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

2

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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.) 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

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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 Si3 N4 . 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 Si3 N4 dielectric layer (0.15 µm) and Teflon® . Top and bottom plates were separated by 370 µm, and silicone oil was

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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 the PCR mix reagents (Figure 1). another for the PCR mix reagents (Figure 1).

Figure (a) Schematic Schematic representation representation of both in in top top view view and and cross-section; cross-section; (b) Photograph Figure 1. 1. (a) of the the chip, chip, both (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, alsowhich provide sensing. Adapted original inoriginal reference platinumwhich heaters, alsotemperature provide temperature sensing.from Adapted from in[17]. reference [17].

The described DMF platform was able to successfully amplify a sequence of the Dengue-II virus (511 bps) volume of of 15 15 µL.µL. Additionally, thisthis approach allowed for low bps) in in55 55min, min,with witha atotal totalsample sample volume Additionally, approach allowed for low droplet actuation (12 V RMS at 3 kHz) and PCR chamber operation voltages (9 V DC ). droplet actuation (12 VRMS at RMS3 kHz) and PCR chamber operation voltages (9 VDC ).DC A partnership Liquid Logic Inc.Inc. (recently acquired by Illumina) and the Duke partnershipbetween betweenAdvanced Advanced Liquid Logic (recently acquired by Illumina) and the University introduced multiplexing in DMF systems, well as as several improvements in architecture. Duke University introduced multiplexing in DMF as systems, well new as several new improvements in In this work, Hua et al.work, developed fully system, comprised of a system, control/detection section architecture. In this Hua aet al. integrated developed a fully integrated comprised of a and a disposable sample sectionsample (Figureprocessing 2) [21]. section (Figure 2) [21]. control/detection section processing and a disposable

Figure 2. 2. (a) (a) Photograph of an digital microfluidics microfluidics (DMF) (DMF) chip; chip; (b) (b) Schematic Schematic of of the the DMF DMF Figure Photograph of an assembled assembled digital chip, evidencing evidencing the heating system, system, as as well well as as the the optical optical detection detection spots. spots. Adapted Adapted from from original original in in chip, the heating reference [21]. reference [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.

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cycle and final reaction product). Also, multiple PCR processes were performed simultaneously,4 of and 14 successful detection of MRSA and M. pneumoniae was possible. As mentioned, mentioned, DMF DMF systems systems usually usually rely rely on on aa two-plate two-plate architecture, architecture, resorting resorting to to fillers fillers to to help help As prevent evaporation, one of the key issues in low-volume reactions. However, Jebrail et al. propose prevent evaporation, one of the key issues in low-volume reactions. However, Jebrail et al. propose a a simpleapproach approachtotouse usea atwo-plate two-plateair-matrix air-matrixDMF DMFplatform platform that that prevents prevents evaporation evaporation on on PCR PCR simple reactions, by by refilling refilling reaction reaction droplets droplets with This DMF DMF reactions, with pre-heated pre-heated solvents solvents whenever whenever necessary necessary [22]. [22]. This platform consists of a PCB bottom-plate with Cu/Ni/Au electrodes and reservoirs, covered by a solder platform consists of a PCB bottom-plate with Cu/Ni/Au electrodes and reservoirs, covered by a ® mask dielectric layer and a Teflon layer, andlayer, includes hole connecting the platformthe to solder mask dielectric layer and a hydrophobic Teflon® hydrophobic anda includes a hole connecting a syringe, which contains refill solvent, through a tube (Figure 3a). Droplet actuation was achieved platform to a syringe, which contains refill solvent, through a tube (Figure 3a). Droplet actuation withachieved 80–100 Vwith at 18 kHz. DMF forallowed PCR-amplification of 200 bp M13mp18 RMS , 80–100 was VRMS,The at 18 kHz.chip Theallowed DMF chip for PCR-amplification of 200 bp bacteriophage DNA in approximately 45 min, for a45 final reaction of 1.5 µL. During M13mp18 bacteriophage DNA in approximately min, for a volume final reaction volume of the 1.5PCR µL. reaction, 33 refill droplets of solvent with 0.5 µL were added to the reaction droplet, as to revert volume During the PCR reaction, 33 refill droplets of solvent with 0.5 µL were added to the reaction droplet, losstodue to evaporation. PCR wastosuccessfully achieved, yet with lower efficiency than bench-top as revert volume loss due evaporation. PCR was successfully achieved, yetthe with lower counterpart (Figure 3b). efficiency than the bench-top counterpart (Figure 3b).

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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, heatingheating areas areas (temperature control relies on on thermoelectric modules reagent reservoirs, (temperature control relies thermoelectric modulesand and resistive resistive temperature detectors placed under the printed circuit boards (PCB) substrate) and platform-refill temperature detectors placed under the printed circuit boards (PCB) substrate) and platform-refill system connections.(b)(b) Electrophoretic analysis of on-chip PCR products for system connections. Electrophoretic analysis of on-chip versusversus off-chipoff-chip PCR products for M13mp18 M13mp18 bacteriophage DNA, in with comparison with aAdapted DNA ladder. Adapted from original bacteriophage DNA, in comparison a DNA ladder. from original in reference [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 device was was assembled assembled on on aa ball-grid-array ball-grid-array (BGA), (BGA), in in which which SU-8 SU-8 polymer polymer was was used used to to define define the the The droplet manipulation region, and actuation electrodes were coated with Parylene C dielectric, covered droplet manipulation region, and actuation electrodes were coated with Parylene C dielectric, ® layer, to ensure the hydrophobicity of the bottom plate. This work shows the introduction by a Teflon ® covered by a Teflon layer, to ensure the hydrophobicity of the bottom plate. This work shows the of flexible components, an ITO-coated naphthalate (PEN) top plate. The device introduction of flexiblenamely components, namely polyethylene an ITO-coated polyethylene naphthalate (PEN) top design includes four reservoirs to store primers, DNA template and PCR reagents, from which 1.2 nL plate. The device design includes four reservoirs to store primers, DNA template and PCR reagents, droplets may For be actuation, a charge pump voltage is able to convert the from which 1.2be nLretrieved. droplets may retrieved. For actuation, a chargeconverter pump voltage converter is able 3.3convert V device power 90 Vsupply applied to electrodes to induce an ON to state. Real-time to the 3.3 Vsupply deviceinto power into 90 V applied to electrodes induce an ONoptical state. detection is performed by an integrated Geiger-mode single-photon avalanche diode (SPAD), located Real-time optical detection is performed by an integrated Geiger-mode single-photon avalanche at a particular in which a window was as to allow the passage diode (SPAD), detection located atpixel a particular detection pixel in opened, which a window was opened, asof tolight allowfrom the an external laser. Several preliminary experiments were performed to evaluate the device, namely passage of light from an external laser. Several preliminary experiments were performed to evaluate temperature calibration, detection performance (nM concentration device lifetime and droplet the device, namely temperature calibration, detection performancescale), (nM concentration scale), device

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.

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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. Rival et presented a DMF platform that allows complete cell expression analysis, Rival et al. al.[24] [24]recently recently presented a DMF platform that allows complete cell expression from cell lysis to real-time nucleic acid amplification. This two-plate configuration device is produced analysis, from cell lysis to real-time nucleic acid amplification. This two-plate configuration device is on a silicon overwafer, whichover an SiO and Ti/AlCu were deposited, 2 insulating produced onwafer, a silicon which an SiO2 layer insulating layer andelectrodes Ti/AlCu electrodes were followed by an Si N dielectric layer and an SiOC hydrophobic coating. The ITO-covered glass 3 by 4 an Si3N4 dielectric layer and an SiOC hydrophobic coating. The ITO-covered deposited, followed top plate contained sand-blasted holesholes for reagent and sample insertion, and was from glass top plate contained sand-blasted for reagent and sample insertion, and separated was separated ® spacer. This DMF platform is accompanied by a bench-top the bottom plate by a 100 µm Ordyl ® from the bottom plate by a 100 µm Ordyl spacer. This DMF platform is accompanied by a instrumentinstrument which allows for allows temperature control (Peltier module), analysis (camera for bench-top which for temperature control (Peltierreaction module), reaction analysis fluorescence detection after sample excitation withexcitation a light emitting and droplet actuation (camera for fluorescence detection after sample with adiode, light (LED)) emitting diode, (LED)) and (Figure 4). With this platform, cell lysis and subsequent mRNA capture (assisted by magnetic beads) droplet actuation (Figure 4). With this platform, cell lysis and subsequent mRNA capture (assisted are magnetic performedbeads) in line,are reducing cross in contamination probability and greatly improving reproducibility. by performed line, reducing cross contamination probability and greatly Platform validation was performed for the detection of the c-MYC gene allowing a limit of detection of improving reproducibility. Platform validation was performed for the detection of the c-MYC gene just one cell, while using a 20-fold volume reduction compared to the traditional benchtop protocols. allowing a limit of detection of just one cell, while using a 20-fold volume reduction compared to the Furthermore, 10 cells protocols. were submitted to the mRNA extraction and amplification procedures, andand the traditional benchtop Furthermore, 10 cells were submitted to the mRNA extraction real-time fluorescence analysis revealed two distinct Ct values, suggesting sub-populations in the amplification procedures, and the real-time fluorescence analysis revealed two distinct Ct values, tested sample. suggesting sub-populations in the tested sample.

Figure Figure 4. 4. DMF DMF device device assembly: assembly: (a) (a) Additional Additional instrumentation instrumentationfor forthe the DMF DMF chip chip control; control; (b) (b) Chip Chip cross section, evidencing all the constituting layers; (c,d) Photographs of the actual DMF chip, cross section, evidencing all the constituting layers; (c,d) Photographs of the actual DMF chip, showing showing most chip relevant chip Adapted regions. Adapted fromin original in reference [24]. the most the relevant regions. from original reference [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 (Figure 5). 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.

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

Fouillet and co-workers successfully successfully demonstrated demonstrated the the use use of of a DMF-based chip for single nucleotide polymorphism polymorphism genotyping genotypingin inhuman humanplacental placentalDNA DNAsamples samples[25]. [25].The Thegroup groupdeveloped developeda ® ®. The aclosed closed configuration device, a polycarbonate top plate, with ITO and Teflon configuration device, withwith a polycarbonate top plate, coatedcoated with ITO and Teflon . The bottom bottom plate allocated Au electrodes coated with a dielectric Si3aNhydrophobic 4 and a hydrophobic plate allocated Au electrodes coated with a dielectric layer of layer Si3 N4 of and Teflon® ® layer. The cartridge is divided into three regions: (1) loading of reagents; (2) storage section, Teflon layer. The cartridge is divided into three regions: (1) loading of reagents; (2) storage section, where where intermediate reactions mayand occur; and (3) for channels droplet Droplets actuation.areDroplets intermediate reactions may occur; (3) channels dropletfor actuation. actuatedare at actuated at 60 VRMS Finally, and 3 kHz. Finally,control temperature controlusing was achieved using aplaced Peltierunder module 60 VRMS and 3 kHz. temperature was achieved a Peltier module the placed the chip for fast thermocycling capability. Thisamplification approach allowed amplification of a chip forunder fast thermocycling capability. This approach allowed of a minimum of 10 DNA minimum of 10 DNA strands, achieving single nucleotide specificity in a total volume of 64 strands, achieving single nucleotide specificity in a total reaction volume of 64reaction nL. Optical detection nL. Optical detection was performed by a standard fluorescence microscope using an intercalating was performed by a standard fluorescence microscope using an intercalating dye. This group also dye. This other groupinteresting also developed other interesting DMF biological chip designs for additional biological developed DMF chip designs for additional processes and applications [26]. processes and applications [26]. DMF–PCR Platform Validation DMF–PCR Validation EffortsPlatform have been made towards the validation of DMF-based platforms for further development of cost-effective diagnostic methodologies. publication the validation of a for DMF-based Efforts have been made towards The thefirst validation ofshowing DMF-based platforms further platform with clinical samples was reported by Wulff-Burchfield et al., who resorted to DMF-based development of cost-effective diagnostic methodologies. The first publication showing the DNA amplification for the detection Mycoplasma in 59 clinicalby samples [27]. In this study, validation of a DMF-based platformofwith clinical pneumoniae samples was reported Wulff-Burchfield et al., the performance an Advanced Liquid Logic DMFfor chip compared to conventional real-timein PCR who resorted to of DMF-based DNA amplification thewas detection of Mycoplasma pneumoniae 59 amplification techniques. As a result, successful amplification was achieved for 3.2 µL and the reaction clinical samples [27]. In this study, the performance of an Advanced Liquid Logic DMF chip was time was reduced by 3-fold, while producing results totechniques. those attained reaction, compared to conventional real-time PCR similar amplification Asvia a bench-top result, successful with 67% sensitivity and 100% amplification was achieved for specificity. 3.2 µL and the reaction time was reduced by 3-fold, while producing Anresults Advanced Liquid Logic via DMF platform reaction, was also employed by Schell etand al. [28] forspecificity. the detection similar to those attained bench-top with 67% sensitivity 100% of Candida albicans inLiquid clinical Logic blood samples. This group real-time PCR onet16al. blood An Advanced DMF platform was performed also employed by Schell [28] samples for the from patients infected with C. albicans (1 mL each) and concluded that the PCR-based diagnostic detection of Candida albicans in clinical blood samples. This group performed real-time PCR on 16 overallsamples performance of on-chip reactions differed from(1the method, yet blood from patients infected with C. albicans mLconventional each) and concluded thatthe thecombination PCR-based of both methods 94% sensitivity. Furthermore, the DMF–PCR lowers both reaction diagnostic overallyielded performance of on-chip reactions differed from theapproach conventional method, yet the time and cost. combination 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—Conventional Hybrid Devicesof channel-based conventional microfluidics (CMF) Another innovative Microfluidics approach is the integration with Another DMF, creating the so-called hybrid devices. These of devices were first developed to fully integrate innovative approach is the integration channel-based conventional microfluidics columnwith separation processes CMF) withThese all the steps were required pre-separation (CMF) DMF, creating the (possible so-called with hybrid devices. devices first for developed to fully (possible with DMF) [29,30]. However, DMF–CMF hybrid devices have also been applied for PCR integrate column separation processes (possible with CMF) with all the steps required for

pre-separation (possible with DMF) [29,30]. However, DMF–CMF hybrid devices have also been

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applied for PCR amplification protocols, either to constrain droplet movement [31] or facilitate amplification protocols, eitherreactions to constrain reagent injection and heated [32]. droplet movement [31] or facilitate reagent injection and heated reactions [32]. Ugsornrat et al. developed a hybrid device where fluids are constrained to a PDMS Ugsornrat et al. microchannel, developed a yet hybrid device fluids are constrained to a PDMS (polydimethylsiloxane) droplets are where still controlled by electrode addressing [31]. (polydimethylsiloxane) microchannel, yet droplets aresubstrate, still controlled by electrode [31]. Briefly, Cr/Au electrodes were deposited on a glass and covered with addressing a layer of SiO 2, ® Briefly, Cr/Au electrodes were deposited on a glass substrate, and covered with a layer of SiO , which which in turn was covered by a hydrophobic layer (Teflon ). The glass cover plate included2 an ITO ® ). The glass cover plate included an ITO layer in turnacting was covered by aelectrode, hydrophobic layer layer as ground as well as (Teflon three different serpentine-shaped Cr/Au microheaters, acting as ground electrode, as well as three different serpentine-shaped Cr/Au microheaters, for precise control of denaturation, annealing and extension temperatures in a reaction volume offor 25 precise control of denaturation, annealing a reaction volumeoperating of 25 µL, µL, where optimized usage of mineral and oil extension preventedtemperatures evaporationinbeyond standard where optimized usage of mineral oil prevented evaporation beyond standard operating conditions. conditions. Kim have developed developed a DMF platform combining PCR Kim et et al. al. have PCR amplification amplification and and capillary capillary microfluidics, sequencing [32].[32]. ThisThis device fully integrates nucleic nucleic acid amplification (usually microfluidics,for forDNA DNA sequencing device fully integrates acid amplification required a pre-sequencing step) with step) the sequencing procedure itself, unlikeitself, other unlike DMF-based (usually as required as a pre-sequencing with the sequencing procedure other sequencing platforms [33,34]. The device is divided into two segments: (1) capillary tubes for reagent DMF-based sequencing platforms [33,34]. The device is divided into two segments: (1) capillary insertion on the DMF hubon and reactions (PCRreactions included)(PCR andincluded) (2) a DMF hub reagent tubes for reagent insertion theheated DMF hub and heated and (2) for a DMF hub mixing and room-temperature reactions, connected to the capillary tubes. The DMF hub consisted for reagent mixing and room-temperature reactions, connected to the capillary tubes. The DMF hub of a two-plate with 40 electrodes and 8 larger reservoirs (Figure 6).(Figure With this consisted of a configuration two-plate configuration with 40 electrodes and 8 larger reservoirs 6). platform, With this Kim et al. achieved PCR amplification of human genomic DNA retrieved from blood from cells platform, Kim et al.successful achieved successful PCR amplification of human genomic DNA retrieved (previously tagged), in about 13 min, for just 2.8 µL, which further enabled the production of a human blood cells (previously tagged), in about 13 min, for just 2.8 µL, which further enabled the DNA library.of a human DNA library. production

Figure 6.6. Schematic Schematic representation representation of of the the DMF DMF platform platform proposed, proposed, evidencing evidencing the the location location of of all all the the Figure reagents required for DNA sequencing. The gap between plates was maintained at either 185 µm or reagents required for DNA sequencing. The gap between plates was maintained at either 185 µm or 400 µm, according to the intended droplet volumes, and no filler medium was added. Adapted from 400 µm, according to the intended droplet volumes, and no filler medium was added. Adapted from original in inreference reference[32]. [32]. original

2.3. DMF DMF for for Isothermal Isothermal Nucleic Nucleic Acid Acid Amplification Amplification 2.3. Even though though PCR PCR is is the the most most widely widely known known nucleic nucleic acid acid amplification amplification methodology, methodology, the the three three Even elevated temperatures needed for thermal cycling add complexity to this technique, and contribute elevated temperatures needed for thermal cycling add complexity to this technique, and contribute to high high energy energyrequirements requirementsand andsophisticated sophisticatedequipment equipment(e.g., (e.g.,thermocyclers). thermocyclers).Thus, Thus,DNA/RNA DNA/RNA to amplification research research has has been been progressing progressing towards towards isothermal isothermal amplification amplification schemes, schemes, enabling enabling amplification low-cost, low-temperature nucleic acid amplification. Recently, isothermal amplification hasbeen also low-cost, low-temperature nucleic acid amplification. Recently, isothermal amplification has also been integrated in DMF devices, envisioning ready-to-use POC molecular diagnostics. integrated in DMF devices, envisioning ready-to-use POC molecular diagnostics. Kalsiet et al. propose anmatrix-based active matrix-based DMF platform the detection of Kalsi al. propose an active DMF platform for the detectionfor of antibiotic-resistance antibiotic-resistance factors in Escherichia coli bacteria (CTX-M gene), through isothermal factors in Escherichia coli bacteria (CTX-M gene), through isothermal recombinase polymerase recombinase polymerase (RPA) [18]. This two-plate platform on a amplification (RPA) [18]. amplification This two-plate configuration platformconfiguration relies on a thin film relies transistor thin film transistor (TFT) backplane, which controls ITO electrodes for droplet actuation and (TFT) backplane, which controls ITO electrodes for droplet actuation and capacitance sensors able 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

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to distinguish between the presence or absence of a droplet, allowing real-time droplet monitoring. Sensors 2017, 17, 8 of 14layer, The backplane is 1495 covered with an Al2 O3 insulator, as well as a disposable Cytop® hydrophobic and the ITO-coated glass top plate is also covered by these two layers (Figure 7). 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– defines a specific distance and droplets are manipulated in anelectrodes, n-Dodecane Derivative (PID) controlbetween system. Aelectrodes, Mylar spacer defines a specific distance between and filler. EWOD actuation voltage is of about 20 V and optical detection is achieved in real-time by fluorescence droplets are manipulated in an n-Dodecane filler. EWOD actuation voltage is of about 20 V and measurements of a Cy5 label added to the by sample. Successful amplification of the was optical detection is achieved in real-time fluorescence measurements of a Cy5 labelgene added to possible the Successful the gene was possible for a minimum 270 nL, limit of for a sample. minimum 270 nL,amplification with a limit of detection of about 1 single template copy, inwith 10 toa 15 min. detection of about 1 single template copy, in 10 to 15 min.

Figure (a) Cross-section of the DMF platform, evidencing the severalmaterials materialsthat thatcomprise comprise the the chip; Figure 7. (a)7.Cross-section of the DMF platform, evidencing the several chip; (b) Schematic top view of the chip, evidencing the reservoirs and the temperature sensor. (b) Schematic top view of the chip, evidencing the reservoirs and the temperature sensor. Adapted from Adapted from original in reference [18]. original in reference [18].

Another example of isothermal nucleic acid amplification performed on a DMF chip is

Another example of isothermal nucleic amplificationof performed a by DMF chip is provided by Kühnemund et al., to detect very acid low concentrations P. aeruginosa on DNA rolling circle by amplification (RCA) followed by circle-to-circle amplification (C2CA), assisted by magnetic provided Kühnemund et al., to detect very low concentrations of P. aeruginosa DNA by rolling [19]. The amplification protocols amplification are performed on a two-plate configuration circlemicroparticles amplification(MMP) (RCA) followed by circle-to-circle (C2CA), assisted by magnetic ® Two top chip, where the bottom plate included Cr electrodes covered by Parylene C and Teflon microparticles (MMP) [19]. The amplification protocols are performed on a two-plate .configuration 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 ® layer. Tape was ITO-covered glass, coveredthe with a Teflon layer and Au-covered glass with a top Teflonof performed by placing chip on a thermocycler plate. This device allows detection 1 aM of P. used aeruginosa as spacer, DNA and droplets were by reaction 120 VACtemperature , at 1 kHz. Heating by placing in only 60 min,actuated for an RCA of 37 °C was and aperformed reaction volume of 5 the chip on µL.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,

alternative techniques developed, mainly based on magnetic, or even acoustic Even though EWOD have is thebeen standard method for controlling dropletoptic motion in DMF systems, forces. Non-EWOD DMF platforms have been explored for a variety of chemical and biological alternative techniques have been developed, mainly based on magnetic, optic or even acoustic forces. procedures, namely PCR-based DNA amplification for POC diagnostics. Non-EWOD DMF platforms have been explored for a variety 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-assisted DMF platforms for PCR have beenthe widely explored at Johns droplets, which in turn are manually controlled byamplification a magnet placed under substrate and allow Hopkins University [35–37], and rely on the addition of superparamagnetic particles to the droplets, droplet motion. Zhang et al. [37] developed a DMF platform for biomarker detection from human whichblood in turn are manually controlled by afrom magnet placedsamples under the substrate allow droplet motion. samples, and pathogen detection biological (E-coli bacteriaand as proof-of-concept). Zhang et al. [37]platform developed a DMF platform for biomarker detection human blood samples, This DMF successfully implemented all steps required forfrom target detection, from solid and phasedetection DNA extraction to real-timesamples PCR or helicase dependent (HDA),This thus DMF proving to pathogen from biological (E-coli bacteria asamplification proof-of-concept). platform be a suitable platform for POC applications. successfully implemented all steps required for target detection, from solid phase DNA extraction to devices based on magnetic forces typically do thus not enable theto full fluidic for real-time DMF PCR or helicase dependent amplification (HDA), proving bespectrum a suitableofplatform operations, namely droplet dispensing and splitting. Zhang et al. [36] surpassed this problem by POC applications.

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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 (Table 1). 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 (Figure 8) [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.

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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.

DMF–PCR platforms

Validation of DMF–PCR platforms

Hybrid platforms

DMF platforms for isothermal amplification

Application

Reaction Volume

Dengue II virus detection

1.46 µL

SNP genotyping

64 nL

POC testing, MRSA, S. aureus and C. albicans detection MRSA, S. aureus, M. pneumoniae and C. albicans detection S. aureus detection Cell genetic expression analysis Bacteriophage M13mp18 detection C. albicans detection on human blood M. pneumoniae detection on human nasopharyngeal wash DNA amplification (only the primers are described)

600 nL

Actuation Voltage 12 VRMS (3 kHz) 60 VRMS (3 kHz) -

Dielectric Material

Hydrophobic Material

Electrode Material

Substrate Material

Filler

Reference

Si3 N4

Teflon® AF

Au

Glass

Silicone oil

[17]

Si3 N4

Teflon® AF

Au

Glass

Silicone oil

[25]

Parylene C

Teflon® AF

Cr

PCB

Hexadecane

[9]

Variable

-

-

-

-

PCB

Hexadecane/silicone oil

[21]

1.2 nL Variable

70 V–250 V 48 V (3 kHz) 80–100 VRMS (18 kHz)

Parylene C Si3 N4

Teflon® AF SiOC

Ti/AlCu

Silicon wafer

n-dodecane Silicone oil

[23] [24]

Solder mask

Teflon® AF

Cu/Ni/Au

PCB

Air

[22]

1.5 µL Variable

-

-

-

-

PCB

Hexadecane/silicone oil

[27]

Variable

-

-

-

-

PCB

Hexadecane/silicone oil

[28]

25 µL

-

SiO2

Teflon® AF

Cr/Au

Glass

Silicone oil

[31]

Pre-DNA sequencing

2.8 µL

80–90 VRMS (15 kHz)

Parylene C

Teflon® AF

ITO

Glass

Air

[32]

CTX-M gene detection in E. coli bacteria

Minimum 270 nL

20 V

Al2 O3

Cytop®

ITO

Glass

n-dodecane

[18]

5 µL

120 VAC (1 kHz)

Cr

Glass

Vapor-Lock oil

[19]

P. aeruginosa detection

Parylene C

Teflon®

AF

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(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. (c) (a)Droplet DMF device screen-printed on paper substrate; (b) from Zoom view inofreference the printed movement on a paper-based DMF device. Adapted original [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.

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