Annals of Biomedical Engineering, Vol. 42, No. 11, November 2014 (Ó 2014) pp. 2289–2302 DOI: 10.1007/s10439-014-1060-2 Magnetic Droplet Manipulation Platforms for Nucleic Acid Detection at the Point of Care DONG JIN SHIN1 and TZA-HUEI WANG1,2,3,4,5 1 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA; 2Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA; 3Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA; 4Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA; and 5 108 Latrobe, 3400 N Charles St, Baltimore, MD 21218, USA (Received 28 February 2014; accepted 19 June 2014; published online 10 July 2014) Associate Editor Tingrui Pan oversaw the review of this article. based microfluidic devices,61 capillary pump-driven microfluidic devices16,19 and droplet-based platforms.16,32 In addition to the traditional benefits of microfluidics like reduced sample consumption and integration, point-of-care diagnostic platforms should also consider factors such as sample preparation, simple end-user operation and portable instrumentation. Among these approaches, droplet-based techniques are of particular interest since aqueous reagents are readily scalable in a droplet format without radical modification to biochemical processes, which makes them easily adaptable to a wide range of current and future benchtop assays. Droplet microfluidic platforms have emerged under various formats, and can be broadly categorized under open-surface and channel-based formats. Furthermore, platforms may be categorized based on the mode of droplet or particle manipulation, some of which include magnetic,2,7,13,27,29,32,34,36,37,39,43,44,52,63–65,67 dielectrophoretic (DEP),15,22,53 electrowetting-ondielectric (EWOD)8,14,20,30,38,47,48,50,55 and optical12,35 approaches. This review focuses specifically on opensurface magnetic droplet platforms that utilize magnetic forces on particles as a means of both droplet and particle manipulation. This is in contrast to other droplet platforms utilizing EWOD and DEP as the primary mechanism for droplet handling, where the incorporation of magnetic particles requires an auxiliary mode of actuation for magnetic manipulation. Magnetic particles are regularly utilized in biochemical assays as solid phase substrates for analyte capture and purification, which highlights the importance of magnetic manipulation in these platforms. Magnetic droplet platforms are particularly well-suited to nucleic acid-based assays for two reasons. Firstly, nucleic acid Abstract—This review summarizes recent developments in the use of magnetically actuated droplets in point-of-care molecular diagnostic platforms. We discuss the fundamentals of magnetic droplet manipulation and the various modes of actuation. The balance of forces acting on a droplet during transport and particle extraction, as well as the devices and instrumentation developed to perform these operations will be presented and discussed. Furthermore, we review some of the recent advances on the diagnostic applications of platforms utilizing magnetic manipulation for genetic assessment of biological samples. Keywords—Magnetofluidics, Droplet manipulation, Electromagnetic actuation, Surface patterning, DNA extraction, Real-time PCR. INTRODUCTION Recent advances in bioanalytical techniques have enabled researchers to develop assays that are both simple and robust. These developments have paved the path towards integrated platforms designed to perform all aspects of molecular diagnostic assays, from sample preparation to detection, on a single device. Taking advantage of these advancements, commercial development of plug-and-play benchtop platforms has already made significant progress in recent years, as demonstrated in products such as Cepheid GeneXpert platform62 and BioFire FilmArray platform.5 Meanwhile, efforts to further miniaturize assays for mobile medicine have also led to shifting paradigms in academic research towards approaches such as paperAddress correspondence to Tza-Huei Wang, 108 Latrobe, 3400 N Charles St, Baltimore, MD 21218, USA. Electronic mail: biomems123@gmail.com, thwang@jhu.edu 2289 0090-6964/14/1100-2289/0 Ó 2014 Biomedical Engineering Society 2290 SHIN purification involves multiple steps of buffer aspiration and addition, which is greatly simplified on a magnetic droplet manipulation platform as a series of particle extraction steps across various buffer droplets.63 Secondly, the issue of contamination with amplificationbased assays such as PCR makes disposable cartridges an essential aspect of device design,32 and the simple layout of a magnetically actuated droplet device confers an advantage over other devices that require components such as electrodes to be fabricated directly on the cartridge. In this review article, the theoretical basis of opensurface magnetic droplet platforms will be discussed in order to highlight the physical basis for magnetofluidic droplet manipulation. Fundamentals of droplet manipulation operations are described as a function of parameters such as droplet volume and particle size, which are important topics in the context of on-chip process design. Principles of device fabrication and various modes of magnetic actuation will be discussed, followed by examples of their application in assessment of genetic biomarkers. PRINCIPLES OF MAGNETOFLUIDIC DROPLET MANIPULATION Magnetically actuated droplet microfluidic platforms have an interdisciplinary origin, as they are the product of recent progress in magnetofluidics, microfabrication and macromolecule purification techniques. From a magnetofluidics perspective, early studies of manipulating liquid containing magnetic particles by using a permanent magnet44 or an electromagnet39 paved the way for other investigators to build on the fundamental mechanisms. Additional progress in device fabrication techniques via surface patterning23 and micro-elevations defined by soft lithography63 enabled integration of multiple reagents in order to recreate the entire workflow of complex bioassays on a single device. A typical magnetically actuated droplet platform as shown in Fig. 1 may include a hydrophobic substrate, on which aliquots of reagents utilized at various stages of a multi-step reaction are primed as sessile droplets. These droplets are separated by passive elements that function as valves that facilitate extraction of magnetic particles from a droplet. The surface-modified magnetic particle serves as a solid phase substrate on which analytes can be purified and transported across reagents at various stages of sample processing. Droplet transport, fusion and magnetic particle extraction are essential operations in any biomolecular assay that involves the addition of reagents and aspiration of buffer solutions from magnetic particles. AND WANG FIGURE 1. Sketch of a typical magnetically-actuated droplet device. Droplets are arranged serially on a hydrophobic substrate and sealed with oil. Passive valves for particle extraction from buffer are realized in this example using topographical barriers. In this example, extraction of nucleic acids and DNA amplification are combined into a singlestream process to integrate all sample processing steps involved in a PCR-based assay on a single device. While the specifics of their implementation may vary across platforms, droplet manipulation on an open surface can be generalized as the interaction of three forces. The balance of these components determines the type of droplet handling operation being performed. The first component is the magnetic force, which is a consequence of paramagnetic bead cluster being attracted by an external magnetic field. For magnetic particles clustered in a nonmagnetic medium, the force acting on the particle cluster can be obtained as follows42:   M B r B: FM ¼ v q l0 Here, M and q represent the mass and density of magnetic particle cluster, x is the magnetic susceptibility of the particles, B is the applied magnetic field, and l0 is the permeability of free space. In magnetbased droplet manipulation, this component is the only parameter that is actively modulated on the device. As such, all operations are facilitated either by modulating travel velocity or by introducing passive structural components that can modify the other two force components on the droplet. Magnetic force is directly proportional to the mass of the magnetic bead cluster, which highlights an important design parameter for magnetic droplet-based platforms. Because magnetic particles also serve a functional role as the solid substrate on which biochemical analytes are captured, large particle load also increases the functional surface area of the system. However, increase in particle load is accompanied by a concomitant increase in the volume of liquid carried by the magnetic particle cluster, which may result in contamination of subsequent steps in complex bioassays.13 The second component is the drag or frictional force, which describes the friction imposed on the droplet by physical impediments such as solid surfaces Magnetic Droplet Manipulation Platforms and barriers in contact with the droplet. This force is described by the following equation29: Ff ffi Kf Rb l U: Here, Kf is the frictional constant, Rb is the radius of contact area between the droplet and the substrate, l is the viscosity of surrounding oil medium and U is the velocity of the droplet. From a design perspective, frictional force can become a hindrance to efficient droplet transport, especially when the magnetic force is smaller than frictional force. This is the case with rugged surfaces as well as substrates with high surface energies, which results in an increase in the frictional constant and the contact area. Open-surface droplet microfluidic platforms address this issue by using planar surfaces coated with commercial fluoropolymers such as amorphous Teflon13,27,36,47,63,67 and Cytop30,55 or chemical vapor deposition with chemicals such as parylene14 and organosiloxane (SiOC).50 Coating the substrate with low surface energy chemicals has additional functional roles as a means of preventing contamination via fouling, as well as preventing loss of biological molecules via adsorption to the surface. A third component is the capillary force, which arises at deformities on the surface of the droplet. This force is particularly relevant for its involvement in droplet fission, which generally takes place after a process of deformation and necking. The maximum capillary force that can be sustained before the droplet of volume V undergoes fission can be followed from derivation by Shikida et al.44:
1 Fc;max ffi c 6p 2 V 3 : Here, c describes the interfacial tension between droplet and the surrounding oil medium. From a design perspective, reducing this component allows the magnetic particles to dissociate from the parent droplet 2291 more easily. This can be facilitated by the addition of surfactants in the oil medium, which may be a necessary condition for fission of magnetic particles from fluids with a high interfacial tension. Optimizing the concentration of surfactants and particle volume is an important process in developing a robust droplet manipulation platform. Chiou et al.13 characterized the effect of varying bead volume and surfactant concentration on the robustness of their droplet fission operation and observed that an increased surfactant concentration is also accompanied by droplet instability, where reagent priming eventually becomes challenging. Designing a droplet microfluidic platform capable of performing multiple fluidic handling operations requires an intuitive understanding of the interplay between the three forces as shown in Fig. 2. These interactions have been investigated by Long et al.29 for open-surface droplet manipulation on a planar hydrophobic substrate, in the form of an ‘operation diagram’ where the relative magnitudes of forces as a function of magnet velocity and particle load result in a particular droplet operation. In this classification scheme, the interaction between the droplet containing magnetic particles and a permanent magnet are categorized under three regimes. The first is particle extraction, which occurs when the magnetic force exceeds the maximum capillary force holding the magnetic plug and the parent droplet together. The second is droplet transport, where the magnetic force is insufficient to overcome the capillary force but is sufficient enough to overcome the friction force acting between the droplet and the device surface. The last regime is droplet disengagement, where the magnetic force is less than both the friction and capillary forces and no further manipulation is achieved by the magnet. In a later work, Zhang and Wang67 described an expanded operation diagram as shown in Fig. 3 to FIGURE 2. (a) Operating diagram developed by Long et al. as a function of particle loading and magnet velocity. OB1, OB2 and OB3 are the operating boundaries separating each operation regions. (b) Top-down schematic of three forces acting on a droplet containing magnetic particles. In general, droplet kinematics involves the relative balance between magnetic force on the particle (FM), friction force (Ff) and capillary force (Fc). (adopted from Ref. 29). 2292 SHIN AND WANG FIGURE 3. Operating diagram developed by Zhang et al. as a function of particle loading, droplet volume and SET dimension. With a fixed travel velocity, droplet manipulation operations depend only on the static parameters including droplet volume, particle amount and SET size (from Ref. 67). include surface patterns that can dramatically enhance frictional forces in order to facilitate a full range of droplet handling operations. The selective patterning of hydrophobic substrate with hydrophilic surface energy traps (SETs) result in an additional force component attributed to surface tension arising from the wetting of the droplet on the SET. In this arrangement, uniform droplet dispensing in a series of SETs becomes possible for applications including serial dilution and aliquoting.65,67 Because surface tension is not a function of velocity but rather the perimeter length of SET, magnet travel velocity can be decoupled from operations such as particle extraction and the operation diagram becomes a function of three static parameters including droplet volume, SET dimension and particle load. This is a significant advantage from a platform design perspective since highly precise instrumentation is no longer essential to achieve particle extraction and droplet transport in a robust manner. DEVICE AND INSTRUMENTATION Designing and realizing an integrated bioassay platform from the basic principles highlighted in the preceding section requires several engineering problems to be addressed. Firstly, the device must have a mechanism to facilitate extraction and transport of magnetic solid substrates across multiple reagents in a robust manner. Secondly, the magnetic particles must be actuated by a mechanism that is robust and operator-independent. Lastly, appropriate supporting instrumentation for thermal incubation and signal acquisition must be in place. In this section, the discussion will focus on the modes of magnetic actuation, device fabrication, thermal control and signal acquisition. Magnetic Actuation Mechanism The most intuitive method of magnetic particle actuation is by using a strong permanent magnet such as neodymium alloy (Nd2Fe14B) to actuate the magnetic particles from the reverse side of the device. In this approach, magnetic particles that are initially suspended in a droplet are concentrated by the applied field into a tight magnetic plug. Once formed, the motion of the magnetic plug along the device surface can be controlled by sliding the magnet on the reverse side. From a theoretical standpoint, the field gradient is essentially varying slowly in time due to the motion of the magnet, such that the force on the magnetic particles is directed towards the motion of the magnet. While manual handling of permanent magnet is feasible in some designs, other designs which depend on the steady control of velocity and position would require motorized instrument with feedback control. Moreover, automation of magnet handling has the added benefit of reducing variability due to the operator. Several modes of automated permanent magnet actuation have been proposed. Translational stages are employed in quantitative investigation of droplet kinematics due to their ability to precisely control the position and velocity of the magnet.29,34,52 Alternatively, a stepper motor with a permanent magnet mounted on the rotor may be employed to facilitate magnetic particle actuation in a circular motion37,43 as shown in Fig. 4. While actuation via a permanent magnet is the most intuitive method, there are some limitations. Firstly, the permanent magnet itself is not capable of switching polarity in a manner that can allow the particles to disperse. Although mixing of constituents in a single droplet may be achieved through internal flow introduced by oscillation of the magnetic particle or by Magnetic Droplet Manipulation Platforms 2293 FIGURE 4. Rotary actuation of magnetic particles using a stepper motor. (a) Device proposed by Shikida et al. incorporates permanent magnets alongside a coil electromagnet to enable particle agitation via magnetic repulsion. (b) Device proposed by Pipper et al. utilizes rotary actuation for thermal cycling between temperature zones H1, H2, H3 and H4 on a micro PCR platform. The length of the scale bar is 5 mm. (from Refs. 43 and 37). droplet transport,30,67 efficient sampling of the entire droplet volume by the magnetic particles would require an active mechanism for agitation. To address this, additional actuation mechanisms may be used in tandem with a single permanent magnet. For example, the instrument proposed by Shikida et al.43 utilizes a stepper motor, with two spokes attached with a permanent magnet and a coil electromagnet respectively, where the electromagnet is able to induce particle agitation and supplement the limitations of the platform solely based on permanent magnet actuation. Zhang and Wang66 developed a magnetic micro gyromixer that spins and balances itself on a droplet surface via a rotating magnetic field to mix the enclosed reagents by stretching and folding internal fluid streamlines in the droplet. In the method proposed by Tsuchiya et al.,52 two linearly actuated magnets on opposite sides of the droplet are used to facilitate agitation of magnetic particles as shown in Fig. 5. Because the magnetic particles can be gathered in two opposing directions inside the droplet, the droplet volume can be sampled more thoroughly by the particles. Another approach to magnetic actuation is by using an array of electromagnets to control the magnetic field across the device surface in an addressable manner. The lack of mechanical components and the ease of access to customized printed circuit boards make this an attractive approach from both miniaturization and commercial standpoints. This mode of magnetic particle actuation is realized using planar coil-induced magnetic field gradients in the presence of a uniform static field as shown in Fig. 6.39 Firstly, a uniform magnetic field is placed perpendicular to the plane of the device, causing the magnetic particles to become strongly polarized perpendicular to the device. Because the magnetic force is scaled by the dot product of magnetic moment and the applied field gradient, force is generated in the direction where the field gradient in alignment with the magnetic particle is maximized. The coils are capable of generating a magnetic field perpendicular to the device in two polarities, and the position of the maximum field gradient can be controlled by changing the polarities of current through each coil element. While a typical neodymium alloy magnet generates strong fields on the order of 102–103 mT, an electromagnet-based platform operates in the presence of approximately 50 mT static field with variations in local field around ±10 mT due to the coil electromagnet.13 Several groups have demonstrated the feasibility of electromagnetic droplet actuation. Among the earlier work, Rida et al.39 demonstrated the use of a coil-based microsystem for the transport of magnetic particles over a distance of millimeters, demonstrating the feasibility of electromagnetic actuation on the order of microliters. Subsequently, Lehmann et al.27 demonstrated transport of magnetic particles through multiple aqueous buffers by performing DNA capture with silica-coated magnetic particles using patterned surfaces combined with coil-based manipulation. Most recently, Chiou et al.13 demonstrated the use of topographical barriers in combination with coil-based magnetic transport in order to recreate the entire workflow for a real-time PCR assay from a biological sample. While electromagnetic manipulation provides great potential in miniaturizing the actuation mechanism, several technical limitations remain. The requirement for generating a field gradient of sufficient magnitude in order to overcome the other two force components acting on the droplet implies the use of high current to drive the coils. This gives rise to two technical bottlenecks that may be applicable to all mobile devices, namely power and thermal management. While a detailed discussion of these topics is beyond the scope of this review, it should be noted that thermal management is strongly linked to reducing the system power requirement since additional components such as thermoelectric cooling must be employed in order to offset the effects of resistive heating. 2294 SHIN AND WANG (a) (b) Forward primer Magnet Reverse primer Magnet handling channel 30mm 45 mm 3mm 3mm 3mm Reaction chamber 7 mm Mineral oil Magnetic beads 4 mm 0.5mm 0.5mm Template DNA or RNA PCR or RT-PCR mixture Magnet Magnetic Beads FIGURE 5. Mixing strategies on a permanent magnet-based platform. (a) In the example demonstrated by Zhang et al., a particle plug is oscillated back and forth in order to generate internal flow. However, the particle is confined to the volume proximal to the substrate and thorough sampling of the reagent by the particles may not be achieved. (b) In the droplet PCR system described by Tsuchiya et al., two magnets are used to sample the entire volume of the droplet more efficiently. (adopted from Refs. 52 and 67). FIGURE 6. Schematic view of electromagnetic manipulation for droplets containing magnetic particles. (a) Uniform magnetic field B generated by placing permanent magnets (3) on a highly field-permeable soft iron plate (4) strongly polarizes magnetic particle (1) perpendicular to the device plane (2). (b) Two layers of overlapping electromagnetic coil array generates magnetic field in the component perpendicular to the device, such that magnetic particle can be actuated along the field gradient. (from Ref. 39). Device Elements and Fabrication Magnetic droplet manipulation can achieve three operations including particle extraction, magnet disengagement and droplet transport as described by Long et al.29 However, velocity is a difficult parameter to control precisely, and a more robust mode of particle extraction is desirable for platforms designed to perform biomolecular assays. In order to aid this process, passive elements can be placed on the device substrate. Passive elements on these platforms are analogous to valves in the sense that reagent transport is controlled such that only magnetic particles can be extracted and transferred to subsequent reagents. Here, two forms of passive elements explored in the literature and their fabrication process are discussed. Topographical patterns are capable of serving dual roles as storage wells for the aqueous reagents and physical barriers to restrain the droplets during particle Magnetic Droplet Manipulation Platforms 2295 FIGURE 7. Demonstration of (a) topography assisted particle extraction and (b) SET-assisted particle extraction. (adopted from Refs. 63 and 67). extraction.63 During particle extraction, the narrow sieve structures formed between topographical barriers provide an outlet port for magnetic particles to be transferred across, as shown in Fig. 7. Because the maximum capillary force at the sieve neck is insufficient to maintain cohesion of the entire droplet past the barrier, droplet fission takes place and particle is extracted. In the regions that are not compartmentalized by topographical barriers, droplet transport can be performed as usual. Fabrication of topographical patterns can be achieved using various techniques. In an earlier work demonstrating the use of physical obstruction for particle extraction, Shikida et al.44 constructed devices by bonding layers of glass plates together with phenol resin, followed by hydrophobic surface treatment. However, glass devices are generally difficult to manufacture and prototype on a regular basis. Using an alternative approach, a more convenient soft lithography-based fabrication58 has been proposed by Zhang et al.63 and also utilized in a similar fashion by Berry et al.7 In this process, master molds with depth on the order of 1024 m are fabricated using SU-8 negative photoresist with planar features defined by a conventional photolithography process. The mold is subsequently cast with poly-dimethylsiloxane (PDMS), which is bonded via oxygen plasma activation to a glass substrate and dip-coated in amorphous Teflon in order to render the device surface hydrophobic. Alternatively, Zhang et al.63 demonstrated the use of a computer numeric controlled (CNC) machined PTFE mold for fabrication of pillar features on the order of 1023 m to be utilized as topographical barriers. More recently, Chiou et al.13 utilized rapid prototyping instrument to generate a casting mold for fabricating an extruded layer of PDMS. These fabrication approaches can significantly reduce the difficulty of generating surface topography and make it easier to prototype new designs. Platforms utilizing magnetically actuated droplets can also incorporate substrates with differential wetting properties in place of topographical barriers. Instead of confining aqueous droplets using structural barriers, hydrophilic spots are used to define regions where the droplets can wet selectively. One key advantage of this approach over the topographical features is the ability to aliquot reagents, since liquid is retained in the hydrophilic spot once wet. This feature is particularly useful in several applications, including spatial multiplexing of reactions and generating dilution series. Moreover, because the processing steps in which patterning takes place is subtractive rather than additive, this process is particularly attractive for fabrication in large batches. In general, substrates from which devices are constructed assume a hydrophilic nature due to the wide usage of glass. In an approach described by Inagaki et al. and utilized by Lehmann et al.,23,27 coating of hydrophilic substrate with hydrophobic film is followed by selective oxygen plasma etching to generate hydrophilic spots. More recently, Zhang and Wang67 developed an improved version of this technique by using lithographically defined SU-8 membrane as a shadow mask. In this work, full-range magnetic manipulation including sample aliquoting was demonstrated for simultaneously performing multiple PCR reactions on a single sample. In another instance, serial dilution was performed by using different sizes of hydrophilic spots followed by merging in diluent droplets.65 In a techniques explored by Hong and Pan, superhydrophobic surfaces have been formed using a mixture of SU-8 photoresist embedded with polytetrafluoroethylene (PTFE) nanoparticles, where large-scale features can be produced using standard soft lithography while taking advantage of extremely low surface energy afforded by 2296 SHIN AND WANG FIGURE 8. Thermal control on droplet platforms. (a) Schematic of a microheater designed for integration with a micro-PCR device, as proposed by by Neuzil et al. (b) A heat map showing temperature profiles on the surface of the micro-PCR device. The device utilizes three microheaters which maintain steady-state temperature at annealing, denaturing and extension temperatures. (c) Schematic of a static droplet micro-PCR device proposed by Angione et al. utilizes a single heating element underneath the reagent which cycles through three temperature zones over time. (d) Temperature profile of the static micro-PCR device during thermal cycling. Black line represents the controlled surface temperature and the red line is the calibrated droplet temperature. (from Refs. 32 and 3). PTFE.18 Alternatively, Schutzius et al.41 demonstrated hydrophilic patterning of a superhydrophobic substrate coated with fluoroacrylic-carbon nanofiber composite using wettable paraffin wax to generate virtual channels on a planar substrate. More recently, a highly facile technique was described by Xing et al.60 utilizing direct laser patterning of PDMS structure to generate superhydrophobic surfaces with contact angle up to 165°. A more comprehensive review of surface preparation for droplet manipulation is available in Ref. 31 Thermal Control Thermal control is an important parameter in assays which require incubation at elevated temperatures for enzymatic reactions. In particular, PCR requires cycling of reagent temperature between approximately 60 and 95 °C for primer annealing and duplex DNA denaturation. Thermal cycling for microdevices has been achieved using various mechanisms including contact heating elements3,9,17,25,28,59 and non-contact modes such as infrared heating21,26 and microwave heating.24 In the context of open-surface droplet platforms, two directions have been explored as shown in Fig. 8. In the first instance, thermal cycling is achieved in the manner of zone heating, where spatially separated zones are maintained at different temperatures required for thermal cycling. This method is more commonly found in channel-based microfluidic platforms17,25,28 since flow-based transport of reagents lends itself to thermal cycling via zone heating, although systems using a static heating chamber have also been reported.9,26 The liquid reagent is transported repeatedly across different temperature zones to alter the temperature. Implementation of this technique on a magnetic droplet platform requires the Magnetic Droplet Manipulation Platforms system to operate under droplet transport mode to ensure that transport of the droplets across temperature zones can be performed without accidental particle extraction. Neuzil et al.32 demonstrated a circularly actuated device with temperature zones maintained by microfabricated heating elements, while Ohashi et al.34 demonstrated a linearly actuated droplet device for thermal cycling reactions with a temperature gradient established by heating one edge of an aluminum plate. In the second instance, thermal cycling is achieved by a single heating element placed in contact with static incubation chamber which transitions through target temperatures in a manner that is analogous to a conventional thermal cycling instrument.3,59 This approach consumes smaller device footprint due to the static nature of the incubation zone and is also suitable in applications where an array of samples must be thermally controlled simultaneously.4 In a micro-PCR platform developed by Angione et al.,3 thermal cycling is achieved directly on a Teflon-coated indium tin oxide substrate via resistive heating. While this type of arrangement requires an integrated heater for each PCR device, the scheme adapted by Xiang et al.59 utilizes a glass-PDMS chamber cartridge that is loaded onto an external thin-film heater for thermal cycling. Similar approaches have been adopted by Zhang et al.63 and Chiou et al.13 for thermal cycling of genomic DNA extracted on their cartridges. There are several considerations in implementing temperature control elements in a magnetic droplet platform. In order to facilitate rapid transition of the reagent between temperatures, it is imperative that the thermal mass of the incubation region is minimized. In contrast to thermal cycling of the entire device, integrated microheaters placed directly in contact with the reagent can minimize the thermal mass involved to the reagent and its immediate vicinity. However, the integration of a microfabricated heater and temperature sensor may contribute significantly to the cost of each assay cartridge, and it may be prudent to incorporate heating elements on the instrument instead. When heating elements are placed outside the microfluidic device, care must be taken to thermally insulate the incubation zone and ensure proper thermal contact between the heating element and the device. Examples demonstrated by Pipper et al.36 and Angione et al.3 both utilize a small droplet of oil encapsulating the PCR reagent such that the reagent is protected from evaporation while maintaining a small thermal mass insulated by surrounding air. Signal Acquisition While open-surface fluidic manipulation can be made compatible with various biosensor technologies, optical modes are conveniently integrated into these 2297 platforms without affecting the droplet kinematics due to their non-contact nature. Furthermore, the prevalence of fluorescent probes and indicator dyes in bioassays has encouraged development of either luminescence or fluorescence-based approaches for signal acquisition. A typical acquisition system involves detectors such as photomultiplier tubes,3 silicon photodiodes63 or charge-coupled devices,59 while illumination sources may range from light-emitting diodes (LED)63 to halogen lamps.3 Optical setups for collection of fluorescence from a single collection spot are configured in an epifluorescence microscope configuration as shown in Fig. 9, where a dichroic beamsplitter is used to route excitation and emission light along the same objective lens. This arrangement lends itself to compact detector design suitable for portable instrument as demonstrated by Novak et al.18 POTENTIAL APPLICATIONS IN MOLECULAR DIAGNOSTICS The fluidic operations handled by magnetically actuated droplet platforms are well-suited to processing of magnetic particles through a sequence of aqueous reagents. In particular, magnetic particles with surfaces coated with glass or modified with silane have been utilized for extraction of nucleic acids.1,6 Solid phase extraction involving the adsorption of duplex DNA to silica is a convenient technique for purifying DNA from biological samples. Initially demonstrated by Vogelstein and Gillespie54 using glass beads, the incorporation of magnetic particles for the ease of handling has given rise to various formulations that are commercially available. In this section, droplet platforms demonstrating nucleic acid based assays will be discussed. For a more comprehensive review of magnetic particle technologies for nucleic acid isolation, readers are encouraged to refer to the following Ref. 6 Nucleic Acid Detection Via Polymerase-Based Signal Amplification Nucleic acid detection via amplification is an effective technique in identifying pathogens, and provides some key advantages over culture-based techniques including shorter turnaround time, assay sensitivity and the ability to assess fastidious samples.49 While DNA amplification-based analysis techniques have traditionally depended on time-consuming processes such as gel electrophoresis and Southern blotting, advancements in fluorescent probes have simplified the workflow such that post-amplification processes could be substituted by optical detection. Earlier designs by Ohashi et al.34 and Neuzil et al.32 demonstrated the feasibility of performing PCR in 2298 SHIN AND WANG FIGURE 9. (a) Epifluorescence microscope configuration. A collimated illumination source is reflected at a right angle by a dichroic beamsplitter and directed towards the objective lens, which focuses directly on the sample droplet. The fluorescence emitted by the droplet is collected by the objective lens and directed through the dichroic beamsplitter, which allows the emitted light to pass into the focusing lens and to be collected at the photodetector and converted into electrical signal. (b) Photograph and (c) schematic of a miniaturized fluorescence detector in epifluorescence microscope configuration as proposed by Novak et al. (adopted from Ref. 33). magnetically actuated droplets. Later platforms demonstrated by Pipper et al.36 and Zhang et al.63 utilize an open-surface design to combine solid phase DNA extraction and PCR amplification on a single device as shown in Fig. 10. Isolation of pathogenic nucleic acid targets and subsequent detection via real-time PCR demonstrates the potential of such platforms in mobile biosurveillance, including pathogen identification and viral load quantification. In addition, these platforms could be extended to perform multiplexed reactions in a single mixture with molecular beacon probes in order to detect a panel of pathogens simultaneously.10 An alternative method of detecting multiple gene targets is by performing amplification reactions from the same DNA isolate in separate reaction wells. To this end, Zhang and Wang67 developed a SET-based integrated platform capable of extracting and aliquoting genomic DNA before performing multiple PCR reactions simultaneously using unique primer sets. Genetic Mutation Detection Via Melting Curve Analysis Alongside pathogen identification, genetic mutation detection is also an important aspect of genetic diagnostics. While sequencing techniques provide the most Magnetic Droplet Manipulation Platforms 2299 FIGURE 10. Real-time PCR droplet platforms for integrated sample processing and detection. Zhang et al.63 (top panel) demonstrates amplification of the 16S ribosomal RNA gene from E. coli using a TaqMan real-time PCR assay. Chiou et al. (bottom panel) demonstrates real-time amplification of the KRAS oncogene target from genomic DNA that was extracted on chip directly from human whole blood13. comprehensive information regarding an unknown nucleic acid target, the high level of fidelity and time required to process sequencing samples preclude their use in point-of-care settings. In light of this, melting curve analysis has recently emerged as a potential alternative to sequencing in rapid diagnostic applications.51 This technique monitors the dissociation of double stranded DNA during heating by measuring the fluorescence emitted by DNA intercalating dyes as shown in Fig. 11.56 By monitoring the fraction of remaining double stranded DNA as the temperature is gradually increased, thermodynamic properties associated with the sequence are resolved as a function of temperature. Since the dissociation characteristics of a DNA strand are a function of base composition and length, samples containing mutations generate melting profiles that are different from their wild-type counterparts. Melting curve analysis is directly compatible with conventional real-time PCR workflow since it requires only a single universal primer pair for product amplification, in contrast with other PCR-based genotyping assays such as allele-specific PCR57 and allele-specific oligonucleotide probes40 which require additional probes for mutation identification. Several droplet platforms utilizing melting curve analysis for detecting variations in DNA sequence have been reported. In a recent study, Shin et al.45 demonstrated identification of a single nucleotide polymorphism in the KRAS oncogene using a melting curve based approach. In this work, open-surface magnetic droplet manipulation was used to combine DNA extraction and amplification via PCR into a single streamlined process. Another study by Athamanolap et al.4 demonstrated the utility of melting curve analysis in a droplet format by obtaining melt signatures from an array of droplets containing PCR amplicons with variations in DNA sequences. CHALLENGES AND PERSPECTIVES The main hurdles in bringing open-surface droplet platforms to commercialization are twofold. The first is reagent storage and transport, which is a challenge shared by many other technologies developed for pointof-care diagnostics. Although many of the buffers containing salts and other soluble, inorganic substances may be kept at room temperature without degradation, fragile organic components such as enzymes may expire when stored outside a freezer. Enzymatic processes are indispensable as they are involved in both the sample preparation stage as well as signal amplification and detection stage. To overcome this challenge, various preservation strategies have been investigated to enhance the shelf life of enzymes.46 One example is desiccation of enzymes for long-term storage, combined with liquid pouches for rehydration of reagents into liquid form prior to using the device.11 2300 SHIN AND WANG FIGURE 11. (a) Principles of melting curve analysis. As the temperature is increased, duplex DNA is dissociated along with double-stranded DNA binding dyes, resulting in a decrease in fluorescence. (b) An example of melting curve analysis for identification of a single-nucleotide polymorphism within a 544-bp fragment of the HTR2A gene. (c) Layout of magnetically actuated droplet cartridge for performing genomic DNA isolation, PCR and melting curve analysis from biological sample. (d) Temperature profile achieved at the incubation chamber, including the initial PCR amplification and thermal ramp for melting curve acquisition. (adopted from Refs. 56 and 45). The second challenge is miniaturization and power management of supporting instrumentation. While microfluidic cartridges themselves are generally small by design, the supporting instrumentation for operating the device including power management and control systems are typically designed for laboratory setting and not suited for portable operation. To this end, miniaturized components for various aspects of device operation have been reported. A miniaturized optical detector for fluorescence detection with sensitivity of 1029 M fluorescein has been proposed by Novak et al.33 using LEDs as an illumination source and silicon photodiode as a detector. Similarly, miniaturization of actuation instruments has also been demonstrated by Shikida et al.,43 utilizing a stepper motor to design a magnetic actuator in a palmtopsized format. Planar electromagnetic coil actuation also lends itself to small form factor, although power requirements remain to be resolved. 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