Digital Microfluidics for Nucleic Acid Amplification

sensors

Review Digital Microﬂuidics for Nucleic Acid Ampliﬁcation

Beatriz Coelho 1 , Bruno Veigas 1,2, Elvira Fortunato 1, Rodrigo Martins 1, Hugo Águas 1, Rui Igreja 1,* and Pedro V. Baptista 2,*

1 i3N|CENIMAT, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal; [email protected] (B.C.); [email protected] (B.V.); [email protected] (E.F.); [email protected] (R.M.); [email protected] (H.A.) 2 UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal * Correspondence: [email protected] (R.I.); [email protected] (P.V.B.)

Received: 27 May 2017; Accepted: 22 June 2017; Published: 25 June 2017

Abstract: Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

Keywords: Digital Microﬂuidics; nucleic acid ampliﬁcation; point-of-care diagnostics

1. Introduction Digital Microfluidics (DMF) is a relatively recent technology for liquid manipulation, which allows the control of discrete droplets on a planar surface, through the use of electric, magnetic, optic or acoustic forces [1,2]. The first DMF device dates back to 1986, when Jean Pesant, Michael Hareng, Bruno Mourey and Jean Perbet submitted a patent describing a device “by means of which it is possible to cause fluid globules to circulate within a capillary space by means of pairs of electrodes establishing capture sites” [3]. Later, in the 2000s, two groups (the Fair group at Duke University [4] and the Kim group at University of California, Los Angeles [5]) rediscovered DMF and disseminated the technique, and demonstrated the impact of this technology. DMF includes all the standard advantages of conventional microfluidics, namely volume reduction, shorter reaction time, increased process sensitivity and decrease in sample cross-contamination [6,7]. Additionally, the devices can be portable and fully automated [6]. Furthermore, DMF offers additional advantages, such as: (1) precise control over unit droplets, (2) easy integration with measurement techniques, (3) multiplex assay capability and (4) there is no need for propulsion devices. In this review, we will focus on the integration of DMF-based approaches designed for nucleic acid amplification-based genetic testing. Specifically, the development of DMF devices where droplets are transported on electrode arrays via electric forces, namely electrowetting-on-dielectric (EWOD) at relatively low frequencies (non-dielectrophoretic). Within the plethora of designs and settings, we shall highlight those that constitute major advances in DMF construction and evolution.

Sensors 2017, 17, 1495; doi:10.3390/s17071495 www.mdpi.com/journal/sensors Sensors 2017, 17, 1495 2 of 13

Digital Microfluidics Configurations DMF devices are mostly derived from two basic configurations: (1) two-plate (or closed) configuration, in which liquid droplets move between two substrates and (2) one-plate (or open) configuration, in which liquid droplets move over one substrate [1]. In the first configuration, the bottom plate includes a substrate (usually glass) where paths of actuation electrodes are deposited, which in turn are covered by a dielectric layer and a hydrophobic layer, preventing sample adhesion to the electrodes. The top plate is generally a single ground electrode, made of transparent, conductive material, allowing for process monitorization (e.g., droplet transportation and reaction visualization by colour change). A filler medium (typically a low viscosity oil) may be added between plates, to reduce evaporation effects and lower the actuation voltage [8]. The second configuration includes all the elements from the previously described bottom-plate, and also allocates the ground electrodes (unless catenae are used as ground electrodes) [2]. Nowadays, most DMF systems are produced in a closed configuration, since it enables all the fluidic operations (dispensing, merging, mixing and splitting), whereas open configurations only allow moving and merging operations [2]. Bottom plates are usually glass, monocrystalline silicon, or printed circuit boards (PCB). Substrate choice is closely related to device architecture, ease of fabrication and production cost [9,10]. Contact electrodes are generally metallic, yet alternative conductors such as Indium–Tin–Oxide and doped silicon have been reported. Commonly used dielectrics are Parylene, SU-8 and Si3N4. Finally, Teflon® (Chemours, Wilmington, DE, USA) and Cytop® (AGCChem, Exton, PA, USA) are standard choices for the hydrophobic layer [2,11].

2. Digital Microfluidics for Nucleic Acid Amplification Nucleic acid amplification is one of the most relevant tools in molecular biology, allowing the monitorization of gene expression, quantification of food-borne pathogens, diagnostic of hereditary and infectious diseases, as well as numerous forensic analysis processes [12]. The identification and characterization of nucleic acid sequences has become a pivotal element in molecular diagnostics of numerous diseases, such as cancer and hereditary conditions, and detection of pathogens via their unique molecular signature. Thus, nucleic acid amplification technologies (NAATs) have been developed in a multitude of strategies focusing on low sample volume, high specificity and selectivity, and preferably in a portable format. Among these NAATs, the polymerase chain reaction (PCR) is still the “gold standard” for most applications [13]. However, the need to miniaturization and the emergence of microfluidics has prompted amplification strategies that are simpler, i.e., do not require complex temperature cycling for detection of amplification. Such technologies include isothermal loop amplification (LAMP) and rolling circle (RCA) that may be coupled to label free detection schemes [14–16]. Particularly, DMF devices have proved to bring several advantages over standard DNA amplification, namely reagent volume reduction, minimization of the analysis time and the possibility of process automation [17–19].

2.1. Development of DMF–PCR Platforms Being the first nucleic acid amplification technique to be developed [20], PCR is today the standard technique for nucleic acid amplification, and its working principles have been widespread across all major molecular biology laboratories. Accordingly, it is also the primary procedure for DMF on-chip amplification found in the literature. The first group to achieve DNA amplification in DMF platforms was the Chang group [17], by amplifying a detection gene for the Dengue II virus via PCR. For this, a two-plate DMF device was developed, where the top-plate was composed of glass covered with an Indium–Tin–Oxide (ITO) ground electrode, which in turn was covered with a Teflon® hydrophobic layer. The bottom plate included a glass substrate, where Au electrodes were deposited and then covered by a Si3N4 dielectric layer (0.15 µm) and Teflon®. Top and bottom plates were separated by 370 µm, and silicone oil was Sensors 2017, 17, 1495 3 of 13

Sensors 2017, 17, 1495 3 of 14 used as a ﬁller. Two separate regions are included, one for the sample DNA template, and another for oil was used as a filler. Two separate regions are included, one for the sample DNA template, and theoil was PCR used mix reagentsas a filler. (Figure Two 1separate). regions are included, one for the sample DNA template, and another for the PCR mix reagents (Figure 1).

Figure 1. a b Figure 1. (a) Schematic representation of the chip, both in top view and cross-section; (b) Photograph of the chip, where the different active regions are visible. Temperature control is based on two platinum of the chip, where the different active regions are visible. Temperature control is based on two heaters, which also provide temperature sensing. Adapted from original in reference [17]. platinum heaters, which also provide temperature sensing. Adapted from original in reference [17].

The described DMF platform was able to successful successfullyly amplify a sequence of the Dengue-II virus (511(511 bps)bps) inin 5555 min, min, with with a a total total sample sample volume volume of of 15 15µL. µL. Additionally, Additionally, this this approach approach allowed allowed forlow for dropletlow droplet actuation actuation (12 V (12RMS VatRMS 3 at kHz) 3 kHz) and and PCR PCR chamber chamber operation operation voltages voltages (9 V (9DC V).DC). A partnershippartnership betweenbetween Advanced Advanced Liquid Liquid Logic Logic Inc. Inc. (recently (recently acquired acquired by Illumina) by Illumina) and the and Duke the UniversityDuke University introduced introduced multiplexing multiplexing in DMF systems,in DMF assystems, well as as several well newas several improvements new improvements in architecture. in Inarchitecture. this work, HuaIn this et al. work, developed Hua aet fully al. integrateddeveloped system, a fully comprised integrated of asystem, control/detection comprisedsection of a andcontrol/detection a disposable samplesection processingand a disposable section sample (Figure processing2)[21]. section (Figure 2) [21].

Figure 2. (a) Photograph of an assembled di digitalgital microfluidics microﬂuidics (DMF) chip; (b) Schematic ofof the DMF chip, evidencing the heating system, as well as thethe opticaloptical detectiondetection spots.spots. Adapted from original in reference [[21].21].

Based on the closed two-plate format, this improved DMF cartridge chip included 4 detection Based on the closed two-plate format, this improved DMF cartridge chip included 4 detection zones for real-time reaction tracking, allowing for multiplex sample analysis. With this DMF zones for real-time reaction tracking, allowing for multiplex sample analysis. With this DMF platform, platform, the Hua group performed real-time PCR detection of methicillin-resistant Staphylococcus the Hua group performed real-time PCR detection of methicillin-resistant Staphylococcus aureus (MRSA), aureus (MRSA), Mycoplasma pneumoniae and Candida albicans. Amplification was possible using just Mycoplasma pneumoniae and Candida albicans. Ampliﬁcation was possible using just one copy of DNA one copy of DNA template (MRSA genomic DNA). Optimization of PCR step times was pursued, template (MRSA genomic DNA). Optimization of PCR step times was pursued, allowing for the allowing for the reduction of the overall reaction time to 18 min, with no significant changes in reduction of the overall reaction time to 18 min, with no signiﬁcant changes in performance (threshold performance (threshold cycle and final reaction product). Also, multiple PCR processes were performed simultaneously, and successful detection of MRSA and M. pneumoniae was possible.

Sensors 2017, 17, 1495 4 of 13 cycle and final reaction product). Also, multiple PCR processes were performed simultaneously, and Sensors 2017, 17, 1495 4 of 14 successful detection of MRSA and M. pneumoniae was possible. As mentioned, DMF systems usually rely on a two-plate architecture, resorting to fillers fillers to help prevent evaporation, one one of of the the key key issues issues in in low-volume low-volume reactions. reactions. However, However, Jebrail Jebrail et et al. al. propose propose a simplea simple approach approach to to use use a atwo-plate two-plate air-matrix air-matrix DMF DMF platform platform that that prevents prevents evaporation evaporation on PCR reactions, by refilling refilling reaction droplets with pre-heatedpre-heated solvents whenever necessary [[22].22]. This DMF platform consistsconsists of of a PCBa PCB bottom-plate bottom-plate with with Cu/Ni/Au Cu/Ni/Au electrodes electrodes and and reservoirs, reservoirs, covered covered by a solder by a ® soldermask dielectric mask dielectric layer and layer a Teflon and a hydrophobicTeflon® hydrophobic layer, and layer, includes and a incl holeudes connecting a hole connecting the platform the to platforma syringe, to which a syringe, contains which refill contains solvent, refill through solvent, a tube through (Figure a3 a).tube Droplet (Figure actuation 3a). Droplet was achievedactuation waswith achieved 80–100 V withRMS, 80–100 at 18 kHz. VRMS, The at 18 DMF kHz. chip The allowed DMF chip for allowed PCR-amplification for PCR-amplification of 200 bp M13mp18of 200 bp M13mp18bacteriophage bacteriophage DNA in approximately DNA in approximately 45 min, for a45 final min, reaction for a volumefinal reaction of 1.5 µvolumeL. During of the1.5 PCRµL. Duringreaction, the 33 PCR refill reaction, droplets of33 solventrefill droplets with 0.5 ofµ Lsolven weret added with 0.5 to theµL reactionwere added droplet, to the as reaction to revert droplet, volume asloss to due revert to evaporation. volume loss PCR due was to successfully evaporation. achieved, PCR was yet withsuccessfully lower efficiency achieved, than yet the with bench-top lower efficiencycounterpart than (Figure the bench-top3b). counterpart (Figure 3b).

Figure 3. Two-plate air-matrix DMF platform. (a) Air-filled DMF platform, featuring a droplet refill Figure 3. Two-plate air-matrix DMF platform. (a) Air-filled DMF platform, featuring a droplet system. The primary regions of the device are highlighted, such as actuation electrodes and reagent refill system. The primary regions of the device are highlighted, such as actuation electrodes and reservoirs,reagent reservoirs, heating heatingareas areas(temperature (temperature control control relies relies on onthermoelectric thermoelectric modules modules and and resistive temperature detectors detectors placed placed under under the printed circ circuituit boards (PCB) substrate) and platform-refillplatform-refill system connections.connections. ( b()b Electrophoretic) Electrophoretic analysis analysis of on-chip of on-chi versusp versus off-chip off-chip PCR products PCR products for M13mp18 for M13mp18bacteriophage bacteriophage DNA, in comparison DNA, in withcomparison a DNA ladder.with a Adapted DNA ladder from original. Adapted in referencefrom original [22]. in reference [22].

Recently, Norian et al. demonstrated a complementary metal–oxide–semiconductor (CMOS) Recently, Norian et al. demonstrated a complementary metal–oxide–semiconductor (CMOS) based DMF device for DNA amplification in point-of-care (POC) diagnostics by real-time PCR [23]. based DMF device for DNA amplification in point-of-care (POC) diagnostics by real-time PCR [23]. The device was assembled on a ball-grid-array (BGA), in which SU-8 polymer was used to define define the droplet manipulationmanipulation region, region, and and actuation actuation electrodes electrodes were were coated coated with Parylenewith Parylene C dielectric, C dielectric, covered by a Teflon® layer, to ensure the hydrophobicity of the bottom plate. This work shows the introduction covered by a Teflon® layer, to ensure the hydrophobicity of the bottom plate. This work shows the introductionof flexible components, of flexible namelycomponents, an ITO-coated namely polyethylenean ITO-coated naphthalate polyethylene (PEN) naphthalate top plate. The(PEN) device top plate.design The includes device four design reservoirs includes to four store reservoirs primers, DNA to store template primers, and DNA PCR template reagents, and from PCR which reagents, 1.2 nL fromdroplets which may 1.2 be nL retrieved. droplets may For be actuation, retrieved. a chargeFor actuation, pump voltagea charge converter pump voltage is able converter to convert is able the to3.3 convert V device the power 3.3 V supply device intopower 90 Vsupply applied into to electrodes90 V applied to induceto electrodes an ON to state. induce Real-time an ON optical state. Real-timedetection isoptical performed detection by an is integrated performed Geiger-mode by an integrated single-photon Geiger-mode avalanche single-photon diode (SPAD), avalanche located diodeat a particular (SPAD), detectionlocated at pixela particul in whichar detection a window pixel was in opened,which a window as to allow was the opened, passage as of to light allow from the passagean external of light laser. from Several an external preliminary laser. experimentsSeveral preliminary were performed experiments to evaluatewere performed the device, to evaluate namely thetemperature device, namely calibration, temperature detection calibration, performance dete (nMction concentration performance scale), (nM concentration device lifetime scale), and dropletdevice lifetime and droplet evaporation (rate of evaporation without filler was found to be 20 pL·s−1 for a droplet of 1.2 nL). This chip was applied to the PCR amplification of a 364 bp sequence from Staphylococcus aureus and allowed amplification of just one copy of template DNA in a 1.2 nL droplet.

Sensors 2017, 17, 1495 5 of 13

evaporation (rate of evaporation without filler was found to be 20 pL·s−1 for a droplet of 1.2 nL). This chip was applied to the PCR amplification of a 364 bp sequence from Staphylococcus aureus and Sensors 2017, 17, 1495 5 of 14 allowed amplification of just one copy of template DNA in a 1.2 nL droplet. RivalRival etet al.al. [ 24[24]] recently recently presented presented a DMF a DMF platform platform that allowsthat allows complete complete cell expression cell expression analysis, analysis,from cell from lysis tocell real-time lysis to real-time nucleic acid nucleic amplification. acid amplification. This two-plate This two-plate configuration configuration device is produceddevice is producedon a silicon on wafer,a silicon over wafer, which over an SiOwhich2 insulating an SiO2 layerinsulating and Ti/AlCulayer and electrodes Ti/AlCu electrodes were deposited, were deposited,followed by followed an Si3N by4 dielectrican Si3N4 dielectric layer and layer an SiOCand an hydrophobic SiOC hydrophobic coating. coating. The ITO-covered The ITO-covered glass glasstop plate top plate contained contained sand-blasted sand-blasted holes holes for reagent for reagent and sampleand sample insertion, insertion, and wasand separatedwas separated from ® fromthe bottom the bottom plate byplate a 100 by µam 100 Ordyl µm spacer.Ordyl® Thisspacer. DMF This platform DMF platform is accompanied is accompanied by a bench-top by a bench-topinstrument instrument which allows which for allows temperature for temperature control (Peltier control module), (Peltier reaction module), analysis reaction (camera analysis for (camerafluorescence for fluorescence detection after detection sample excitationafter sample with exci a lighttation emitting with a diode, light (LED))emitting and diode, droplet (LED)) actuation and droplet(Figure 4actuation). With this (Figure platform, 4). With cell lysisthis andplatform, subsequent cell lysis mRNA and subsequent capture (assisted mRNA by capture magnetic (assisted beads) byare magnetic performed beads) in line, are reducing performed cross in contamination line, reducing probability cross contamination and greatly improving probability reproducibility. and greatly improvingPlatform validation reproducibility. was performed Platform for validati the detectionon was ofperformed the c-MYC forgene the allowing detection a limitof the of c-MYC detection gene of allowingjust one cell,a limit while of detection using a 20-fold of just volumeone cell, reduction while using compared a 20-fold to volume the traditional reduction benchtop compared protocols. to the traditionalFurthermore, benchtop 10 cells protocols. were submitted Furthermore, to the mRNA 10 cells extraction were submitted and amplification to the mRNA procedures, extraction and and the amplificationreal-time fluorescence procedures, analysis and the revealed real-time two fluorescence distinct Ct values,analysis suggesting revealed two sub-populations distinct Ct values, in the suggestingtested sample. sub-populations in the tested sample.

FigureFigure 4. DMFDMF device device assembly: assembly: ( (aa)) Additional Additional instrumentation instrumentation for for the the DMF DMF chip chip control; control; ( (bb)) Chip Chip crosscross section,section, evidencingevidencing all all the the constituting constituting layers; layers; (c,d )( Photographsc,d) Photographs of the of actual the DMFactual chip, DMF showing chip, showingthe most the relevant most chiprelevant regions. chip Adaptedregions. Adapted from original from inoriginal reference in reference [24]. [24].

Sista et al. [9] developed a DMF platform for POC testing, including PCR-based amplification of Sista et al. [9] developed a DMF platform for POC testing, including PCR-based amplification of pathogenic DNA, with real-time measurement capability. In this closed platform, the bottom plate pathogenic DNA, with real-time measurement capability. In this closed platform, the bottom plate consisted of an array of independently addressable control electrodes deposited on a printed circuit consisted of an array of independently addressable control electrodes deposited on a printed circuit board (Figure 5). Optical real-time tracking of the PCR reaction was made by using a miniature board (Figure5). Optical real-time tracking of the PCR reaction was made by using a miniature in-house in-house designed fluorimeter, which consisted of an LED and a photodiode aligned with the designed fluorimeter, which consisted of an LED and a photodiode aligned with the detection spot. detection spot. The design also included a heating system made by two aluminium heating bars The design also included a heating system made by two aluminium heating bars placed underneath placed underneath the platform. A considerable reduction in reaction time, down to 12 min was the platform. A considerable reduction in reaction time, down to 12 min was achieved. Furthermore, achieved. Furthermore, it was demonstrated that a single copy of DNA template is enough to it was demonstrated that a single copy of DNA template is enough to achieve amplification in this achieve amplification in this DMF platform. DMF platform.

Sensors 2017, 17, 1495 6 of 13 Sensors 2017, 17, 1495 6 of 14

Figure 5. Schematic of the in-house fabricated DMF device for DNA ampliﬁcation.amplification. Top Top glass plate included drilled ports for sample insertion/withdrawal, and and both both plates plates were were spaced spaced via via a a 185 185 µmµm polymer ﬁlm.film. Adapted from original in reference [[9].9].

Fouillet and co-workers successfully demonstrated demonstrated the the use use of a DMF-based chip for single nucleotide polymorphism genotypinggenotyping inin humanhuman placentalplacental DNA DNA samples samples [ 25[25].]. The The group group developed developed a aclosed closed configuration configuration device, device, with with a polycarbonate a polycarbonate top plate,top plate, coated coated with ITOwith and ITO Teflon and® Teflon. The bottom®. The ® bottomplate allocated plate allocated Au electrodes Au electrodes coated withcoated a dielectricwith a dielectric layer of layer Si3N4 ofand Si3 aN hydrophobic4 and a hydrophobic Teflon Teflonlayer. The® layer. cartridge The cartridge is divided is divided into three into regions: three region (1) loadings: (1) loading of reagents; of reagents; (2) storage (2) storage section, section, where whereintermediate intermediate reactions reactions may occur; may and occur; (3) channelsand (3) forchannels droplet for actuation. droplet Dropletsactuation. are Droplets actuated are at actuated60 VRMS andat 60 3 kHz.VRMS Finally,and 3 kHz. temperature Finally,control temperature was achieved controlusing was achieved a Peltier module using a placed Peltier under module the placedchip for under fast thermocycling the chip for fast capability. thermocycling This approach capability. allowed This amplification approach allowed of a minimum amplification of 10 DNA of a minimumstrands, achieving of 10 DNA single strands, nucleotide achieving specificity single nucleotide in a total reaction specificity volume in a total of 64 reaction nL. Optical volume detection of 64 wasnL. Optical performed detection by a standardwas performed fluorescence by a standard microscope fluorescence using an intercalatingmicroscope using dye. Thisan intercalating group also dye.developed This othergroup interesting also developed DMF chip other designs interesting for additional DMF biologicalchip designs processes for additional and applications biological [26]. processes and applications [26]. DMF–PCR Platform Validation DMF–PCREfforts Platform have been Validation made towards the validation of DMF-based platforms for further development of cost-effectiveEfforts have diagnostic been made methodologies. towards Thethe first validation publication of showingDMF-based the validation platforms of afor DMF-based further developmentplatform with clinicalof cost-effective samples was diagnostic reported bymethodologies. Wulff-Burchfield The et first al., whopublication resorted toshowing DMF-based the validationDNA amplification of a DMF-based for the detection platform of withMycoplasma clinical pneumoniaesamples wasin 59reported clinical by samples Wulff-Burchfield [27]. In this study,et al., whothe performance resorted to ofDMF-based an Advanced DNA Liquid amplification Logic DMF for chip the was detection compared of Mycoplasma to conventional pneumoniae real-time in PCR 59 clinicalamplification samples techniques. [27]. In this As astudy, result, the successful performance amplification of an Advanced was achieved Liquid for 3.2LogicµL andDMF the chip reaction was comparedtime was reduced to conventional by 3-fold, whilereal-time producing PCR similaramplification results totechniques. those attained As viaa bench-topresult, successful reaction, withamplification 67% sensitivity was achieved and 100% for specificity.3.2 µL and the reaction time was reduced by 3-fold, while producing similarAn results Advanced to those Liquid attained Logic via DMF bench-top platform reaction, was also employedwith 67% sensitivity by Schell et and al. [100%28] for specificity. the detection of CandidaAn Advanced albicans in Liquid clinical Logic blood samples.DMF platform This group was performedalso employed real-time by Schell PCR on et 16 al. blood [28] samplesfor the detectionfrom patients of Candida infected albicans with C.in albicansclinical blood(1 mL samples. each) and This concluded group performed that the PCR-based real-time PCR diagnostic on 16 bloodoverall samples performance from patients of on-chip infected reactions with differed C. albicans from (1 the mL conventional each) and concluded method, yetthat the the combination PCR-based diagnosticof both methods overall yielded performance 94% sensitivity. of on-chip Furthermore, reactions differed the DMF–PCR from the approachconventional lowers method, both reaction yet the combinationtime and cost. of both methods yielded 94% sensitivity. Furthermore, the DMF–PCR approach lowers both reaction time and cost. 2.2. DMF—Conventional Microfluidics Hybrid Devices 2.2. DMF—ConventionalAnother innovative Microfluidics approach is the Hybrid integration Devices of channel-based conventional microfluidics (CMF) withAnother DMF, creating innovative the so-called approach hybrid is the devices. integratio Thesen of devices channel-based were first developedconventional to fullymicrofluidics integrate (CMF)column with separation DMF, creating processes the (possibleso-called withhybrid CMF) devices. with These all the devices steps were required first fordeveloped pre-separation to fully (possibleintegrate withcolumn DMF) separation [29,30]. However, processes DMF–CMF (possible hybridwith CMF) devices with have all also the been steps applied required for PCRfor pre-separation (possible with DMF) [29,30]. However, DMF–CMF hybrid devices have also been

SensorsSensors2017 2017,,17 17,, 14951495 77 ofof 1314

applied for PCR amplification protocols, either to constrain droplet movement [31] or facilitate amplificationreagent injection protocols, and heated either reactions to constrain [32]. droplet movement [31] or facilitate reagent injection and heatedUgsornrat reactions [et32 ].al. developed a hybrid device where fluids are constrained to a PDMS (polydimethylsiloxane)Ugsornrat et al. microchannel, developed a yet hybrid droplets device are wherestill controlled fluids are by electrode constrained addressing to a PDMS [31]. (polydimethylsiloxane)Briefly, Cr/Au electrodes microchannel, were deposited yet dropletson a gla aress substrate, still controlled and covere by electroded with addressinga layer of SiO [31].2, Briefly,which in Cr/Au turn was electrodes covered were by depositeda hydrophobic on a glass layer substrate, (Teflon®). and The covered glass cover with plate a layer included of SiO2 ,an which ITO inlayer turn acting was coveredas ground by aelectrode, hydrophobic as well layer as (Teflonthree different®). The glassserpentine-shaped cover plate included Cr/Au microheaters, an ITO layer actingfor precise as ground control electrode, of denaturation, as well annealing as three differentand extension serpentine-shaped temperatures in Cr/Au a reaction microheaters, volume of for 25 preciseµL, where control optimized of denaturation, usage of annealing mineral andoil extensionprevented temperatures evaporation inbeyond a reaction standard volume operating of 25 µL, whereconditions. optimized usage of mineral oil prevented evaporation beyond standard operating conditions. KimKim etet al.al. have have developed a DMF platform combining PCR amplificationamplification andand capillarycapillary microfluidics,microfluidics, for for DNA DNA sequencing sequencing [32 ].[32]. This This device device fully integratesfully integrates nucleic nucleic acid amplification acid amplification (usually required(usually asrequired a pre-sequencing as a pre-sequen step)cing with step) the sequencing with the sequencing procedure itself,procedure unlike itself, other unlike DMF-based other sequencingDMF-based platforms sequencing [33 ,platforms34]. The device [33,34]. is dividedThe dev intoice twois divided segments: into (1) two capillary segments: tubes (1) for capillary reagent insertiontubes for reagent on the DMFinsertion hub on and the heated DMF hub reactions and heated (PCR reactions included) (PCR and included) (2) a DMF and hub (2) fora DMF reagent hub mixingfor reagent and mixing room-temperature and room-temperature reactions, connected reactions, toconnected the capillary to the tubes. capillary The tubes. DMF hubThe consistedDMF hub ofconsisted a two-plate of a configurationtwo-plate configuration with 40 electrodes with 40 electr and 8odes larger and reservoirs 8 larger reservoirs (Figure6). (Figure With this 6). platform, With this Kimplatform, et al. achievedKim et al. successful achieved successful PCR amplification PCR amplification of human genomicof human DNA genomic retrieved DNA from retrieved blood from cells (previouslyblood cells tagged),(previously in about tagged), 13 min, in for about just 2.813µ L,min, which for furtherjust 2.8 enabled µL, which the production further enabled of a human the DNAproduction library. of a human DNA library.

FigureFigure 6.6. SchematicSchematic representationrepresentation ofof thethe DMFDMF platformplatform proposed,proposed, evidencingevidencing thethe locationlocation ofof allall thethe reagentsreagents requiredrequired forfor DNADNA sequencing.sequencing. TheThe gapgap betweenbetween platesplates waswas maintainedmaintained atat eithereither 185185 µµmm oror 400400 µµm,m, accordingaccording toto thethe intendedintended dropletdroplet volumes,volumes, andand nono ﬁllerfiller mediummedium waswas added.added. AdaptedAdapted fromfrom originaloriginal inin referencereference [[32].32].

2.3.2.3. DMFDMF forfor IsothermalIsothermal NucleicNucleic AcidAcid AmplificationAmplification EvenEven thoughthough PCRPCR isis thethe mostmost widelywidely knownknown nucleicnucleic acidacid amplificationamplification methodology,methodology, thethe threethree elevatedelevated temperaturestemperatures neededneeded forfor thermalthermal cyclingcycling addadd complexitycomplexity toto thisthis technique,technique, andand contributecontribute toto highhigh energyenergy requirementsrequirements and and sophisticated sophisticated equipment equipment (e.g., (e.g., thermocyclers). thermocyclers). Thus, Thus, DNA/RNA DNA/RNA amplificationamplification researchresearch hashas beenbeen progressingprogressing towardstowards isothermalisothermal amplificationamplification schemes,schemes, enablingenabling low-cost,low-cost, low-temperaturelow-temperature nucleic nucleic acid acid amplification. amplificatio Recently,n. Recently, isothermal isothermal amplification amplification has also has beenalso integratedbeen integrated in DMF in devices,DMF devices, envisioning envisioning ready-to-use ready-to-use POC molecularPOC molecular diagnostics. diagnostics. KalsiKalsi etet al. proposeal. propose an active an matrix-basedactive matrix-based DMF platform DMF for theplatform detection for of antibiotic-resistancethe detection of factorsantibiotic-resistance in Escherichia factors coli bacteria in Escherichia (CTX-M gene),coli bacteria through ( isothermalCTX-M gene), recombinase through polymeraseisothermal amplificationrecombinase polymerase (RPA) [18]. amplification This two-plate (RPA) configuration [18]. This two-plate platform configuration relies on a thinplatform film relies transistor on a (TFT)thin film backplane, transistor which (TFT) controls backplane, ITO electrodes which co forntrols droplet ITO actuation electrodes and for capacitance droplet actuation sensors ableand capacitance sensors able to distinguish between the presence or absence of a droplet, allowing real-time droplet monitoring. The backplane is covered with an Al2O3 insulator, as well as a

Sensors 2017, 17, 1495 8 of 13 to distinguish between the presence or absence of a droplet, allowing real-time droplet monitoring. ® The backplaneSensors 2017, 17 is, 1495 covered with an Al2O3 insulator, as well as a disposable Cytop hydrophobic8 of 14layer, and the ITO-coated glass top plate is also covered by these two layers (Figure7). The top plate acts disposable Cytop® hydrophobic layer, and the ITO-coated glass top plate is also covered by these simultaneously as ground electrode and Joule heater (4 W dissipation power), being connected to two layers (Figure 7). The top plate acts simultaneously as ground electrode and Joule heater (4 W an off-chip thermistor and a Proportional Integral–Derivative (PID) control system. A Mylar spacer dissipation power), being connected to an off-chip thermistor and a Proportional Integral– definesDerivative a specific (PID) distance control between system. A electrodes, Mylar spacer and def dropletsines a specific are manipulated distance between in an electrodes, n-Dodecane and filler. EWODdroplets actuation are manipulated voltage is of in about an n-Dodecane 20 V and optical filler. EWOD detection actuation is achieved voltage in real-timeis of about by 20 fluorescence V and measurementsoptical detection of a Cy5 is achieved label added in real-time to the by sample. fluorescence Successful measurements amplification of a Cy5 of thelabel gene added was to possiblethe for asample. minimum Successful 270 nL, amplification with a limit of detectionthe gene was of aboutpossible 1 singlefor a minimum template copy,270 nL, in with 10 to a 15limit min. of detection of about 1 single template copy, in 10 to 15 min.

FigureFigure 7. (a )7. Cross-section (a) Cross-section of the of the DMF DMF platform, platform, evidencing evidencing the the several several materials materials that that comprise comprise thethe chip; (b) Schematicchip; (b) topSchematic view of top the view chip, of evidencing the chip, evidencing the reservoirs the andreservoirs the temperature and the temperature sensor. Adapted sensor. from Adapted from original in reference [18]. original in reference [18]. Another example of isothermal nucleic acid amplification performed on a DMF chip is Anotherprovided by example Kühnemund of isothermal et al., to detect nucleic very acidlow concentrations amplification of performedP. aeruginosa onDNA a by DMF rolling chip is providedcircle byamplification Kühnemund (RCA) et al.,followed to detect by circle-to-ci very lowrcle concentrations amplification (C2CA), of P. aeruginosa assisted byDNA magnetic by rolling circlemicroparticles amplification (MMP) (RCA) [19]. followed The amplification by circle-to-circle protocols amplificationare performed on (C2CA), a two-plate assisted configuration by magnetic ® microparticleschip, where (MMP) the bottom [19]. plate The amplificationincluded Cr electrodes protocols covered are performed by Parylene on C a and two-plate Teflon . configuration Two top plate configurations were developed, as to study the influence of the top plate thickness on MMP chip, where the bottom plate included Cr electrodes covered by Parylene C and Teflon®. Two top plate extraction: ITO-covered glass, covered with a Teflon® layer and Au-covered glass with a top Teflon® configurations were developed, as to study the influence of the top plate thickness on MMP extraction: layer. Tape was used as spacer, and droplets were actuated by 120 VAC, at 1 kHz. Heating was ® ® ITO-coveredperformed glass, by placing covered the with chip a Teflonon a thermocyclerlayer and Au-coveredplate. This device glass withallows a topdetection Teflon of layer.1 aM of Tape P. was usedaeruginosa as spacer, DNA and droplets in only 60 were min, actuated for an RCA by reaction 120 VAC temperature, at 1 kHz. Heating of 37 °C wasand a performed reaction volume by placing of 5 the chip onµL. a thermocycler plate. This device allows detection of 1 aM of P. aeruginosa DNA in only 60 min, for an RCA reaction temperature of 37 ◦C and a reaction volume of 5 µL. 3. Alternatives to EWOD for DMF-Assisted Nucleic Acid Amplification 3. Alternatives to EWOD for DMF-Assisted Nucleic Acid Amplification Even though EWOD is the standard method for controlling droplet motion in DMF systems, Evenalternative though techniques EWOD have is the been standard developed, method main forly controllingbased on magnetic, droplet optic motion or even in DMF acoustic systems, alternativeforces. techniquesNon-EWOD have DMF been platforms developed, have been mainly explor baseded for on a magnetic, variety of optic chemical or even and acousticbiological forces. Non-EWODprocedures, DMF namely platforms PCR-based have DNA been amplification explored for for a varietyPOC diagnostics. of chemical and biological procedures, Magnetically-assisted DMF platforms for PCR amplification have been widely explored at namely PCR-based DNA amplification for POC diagnostics. Johns Hopkins University [35–37], and rely on the addition of superparamagnetic particles to the Magnetically-assisteddroplets, which in turn are DMF manually platforms controlled for PCR by amplification a magnet placed have under been the widely substrate explored and allow at Johns Hopkinsdroplet University motion. Zhang [35–37 et], andal. [37] rely developed on the addition a DMF platform of superparamagnetic for biomarker detection particles from to the human droplets, whichblood in turn samples, are manually and pathogen controlled detection by a from magnet biological placed samples under the(E-coli substrate bacteria and as proof-of-concept). allow droplet motion. ZhangThis et al.DMF [37 ]platform developed successfully a DMF platformimplemented for biomarker all steps required detection for from target human detection, blood from samples, solid and pathogenphase detection DNA extraction from biologicalto real-time samples PCR or helicase (E-coli bacteriadependent as amplification proof-of-concept). (HDA), Thisthus DMFproving platform to successfullybe a suitable implemented platform for all POC steps applications. required for target detection, from solid phase DNA extraction to real-timeDMF PCR ordevices helicase based dependent on magnetic amplification forces typically (HDA), do thusnot enable proving the tofull be spectrum a suitable of platform fluidic for POCoperations, applications. namely droplet dispensing and splitting. Zhang et al. [36] surpassed this problem by

Sensors 2017, 17, 1495 9 of 13

DMF devices based on magnetic forces typically do not enable the full spectrum of fluidic operations, namely droplet dispensing and splitting. Zhang et al. [36] surpassed this problem by creating surface energy traps to which droplets bind, thus allowing droplet separation. Droplet splitting enabled multiplex detection of three different biomarkers (TP53, HER2 and RSF1) from human blood samples, and once again the entire procedure (from sample to result) was performed on the DMF platform. Furthermore, Chiou et al. [35] created a fully automated platform for sample-to-result detection of the KRAS (Kirsten rat sarcoma viral oncogene homolog) biomarker in human blood, which relies on an electromagnet for transport of the sample droplet through several reaction sports, separated by strictures. Apart from human blood, saliva has also been used as a sample by Pipper et al. [38], to detect the avian inﬂuenza virus H5N1 on a magnetically-assisted DMF platform, by real-time PCR. This device also performed all the steps required for detection, including RNA solid-phase extraction from the biological sample. Finally, surface acoustic waves (SAW) and photo-actuation have also emerged as alternatives to EWOD for droplet actuation in DMF platforms for nucleic acid testing. Using SAWs allows low power actuation, however, SAW-mediated devices are still not cost-effective, due to the excessive cost of piezoelectric materials. Photo-actuation eliminates the need for electrodes, therefore simplifying the production process, however, for low-volume droplet actuation (nL scale), expensive laser equipment may be required [39–41].

4. Future Prospects DMF development has been gaining momentum strongly focused on nucleic acid amplification, which is at the basis of genetic testing, and plays a fundamental part in POC applications. Joining DMF and nucleic acid amplification represents a major step in reinventing the paradigm of molecular diagnostics, bringing it closer to actual POC. So far, several DMF platforms have been developed for DNA/RNA amplification, mainly resorting to PCR, the standard technique in genetic testing (Table1). Real-time reaction monitoring is now a standard requirement, and a variety of target samples were tested, from biological samples to device validation using clinical samples. Others have focused on digital PCR approaches for increased sensitivity in mutation analysis [42,43]. However, efforts have been made to pursue isothermal amplification methodologies, which are intrinsically simpler, with lower energy and equipment requirements than PCR. Recent publications have focused on creating DMF platforms for nucleic acid amplification testing using low cost materials and mass production technologies. Accordingly, simpler reactions lead to simpler devices and economic materials and processes that facilitate commercialization. Indeed, industry is beginning to realize the enormous potential of DMF, and some of the most ground-breaking works in the field have resulted from academia–industry collaborations. More importantly, the first commercial DMF-based devices have been presented, such as Sandia Digital Microfluidics Hub (https://ip.sandia.gov/technology.do/techID=102) or the Illumina NeoPrep sequencing platform (https://www.illumina.com/systems/neoprep-library-system.html). Nevertheless, we strongly believe that DMF for genetic testing will exponentially evolve in the next years, when simulation and control studies (already developed) meet actual platforms, thus empowering fully automated processes, where samples are simply inserted in the platform, and final results are obtained a few minutes later. Furthermore, considering previous experience from our group [44–49], we believe that DMF will evolve towards fully disposable devices, with an even lower production cost, resorting to paper-based substrates and printing technologies, such as inkjet [50,51] or screen-printing (Figure8)[ 52]. To conclude, in a near future, no specialized laboratory technicians will be required to prepare the samples, perform the assays or read the results, and testing platforms will be portable, low-cost and disposable, thus enabling the true ideal of POC: devices available anytime, anywhere, for everyone. Sensors 2017, 17, 1495 10 of 13

Table 1. Summary of DMF platforms for nucleic acid amplification. ITO—Indium–Tin–Oxide; PCB—Printed Circuit Board; PEN—Polyethylene Naphthalate; qRT-PCR—Quantitative Real-Time Polymerase Chain Reaction; SNP—Single Nucleotide Polymorphism; POC—Point-of-care; RCA—Rolling Circle Amplification; C2CA—Circle-to-circle Amplification; RPA—Recombinase Polymerase Amplification; MRSA—methicillin-resistant Staphylococcus aureus (MRSA); M pneumoniae—Mycoplasma pneumoniae; C. albicans—Candida albicans; E. coli—Escherichia coli; P. aeruginosa—Pseudomonas aeruginosa.

Reaction Actuation Dielectric Hydrophobic Electrode Substrate Application Filler Reference Volume Voltage Material Material Material Material 12 V Dengue II virus detection 1.46 µL RMS Si N Teflon® AF Au Glass Silicone oil [17] (3 kHz) 3 4 60 V SNP genotyping 64 nL RMS Si N Teflon® AF Au Glass Silicone oil [25] (3 kHz) 3 4 DMF–PCR platforms POC testing, MRSA, S. aureus 600 nL - Parylene C Teflon® AF Cr PCB Hexadecane [9] and C. albicans detection MRSA, S. aureus, M. pneumoniae Variable - - - - PCB Hexadecane/silicone oil [21] and C. albicans detection S. aureus detection 1.2 nL 70 V–250 V Parylene C Teflon® AF - - n-dodecane [23] Cell genetic expression analysis Variable 48 V (3 kHz) Si3N4 SiOC Ti/AlCu Silicon wafer Silicone oil [24] Bacteriophage M13mp18 80–100 V 1.5 µL RMS Solder mask Teflon® AF Cu/Ni/Au PCB Air [22] detection (18 kHz) C. albicans detection on Variable - - - - PCB Hexadecane/silicone oil [27] Validation of DMF–PCR human blood platforms M. pneumoniae detection on Variable - - - - PCB Hexadecane/silicone oil [28] human nasopharyngeal wash DNA amplification (only the 25 µL - SiO Teflon® AF Cr/Au Glass Silicone oil [31] Hybrid platforms primers are described) 2 80–90 V Pre-DNA sequencing 2.8 µL RMS Parylene C Teflon® AF ITO Glass Air [32] (15 kHz) CTX-M gene detection in Minimum 20 V Al O Cytop® ITO Glass n-dodecane [18] DMF platforms for E. coli bacteria 270 nL 2 3 isothermal amplification 120 V P. aeruginosa detection 5 µL AC Parylene C Teflon® AF Cr Glass Vapor-Lock oil [19] (1 kHz) Sensors 2017, 17, 1495 11 of 13

Sensors 2017, 17, 1495 10 of 14

(a) (b)

Droplet dispensing

Droplet merging

(c)

Figure 8. (a) DMF device screen-printed on paper substrate; (b) Zoom view of the printed electrodes; Figure 8. ((ca) )Droplet DMF devicemovement screen-printed on a paper-based on paperDMF device substrate;. Adapted (b) from Zoom original view in of reference the printed [52]. electrodes; (c) Droplet movement on a paper-based DMF device. Adapted from original in reference [52].

Acknowledgments: This work was funded by FEDER funds through the COMPETE 2020 Program (POCI-01-0145-FEDER-007728) and National Funds through FCT—Portuguese Foundation for Science and Technology under the project UID/CTM/50025/2013 for I3N and UID/Multi/04378/2013 for Unidade de Ciências Biomoleculares Aplicadas (UCIBIO). BV thanks SFRH/BPD/124311/2016. PVB thanks Water JPI Proj TRACE (Ref 281715). Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Jebrail, M.J.; Wheeler, A.R. Let’s get digital: Digitizing chemical biology with microfluidics. Curr. Opin. Chem. Biol. 2010, 14, 574–581. [CrossRef][PubMed] 2. Choi, K.; Ng, A.H.C.; Fobel, R.; Wheeler, A.R. Digital Microfluidics. Annu. Rev. Anal. Chem. 2012, 5, 413–440. [CrossRef][PubMed] 3. Pesant, J.; Hareng, M.; Mourey, B.; Perbet, J. Electrodes for a Device Operating by Electrically Controlled Fluid Displacement. U.S. Patent US4569575 A, 11 February 1986. 4. Pollack, M.G.; Fair, R.B.; Shenderov, A.D. Electrowetting-based actuation of liquid droplets for microfluidic applications. Appl. Phys. Lett. 2000, 77, 1725–1726. [CrossRef] 5. Lee, J.; Moon, H.; Fowler, J.; Schoellhammer, T.; Kim, C.-J. Electrowetting and electrowetting-on-dielectric for microscale liquid handling. Sens. Actuators A Phys. 2002, 95, 259–268. [CrossRef] 6. Berthier, J. Micro-Drops and Digital Microfluidics, 2nd ed.; Elsevier Inc.: Waltham, MA, USA, 2013. 7. Millington, D.S.; Sista, R.; Eckhardt, A.; Rouse, J.; Bali, D.; Goldberg, R.; Cotten, M.; Buckley, R.; Pamula, V. Digital Microfluidics: A Future Technology in the Newborn Screening Laboratory? Semin. Perinatol. 2010, 34, 163–169. [CrossRef][PubMed] 8. Fair, R.B. Digital microfluidics: Is a true lab-on-a-chip possible? Microfluid. Nanofluid. 2007, 3, 245–281. [CrossRef] 9. Sista, R.; Hua, Z.; Thwar, P.; Sudarsan, A.; Srinivasan, V.; Eckhardt, A.; Pollack, M.; Pamula, V. Development

of a digital microﬂuidic platform for point of care testing. Lab Chip 2008, 8, 2091–2104. [CrossRef][PubMed] 10. Abdelgawad, M.; Freire, S.L.S.; Yang, H.; Wheeler, A.R. All-terrain droplet actuation. Lab Chip 2008, 8, 672–677. [CrossRef][PubMed] 11. Nelson, W.C.; Kim, C.-J. “Cj” Droplet Actuation by Electrowetting-on-Dielectric (EWOD): A Review. J. Adhes. Sci. Technol. 2012, 26, 1747–1771. 12. Nolan, T.; Bustin, S. PCR Technology: Current Innovations, 3rd ed.; CRC Press: Boca Raton, FL, USA, 2013. 13. Ahrberg, C.D.; Manz, A.; Chung, B.G. Polymerase chain reaction in microﬂuidic devices. Lab Chip 2016, 16, 3866–3884. [CrossRef][PubMed] Sensors 2017, 17, 1495 12 of 13

14. Veigas, B.; Pinto, J.; Vinhas, R.; Calmeiro, T.; Martins, R.; Fortunato, E.; Baptista, P.V. Quantitative real-time monitoring of RCA amplification of cancer biomarkers mediated by a flexible ion sensitive platform. Biosens. Bioelectron. 2017, 91, 788–795. [CrossRef][PubMed] 15. Veigas, B.; Branquinho, R.; Pinto, J.V.; Wojcik, P.J.; Martins, R.; Fortunato, E.; Baptista, P.V. Ion sensing (EIS) real-time quantitative monitorization of isothermal DNA amplification. Biosens. Bioelectron. 2014, 52, 50–55. [CrossRef][PubMed] 16. Bernacka-Wojcik, I.; Águas, H.; Carlos, F.F.; Lopes, P.; Wojcik, P.J.; Costa, M.N.; Veigas, B.; Igreja, R.; Fortunato, E.; Baptista, P.V.; et al. Single nucleotide polymorphism detection using gold nanoprobes and bio-microfluidic platform with embedded microlenses. Biotechnol. Bioeng. 2015, 112, 1210–1219. [CrossRef] [PubMed] 17. Chang, Y.H.; Lee, G.-B.; Huang, F.-C.; Chen, Y.-Y.; Lin, J.-L. Integrated polymerase chain reaction chips utilizing digital microfluidics. Biomed. Microdevices 2006, 8, 215–225. [CrossRef][PubMed] 18. Kalsi, S.; Valiadi, M.; Tsaloglou, M.; Parry-jones, L.; Jacobs, A.; Watson, R.; Turner, C.; Amos, R.; Hadwen, B.; Buse, J.; et al. Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform. Lab Chip 2015, 15, 3065–3075. [CrossRef][PubMed] 19. Kühnemund, M.; Witters, D.; Nilsson, M.; Lammertyn, J. Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection. Lab Chip 2014, 14, 2983–2992. [CrossRef][PubMed] 20. Mullis, K.; Faloona, F.; Scharf, S.; Saiki, R.; Horn, G.; Erlich, H. Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction. Cold Spring Harb. Symp. Quant. Biol. 1986, 51, 263–273. [CrossRef][PubMed] 21. Hua, Z.; Rouse, J.L.; Eckhardt, A.E.; Srinivasan, V.; Pamula, V.K.; Schell, W.A.; Benton, J.L.; Mitchell, T.G.; Pollack, M.G. Multiplexed real-time polymerase chain reaction on a digital microfluidic platform. Anal. Chem. 2010, 82, 2310–2316. [CrossRef][PubMed] 22. Jebrail, M.J.; Renzi, R.F.; Sinha, A.; Van De Vreugde, J.; Gondhalekar, C.; Ambriz, C.; Meagher, R.J.; Branda, S.S. A solvent replenishment solution for managing evaporation of biochemical reactions in air-matrix digital microfluidics devices. Lab Chip 2015, 15, 151–158. [CrossRef][PubMed] 23. Norian, H.S.; Shepard, K.; Kymissis, J.; Field, R. An integrated CMOS quantitative-polymerase-chain-reaction lab-on-chip for point-of-care diagnostics. Lab Chip 2014, 14, 4076–4084. [CrossRef][PubMed] 24. Rival, A.; Jary, D.; Delattre, C.; Fouillet, Y.; Castellan, G.; Bellemin-Comte, A.; Gidrol, X. An EWOD-based microfluidic chip for single-cell isolation, mRNA purification and subsequent multiplex qPCR. Lab Chip 2014, 14, 3739–3749. [CrossRef][PubMed] 25. Fouillet, Y.; Jary, D.; Brachet, A.; Berthier, J.; Blervaque, R.; Davous, L.; Roux, J.; Achard, J.; Peponnet, C. EWOD digital microfluidics for Lab on a Chip. In Proceedings of the ASME 4th International Conference on Nanochannels, Microchannels and Minichannels, Limerick, Ireland, 19–21 June 2006; ASME Press: Limerick, Ireland, 2006; pp. 1255–1264. 26. Berthier, J.; Mourier, V.; Sarrut, N.; Jary, D.; Fouillet, Y.; Pouteau, P.; Caillat, P.; Peponnet, C. Some examples of micro-devices for biotechnology developed at the Department of Technologies for Life Sciences and Healthcare of the LETI. Int. J. Nanotechnol. 2010, 7, 802–818. [CrossRef] 27. Wulff-Burchfield, E.; Schell, W.A.; Eckhardt, A.E.; Pollack, M.G.; Hua, Z.; Rouse, J.L.; Pamula, V.K.; Srinivasan, V.; Benton, J.L.; Alexander, B.D.; et al. Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens. Diagn. Microbiol. Infect. Dis. 2010, 67, 22–29. [CrossRef][PubMed] 28. Schell, W.A.; Johnson, M.D.; Alexander, B.D.; Perfect, J.R.; Smith, P.B.; Benjamin, D.K.; Mitchell, T.G.; Benton, J.L.; Poore, M.; Rouse, J.L.; et al. Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood. Eur. J. Clin. Microbiol. Infect. Dis. 2012, 31, 2237–2245. [CrossRef] [PubMed] 29. Abdelgawad, M.; Watson, M.W.L.; Wheeler, A.R. Hybrid microfluidics: A digital-to-channel interface for in-line sample processing and chemical separations. Lab Chip 2009, 9, 1046–1051. [CrossRef][PubMed] 30. Watson, M.W.L.; Jebrail, M.J.; Wheeler, A.R. Multilayer hybrid microfluidics: A digital-to-channel interface for sample processing and separations. Anal. Chem. 2010, 82, 6680–6686. [CrossRef][PubMed] 31. Ugsornrat, K.; Afzulpurkar, N.V.; Wisitsoraat, A.; Tuantranont, A. Design, Simulation, and Experimental Study of a Droplet-Based PCR by EWOD. Sens. Mater. 2010, 22, 271–284. 32. Kim, H.; Jebrail, M.J.; Sinha, A.; Bent, Z.W.; Solberg, O.D.; Williams, K.P.; Langevin, S.A.; Renzi, R.F.; Van De Vreugde, J.L.; Meagher, R.J.; et al. A Microfluidic DNA Library Preparation Platform for Next-Generation Sequencing. PLoS ONE 2013, 8, e68988. Sensors 2017, 17, 1495 13 of 13

33. Welch, E.R.F.; Lin, Y.Y.; Madison, A.; Fair, R.B. Picoliter DNA sequencing chemistry on an electrowetting-based digital microfluidic platform. Biotechnol. J. 2011, 6, 165–176. [CrossRef][PubMed] 34. Boles, D.J.; Benton, J.L.; Siew, G.J.; Levy, M.H.; Thwar, P.K.; Sandahl, M.A.; Rouse, J.L.; Perkins, L.C.; Sudarsan, A.P.; Pamula, V.; et al. Droplet-Based Pyrosequencing Using Digital Microfluidics. Anal. Chem. 2011, 83, 8439–8447. [CrossRef][PubMed] 35. Chiou, C.H.; Jin Shin, D.; Zhang, Y.; Wang, T.H. Topography-assisted electromagnetic platform for blood-to-PCR in a droplet. Biosens. Bioelectron. 2013, 50, 91–99. [CrossRef][PubMed] 36. Zhang, Y.; Wang, T.H. Full-range magnetic manipulation of droplets via surface energy traps enables complex bioassays. Adv. Mater. 2013, 25, 2903–2908. [CrossRef][PubMed] 37. Zhang, Y.; Park, S.; Liu, K.; Tsuan, J.; Yang, S.; Wang, T.-H. A surface topography assisted droplet manipulation platform for biomarker detection and pathogen identification. Lab Chip 2011, 11, 398–406. [CrossRef][PubMed] 38. Pipper, J.; Inoue, M.; Ng, L.F.-P.; Neuzil, P.; Zhang, Y.; Novak, L. Catching bird flu in a droplet. Nat. Med. 2007, 13, 1259–1263. [CrossRef][PubMed] 39. Reboud, J.; Bourquin, Y.; Wilson, R.; Pall, G.S.; Jiwaji, M.; Pitt, A.R.; Graham, A.; Waters, A.P.; Cooper, J.M. Shaping acoustic fields as a toolset for microfluidic manipulations in diagnostic technologies. Proc. Natl. Acad. Sci. USA 2012, 109, 15162–15167. [CrossRef][PubMed] 40. Guttenberg, Z.; Müller, H.; Habermüller, H.; Geisbauer, A.; Pipper, J.; Felbel, J.; Kielpinski, M.; Scriba, J.; Wixforth, A. Planar chip device for PCR and hybridization with surface acoustic wave pump. Lab Chip 2005, 5, 308–317. [CrossRef][PubMed] 41. Pei, S.N.; Valley, J.K.; Wang, Y.; Wu, M.C. Distributed Circuit Model for Multi-Color Microfluidic Device. J. Lightware Technol. 2015, 33, 3486–3493. [CrossRef] 42. Vogelstein, B.; Kinzler, K.W. Digital PCR. Proc. Natl. Acad. Sci. USA 1999, 96, 9236–9241. [CrossRef][PubMed] 43. Dong, L.; Meng, Y.; Sui, Z.; Wang, J.; Wu, L.; Fu, B. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material. Sci. Rep. 2015, 5, 13174. [CrossRef][PubMed] 44. Coelho, B.J.; Veigas, B.; Águas, H.; Fortunato, E.; Martins, R.; Baptista, P.V.; Igreja, R. Digital Microfluidics Devices for Nucleic Acid Amplification: Redifining the Paradigm of Molecular Diagnostics. In Proceedings of the Materiais 2017: VIII International Symposium on Materials, Aveiro, Portugal, 9–12 April 2017. 45. Marques, A.C.; Santos, L.; Costa, M.N.; Dantas, J.M.; Duarte, P.; Goncalves, A.; Martins, R.; Salgueiro, C.A.; Fortunato, E. Office Paper Platform for Bioelectrochromic Detection of Electrochemically Active Bacteria using Tungsten Trioxide Nanoprobes. Sci. Rep. 2015, 3, 1–7. [CrossRef][PubMed] 46. Costa, M.N.; Veigas, B.; Jacob, J.M.; Santos, D.S.; Gomes, J.; Baptista, P.V.; Martins, R.; Inácio, J.; Fortunato, E. A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: Lab-on-paper. Nanotechnology 2014, 25, 1–12. [CrossRef][PubMed] 47. Veigas, B.; Jacob, J.M.; Costa, M.N.; Santos, D.S.; Viveiros, M.; Inácio, J.; Martins, R.; Barquinha, P.; Fortunato, E.; Baptista, P.V. Gold on paper-paper platform for Au-nanoprobe TB detection. Lab Chip 2012, 12, 4802–4808. [CrossRef][PubMed] 48. Martins, R.; Ferreira, I.; Fortunato, E. Electronics with and on paper. Phys. Status Solidi Rapid Res. Lett. 2011, 5, 332–335. [CrossRef] 49. Coelho, B.J. A Digital Microfluidics Platform for Loop-Mediated Isothermal Amplification of DNA. Master’s Thesis, Faculdade de Ciências e Tecnologia—Universidade Nova de Lisboa, Caparica, Portugal, 2016. 50. Bernacka-Wojcik, I.; Senadeera, R.; Wojcik, P.J.; Silva, L.B.; Doria, G.; Baptista, P.; Aguas, H.; Fortunato, E.;

Martins, R. Inkjet printed and “doctor blade” TiO2 photodetectors for DNA biosensors. Biosens. Bioelectron. 2010, 25, 1229–1234. [CrossRef][PubMed] 51. Fobel, R.; Kirby, A.E.; Ng, A.H.C.; Farnood, R.R.; Wheeler, A.R. Paper microﬂuidics goes digital. Adv. Mater. 2014, 26, 2838–2843. [CrossRef][PubMed] 52. Matos, D. Digital Microﬂuidics on Paper. Master’s Thesis, Faculdade de Ciências e Tecnologia—Universidade Nova de Lisboa, Caparica, Portugal, 2014.

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).