Microfluidics for medical diagnostics and biosensors

Chemical Engineering Science 66 (2011) 1490–1507

Contents lists available at ScienceDirect

Chemical Engineering Science

0009-25

doi:10.1

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journal homepage: www.elsevier.com/locate/ces

Microfluidics for medical diagnostics and biosensors

Catherine Rivet b,1, Hyewon Lee a,1, Alison Hirsch a,1, Sharon Hamilton a,1, Hang Lu a,b,n

a School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USAb Interdisciplinary Program in Bioengineering, Georgia Institute of Technology, Atlanta, GA, USA

a r t i c l e i n f o

Article history:

Received 2 February 2010

Received in revised form

21 July 2010

Accepted 10 August 2010Available online 27 August 2010

Keywords:

Microfluidics

Sensors

Diagnostics

Point-of-care

Materials

Integration

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esponding author at: 311 Ferst Dr. NW, At

404 894 8437; fax: +1 404 894 4200.

ail address: [email protected] (H. Lu).

ese authors contributed equally.

a b s t r a c t

This article reviews the recent development in microfluidics for medical diagnostics and integrations

with biosensors. Diagnostic and sensing applications have been the focus of much of the development

of the micro-Total-Analysis-Systems (MicroTAS), and have recently enjoyed further development in

new fabrication technologies, integrations, and utilities in field- and medical-applications. The

challenges for these applications have been to reduce cost, to meet the sensitivity requirements while

providing throughput and speed, and to expand the repertoire of applications. This review focuses

mostly on new developments in the last 5–10 years in materials development, chip architecture and

integration, different sensing modes that can be used in conjunction with microfluidics, and new

applications that have emerged or have been demonstrated; it also aims to point out where future

research can be directed to in these areas.

& 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Medical diagnostics and bio-sensing are two important appli-cation areas of microfluidic technologies. Many early techniquesthat were successfully miniaturized were in DNA separation anddiagnostics. Since the field of microfluidics has been maturing inthe last decade, many more applications and integration ofcomplex functions on-chip have been demonstrated. In general,microfluidics seek to overcome difficulties or challenges intraditional assays in medical diagnosis such as cell-based assaysand disease screens, drug screens, and in some cases fundamentalbiological studies. The obvious advantage of using microfluidics isthe ability to handle small sample sizes and also as a result savingvaluable reagents used in the assays. Other advantages are in thepotential applications in field use or point-of-care (POC) usage.Increasingly, many microfluidic systems are also integrated withsensing modules or sample-pre-treatment modules, which in-creases the efficiency of the assays and reduces cross-contamina-tion. Overall, these advantages could potentially bring the cost ofthe assays down as well as provide a faster diagnosis. Severalseminal reviews in microfluidics have addressed the generaldevelopment of the field in the last two decades (Beebe et al.,2002; deMello, 2006; Dittrich et al., 2006; El-Ali et al., 2006;Janasek et al., 2006; Psaltis et al., 2006; Squires and Quake, 2005;

ll rights reserved.

lanta, GA 20221-0100, USA.

Stone et al., 2004; Whitesides, 2006; Yager et al., 2006). Thepresent review focuses on new technologies (including newmaterials such as paper-based chips, new fabrication processes,and novel uses), new architectures and integration with sensingtechnologies to improve controllability, throughput, and reliabil-ity, and a large range of diagnostic applications that have beendeveloped in the last three to five years.

Section 2 covers new material development, such as usingpaper and thermoplastics for POC applications, and new devel-opment in chip designs and component designs, such as fluiddelivery schemes, fluid control/valves, and using digital anddroplet-based microfluidics. Section 3 covers many differentmodes of sensing that have been integrated with microfluidics,including optical, electrical, and mechanical means; this sectionalso provides examples of recent development in cell-basedassays for clinical or biomedical science applications.

2. New developments in microfluidic platforms

Major components of the microfluidic platforms are thematerials they are made of and the methods used to controlthe fluid flow. While microfluidics hold great promises to theapplications of POC medical diagnostics, limitations to itsapplicability still exist. First, fabrication costs and materialcompatibility are two major considerations in material develop-ment; novel materials and processing that overcome theselimitations are addressed in Section 2.1. Second, most currentmethods for fluid control require expensive and complicated off-

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C. Rivet et al. / Chemical Engineering Science 66 (2011) 1490–1507 1491

chip components that are not directly amenable to use outside ofthe laboratory setting. Some new developments in techniquesfor driving flow and reducing valve complexity, reviewed inSection 2.2.1, address the issue of reducing the need for use ofexpensive and bulky off-chip equipment.

2.1. New material development

The elastomer polydimethylsiloxane (PDMS) is a popularmaterial used for various diagnostic and biochemical assays sinceits introduction (Whitesides, 2006). Since it is optically transpar-ent and exhibits low autofluorescence, PDMS is suitable for a largenumber of biological analyses (Psaltis et al., 2006). Besides itsphysical properties, the ease of PDMS fabrication methodscontributes to its wide applicability as a material for rapidprototyping of microfluidic chips. One of the most common PDMSfabrication methods is replica molding, which allows for sub-micron size patterning; it is fast, simple and does not requireexpensive facilities. Moreover, its applicability is further en-hanced by the development of simple methods for integratingactive components such as valves and pumps (Thorsen et al.,2002; Unger et al., 2000). However, currently commercializationis rare because of lab-scale fabrications and thus a gap betweenacademic and industrial practices still exists. In addition, PDMS’schemical properties, e.g. organic solvent swelling and hydropho-bic surface, limit the range of its applications (Lee et al., 2003a;Rolland et al., 2004b).

To address some of these limitations, new materials formicrofluidic chips such as paper and engineered plastics, likecyclo-olefin copolymers (COC) and photocurable perfluoropo-lyether (PFPE), have been developed in the last few years.Compared to PDMS chip fabrication, manufacture for paper-basedor thermoplastic chips does not require the clean-room infra-structure for the construction of masters. Thus, these newtechnologies are easy to apply in large scale fabrication, whichis advantageous for commercialization.

In addition, these new technologies have several advantagesfor point of care (POC) diagnostics. First, the materials themselvesand the fabrication methods are inexpensive when compared toglass and silicon and thus can be manufactured in large volumesat low cost. Second, because of the low cost, these chips aredisposable, which helps eliminate cross-contamination of biolo-gical samples. Because of the single-use, they do not requirerecalibration or maintenance. Third, both paper and plastics arelightweight and easy to transport and store. Thus, these newlydeveloped devices are expected to improve health care indeveloping countries where expensive and large equipment isnot available. Moreover, the merits of a paper-on-chip device arenot limited to applications in developing countries but couldextend to applications elsewhere because of the convenience ofthe POC devices.

2.1.1. Paper-based chips

Recently, paper-based microfluidics has been developed as analternative to PDMS or other inexpensive materials for a variety ofapplications (Chin et al., 2007; Martinez et al., 2008b). In additionto advantages mentioned in the previous paragraph, paper-baseddiagnostics usually do not require sample processing and externalpower sources or equipment to drive fluid movement; flow insuch systems is mostly driven by capillary actions and fluidwicking into the inter-fiber space of the paper. Although paperhas useful material properties, currently commercialized paperstrips for diagnostics, such as immunochromatographic strips, arelimited in their capabilities to perform quantitative assays andcannot be multiplexed (Chin et al., 2007), and yet multiplexing is

common and useful especially in screening to improve efficiency.Therefore, recent developments have focused on integrated POCmicrofluidic devices for multiplex assays on paper.

One of the typical paper-based multiplex systems is the 3Dmicrofluidic paper analytical device (3D mPAD) (Martinez et al.,2008b) (Fig. 1A). Martinez et al. (2008b) fabricated 3D mPAD bystacking layers of patterned paper and water-impermeabledouble-sided tape. The tape, patterned with holes, connectschannels in different layers of paper and thus allows vertical-flowdistribution in addition to lateral flow. This 3D mPAD wicks samplesolution from single-inlet points and distributes the solution intoarrays of detection zones in 2 min. This device has been applied toglucose and protein assays, which test 4 different samples for upto 4 different analytes. In another example, Derda et al. (2009)developed a paper-supported 3D cell culture system by stackingand de-stacking layers of paper impregnated with cell suspensionsin extracellular matrix hydrogel (Fig. 1B). This multilayeredconstruct allows the culturing of multiple cell types withcontrolled oxygen and nutrient gradients in 3D geometries. Thissystem was used to analyze biochemical and genetic responses incells for tissue-based bioassays, and as the authors argue, can befurther used in high-throughput screening and drug discovery.One advantage shown by this literature is that the assay capacitiesof paper-based 3D chips can be increased by simply assemblingmultiple types of paper possessing different functionalities.

2.1.1.1. Paper-based microfluidic fabrication and surface pattern-

ing. The most common way to create microchannels in paper is topattern hydrophilic paper with hydrophobic photoresist such asSU-8 (Martinez et al., 2007, 2008a, 2008b, 2008c). The patternedregions act as hydrophobic barriers while the resulting channelsare hydrophilic and allow aqueous samples to wick into thedevice. This photolithography step is simple and easy to use;however, the photoresist barriers are less flexible and thus sus-ceptible to mechanical damage. To improve the barrier flexibility,Bruzewicz et al. (2008) reported a process to generate hydrophilicchannels by printing an ink of PDMS solution onto paper. PDMSpenetrates into the paper substrate and forms hydrophobic bar-riers to define microchannels. Therefore, the flexibility of PDMSenables the printed paper device to be folded easily withoutdamage. Recently two groups further improved the process byusing wax printing as an alternative to PDMS printing (Carrilhoet al., 2009; Lu et al., 2009). The new wax-based process enablescomplete hydrophobic barriers in paper, which was not easilyachieved in PDMS printing because the penetration of PDMScannot be well controlled due to the irregular porosity of paper.Although the patterned hydrophobic barriers are not isotropic andneed further improvement, Carrilho et al. (2009) developed asimple equation to understand the spreading of wax in paper fordesign optimization of microchannels. Moreover, the wax-basedprocess is rapid and especially suitable for mass production ofpaper devices because it does not require a clean room, UV lamp,or organic solvents.

In addition to hydrophobic patterning, hydrophilic patterning isalso used to fabricate paper-based microfluidic devices (Abe et al.,2008; Lin et al., 2008). Abe et al. (2008) soaked paper in a solution ofpoly(styrene) in toluene to make it hydrophobic. They subsequentlyused an inkjet printing method using toluene to locally dissolve thehydrophobic poly(styrene) layer and thus fabricate hydrophilicpatterns (Abe et al., 2008). In a similar approach, Li et al. madepaper hydrophobic with a wax solution and then created hydrophilicchannels using plasma treatment (Lin et al., 2008).

Another interesting method for creating paper-based devices ispatterning superhydrophobic paper to control the mobility ofmicroliter drops (Balu et al., 2009) (Fig. 1C). Commercially

Fig. 1. Examples of 3D paper-based systems. (A) 3D microfluidic paper analytical device (3D mPAD) (Martinez et al., 2008b)—reprinted with permission from American

Chemical Society. (B) 3D cell culture system (Derda et al., 2009)—copyright (2009) National Academy of Sciences, U.S.A.; reproduced with permission.

(C) Superhydrophobic-patterned paper to control the mobility of microliter drops (Balu et al., 2009)—reproduced by permission of The Royal Society of Chemistry.

C. Rivet et al. / Chemical Engineering Science 66 (2011) 1490–15071492

available desktop printing technology was used to pattern asuperhydrophobic paper substrate with high-surface-energy ink.The shape and size of the ink patterns control the adhesion forceson water drops in two directions, parallel and perpendicular tothe substrate. This device performs not only qualitative analyticalfluidic testing but also droplet manipulation on paper via storage,transfer, mixing, and sampling for further quantitative analysis.

2.1.1.2. Readout and display. In order to be used as an assayplatform, e.g. quantitative detection of disease markers, a paper-based chip is commonly integrated in colorimetric assays (Abeet al., 2008; Lu et al., 2009; Martinez et al., 2007, 2008a, 2008b). Ineach assay, reagents react with the target analytes in the samplesolution and the products from the reaction change color in theassay solution. For readout of results of the assay, the intensitiesof the colors are compared with those of calibration curves to

quantify the concentration of analytes. These simple readouts areeasily recorded as digital images and transferred to off-site di-agnosticians by portable consumer-grade equipment such ascamera phones, scanners, and digital cameras (Breslauer et al.,2009; Martinez et al., 2008a) (Fig. 2A). With the use of this ap-proach, colorimetric-assay-based paper-on-chip devices havesuccessfully quantified glucose, pH, and protein levels, which areuseful for POC monitoring. Thus, colorimetric detection is ideal forsimple POC diagnosis because of their visual readouts, low cost,and stability.

Despite advantages of colorimetric assays, requirements of anexpert or a device to analyze and interpret the data may limit theusage of colorimetric-assay-based devices. Moreover, the sensi-tivity of the assay depends on the portable imaging device andlaboratory analytical instrumentation, which usually lack thesensitivity. To overcome this limitation of colorimetric assays, twogroups have integrated electrochemical detection (ECD) and

Fig. 2. Examples of system display and readout for paper-based devices. (A) A paper-based chip integrated with the colorimetic assay for simple telemedicine. Adapted

with permission from Martinez et al. (2008a). Copyright 2008 American Chemical Society. (B) Thermochromatic display. Siegel et al. (2009)—reproduced with permission

of the Royal Society of Chemistry.


paper-based microfluidic devices, as an alternative detectionscheme (Dungchai et al., 2009; Nie et al., 2009). ECD produceselectrical signal from chemical reactions, which is easy toquantify. ECD is attractive for paper-on-chip because of its smallsize, portability, simplicity of the instrumentation, low cost, highsensitivity, and high selectivity by proper choice of detectionpotential and/or electrode material. ECD-based paper deviceshave thus far been used for quantifying glucose, lactate, and uricacid in biological samples by employing screen-printed carbonelectrodes, which are useful for disease diagnoses.

Siegel et al. (2009) developed another quantitative displayscheme by introducing the simple thermochromatic display thatserves as a basis for colored and colorless state (Fig. 2B). Thisdisplay scheme is fabricated at low cost by patterning thermo-chromic ink on the front side of the paper, and electricallyconductive wires on the opposite side. Heat generated by theelectrical current through the wires causes the colored thermo-chromic ink to disappear and reveals any messages printed on thepaper underneath the ink. While its response time and powerconsumption vary with environmental temperature, this displayis useful when clear information needs to be delivered withoutsophisticated instruments utilizing limited electrical power, as inmany POC situations. The authors suggest that in the future, thisdisplay scheme can be integrated in paper-on-chip devices toclearly visualize the results of assays as instructive messages suchas ‘‘Hepatitis B positive’’ and ‘‘Do not drink’’.

2.1.2. Thermoplastic chips

Thermoplastics are another class of materials that can beutilized for sensor fabrication (Becker and Gartner, 2008). Likepaper-on-chip devices, thermoplastics have several advantagesfor recent and possible future developments of microfluidics forPOC because of thermoplastic’s low cost and disposability (Attiaet al., 2009; Becker and Gartner, 2008). Additionally, the polymer

can be fabricated using replication molding techniques likeinjection molding or hot embossing, which are simple and exhibitaccurate repeatability in mass production.

Application of thermoplastic chips can be extended to high-temperature applications such as polymerase chain reaction (PCR)because they typically have high melting points. One of therecent applications for thermoplastic devices is solid-phaseextraction (SPE)-based isolation of nucleic acids to detect bacteria(Mahalanabis et al., 2009; Sauer-Budge et al., 2009). Mahalanabiset al. fabricated a polymeric microfluidic device in a cyclicpolyolefin by hot embossing. This device was used for samplepreparation and rapid detection of infectious organisms on asingle chip (Mahalanabis et al., 2009). To identify gram-negative(Escherichia coli) and gram-positive (Bacillus subtilis and Enter-

ococcus faecalis) bacteria, genomic DNA was isolated in amicroscale SPE column after lysing bacteria on-chip. Similarly,Sauer-Budge et al. (2009) developed a polymeric microfluidicdevice fabricated in cyclo-olefin polymer (COP) by injectionmolding. This system is a fully integrated lab-on-a-chip conduct-ing bacterial lysis, nucleic acid isolation and concentration, PCR,and end-point fluorescent detection. Unlike typical multi-func-tional devices, this polymeric chip does not require multiplematerial types and numerous assembly steps, which ultimatelywill reduce the cost of the chips and improve the feasibility in POCapplications. Additionally, this system automatically controlsfluids, temperature cycling, and optical detection by utilizing aremote valve switching technique.

For thermoplastic chips, it is important to select appropriatematerials and fabrication methods. Two commonly used thermo-plastics are poly(methyl methacrylate) (PMMA) and polycarbonate(PC) (Becker and Gartner, 2008). PMMA microchips can be used inthe analysis of DNA, proteins, and oligo-saccharides, because PMMAis mechanically stable and optically transparent, it has lowautofluorescence, and surface modification for separation applica-tions is relatively simple (Dang et al., 2005; Thamdrup et al., 2008).


Similarly PC is also applied in DNA analysis, especially for PCRbecause of its low thermal conductivity compared with silicon orglass. However, PMMA and PC are easily degraded by most organicsolvents; additionally, PC delaminates at high pressures. COP(Bhattacharyya and Klapperich, 2006; Mahalanabis et al., 2009)and COC (Mair et al., 2006; Pu et al., 2007) have been attractive tomicrofluidic devices because they have excellent optical propertiesand low water uptake (Becker and Gartner, 2008).

The fabrication methods of polymeric microfluidic devices aremostly based on the replication of a metal (e.g. nickel alloy) orsilicon master, mainly injection molding and hot embossing (Beckerand Gartner, 2008). Bhattacharyya and Klapperich (2006) recentlydeveloped a fabrication process of direct embossing from an SU-8master, making rapid prototyping also possible with these materials.Both injection molding and hot embossing play a major role in thecommercialization of microfluidic devices, particularly for POCdiagnostics, because of the well-established processes and widelyavailable commercial tools: injection molding is highly developedand inexpensive, and a large range of equipment and automationsolutions are available for mass production of microfluidic chips(Attia et al., 2009); similarly, hot embossing is comparatively simpleand widely used both in academia and industry (Becker and Gartner,2008). A wide variety of materials can also use these fabricationmethods should other materials be desirable.

2.1.3. Microfluidic devices using other polymers

As previously mentioned in the introductory part of Section2.1, one of the challenges use PDMS devices in analyticalchemistry and biochemistry is that PDMS swells and sometimesdisintegrate in organic solvents. Like PDMS, the applications ofthermoplastic chips are limited in some chemical and biologicalassays involving non-polar organic solvents because of theirsolubility (PMMA and COCs). Rolland et al. (2004b) developedmicrofluidic devices with PFPE that exhibits increased resistanceto swelling in common organic solvents with enhanced structuralstability. Furthermore, like PDMS, PFPE has high gas permeability,high flexibility, and low toxicity, which are good for biologicalassays. To construct microchips based on photocurable polymers,a high resolution nanoimprint method is commonly used (Rollandet al., 2004a). Bartolo et al. (2008) developed a simple prototypingmethod to make micropatterned stickers and assembled them toform microfluidic plastic microfluidic devices in a few minutes.This method is simple and versatile for cell- and tissue-basedassays (Morel et al., 2009).

Fig. 3. A compact disc (CD)-based platform uses centrifugal force to drive flow.

(A) A schematics of a disc with a set of microfluidic devices. The disc rotates at

angular velocity (o). (B) A close-up schematics of one device on the chip. The force

2.2. New architectures for fluid manipulation on-chip

New architectures for fluid control are necessary in order formicrofluidic devices to have a large presence in small medicalclinics, in areas without clinics, or in the field by militaryand emergency response teams. Many currently available archi-tectures rely on large, expensive laboratory equipment for fluiddelivery and control, making them impractical in many of thoseareas. This section will cover recent advances toward this goalwith respect to fluid delivery, manipulation, and control. Fluiddelivery methods, control of valves (active and passive), and theuse of droplets for digital microfluidics are discussed in thecontext of use outside of the conventional laboratory settings.

is a function of the absolute velocity, the angular velocity and the radial position

(R) on the chip. C–E show possible valving in at end of the channel in B (inside the

red box). (C) A capillary valve, where the fluid flow is controlled by a balance

between the capillary pressure PC and the force created by the spinning CD per

unit area (Po). (D) A valve created using a patterned hydrophobic patch (the darker

gray area). The hydrophobic effects prevent aqueous solutions from passing

through below a certain Po. (E) Using geometry to constrict the channel will

increase the effect of capillary forces.

2.2.1. Fluid delivery methods

Many early microfluidic devices use pressure-driven flow froma syringe pump or pressure injector, or electroosmotic flow usingpower supplies (Park et al., 2009). While these methods arewidely applicable in laboratory settings, they require external

equipment, which are bulky and not always available in theclinical setting. For POC diagnostics, methods for fluid deliverythat are inexpensive and easy to implement have recently beendeveloped. Methods include passive delivery, mechanical pump-ing, and even using light to manipulate fluids. Passive delivery bycapillary action is a simple way of delivering fluids without anymechanical pumping (Juncker et al., 2002; Walker and Beebe,2002). An aliquot of solution is dispensed into the service portusing a conventional pipette and the liquid spontaneously flowsby capillary action. To use this technique, the surface tension ofthe liquid and the chemistry and geometry of the solid interfacesmust be optimized to obtain the desired flow rate. In generalthese are non-continuous systems limited to sub-mL per minuteflow rates. Repeatability is limited by the consistency of manualpipetting of fluids into the service port. More recently, Khnoufet al. (2009) adapted this to an existing multi-well plate platformfor simple multiplexing of cell-free protein expression experi-ments. Other more recent advances have also been made in thevalving of these systems (see Section 2.2.2).

In scenarios where capillary action is not enough to meet theflow requirement, a compact disc (CD)-based platform usescentrifugal force to drive flow (Lee et al., 2001) as shownin Fig. 3. Valving, decanting, calibration, mixing, metering, samplesplitting, and separation can all be accomplished by balancing thegeometry (resistance) of the flow channel with the centrifugalforce generated using an inexpensive CD drive. Flow rates of10 nL s�1 to greater than 100 mL s�1 can be achieved dependingon the channel geometry, disc rotational rate, and fluid properties(density and viscosity), which makes it more versatile thanrelying solely on capillary forces. Furthermore, this method hasthe advantage that pumping is relatively insensitive to chemicalproperties such as pH, ionic strength, or chemical composition.


Other common household products can be used to drive the flow,which is amenable for field use due to the easy accessibility and therelatively low cost. In a recent example, Wong et al. (2008) used anegg beater to isolate human blood plasma from whole blood. A tubecontaining the blood sample was attached to the paddle of the eggbeater. As the paddle spun, it worked as a centrifuge causing thecells to pellet at the end of the tubing. This approach is an alternativeto using a large, electrically powered centrifuge to separate plasmafrom blood cells by density. It uses readily available products (eggbeater, PE tubing, and tape) to facilitate the usage far from thelaboratory in resource-poor settings. With this method however, thepace of spinning and thus the centrifugal force will vary betweenusers; a very fine level of control may be difficult.

For other applications, light can also be used to manipulatefluid without direct contact. In general light-based techniquesproduce relatively small flow rates, so they are better suited forspecialty applications. For example, Leach et al., 2006 created asmall mechanical pump inside a microfluidic channel usingrotating particles driven by optical tweezers (Leach et al., 2006).The transfer of angular momentum from a circularly polarizedlaser beam rotates the two particles on opposite sides of thechannels at up to 10 Hz, creating flow rates of up to 2 nL s�1. Liuet al. (2006) also used light to control the movement of dropletscontaining photothermal nanoparticles through an evaporation/condensation process as shown in Fig. 4A. The focused lightillumination on particles increased the local temperature of theliquid and caused water evaporation at the liquid–air interface.The vapor then condensed into droplets in front of that liquid–air

Fig. 4. Digital microfluidic methods for manipulating droplets and a method of using dr

selectively heat up the front of a droplet containing photothermal nanoparticles. As the

by permission from Macmillan Publishers Ltd: (Liu et al., 2006), copyright (2006). (B) In

the sample of interest (bound to magnetic beads) is immobilized by a magnet and

(iii) immobilization of magnetic beads, (iv) removal of supernatant and washing, and (

Royal Society of Chemistry.

interface. As the droplets coalesce with the original bulk liquidbody, the droplets advance. This processes is repeated as the lightis translated, creating continuous creeping flow. The surface mustbe hydrophobic such that the solution forms a droplet and doesnot spread out. In addition, because of the high temperaturesinduced, this technique is not applicable to temperature sensitivesamples (e.g. proteins). While these optical methods still require alarge microscope setup, there is potential for future integrationinto lab-on-a-chip devices (see Section 3.1).

2.2.2. Valves for fluid control

Often diagnostic assays require multiple-step processes in-cluding washing, incubation, metering, and dilution. These canbe accomplished with the use of valves. Microfabricated pneu-matic valves using the elastomeric properties of poly(dimethyl-siloxane) (PDMS) were developed by Unger et al. (2000) and havebeen used extensively to perform many chemical and biologyassays. However, significant off-chip hardware and a pressuresource are necessary thus limiting their usage to the laboratorysetting currently. To overcome the difficulty of valve control,several groups have endeavored to reduce and optimize theactuation effort. Recent work by Leslie et al. (2009), in controllingmultiple valves with minimal off-chip control, uses frequency-specific valves. Fluid flow patterns are controlled with bycontrolling the actuation frequency of a single pressureinput. Operation of these frequency-specific valves is similar tothe analog responses of passive electrical circuits composed

oplet to perform serial operations (sandwiched immunoassay). (A) Light is used to

vapor condenses on the surface in front of the droplet, it moves forward. Reprinted

the digital sandwiched immunoassay the droplets are manipulated via EWOD and

analyzed. The steps are as follows: (i) dispensing of reagents, (ii) incubation,

v) adding fresh wash buffer. Sista et al. (2008)—reproduced by permission of The


of resistors, capacitors, and diodes, where the frequency isconverted differently by each component depending on the sizeand mechanical properties of a deformable membrane. Anotherexample (Fig. 5) uses valves with a normally closed nature;latching pneumatic valves were created by using microfluidiclogical structures (Grover et al., 2006). This device featured a de-multiplexer for controlling large arrays of independent latchingvalves. It uses only n off-chip pneumatic inputs to control 2(n–1)

multiplexed latching valves, thus maximizing operation capabil-ities while minimizing off-chip actuation.

In assays where cost needs to be minimized, valve actuationsare infrequent, and automation is not necessary, more simplevalve control is desired. In one approach, Weibel et al. (2005)developed macroscale, manually controllable ‘‘TWIST valves’’,which are easy to use and do not require additional equipment tohold the valve closed. A small screw imbedded just above amicrofluidic channel in a PDMS device works as a valve byconverting torque (using a screwdriver or by hand) into down-ward compression of the underlying microfluidic channel, closingit off. The advantage here is that these valves are not powered andare inexpensive to implement, hence most suitable for use inareas with minimal resources.

Yet another simple way to control and drive flow is throughpassive valving. The advantage is that it does not require any off-chip actuation. This is especially useful for situations where thecomplexity of the system makes the integration of off-chipcomponents practically impossible. These valves are single-use,influencing how a fluid front breaks past the valve, not how itflows through afterwards. Passive valves can be used to defineunit operations using capillary forces (surface tension andchannel resistance) to control the flow in situ. In one general

Fig. 5. Actuating latching valves to control many channels in a multiplex fashion. (A) A

opens the valve. (B) One set of latching valve. A short vacuum or pressure pulse is applie

open or closed, respectively. These can be combined in series to control many channels

input to control 16 valves. Grover et al. (2006)—reproduced by permission of The Roy

approach, an aqueous solution would flow freely through achannel with a hydrophilic surface and stop at a patternedhydrophobic interface (Handique et al., 2000). The time the fluidis stopped for is a function of capillary pressure and the pressuredrop across the flow channel. Londe et al. (2008) developed athermoswitchable interface where the patterned surface ishydrophilic at room temperature (allowing flow of aqueoussolutions), and hydrophobic above 65 1C, resisting the flow ofwater. Another more sophisticated example, shown by Zimmer-mann et al. (2008), incorporates varying channel geometries tocreate delay lines and stopping of liquids in microchannels. Thevalves employ an abruptly changing geometry of the flow path todelay a moving liquid front in a wettable microchannel. They alsoincorporated trigger valves, where at least two liquids must meetin the valve to trigger it and move forward in a common outlet.These passive valving mechanisms allow multiplexed sampleanalysis without any off-chip hardware.

2.2.3. Digital microfluidics and droplet-based microfluidics

In microfluidic systems, droplets or fluid plugs can be used fordigital fluidic operations with chemical and biological reagents(Teh et al., 2008). Creating, transporting, and cutting and mergingof droplets can be all accomplished using electrowetting-on-dielectric (EWOD) (Cho et al., 2003). An electrode coated withdielectrics controls the wetting properties of the surface throughcontrolling the electric potential. Typically the fluidic movementis confined between two plates or on an open surface rather thanthrough closed channels. Srinivasan et al. (2004) use electrowet-ting on a surface for dispensing, transporting, splitting, merging,and mixing of nanoliter aqueous droplets to analyze glucose

schematic of a valve cross-section. Applying vacuum to the displacement chamber

d to the input channel. The valve uses trapped vacuum or pressure to hold the valve

with one input. (C) A representative device using a combination of valves with 1

al Society of Chemistry.


levels in biological fluids (blood, serum, plasma, and urine).Electric field-induced interfacial tension changes between a liquidand a solid conductor control the movement of the droplets. Thedroplet solution must be somewhat conductive; the thresholdvoltage for movement decreases with increasing electrolyteconcentrations. Additionally, by using magnetic beads to holdthe molecules or particles of interest in place, dilution-basedwashing could be added to the process, facilitating morecomplicated biochemical analysis (Sista et al., 2008) as shown inFig. 4B. In this technique, aqueous droplets move by electrowet-ting on an open hydrophobic surface. The open surface architec-ture is advantageous because many biological samples containcells and/or protein aggregates that would clog conventionalmicrofluidic channels. However, evaporation of the fluids duringthe experiment using electrowetting must be considered. Themore successful applications of method of digital microfluidicsare in sample preparation. For example, Mousa et al. (2009)recently used an open surface digital microfluidic device forsample cleanup and extraction of estradiol from breast tissue,serum, and whole blood samples of only 1 mL total volume.

Similarly, plug-based-microfluidics employs nanoliter dropletsof fluid suspended in another immiscible fluid for dispensing,transporting, splitting, merging, and mixing nanoliter aqueousdroplets in microfluidic channels (Sassa et al., 2008). These tinyreactors have been used successfully as tiny PCR chambers(Tewhey et al., 2009), reducing sample volume needs anddiffusion limitations. These systems have the added benefit ofthe ability to retain the droplets suspended in the immiscible fluidfor long periods of time (without evaporation), allowing preload-ing of the device with nanoliter plugs of reagents for high-throughput screening (Agresti et al., 2010; Chen and Ismagilov,2006) or incubation of cells in the well-controlled droplets beforeanalysis (Koster et al., 2008).

3. New developments in sensor integration and applications

To enable the development of fast, sensitive, and portable POCdevices, recent progress (in the last five years or so) have beenmade towards a complete integration of the sensing mechanism.Therefore, devices can be completely portable and achieve therequired sensitivity for a reliable detection and diagnostics.The advantage of using microfluidics is that it allows for confiningthe preprocessed biomolecules or cells in a region of interest, andas a result, facilitating sensing from smaller sample volumes. Thissection will focus on new sensing mechanisms that have beenintegrated in a microfluidic setting, including optical, plasmonic,mechanical, and electrical sensing.

3.1. Integrated detection methods

Microscale detection of biomolecules is necessary for fullyfunctional diagnostics. A variety of methods are discussed in thissection, ranging from image-based methods that generally requirelabeling to label-free plasmonic or electrical properties-baseddetection. Some of these techniques are not unique to micro-fluidics; however interfacing these methods with microfluidicshelps decrease the risks of contamination, minimize the analysistime and enable portability for point of care diagnosis systems.

3.1.1. Microscope-on-chip and optical components on-chip

Most imaging applications on-chip require a conventionalmicroscope, which can be expensive, bulky, and not compatiblewith completely portable devices. To address this commonlimitation, Heng et al. (2006) reported an on-chip high-resolution

and high-throughput imaging technique, termed optofluidicmicroscopy (OFM), to replace the use of a conventional microscope.In OFM devices, an opaque metal film with an etched array ofsubmicron apertures is bonded to a PDMS microfluidic chip. Thedevice can be fabricated directly onto a CCD or CMOS array tocreate a truly compact microscope system. Imaging is achieved byflowing the specimen on an aperture array. The newest designsfrom the same group can achieve submicron resolution without theuse of a large, expensive microscope setup (Cui et al., 2008) and canperform two-color imaging (Pang et al., 2010) (Fig. 6A).

Other ways of coupling optical functionalities on-chip includeusing fluids of different refractive indices as waveguides or lensesto manipulate light. This ability to directly excite and detectphotoactive molecules or entities on-chip makes the system moretunable and faster for analysis. Mao et al. (2007, 2009) developeda tunable microfluidic lens using the interface of two misciblefluids of different refractive indices, e.g. water and calciumchloride. As the fluids flow around a curve, the calcium chlorideinterface bows outward in the water solution, creating anoptofluidic lens. The shape of the lens can be controlled bymerely changing the flow rates of the liquids, thereby controllingthe focus of an on-chip laser in real-time. For instance, higher flowrates in their device generate a lens with a larger curvature andtherefore a shorter focal length. The same group developed a lensthat can change the focal plane and the output direction of thelaser, thus adding another degree of freedom. This is accom-plished by using a gradient of refractive indices in the fluid; lighttravelling along the optical axis is gradually bent and brought to afocusing point (Mao et al., 2009). This lens also operates at aslower flow rate, thus reducing reagent consumption. Addition-ally, a microfluidic waveguide can be created by hydrodynami-cally focusing the core liquid with a high refractive index betweenthe cladding (with low refractive index; Brennan et al., 2008). Themicrofluidic waveguide benefits from the smooth interfacebetween the two liquids in laminar flow, resulting in low opticalloss due to scattering. In addition, it allows dynamic control ofoptical functionalities.

To increase sensitivity, waveguide-based interferometry issometimes used (Lukosz, 1991). As a detection scheme, thesesystems rely on the phase change upon recombination of split-beam coherent light that has been directed along two opticalpaths. The composition and concentrations of biomoleculesmodify the optical properties of the fluid, e.g. the refractive indexof the solution, thus inducing the phase change. The resultingphase change may be viewed either as a change in fringe patternwhen adjacent coherent sources are brought together, as whenbased on Young’s interferometry, or as in a change in outputintensity in the case of a Mach–Zehnder geometry. The principleof Young’s interferometry was utilized by Ymeti et al. (2007) toproduce a multiplexed, label-free viral particle detector withsensitivity in PBS down to 850 particles mL�1. Blanco et al. (2006)developed a biosensor based on a Mach–Zehnder interferometerwhich was implemented in a CMOS-compatible silicon processand was integrated with SU-8 microfluidic channels.

For fluorescence detection, chip-integrated photodiode detectorshave been developed (Irawan et al., 2007). This schemeof integration enables the creation of compact fluorescentsystems that are easy to fabricate, with low background fluores-cence and low cost. Novak et al. (2007) added a built-in lock-inamplifier to the chip integrated photodiode detection system toimprove the signal to noise ratio of the output signal so thatmeasurements could be performed under ambient light (outside ofthe laboratory environment) while maintaining sensitivity in thelow nanomolar range. Pipper et al. (2007) developed a completemicrofluidic platform, to detect the avian influenza virus H5N1.Sample preparation, RT-PCR and fluorescence detection were done

Fig. 6. Recent advances in integrated detection schemes. (A) Implementation of a color-capable optofluidic microscope on a RGB CMOS color sensor chip substrate.

(i) Schematic plot of gravity driven color OFM. (ii) Bayer pattern for the color filter mask of the CMOS sensor. (iii) Color OFM device comparing its size with a 10�

microscope objective. (iv) Schematic plot for the aperture array and the microscope image of an aligned PDMS channel on top of the aperture array. Scale bar is 20 mm. Pang

et al. (2010)—reproduced by permission of The Royal Society of Chemistry. (B) Printed circuit board for droplet arrangement for bird flu virus detection. Pipper et al.

(2007)—reprinted by permission from Macmillan Publishers Ltd. (C) (i–iv) Stop-flow lithography to synthesize coded particles by using co-flow of laminated streams.

(v) Reading coded particles for analyte detection. Pregibon et al. (2007)—reprinted with permission from AAAS. (D) Suspended silicon microchannel resonators in a sealed

vacuum cavity for label free biomarker detection in serum. von Muhlen et al. (2010)—reprinted with permission from American Chemical Society.


in droplets on a Teflon coated disc, which is placed on amicrofabricated heater with an integrated modulated LED/demo-dulated photodetector (Fig. 6B). In these examples, integration ofdetection methods on-chip can minimize the expense associatedwith large laboratory equipment and allow for complete deviceportability. The major challenge for future development is indesigning on-chip components that have the same sensitivity asthat of (or better than) conventional techniques.

3.1.2. Optical detection techniques integrated with microfluidics

Optical detection schemes integrated on the microfluidic chipcan offer many benefits. Labeling techniques are relativelysensitive for detecting biomolecules in traditional bioassays.Shrinking the sensing scheme to on-chip operation can potentiallysave reagents due to the small volumes required for microfluidics,offer integration (with fluidic and optical components) andtherefore fewer steps in assays as well as the possibility for

automation, and potential field- or bed-side point-of-use of theseassays. In this section, we review labels of different natures ormechanisms such as small-molecule fluorescent dyes, quantumdots, and magnetic or gold particles that have been integrated inmicrofluidic devices.

3.1.2.1. Fluorescent labels. Fluorescent labels are typically smallmolecules or proteins that can be excited at a particular wave-length and emit at a longer wavelength. Their use is common inbiology and chemistry due to the simplicity of the method, buttheir drawbacks include potential pH sensitivity and suscept-ibility to photobleaching over time. The advantage in integratingfluorescent labels with microfluidics is the reduction in thesample volume, which reduces background signal noise, henceincreasing the sensitivity, signal-to-noise ratio, Rayleigh straylight, and Raman scattering (Dittrich and Manz, 2005). However,using fluorescent labels with microfluidics can also present some


challenges. The chip material needs to be microscopy-compatible,e.g. low autofluorescence and non-adsorbent to the molecules.The off-chip optics required for signal acquisition can be bulky;hence efforts are currently devoted to developing fully integratedsystems for fluorescence detection in point-of-care systems.

Typically, samples are labeled with a specific fluorophore, andthe fluorescence signal induced by a laser source (as in laserinduced fluorescence, LIF) is detected as samples flow througha channel. A more sensitive technique, fluorescence cross-correlation spectroscopy (FCCS), uses cross-correlation of thetemporal fluorescence fluctuations from dual-labeled molecules,which allows detection of very low concentrations of proteins(10�18–10�21 M) in microliter volumes of samples. One suchapplication is for early diagnosis of diseases. Zhang et al. (2006b)used FCCS to probe and compartmentalize single dengue virusesin an array of nanoscale fluidic chambers after specific associationof dengue antibody.

Another technique, Forster/Fluorescence Resonance EnergyTransfer (FRET) is used to measure molecular interactions. Twodifferent fluorophores are tethered to two molecules of interest.When the molecules interact and bring the fluorophores closetogether, the emitted photon from the donor is transferred to theacceptor to excite the acceptor, which emits at a differentwavelength from the donor. Changes in ratiometric measurementsof the two colors suggest molecular interactions. Jung et al. (2007)show the integration of this technique with microfluidics for DNAanalysis. This technique enables the distinction between hybridiz-ing and non-hybridizing DNA oligomers, with an improvedspatial and temporal resolution compared to simple fluorescentlylabeled molecules. The study of intermolecular interactions isimproved in microfluidic devices, due to enhanced mixing thatovercomes slow hybridization caused by limited diffusion inregular microarray chips.

To overcome photobleaching and pH sensitivity issues oftraditional labels, quantum dots (QD) are used to provide betterstability and a higher signal. They are bright, and their emissionwavelength can be easily tuned by varying size and composition,therefore allowing multiplexing with labels across a wide range ofaccessible wavelengths. QDs have been used in microfluidicassays for microbe and virus detection (Liu et al., 2005; Zhanget al., 2006a), sensing of single molecules (Agrawal et al., 2006;Stavis et al., 2005), or in combination with FRET for applicationssuch as signal amplification during DNA sensing and detection ofmolecular orientation, size, and binding (Ho et al., 2006; Medintzet al., 2003; Puleo et al., 2008; Zhang et al., 2005).

3.1.2.2. Chemiluminescence. Chemiluminescence is the emissionof energy in the form of light (with limited emission of heat) asthe result of a chemical reaction. Specifically, the analyte bindingto a substrate or with an enzyme causes light emission. The ad-vantage of this technique is that no excitation instrumentation isrequired, and therefore background interference is virtuallyeliminated. However, emission is generally low which requires amore sensitive detection mechanism (Kim et al., 2009b). To cap-ture the low chemiluminescent signal, Vykoukal et al. (2009b)developed a low-cost and readily available CMOS transistorssensor chip as a microscale contact imager for droplet-basedmicrofluidics and as a quantitative photometer for the integratedoptical detection of chemiluminescent assays implemented on amicrofluidic platform. Their sensor shows similar sensitivities toconventional spectrophotometers, while being significantlycheaper with disposable microfluidic cartridges. Hatakeyamaet al. (2009) used a thin film transistor photosensor with anssDNA-modified surface in a microfluidic channel to discriminatesingle nucleotide polymorphisms (SNPs). Microarrays are the

conventional method for SNPs detection and require expensiveapparatus and long incubation times. The integrated chip devel-oped by Hatakeyama et al. is able to detect SNPs in less than anhour, with a sensitivity limit of 0.5 nM, a DNA concentrationmuch lower than that typically required for this application.Micro-analysis systems with chemiluminescence detection havealso been developed for the determination of cancer markers(Tsukagoshi et al., 2005), for detecting C reactive protein in serumwith a reduction in the assay time (Bhattacharyya and Klapperich,2007) and multiplexed allergen screening (Tai et al., 2009).

3.1.2.3. Nanoparticles. Nanoparticles, including magnetic (e.g. ironoxides, cobalt, or alloys) and gold particles coated with antibodiesor aptamers can be used as labels for the separation and detectionof cells and specific biomolecules. In standard bench top proto-cols, magnetic particles have been used extensively for molecularand cell separation purposes due to their simplicity of use andrelatively high efficiency. Examples of their integration with mi-crofluidic devices include those for cell sorting, use of magneticparticles as traps (Han et al., 2004; Smistrup et al., 2005) withexternal permanent magnets (Bronzeau and Pamme, 2008) ormagnetic field guides. For a more detailed review, the reader canrefer to the recent reviews by Liu et al. (2009) or Gijs et al. (2009).These magnetic beads can also be used to detect and quantifybiomolecules. Leary et al. (2006) developed a fully automatedplatform to detect and quantify the hepatitis C virus core proteinantigen in blood with a magnetic microparticle-based sandwichassay. External permanent magnets are frequently used, butseveral groups have integrated sources of magnetic field on-chipfor better control. Kim and Soh (2009) developed an integrateddielectrophoretic–magnetic activated cell sorter for multiplebacterial targets using an array of microfabricated nickel ferro-magnetic strips. These strips create large magnetic field gradientsafter application of an external magnetic field. Lee et al. (2007a)designed a hybrid CMOS/microfluidic chip with arrays of micro-coils to generate microscopic magnetic field patterns to trap andmanipulate magnetically tagged cells. In a subsequent study,these microcoils, in conjunction with a small permanent magnetwere used for both radio frequency excitation and nuclear mag-netic resonance (NMR) signal detection in a completed integratedNMR microfluidic chip (Lee et al., 2008). This telephone sized POCdevice enables detection of bacteria or cancer biomarkers.

Gold particles are commonly used nanoscale materials inmolecular diagnostics, because of their low toxicity, easy conjuga-tion to biomolecules and their versatility in detection methodsemployed for their analysis. Readouts based on optical absorption(Luo et al., 2005), fluorescence (Li et al., 2005; Lin et al., 2005),Raman scattering (Huh et al., 2009), and electrical conductivity(Maeng et al., 2008) have all been implemented in microfluidicdevices. Bailey et al. (2007) introduced a method called DNA-encoded antibody libraries (DEAL) for rapid multiple proteindetection on a single microchannel utilizing a gold nanoparticle-labeled secondary antibodies to amplify the signal from theprotein to detect. The DEAL method can achieve a detection limitof 10 fM interleukin-2 (IL-2), which is 150 times more sensitivethan the analog enzyme-linked immunosorbent assay (ELISA). Thelow sample volumes used in microfluidic chips enables the deviceto concentrate and amplify signal from gold particles.

3.1.2.4. Barcodes. One of the main advantages of microfluidics isthat it enables high throughput, parallel and multiplexed experi-ments. In order to simplify the readout of these assays and reducethe total time, several groups have developed methods to createbarcodes on-chip. Encoded barcodes also overcome the intrinsic


limitation of the number of available colors offered by fluorescentlabels. Fan et al. (2008) presented an integrated microfluidic bloodbarcode chip that can sample a large panel of protein biomarkersover broad concentration ranges with high sensitivity and within10 min of sample collection. It enables on-chip blood separationand rapid measurement of a panel of plasma proteins fromquantities of whole blood as small as those obtained by a fingerprick. Pregibon et al. (2007) showed a multiplexed, single fluor-escence detection of DNA oligomers with encoded particle li-braries that can be scanned rapidly in a flow-through microfluidicchannel (Fig. 6C). By exploiting laminar flows characteristic ofmicrofluidics, multi-functional particles with distinct regions foranalyte barcoding and target capture were created in a device. Anoptimized design of these hydrogel microparticles enabled smallRNA detection with sub-attomole sensitivity and single-nucleotidespecificity (Pregibon and Doyle, 2009). To further improve thedetection and quantification of biomolecules, a microfluidic sys-tem with side focusing streams was designed to align the barcodesfor proper and faster decoding, thus improving throughput(40 particles/s) and sensitivity (Chapin et al., 2009).

Optical detection is currently the preferred detection techni-que, due to its simplicity, high sensitivity, continuous monitoringcapacity, and the absence of contact with the target analytes. Thecombination of microfluidics with conventional optical detectionoffers several advantages: (1) The tight control of liquid flowenables the concentration of analytes and labels of interest in asmall volume, therefore enhancing binding kinetics and over-coming sensitivity issues. (2) Microfluidics save precious reagents(labels, cells) and time. (3) Microfluidics offer automated proces-sing of samples, reducing human error, loss of samples andcontamination with the added ability to process samples inparallel. (4) Microfluidics can bring the target close to thedetection area, herein enhancing the signal-to-noise ratio andimproving the sensitivity. However, optical detection does notseem to be an optimal solution for detection in POC devices. It isstill difficult to produce low-cost, sensitive, and portable optics.The necessary precise alignment between microfluidic and opticaldetection components makes it hard for mass production. Theshelf life of fluorescent reagents (dyes or quantum dots), orfunctionalized nanoparticles are limited, hence effort has beendevoted in the development of label free detection methods.

3.1.3. Label-free detection plasmonic-based detection techniques

While label-based methods provide high sensitivity, the addi-tional steps required for the labeling process increase the probabilityof error, and may also induce conformational changes, resulting invariations in the binding affinities or functions of the biomoleculesto be detected. Label-free methods therefore offer the alternative ofa simplified assay, with good sensitivity, and integrated detectionsystems. The assay simplification as well as its high throughputcapabilities could potentially facilitate point of care diagnoses aswell as sensor applications in other microfluidic systems.

3.1.3.1. Surface plasmon resonance (SPR). SPR is an optical detec-tion method that senses the change in the refractive index of avery thin metallic film (�50 nm); in sensing applications, thechange in the refractive index is induced by the binding or ad-sorption of molecules to the surface. It can be used to detect ad-sorption of molecules, such as polymers, DNA, or proteins withgood sensitivity (but not as good as sensitivities achieved byfluorescence techniques) and fairly good temporal resolution inreal-time. Completely integrated SPR systems have been devel-oped to measure binding kinetics in a protein microarray(Natarajan et al., 2008) or to perform immunoassays (Lee et al.,2007b; Rich et al., 2008; Suzuki et al., 2002). SPR has also been

coupled with digital microfluidics, thus creating a high-through-put screening system with reliable quantification (Galopin et al.,2007). Localized SPR (LSPR) improves the sensitivity of themethod and requires a smaller sample volume. This techniquerelies on a wavelength shift due to a change in the dielectricconstant of the substrate. Noble metal nanoparticles, e.g. Au or Agparticles, exhibit selective photon absorption in the ultra-violet�visible regime. The maximum extinction wavelength,lmax, of LSPR is dependent upon the composition, size, shape, andinter-particle spacing of the nanoparticles (Haes et al., 2004). Thistechnique has been applied in microfluidic devices for im-munoassays with good results (Endo et al., 2006; Huang et al.,2009). Disadvantages to note include the potential difficulty tofunctionalize metal surfaces, nonspecific adsorption, and im-mobilized biomolecules potentially losing their native bioactivity.

SPR with integrated microfluidics is a widely used technique tostudy molecular interactions and enables the characterization ofmolecules and biochemical interactions occurring in diseases. Ithas recently emerged as a platform for disease biomarkerdiscovery (e.g. Biacore platforms). It is a good alternative tooptical detection for bio-diagnostics, as it does not requirelabeling and target modification prior to analysis. Low-cost SPRbased bio-sensing platforms are also commercially available forbio-diagnostics, food safety, environmental testing, or point-of-care diagnostics (e.g. Spreeta by Sensata Technologies). Thesetechniques are continuously being improved for their sensitivityto enable additional diagnostic applications, for instance, for thedetection of proteins characteristic of early disease states that areexpressed in low concentrations.

3.1.3.2. Surface enhanced Raman scattering (SERS). Raman spec-troscopy is a technique based on scattering due to molecular vi-bration and rotation and does not require labeling. The uniquevibrational spectra of specific molecular bonds enable the distinc-tion of molecules in a multiplexed manner. Raman signals, how-ever, are relatively weak and need to be amplified for most sensingapplications. Plasmonic interactions between surface chemistry anda nanostructured metallic surface are used to amplify the Ramansignals. Qian and Nie (2008) developed a method to improve thesensitivity and the amplitude of the signal by factors up to 1015times, enabling single molecule detection. Further enhancementcan be achieved by the additional labeling of targets with a dye ormetal nanoparticles or by controlling a target’s size and shape.

SERS detection in a microfluidic channel has some advantagesover conventional SERS detection under static conditions. Simi-larly, the measurement under flowing conditions preventsproblems of variable mixing times, scattering geometry, localizedheating, and photo-dissociation. Flow conditions yield morereproducible results than static conditions because of the moreconsistent geometries and heat dissipation (Chen and Choo,2008). In one example, Park et al. (2005) developed an alligatorteeth-shaped PDMS channel to promote mixing between thetarget analyte and the metallic particles used as SERS enhancers.This technique increases the signal-to-noise ratio by excludingbackground noise due to the Raman signals generated by PDMS(Park et al., 2005). In another example, Zhang et al. successfullyperformed cellular assays with SERS in a microfluidic chip. Theycharacterized cellular drug response of a single Chinese hamsterovary (CHO) cell (Zhang et al., 2008). Additionally, airbornemolecules have been detected with a SERS-based gas sensor. Thedevice consists of an open microchannel (free-surface fluidics),designed for confining a silver nanoparticle solution flowing at aconstant rate by surface tension (Piorek et al., 2007). For moreextensive reviews on the combined use of SERS in microfluidicdevices, readers should consult recently published reviews on thisspecific subject (Chen and Choo, 2008; Huh et al., 2009).


SERS is ideal to simultaneously detect multiple species and hasvery good sensitivity and specificities, which make it a viablecompetitor for fluorescence detection. Microfluidics combinedwith SERS detection enables accurate and reproducible results,due to its ability to maintain continuous flow and homogeneousmixing conditions. The main bottleneck of this technique is thedifficulty to prepare homogeneous SERS enhancers nanoparticlesand the size of Raman systems, usually composed of bulkycomponents. This technique shows great promise for highlysensitive detection of chemical and biological analyses, and hasbeen successfully translated into a commercial handheld sensorfor analyte detection relevant to homeland security (e.g. Spectra-Fluidics). The development of SERS-active nanoparticles fordetection of cancer markers on circulating cells (Sha et al.,2008) or pathogen DNA (Kang et al., 2010) makes this techniquevery attractive for clinical diagnostics.

3.1.4. Mechanical and electrical detections

3.1.4.1. Mass-sensors based on resonance-frequency shifts.

Cantilevers and quartz crystal micro-balance are two techniquesrelying on a change in resonant frequency to detect a mass change.Cantilevers can be used to detect molecular binding by measuringchanges in cantilever bending or resonance frequency. Althoughdamping, viscosity effects and thermal fluctuations make it moredifficult to use in fluids, integration of cantilevers with micro-fluidics has some advantages. A microfluidic-based multiplexedassay format enables faster sample delivery to the integratedcantilevers due to the small diffusion lengths in micro-systems,and facilitates the monitoring of multiple cantilevers in parallel.Each chamber can be addressed independently by a differentanalyte. Each cantilever-based sensor can also be separatelyfunctionalized by flowing different solutions through each cham-ber (Lechuga et al., 2006). To overcome the fluid damping draw-back, Burg et al. (2007) designed a hollow cantilever and instead ofhaving cantilevers in fluids, had fluids inside cantilevers andcantilevers in vacuum; they successfully demonstrated the abilityto measure the weight of single cells and single nanoparticles inthis system. Using a similar approach and dynamic microfluidiccontrol to trap living cells, the instantaneous growth rate of in-dividual cells has been measured for the first time (Godin et al.,2010). In another work by the same group, a cancer biomarker wasdetected with a detection limit of 10 ng/mL in undiluted serum,using microcantilevers functionalized with carboxybetaine-de-rived polymers with embedded microchannels (von Muhlen et al.,2010) (Fig. 6D). The functionalization of the surface of the re-sonator reduces unspecific binding and the sensitivity of thistechnique is comparable to conventional ELISA assays.

Quartz crystal microbalance (QCM), more widely known asacoustic wave sensors (AW), is an established, commerciallyavailable, low-cost mass sensor. For the purpose of bio-detection,the surface of a quartz crystal microchip is coated with antibodiesagainst the target biomolecules. The binding of the target agentsresults in an increase in mass that lowers the chip bulk resonantfrequency. Compared to some common bio-sensing technologies,such as SPR, optical fibers, and sensors based on field effecttransistors or cantilever-based detectors, AW-based technologieshave the combined advantages of simple operation (electricalsignal), high sensitivity, small size, low cost, and no need for bulkyoptical detection systems (Lange et al., 2008). Hayden et al. used aQCM for the detection of tobacco mosaic virus, using artificialreceptors based on a molecularly imprinted polymer (MIP)technique (Dickert and Hayden, 2002). Such polymer receptorshave greater stability and thus much longer shelf life thantraditional antibody-functionalized QCM chips, making them aviable alternative for point-of-care use. To increase sensitivity,Cooper et al. (2001) found a novel mode of operation for QCM in a

technique called rupture event scanning, enabling the detection ofa single infectious particle on a chip. To further improve therobustness of the devices and the detection sensitivity, surfaceacoustic wave (SAW) sensors have been developed which exploitshear surface wave techniques. Integrating microfluidic channelson a SAW surface allows multi-analyte detection on a single SAWdevice (Mitsakakis et al., 2008).

As these examples demonstrate, the sensitivity of cantileversand QCM sensor is limited by unspecific binding and dampening inliquids, but new functionalization processes and clever designsmay overcome these limitations. The sensing mechanism of bothtechniques relies on the accurate measurement of frequency shiftsin resonance, requiring complex electronic circuitry that could beeasily completed with additional circuitry. The inherent small sizeof the resonator beams and further integration to electronics couldmake it an ideal candidate for cheap POC diagnostics.

3.1.4.2. Nanowires. Nanowires can be used to detect molecularbinding events through changes in the binding that induces in thewire’s conductance. They require low volumes of samples, enablereal-time sensing with a very high sensitivity (down to the fM)and can be very easily multiplexed. Using a microfluidic chip in-tegrated with DNA-coated nanowires, researchers have measuredDNA hybridization kinetics (Bunimovich et al., 2006; Nie et al.,2007) and detected cancer biomarkers (Zheng et al., 2005). Formore details, the reader can refer to a recent review (He et al.,2008). Nanowires need to be integrated with microfluidics, so thatthe fluid containing the target molecules can be delivered to thenanowire, and to protect the detection electronic circuits from theliquid. An interesting theoretical study by Kim and Zheng (2008)shows how modifying the microfluidic channel to allow bettermixing can further improve the response time of nanowires.

3.1.4.3. Dielectric spectroscopy and field flow fractionation. Di-electric spectroscopy relies on the measurement of the dielectricproperties of a material as a function of frequency. It is a powerfultechnique to elucidate a number of important biophysical cellproperties, with the ability to provide information about the cell,including morphology, physiological state, and viability. Vykoukalet al. (2009a) used a microscale device to determine dielectricproperties for each of the six main leukocyte subpopulations.They showed differences in the dielectric properties of the variouswhite blood cell subpopulations. This label-free method could bea good addition to tools for flow cytometry in drug screening orcell separation (Vykoukal et al., 2009a).

Vykoukal et al. (2009a) also worked on dielectrophoretic fieldflow fractionation (DEP-FFF) to isolate circulating tumor cells(CTCs) from clinical blood specimens. This method is not only ableto exploit intrinsic tumor cell properties so that labeling isunnecessary, but also delivers unmodified, viable tumor cells forculture and/or all types of molecular analysis (Gascoyne et al.,2009). Kim et al. (2007b) used a similar method to select cellsbased on cell cycle phases. They created the dielectrophoresisactivated cell synchronizer (DACSync) device, which accepts anasynchronous mixture of cells at the inlet, fractionates the cellpopulations by exploiting the relationship between the cell’svolume and its phase in the cell cycle (G(1)/S and G(2)/M), andelutes them through different outlets. Such a device could beuseful in cancer drug studies, where the tumor cell response to adrug depends on its phase in the cycle.

Vahey and Voldman (2008, 2009) developed a new micro-fluidic method called isodielectric separation (IDS) for simulta-neous separation and particle characterization. Particles areseparated according to their isodielectric points in a buffergradient. This technique is similar to isoelectric focusing (IEF),where particles can be separated depending on the net charge at

Fig. 7. (A) Diagram of layered membrane-based LOC sensor designed to separate cells according to size. (b) Cross-sectional view of membrane-based LOC sensor shows

simple fluid flow path allowing cell capture and delivery of reagents for on-membrane assays. (A–B) Weigum et al. (2007)—reproduced by permission of The Royal Society

of Chemistry. (C) An integrated blood barcode chip (IBBC) that separates plasma from whole blood, allowing for in situ protein measurement. Reprinted by permission from

Macmillan Publishers Ltd: Fan et al. (2008), copyright 2008. (D) A complex polycarbonate microfluidic system for saliva-based bacteria detection contains many

compartments for on-chip analysis, including fluid modules for cell lysis, and nucleic acid analysis and labeling as well as lateral flow strips for nucleic acid detection. Chen

et al. (2007)—reproduced by permission from John Wiley and Sons.


different pH conditions. IDS enables separation of cells based ondifferences in the electrical properties of the cell membrane orcytoplasm, such as live versus dead bacteria and mammalian cells,and flow fractionation enables such separate to be continuousrather than batch-wise.

In summary, integrated electromechanical sensing with micro-fluidics offers great promise towards portable, cheap, and reliablePOC devices. The main bottleneck is the difficulty to fabricate thesensors en masse, with its integration with microfluidic sub-strates, as the fabrication process is not necessarily compatiblewith conventional microelectronic fabrication processes.

3.2. Cell-based assays

One of the major application areas of microfluidics is in cell-based assays for disease diagnostics. Cell analysis and processingtechniques for cell-based assays and biosensors include cellsorting, separating sera from cells for immunoassays, and celllysis for immunoassays or for nucleic acid amplification in avariety of disease detections (Daniel et al., 2009; Du et al., 2006;Fan et al., 2008; Weigl et al., 2008). These devices (as exemplifiedin Fig. 7) allow for the miniaturization of equipment, improvedresponse times, and simplified analysis procedures (Hennig et al.,2009; Park and Shuler, 2003; Sung et al., 2009; Zurgil et al., 2010).While cell-based biosensors are related devices, which are usedfor monitoring physiological changes induced by exposure toenvironmental perturbations (Stenger et al., 2001), they will notbe the focus of this review.

3.2.1. Cell separation by physical characteristics

Cell sorting is of interest for many diagnostic applications (e.g.identifying diseased cells from blood) as well as for fundamentalbiological research. Sorting can be based on physical parametersincluding cell motility, size, deformability, and biochemicalcharacteristics such as surface markers. Many groups havesuccessfully separated cells in small volume samples usingmicrofluidic devices. For example, one device used to study andsort motile cells has proven effective as a cell-based cancer assay(Mahmud et al., 2009). Ratchet-shaped micropatterns in thedevice and the biased motions of different cells in response toenvironmental and geometric cues resulted in the partial sortingof mixed cell populations. This device has been used to sortcancerous versus non-cancerous cells into separate microreser-voirs. Similarly, Huang et al. (2008) developed a microfluidicdevice to assess fetal anomalies or pregnancy complications inpregnant women by efficiently isolating nucleated red bloodcells (NRBCs) from a small blood sample. The separation isbased on three parameters: (1) cell size, (2) ability to deform, and(3) hemoglobin content and relies on the paramagnetic propertiesof NRBCs. This high-throughput and highly efficient microchipprovides a viable alternative to other complex strategies forisolating NRBCs that often result in low NRBC recovery and lessthan optimal cell purity. Another example is a simple andcompact device to be used as a POC tool to detect clinicalproblems like anemia (Kim et al., 2009a). The glass microfluidicchip is a cell counter that evaluates the red blood cell (RBC) countin a small volume of blood to obtain information about thenumber and size of RBCs in the sample. In these examples, the


ability to control device geometry and design while minimizingshear and the incorporation of standard laboratory analysistechniques into microscale features that are in the same orderof magnitude as cells are what enabled the successful separationsfor diagnosis.

3.2.2. Cell capture on-chip

3.2.2.1. Cell capture based on cell-specific surface markers. Cell-specific surface markers can be exploited in cell detection for thediagnosis of a variety of diseases such as cancer or immunologicaldiseases. Many microfluidic devices use surfaces coated withantibodies specific to cell surface markers to capture cells of in-terest. Nagrath et al. (2007) demonstrated a device with antibody-coated pillars that can capture circulating tumor cells (CTCs)overexpressing epithelial cell adhesion molecule (EpCAM)from blood samples. A PMMA-based lab-on-a-chip sensorused a size-selective membrane to trap cells that were thenimmunofluorescently assayed for cancer biomarkers, such as theepidermal growth factor receptor (EGFR) (Weigum et al., 2007)(Fig. 7A,B). Cell presence and isotype can be detected via auto-mated microscopy and fluorescent image analysis. This techniqueallows for the rapid analysis of biomarkers associated with earlytumorigenesis and aggressive cancer phenotypes with resultscomparable to conventional labeling methods. This LOC sensorsystem provides rapid detection of cancer biomarkers and may beclinically useful in cancer diagnostics for early detection andprognostic evaluation. Du et al. (2009) has developed a PDMS-based device that includes a6-integrin treated microchannels thatbind up-regulated a6-integrin cell surface receptors on humanpapillomavirus (HPV) type-16 infected cells. In this way, humancervical cancer cells are preferentially captured in the micro-channels when a suspension of human cancer cells with up-regulated a6-integrin receptors and normal cells are flowedthrough the device.

One design employed poly(ethylene glycol) (PEG) hydrogelmicrowells coated with specific antibodies to capture T-cells fromRBC depleted human blood (Zhu et al., 2009). Another microfluidicsensor captures CD4+ T-cells in an antibody-coated channeldownstream from a CD14+ monocyte trapping chamber. Thisdouble-stage cascaded device offers a simple and cheaper viablealternative to flow cytometry for efficiently assessing T-cell countsin HIV-positive patients. This device could be especially useful as adiagnostic tool in the field as it eliminates the need for acumbersome flow cytometer (Cheng et al., 2009). While both ofthese devices require very little blood volume for analysis, it shouldbe noted that the blood sample must be preprocessed, either on-chip or off-chip, before the cell separation can be performed.Comparatively, Adams et al. (2008) developed a PMMA-basedmicrofluidic device able to analyze whole blood samples to detectcells overexpressing EpCAM. While the captured cells must undergooff-chip analysis to further diagnose the exact cancerous cells, thissimple device is cost-effective and requires no sample preproces-sing, and therefore could prove to be a potentially useful diagnostictool for monitoring cancer cell levels in blood.

3.2.2.2. Using aptamers for cell detection. Besides antibodies, ap-tamers have been recently added to the repertoire of molecularrecognition tools for detection and separation applications. Xuet al. (2009) developed a simple and inexpensive PDMS-baseddevice that can detect multiple cancer cell types based on theselective cell capture of immobilized DNA-aptamers. The devicehas aptamers for various leukemia cell lines at different regionswithin a channel to create up to three isolated specific cell type-capture zones. Notably this device does not require sample pre-treatment before analysis due to the high selectivity and specifi-

city of the aptamers to target and bind only cancer cells, which isimportant in POC sensor usage. In general, the detection of mul-tiple cancer cells can be used to detect cancers at early stages,diagnose metastatic relapse, stratify patients for therapeuticpurposes, monitor response to drugs and therapies, track tumorprogression, and gain a deeper understanding of the biology ofCTCs. Although significant progress has been made in the past fewyears to develop more selective isolation methods, few of thesesensors have been fully integrated with sample pre-processingchambers in a completely portable fashion. Furthermore, the shelflife and accessibility of such sensors is limited by the stability,availability, and cost of the capturing agents.

3.2.3. Integrated molecular detection for diagnostics

3.2.3.1. Detecting protein concentration in plasma. Plasma proteinprofiles are often associated with many human diseases. To detectthe presence of specific markers, some methods require the removalof cells from whole blood, while others require the additional se-paration of plasma (Fan et al., 2008; VanDelinder and Groisman,2006). Fan et al. (2008) has engineered a device that enables on-chipblood separation and rapid measurement of a panel of plasmaproteins from small quantities of whole blood. The plasma is furtheranalyzed in an immunoassay region of the channel customized forthe detection and quantification of various proteinds (Fig. 7C). It hasbeen used to detect human chorionic gonadotropin (hCG) fromhuman serum and to stratify 22 cancer patients. This device po-tentially represents a realistic clinical diagnostic platform for rapidmeasurement of a panel of plasma proteins from a small amount ofblood in a minimally invasive, low-cost, and robust procedure. Thischip can also be altered to assay a multiplexed panel of plasmabiomarker proteins to provide a high throughput and accurate de-vice that can be utilized in multiplexed blood diagnostic measure-ments in remote locations by minimally trained individuals (Qinet al., 2009; Tonisson et al., 2000).

3.2.3.2. Genetic analysis. While proteins are good disease markers,DNA analysis on-chip can be used in disease diagnosis with agenetic basis. Polymerase chain reaction (PCR)-based amplifica-tion and analysis can provide information on patients’ gene ex-pression profiles. PCR is typically coupled with reversetranscription (RT) and capillary electrophoresis (CE), all of whichhave been the subjects of miniaturization in the last couple ofdecades (Bianchi et al., 1997; Burns et al., 1998; Harrison et al.,1993; Livache et al., 1998; Noble, 1995; Yoon et al., 2000). Recentprogress has been made on applying these techniques for specificdiseases. For instance, Kaigala et al. (2008) designed a systemto amplify specific genes in a cancer cell line. This system isboth portable and cost-effective and can be used to perform ge-netic amplification and analysis — the basis of many medicaldiagnostics. This system does not include a means to extractand concentrate nucleic acids from raw clinical samples likewhole blood; however, a sample preparation technique couldbe integrated into the system along with strategies to increasethe rapidity of tests to make this device suitable for a wide varietyof real-time diagnostics in the clinic or in the field. In a differentexample, a PCR amplification-based method has been developedand coupled with genomic DNA extraction from whole bloodand microchip separation of the PCR fragments for Duchennemuscular dystrophy (DMD) diagnosis (Ferrance et al., 2003).This method provides a faster and less labor-intensive techniqueas compared to the traditional Southern blot diagnosis ofDMD.

As suggested above, DNA analysis can be used to detectpathogens by genetic finger-printing (Lagally et al., 2004; Leeet al., 2003b). Currently, one challenge of the early detection of


pathogens is the limited ability to detect pathogens at extremelylow concentrations. In some cases, culturing the pathogens isdifficult and time consuming. The use of microfluidic diagnosticsfor pathogen detection by DNA-amplification and analysis wouldenable quick diagnosis, treatment, and isolation of infectedpatients (Koh et al., 2003; Lagally et al., 2004). These microfluidicdevices efficiently and effectively detect pathogens while provid-ing many advantages over typical pathogen detection methods byreducing the sample volume, reducing the risk of samplecontamination and clinician infection, and improving analysisand diagnosis speed. For instance, a system has been developed inwhich reagents can be introduced to trapped cells for cell lysisand reactions for on-chip genetic detection (Lagally et al., 2005).The detection scheme makes use of sequence-specific hybridiza-tion of an rRNA-directed optical molecular beacon. The research-ers demonstrated the detection of specific strains of Escherichia

coli cells. One advantage of this approach is that this system couldbe combined with PCR for detecting pathogens based on multiplecriteria simultaneously. Chen et al. (2007) designed a polycarbo-nate system for detecting bacterial pathogens in oral fluidsamples (Fig. 7D). This integrated system has fluid modules forcell lysis, nucleic acid extraction and isolation, PCR, and labelingof the PCR product for detection on a lateral flow strip. The lateralflow strips provide fast, convenient and low-cost POC and hometesting. This technology has been shown to label and detectmicrofluidicPCR products from Gram-positive bacterial targets; it could easilybe adapted for the detection of other viral and bacterialpathogens, and potentially used for multiplexed analysis ofseveral pathogens. Another example is a plastic cyclic polyolefinlab-on-a-chip platform that purifies viral RNA (Bhattacharyya andKlapperich, 2008). The device can lyse viral particles andsubsequently concentrate and then elute nucleic acids fromlysate of influenza A (H1N1) infected mammalian cells. Thepurification process is fast (�10 min), simple, requires minimaluser-handling and could potentially be performed by a minimallytrained individual.

The ultimate large-scale genetic analysis is whole genomesequencing, which has benefited from the development of micro-and nanofluidic technologies (Blazej et al., 2007; Huang et al., 2002;Thaitrong et al., 2009; Toriello et al., 2007). Several academic andcommercial groups have now used second-generation sequencingtechnologies to drive down the cost and increase the throughput ofgenomic sequencing (Mardis, 2008; Shendure and Ji, 2008). AsShendure and Ji point out, microfluidic chip based electrophoreticsequencing when integrating all sample processing steps, couldpotentially impact point-of-care diagnostics. Alternatively, droplet-based microfluidics (such as those developed by academiclaboratories (Ahn et al., 2006; Baret et al., 2009; Kim et al., 2007a;Link et al., 2004; Utada et al., 2005) and Raindance Technologies) incombination with PCR for targeted sequencing could potentiallycompete with current sequencing technologies.

In summary, medical diagnostic tests provide a result, usuallyof the presence (or absence) or concentration of a molecularspecies contained in a tissue or bodily fluid. Simple POCdiagnostics typically analyze blood, saliva, or other cellularmaterial. They must be capable of measuring a parameter directlyin a sample with relatively minimal interference from othercomponents of the fluid or without requiring the performance of avariety of preparative steps prior to analysis (Weigl et al., 2001).As shown through the examples above, the incorporation ofmicrofluidics into diagnostic devices can elegantly reduce thecomplexity of a diagnostic test by combining many steps towardspoint-of-care testing for the diagnosis or management of disease.To increase the number of these devices available in field use,future research efforts should focus on incorporating both sample

pre-processing, detection methods on-chip, and continue to drivedown the cost.

4. Conclusions

Here we have reviewed some recent developments in micro-fluidics for medical diagnostics and bio-sensing. Although muchprogress has been made towards applications in the medical fieldsince the conception of microfluidics, the current state-of-the-arttechnologies in this field are not yet capable of field- or point-of-useapplications; in contrast, microfluidics developed in academic labsover the years made tangible contributions to basic sciences. As thehealth care costs continue to escalate, inexpensive, reliable, andeasy-to-use devices are likely the next-generation tools thatbiomedical diagnostic industries and field users anticipate. Webelieve academic research in microfluidics should continue to pushthe frontiers of the development in new materials, processes, andfunctionalities; furthermore, research should be directed towardsstandardization, automation, and high throughput.

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Microfluidics for medical diagnostics and biosensors

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