5. On-chip manipulation of proteins

The density of intracellular proteins is much higher than that of DNAor RNA. Despite this fact, measuring the expression level of the concen-tration of a protein of interest, especially in small cell samples is chal-lenging due to the limitations of protein amplication. Heterogeneoussubsets of immune cells are present during immune responses to infec-tion. These cells harbour both phenotypic and functional differences.Thus, capturing a broad functional spectrum of given heterogeneouscell populations remains challenging, and requires the analysis of alarge 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 interac-tions have beenwidely used for protein detection. A variety of immuno-assays are implemented in clinical laboratories. This includes ELISA todetect and quantify the target antigen in a sample, immuno-blottingto detect and approximate the size of a protein, ow cytometry to mea-sure the expression of cell surface or intracellular proteins, and immu-nohistochemistry to detect and localize antigens in tissue samples. Themain disadvantage of these methods is that they are labour intensiveand time consuming. Additionally, they all rely on the availability ofcommercial antibody conjugates to bind to target proteins and oftenrequire 10 to 100 l of the sample (Hauss and Mller, 2007).

Microuidic platforms enable the transportation, mixing, separationand detection of proteins in a 11000 picoliter range of sample volumes(Freire and Wheeler, 2006). For example, a microuidic system wasdeveloped to detect the cytokine tumour necrosis factor (TNF-)(Cesaro-Tadic et al., 2004). In this work, a PDMS cover precoated withcapture antibodies was placed onto an array of parallel microchannelsto capture the moving analyte molecules. The PDMS cover wasthen peeled off and orthogonally placed onto an array of parallelmicrochannels where uorescently labelled detection antibodies weremoving. This led to the formation of a mosaic of uorescent signals

on the surface of PDMS. Moreover, researchers have succeeded in


developing microuidic-based ELISA chips for the quick detection andquantication of target proteins within a sample. One example is thedisc-based microuidic ELISA system for the detection of Hepatitis Bvirus (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. Thesystem enabled the automated separation of plasma from the wholeblood, incubation of plasma with target specic antigen or antibodyconjugated polystyrene beads, several washing steps, mixing andreacting with chemiluminescent enzyme substrates, and absorbancedetection at 450 and 630 nm within 30 min (Lee et al., 2009) (Fig. 5A).

5.2. Purication of proteins

Antibody capture of target proteins is an applicable method forpurifying them. Conventional methods of purifying proteins includechromatography, which separates a large pool of proteins in to a smallpool, based on size, charge, hydrophobicity and afnity. However, con-ventional chromatography is labour intensive, is not suitable for pro-teins in the microgram scale. Microuidic platforms have enormouscapabilities with regard to the purication of small amount of proteinsfrom samples. The capturing efciency of microuidic systems can beimproved in several ways. For example, Sandison et al. employed aPDMS column lled with an array of 50 m diameter pillars, whichwere functionalized with covalently bound antibodies to purify recom-binant afnity-tagged proteins from a bacterial lysate (Sandison et al.,2010) (Fig. 5B). An alternative method to increase the capturing ef-ciency of proteins is to ll the microchannels with antibody conjugatedmicrobeads. For instance, a capillary column packed with antibody-coated silica beads has been applied to capture C-reactive protein

(CRP) (Peoples and Karnes, 2008). A buffer of uorescently labelledantibody was introduced into the chamber to bind to the capturedCRP. This was followed by the addition of an acidic elution buffer to dis-sociate the antibodyCRP complexes and measure the concentration ofthe labelled antibody. In another work, RNA aptamer conjugated beadswere applied to purify carcinoembryonic antigen, a cancermarker, fromhuman serum (Koh et al., 2012). The beads were packedwithin a PDMSchamber lled with an array of micropillars. The chamber was thenheated to 85 C for 5 min to unfold the aptamers, enabling the captureof passing antigens. The chamber was washed to remove the unboundantigens, and its temperature was increased to 85 C for 35 s to dena-ture and release the puried antigens.

5.3. Separation and analysis of proteins

The common method to analyse the proteins is gel electrophoresis(GE), which enables the separation of proteins in gel media based ontheir electrophoreticmobility aswell asmolecularweight (size). As pro-teins electro-migratewithin a gel, the smaller proteinsmove faster thanthe larger ones, and therefore the gel acts as a size-selective molecularsieve. Photo-polymerized sieving gels such as polyacrylamide (PA) arequite 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 andtetanus toxin C-fragment levels in serum samples (Herr et al., 2005).

Immuno-blotting is a powerful technique to detect and analysespecic proteins. This method uses gel electrophoresis to separate theproteins based on their size. The proteins are then transferred to themembrane where they are then stained with antibodies specic forthe targeted protein. On-chip immuno-blotting of proteins has been

ly incilith anne Psateface an

10 S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxFig. 5.Microuidic platforms for detection, purication and separation of proteins: (A) A fulfrom blood samples (Lee et al., 2009). The chip operation relies on spining of the disc to fasystem enables the separation of plasma from the whole blood, incubation of plasma witsubstrates, and the absorbance detection at 450 and 630 nmwithin 30 min, (B) a serpentibound antibodies for purication of recombinant afnity-tagged proteins from a bacterial lynoblotting of proteins realized by photopatterning of polyacrylamide gels on glass. The chipas indicated by blue and yellowbands. The direction of the electric current is shownby i (H

is referred to the web version of this article.)

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008tegrated lab-on-a-disc ELISA system for detection of Hepatitis B virus antigen and antibodyate the transporation of liquid and laser controllable microvalves to control the ow. Thetibody conjugated beads, several washing steps, mixing and reacting with TMB enzymeDMS column lled with an array of 50 m diameter pillars functionalized with covalently(Sandison et al., 2010), (C) the layout and operation of a microchip for automated immu-

ilitates the rapid loading, stacking, PAGE separation, transfer and in gel blotting of proteins,d Herr, 2010) (For interpretation of the references to colour in this gure legend, the readerand opportunities, Biotechnol Adv (2013), http://dx.doi.org/10.1016/

11S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxdemonstrated by photo-patterning several in-line PA gel elements ontospecic regions of a glass slide to enable the loading and stacking ofsamples via the large pore-size loading gel, followed by electrophoreticseparation of proteins via the small pore-size separation gel, and subse-quent identication of separated proteins via antibody-functionalizedblotting gels patterned in series (He and Herr, 2009). The performanceof the above system was further improved by patterning the blottinggel in parallel with the PAGE separation region in such a way that theseparated proteins could be laterally transferred onto the blottingregion. This enabled running several PAGE separations to optimize theexperimental conditions, prior to transferring to the blotting region(He and Herr, 2010) (Fig. 5C).

The efciency of protein separation can be improved byimplementing multi-dimensional separation strategies. One exampleis the two-dimensional (2D) separation of proteins by integrating iso-electric focusing (IEF) with PAGE on a polycarbonate microuidic chip(Li et al., 2004). In this work, the designed system consisted of a longhorizontal channel and an array of orthogonal parallel channels. TheIEF separation was achieved by establishing a pH gradient along thehorizontal channel to discriminate the proteins based on their charge.Alternatively, the PAGE separation was achieved by electrophoresis ofproteins along the parallel channels to discriminate them based ontheir molecular weight. A similar concept has been used by Emrichet al. (Emrich et al., 2007), in which the horizontal and orthogonalseparation channels were connected via smaller channels to minimizethe diffusion of analytes at the intersection of channels. The 2D separa-tion of proteins was also achieved by integrating isotachophoresiswith CE on a poly(methyl methacrylate) chip (Olvecka et al., 2004).Isotachophoresis created a sharp band of concentrated proteinselectro-migrating along the channel while CE destacked and separatedthe 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 massspectrometer to obtain the mass spectrum of their constituent peptidefragments. For example, Mellors et al. reported a glass microchip capa-ble 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 serveas the electrospray source. This enabled the direct coupling of separatedsamples into a mass spectrometer without the use of external pressureor vacuum sources or the addition of capillary spray tips.

Alternatively, a PDMS microuidic system was developed for cou-pling CE and matrix assisted laser desorption ionization-mass spec-trometry (MALDI-MS) (Luo et al., 2009). The samples were separatedalong a CE channel and compartmentalized by means of fractionationvalves before being transported into the open reservoirs through theuse of a monolithic peristaltic pump. The analyte solutions were thenmixed with a matrix solution and deposited onto a MALDI target platefor MALDI-MS. In another work, CE separation was coupled withMALDI-MS by means of electrowetting mechanism (Gorbatsova et al.,2012).

6. Outlook

The continuing developments in microuidic technology enable usto improve our understanding of biological processes, and in particularfor analytical studies of the immune system. Microuidic platformsoffer myriad advantages including the potential for fast analysis, theincorporation of multiple processing steps for different applications,and importantly, the ability to reduce sample volume and reagentsusage. Furthermore, microuidic systems allow the undertakingof experiments under accurately controlled conditions, limiting theuser interaction and in some instances, improving the accuracy of the

obtained results.

Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008The utility of microuidic platforms for a variety of cell-, nucleicacid- and protein-based experiments has been demonstrated in severalstudies, as summarized in Sections 3 to 5 of this review. Despite theunique advantages offered by microuidic platforms, signicant chal-lenges lie ahead for the successful and full integration of such systemsin routine clinical and research laboratory procedures to address differ-ent aspects of immunological problems. Someof these challenges can besummarized as below:

First, most microuidic platforms have been operated using simpleone-step procedures (e.g. purication of nucleic acids) but no con-certed effort has been made to integrate multiple steps for analysisof clinical samples. Only a small portion of the papers in the litera-ture have demonstrated the multistep processing of the samples.Among those is the microchip developed by Chen et al. (Chenet al., 2010a) to detect the presence of bacteria and viruses in salivasamples. This microchip enabled chemical lysis of cells and viruses,followed by SPE-based extraction, PCR amplication, labelling andchromatographic-based detection of nucleic acids from the celllysates. Another example is the microchip developed by Irimiaet al. (2009) for genome wide expression analysis of samples asfew as 150 cells. The microchip facilitated the chemical lysis ofcells, followed by degrading of cellular proteins, inactivationof RNAses, SPE-based capturing of nucleic acids, enzymatic removalof contaminant DNA and releasing the captured RNA to be ampliedoff the chip.Second, inmost 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 forconducting fundamental and applied research in the eld of immu-nology still remains a challenge due to the complexity and heteroge-neity of the samples. Integration of components relies on thesuccessful implementation of microvalves. A variety of microvalveshave been reported in the literature including the pneumatic-basedones developed by Quake's group (Unger et al., 2000). However,the fabrication and operational issues surrounding these microvalveshave limited their widespread implementation. One way to resolvethese issues is to design innovate microvalves which are less com-plex. One example is the disc-based ELISA microchip developed byLee et al. (2009) to detect viral infections in blood samples. Themicrochip took advantage of laser controllablemicrovalves to controltheowwithin the different compartments of themicrochip. This en-abled the serial separation of plasma fromwhole blood, incubation ofplasmawith antibody conjugated beads followed bywashing,mixingand reaction with chemiluminescent enzyme substrates.Third, most microuidic systems rely on bulky and expensive off-chip systems such as uorescent microscopes, syringe pumps, andsignal generators. Therefore, the next challenge in the eld is to beable to miniaturize those systems to realize fully independentimmuno lab-on-a-chip platforms, which are small, transportable,and low-cost. For example, the mini-microscope developed by Kimet al. (2012) enables in situ monitoring of cells. Alternatively, inno-vative approaches can be utilized to replace the bulky off-chip com-ponents with smaller ones. For example, the microuidic-basedcytometer developed by Holmes et al. (2009), takes advantage ofimpedance measurement of passing cells to achieve a label-free dif-ferentiation of leukocytes sub-populations based on their size andmembrane capacitance. This makes the cytometer independent ofbulky optical components such as laser sources, lters, lenses and

PMTs.


12 S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxFourth,most studies demonstrate only the proof-of-concept capabil-ity of microuidic platforms for conducting biological experimentswhile there are only a few reported works in the literature, wherethe utility of microuidic chips has been demonstrated in real clini-cal procedures. Among these is themicrochip developed by Nagrathet al. (2007) to capture CTCs in the peripheral blood of patients withmetastatic lung, prostate, pancreatic, breast and colon cancer in asingle step without any processing steps. Another example is themicrochip developed by Rosenbach et al. (2011) for rapid RNA ex-traction from highly pure T lymphocytes of burn-injured patients,which was used for downstream evaluation of the gene expressionof cytokines via quantitative real-time PCR.Fifth, to ensure that microuidic platforms can be routinely used inclinical laboratories, they must be highly developed and free fromtechnical challenges that can present obstacles to users who arenot familiar with microuidic devices. Most devices are customdesigned for each application and thus may be prone to operationalissues such as leaking of microchannels, clogging of microchannelsby entrapped bubbles, and difculty in interfacing with severalinlet/outlet tubes. There exist several excellent technological solu-tions to these problems and it is suggested that standards be devel-oped to ensure that all microuidic devices benet from thesesolutions.

In conclusion, immuno lab-on-a-chip systems could be equippedwith 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 oftarget nucleic acids, (v) detection, capture, and separation of targetproteins, and (vi) interfacing target immobilized samples with off-chip imaging or spectroscopy technologies. Such systems require thedesign and implementation of modular microuidic components,which can be easily laid in series to form highly integrated multi-purpose immuno lab-on-a-chip platforms. These integrated systemswould enable the routine laboratory techniques to be performed withsmaller sample volumes, a smaller volume but larger array of consum-ables, and vastly reduced infrastructure scale and cost. A positive out-look is that in the future, more effective, robust, low-cost and portablemicro devices will be available for routine clinical procedures.

Acknowledgements

K. Khoshmanesh acknowledges the Australian Research Council(project number DE120101402), D. Wlodkowic acknowledges theAustralian Research Council and RMIT Vice Chancellor's Senior Fellow-ship, P.McIntyre acknowledges theNationalHealth andMedical ResearchCouncil (project grant APP1046860) and A. Mitchell acknowledges theAustralian Research Council for funding.

References

Abhyankar VV, Lokuta MA, Huttenlocher A, Beebe DJ. Characterization of a membrane-based gradient generator for use in cell-signaling studies. Lab Chip 2006;6:38993.

Ambravaneswaran V, Wong IY, Aranyosi AJ, Toner M, Irimia D. Directional decisionsduring neutrophil chemotaxis inside bifurcating channels. Integr Biol 2010;2:63947.

Azimi SM, Nixon G, Ahern J, BalachandranW. Amagnetic bead-based DNA extraction andpurication microuidic device. Microuid Nanouid 2011;11:15765.

Baker M. Clever PCR: more genotyping, smaller volumes. Nat Methods 2010;7:3515.Baret J-C, Miller OJ, Taly V, Ryckelynck M, El-Harrak A, Frenz L, et al. Fluorescence-

activated droplet sorting (FADS): efcient microuidic cell sorting based on enzy-matic activity. Lab Chip 2009;9:18508.

Brian AA, McConnell HM. Allogeneic stimulation of cytotoxic T cells by supported planarmembranes. Proc Natl Acad Sci U S A Biol Sci 1984;81:615963.

Cady NC, Stelick S, Batt CA. Nucleic acid purication using microfabricated silicon struc-tures. Biosens Bioelectron 2003;19:5966.

Cesaro-Tadic S, Dernick G, Juncker D, Buurman G, Kropshofer H, Michel B, et al.High-sensitivity miniaturized immunoassays for tumor necrosis factor a usingmicrouidic systems. Lab Chip 2004;4:5639.Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008Chen D, Mauk M, Qiu X, Liu C, Kim J, Ramprasad S, et al. An integrated, self-containedmicrouidic cassette for isolation, amplication, and detection of nucleic acids.Biomed Microdevices 2010a;12:70519.

Chen GD, Alberts CJ, RodriguezW, Toner M. Concentration and purication of human im-munodeciency virus type 1 virions bymicrouidic separation of superparamagneticnanoparticles. Anal Chem 2010b;82:7238.

Chen Y, Zhang B, Feng H, ShuW, Chen GY, Zhong JF. An automated microuidic device forassessment of mammalian cell genetic stability. Lab Chip 2012;12:39305.

Chen W, Huang N-T, Li X, Yu ZTF, Kurabayashi K, Fu J. Emerging microuidic toolsfor functional cellular immunophenotyping: a new potential paradigm for immunestatus characterization. Front Oncol 2013a:3.

Chen W, Huang N-T, Oh B, Lam RHW, Fan R, Cornell TT, et al. Surface-micromachinedmicroltrationmembranes for efcient isolation and functional immunophenotypingof subpopulations of immune cells. Adv Healthc Mater 2013b:96575.

Chung YC, Jan MS, Lin YC, Lin JH, Cheng WC, Fan CY. Microuidic chip for high efciencyDNA extraction. Lab Chip 2004;4:1417.

Crews N, Wittwer C, Gale B. Continuous-ow thermal gradient PCR. BiomedMicrodevices2008;10:18795.

Davis JA, Inglis DW, Morton KJ, Lawrence DA, Huang LR, Chou SY, et al. Deterministichydrodynamics: taking blood apart. Proc Natl Acad Sci U S A 2006;103:1477984.

Di Carlo D, Jeong KH, Lee LP. Reagentless mechanical cell lysis by nanoscale barbs inmicrochannels for sample preparation. Lab Chip 2003;3:28791.

Dustin ML, Groves JT. Receptor signaling clusters in the immune synapse. In: ReesDC, editor. Annual review of biophysics, vol. 41. 2012. p. 54356.

Dykes J, Lenshof A, Astrand-Grundstrom I-B, Laurell T, Scheding S. Efcient removal ofplatelets from peripheral blood progenitor cell products using a novel micro-chipbased acoustophoretic platform. PLoS ONE 2011:6.

Easley CJ, Karlinsey JM, Bienvenue JM, Legendre LA, Roper MG, Feldman SH, et al. Afully integrated microuidic genetic analysis system with sample-inanswer-outcapability. Proc Natl Acad Sci U S A 2006;103:192727.

El-Ali J, Sorger PK, Jensen KF. Cells on chips. Nature 2006;442:40311.Emrich CA, Medintz IL, Chu WK, Mathies RA. Microfabricated two-dimensional electro-

phoresis device for differential protein expression proling. Anal Chem 2007;79:73606.

Faley S, Seale K, Hughey J, Schaffer DK, VanCornpernolle S, McKinney B, et al. Microuidicplatform for real-time signaling analysis of multiple single T cells in parallel. Lab Chip2008;8:170012.

Fan A, Byrnes S, Klapperich C. Purication of DNA/RNA in a microuidic device #.T Microuidic Diagnostics; 201240311.

Frankowski M, Bock N, Kummrow A, Schaedel-Ebner S, Schmidt M, Tuchscheerer A, et al.A microow cytometer exploited for the immunological differentiation of leukocytes.Cytometry A 2011;79A:61324.

Freire SLS,Wheeler AR. Proteome-on-a-chip: mirage, or on the horizon? Lab Chip 2006;6:141523.

Garcia-Schwarz G, Santiago JG. Integration of on-chip isotachophoresis and functionalizedhydrogels for enhanced-sensitivity nucleic acid detection. Anal Chem 2012;84:63669.

Gascoyne P, Mahidol C, Ruchirawat M, Satayavivad J, Watcharasit P, Becker FF. Microsamplepreparation by dielectrophoresis: isolation of malaria. Lab Chip 2002;2:705.

Gascoyne PRC, Noshari J, Anderson TJ, Becker FF. Isolation of rare cells from cell mixturesby dielectrophoresis. Electrophoresis 2009;30:138898.

Gorbatsova J, Borissova M, Kaljurand M. Electrowetting on dielectric actuation of dropletswith capillary electrophoretic zones for MALDI mass spectrometric analysis. Electro-phoresis 2012;33:26828.

Gossett DR,WeaverWM,Mach AJ, Hur SC, Tse HTK, LeeW, et al. Label-free cell separationand sorting in microuidic systems. Anal Bioanal Chem 2010;397:324967.

Grow AE, Wood LL, Claycomb JL, Thompson PA. New biochip technology for label-freedetection of pathogens and their toxins. J Microbiol Methods 2003;53:22133.

Hatch A, Pesko DM, Murthy SK. Tag-free microuidic separation of cells against multiplemarkers. Anal Chem 2012;84:461821.

Hauss O, Mller O. The protein truncation test in mutation detection and molecular diag-nosis. In: Grandi G, editor. In vitro transcription and translation protocols. HumanaPress 2007. p. 15164.

He M, Herr AE. Microuidic polyacrylamide gel electrophoresis with in situ immunoblot-ting for native protein analysis. Anal Chem 2009;81:817784.

He M, Herr AE. Automated microuidic protein immunoblotting. Nat Protoc 2010;5:184456.

Herr AE, Throckmorton DJ, Davenport AA, Singh AK. On-chip native gel electrophoresis-based immunoassays for tetanus antibody and toxin. Anal Chem 2005;77:58590.

Heyries KA, Tropini C, Vaninsberghe M, Doolin C, Petriv OI, Singhal A, et al. Megapixeldigital PCR. Nat Methods 2011;8:64951.

Holmes D, Pettigrew D, Reccius CH, Gwyer JD, van Berkel C, Holloway J, et al. Leukocyteanalysis and differentiation using high speed microuidic single cell impedancecytometry. Lab Chip 2009;9:28819.

Hua Z, Rouse JL, Eckhardt AE, Srinivasan V, Pamula VK, Schell WA, et al. Multiplexedreal-time polymerase chain reaction on a digital microuidic platform. Anal Chem2010;82:23106.

Huang FC, Liao CS, Lee GB. An integrated microuidic chip for DNA/RNA amplication,electrophoresis separation and on-line optical detection. Electrophoresis 2006;27:3297305.

Huang N-T, ChenW, Oh B-R, Cornell TT, Shanley TP, Fu J, et al. An integrated microuidicplatform for in situ cellular cytokine secretion immunophenotyping. Lab Chip2012;12:4093101.

Huppa JB, Axmann M,Moertelmaier MA, Lillemeier BF, Newell EW, Brameshuber M, et al.TCR-peptide-MHC interactions in situ show accelerated kinetics and increasedafnity. Nature 2010;463:9637.and opportunities, Biotechnol Adv (2013), http://dx.doi.org/10.1016/

13S. Baratchi et al. / Biotechnology Advances xxx (2013) xxxxxxIrimia D, Tompkins RG, Toner M. Single-cell chemical lysis in picoliter-scale closedvolumes using a microfabricated device. Anal Chem 2004;76:613743.

Irimia D, Mindrinos M, Russom A, Xiao W, Wilhelmy J, Wang S, et al. Genome-widetranscriptome analysis of 150 cell samples. Integr Biol 2009;1:99107.

Ji HM, Samper V, Chen Y, Heng CK, Lim TM, Yobas L. Silicon-based microlters for wholeblood cell separation. Biomed Microdevices 2008;10:2517.

Jing Y, Moore LR, Williams PS, Chalmers JJ, Farag SS, Bolwell B, et al. Blood progenitor cellseparation from clinical leukapheresis product by magnetic nanoparticle binding andmagnetophoresis. Biotechnol Bioeng 2007;96:113954.

Kam L, Boxer SG. Formation of supported lipid bilayer composition arrays by controlledmixing and surface capture. J Am Chem Soc 2000;122:129012.

Khoshmanesh K, Akagi J, Nahavandi S, Kalantar-Zadeh K, Baratchi S, Williams DE, et al.Interfacing cell-based assays in environmental scanning electron microscopy usingdielectrophoresis. Anal Chem 2011a;83:321721.

Khoshmanesh K, Akagi J, Nahavandi S, Skommer J, Baratchi S, Cooper JM, et al. Dynamicanalysis of drug-induced cytotoxicity using chip-based dielectrophoretic cell immo-bilization technology. Anal Chem 2011b;83:213344.

Khoshmanesh K, Nahavandi S, Baratchi S, Mitchell A, Kalantar-zadeh K. Dielectrophoreticplatforms for bio-microuidic systems. Biosens Bioelectron 2011c;26:180014.

Kim D, Haynes CL. Neutrophil chemotaxis within a competing gradient of chemoattractants.Anal Chem 2012;84:60708.

Kim D, Herr AE. Protein immobilization techniques for microuidic assays. Biomicrouidics2013:7.

Kim J, Johnson M, Hill P, Gale BK. Microuidic sample preparation: cell lysis and nucleicacid purication. Integr Biol 2009a;1:57486.

Kim J, Lee H-H, Steinfeld U, Seidel H. Fast capturing on micromagnetic cell sorter. IEEESens J 2009b;9:90813.

Kim S-B, Koo K-i, Bae H, Dokmeci MR, Hamilton GA, Bahinski A, et al. A mini-microscopefor in situ monitoring of cells. Lab Chip 2012;12:397682.

Koh Y, Lee B-R, Yoon H-J, Jang Y-H, Lee Y-S, Kim Y-K, et al. Bead afnity chromatographyin a temperature-controllable microsystem for biomarker detection. Anal BioanalChem 2012;404:226775.

Lee BS, Lee J-N, Park J-M, Lee J-G, Kim S, Cho Y-K, et al. A fully automated immunoassayfrom whole blood on a disc. Lab Chip 2009;9:154855.

Lenshof A, Laurell T. Emerging clinical applications of microchip-based acoustophoresis.Jala 2011;16:4439.

Lenshof A, Ahmad-Tajudin A, Jaras K, Sward-Nilsson A-M, Aberg L, Marko-Varga G, et al.Acoustic whole blood plasmapheresis chip for prostate specic antigen microarraydiagnostics. Anal Chem 2009;81:60307.

Li XF, Kolega J. Effects of direct current electric elds on cell migration and actinlament distribution in bovine vascular endothelial cells. J Vasc Res 2002;39:391404.

Li J, Lin F. Microuidic devices for studying chemotaxis and electrotaxis. Trends Cell Biol2011;21:48997.

Li Y, Buch JS, Rosenberger F, DeVoe DL, Lee CS. Integration of isoelectric focusing withparallel sodium dodecyl sulfate gel electrophoresis for multidimensional proteinseparations in a plastic microudic network. Anal Chem 2004;76:7428.

Li J, Zhu L, Zhang M, Lin F. Microuidic device for studying cell migration in single orco-existing chemical gradients and electric elds. Biomicrouidics 2012:6.

Lin F, Baldessari F, Gyenge CC, Sato T, Chambers RD, Santiago JG, et al. Lymphocyteelectrotaxis in vitro and in vivo. J Immunol 2008;181:246571.

Liu C, Qu Y, Luo Y, Fang N. Recent advances in single-molecule detection on micro- andnano-uidic devices. Electrophoresis 2011;32:330818.

Luo Y, Xu S, Schilling JW, Lau KH, Whitin JC, Yu TTS, et al. Microuidic device for couplingcapillary electrophoresis and matrix-assisted laser desorption ionization-mass spec-trometry. Jala 2009;14:25261.

Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the HumanImmunology Project. Nat Rev Immunol 2012;12:191200.

Male DK, Brostoff J, Roth DE, Roitt IM. Immunology. 8th ed. Elsevier; 2012.Marshall LA, Han CM, Santiago JG. Extraction of DNA from malaria-infected erythrocytes

using isotachophoresis. Anal Chem 2011;83:97158.Marshall LA, Wu LL, Babikian S, Bachman M, Santiago JG. Integrated printed circuit board

device for cell lysis and nucleic acid extraction. Anal Chem 2012;84:96405.Mattila PK, Feest C, Depoil D, Treanor B, Montaner B, Otipoby KL, et al. The actin and

tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling. Immunity 2013;38:46174.

McClain MA, Culbertson CT, Jacobson SC, Allbritton NL, Sims CE, Ramsey JM. Microuidicdevices for the high-throughput chemical analysis of cells. Anal Chem 2003;75:564655.

Medzhitov R, Shevach EM, Trinchieri G, Mellor AL, Munn DH, Gordon S, et al. Highlights of10 years of immunology in Nature Reviews Immunology. Nat Rev Immunol 2011;11:693702.

Mellors JS, Gorbounov V, Ramsey RS, Ramsey JM. Fully integrated glass microuidicdevice for performing high-efciency capillary electrophoresis and electrospray ion-ization mass spectrometry. Anal Chem 2008;80:68817.

Melville D, Paul F, Roath S. Direct magnetic separation of red cells from whole blood.Nature 1975;255:706.

Mohamed H, Turner JN, Caggana M. Biochip for separating fetal cells from maternalcirculation. J Chromatogr A 2007;1162:18792.

Mun BP, Jung SM, Yoon SY, Kim SH, Lee JH, Yang S. Continuous cell cross over and lysis ina microuidic device. Microuid Nanouid 2010;8:695701.

Munce NR, Li JZ, Herman PR, Lilge L. Microfabricated system for parallel single-cellcapillary electrophoresis. Anal Chem 2004;76:49839.

Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, et al. Isolation of rare cir-culating tumour cells in cancer patients by microchip technology. Nature 2007;450:12359.Please cite this article as: Baratchi S, et al, Immunology on chip: Promisesj.biotechadv.2013.11.008Olvecka E, Kaniansky D, Pollak B, Stanislawski B. Separation of proteins by zone electro-phoresis on-line coupled with isotachophoresis on a column-coupling chip withconductivity detection. Electrophoresis 2004;25:386574.

Peoples MC, Karnes HT. Microuidic capillary system for immunoafnity separations ofC-reactive protein in human serum and cerebrospinal uid. Anal Chem 2008;80:38538.

Perroud TD, Kaiser JN, Sy JC, Lane TW, Branda CS, Singh AK, et al. Microuidic-based cellsorting of Francisella tularensis infected macrophages using optical forces. Anal Chem2008;80:636572.

Persat A, Marshall LA, Santiago JG. Purication of nucleic acids from whole blood usingisotachophoresis. Anal Chem 2009;81:950711.

Pethig R, Menachery A, Pells S, De Sousa P. Dielectrophoresis: a review of applications forstem cell research. J Biomed Biotechnol 2010.

Quinto-Su PA, Lai H-H, Yoon HH, Sims CE, Allbritton NL, Venugopalan V. Examinationof laser microbeam cell lysis in a PDMS microuidic channel using time-resolvedimaging. Lab Chip 2008;8:40814.

Robert D, Pamme N, Conjeaud H, Gazeau F, Iles A, Wilhelm C. Cell sorting by endocytoticcapacity in a microuidic magnetophoresis device. Lab Chip 2011;11:190210.

Rogacs A, Qu Y, Santiago JG. Bacterial RNA extraction and purication from whole humanblood using isotachophoresis. Anal Chem 2012;84:585863.

Root BE, Agarwal AK, Kelso DM, Barron AE. Purication of HIV RNA from serumusing a polymer capture matrix in a microuidic device. Anal Chem 2011;83:9828.

Rosenbach AE, Koria P, Goverman J, Kotz KT, Gupta A, Yu M, et al. Microuidics forT-Lymphocyte cell separation and inammation monitoring in burn patients. ClinTransl Sci 2011;4:638.

Rotman B. Measurement of activity of single molecules of beta-D-galactosidase. Proc NatlAcad Sci U S A 1961;47:198191.

Ruan CM, Yang LJ, Li YB. Immunobiosensor chips for detection

immunology on chip

Documents

rmit university

cell analysis

chip manipulation of

separation of nucleic

chip manipulation of

purication of nucleic

r t i c

customized c