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A passive micromixer for enzymatic digestion of DNA

A passive micromixer for enzymatic digestion of DNA
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  A passive micromixer for enzymatic digestion of DNA V.E. Papadopoulos a , I.N. Kefala a , G. Kaprou a , G. Kokkoris a, ⇑ , D. Moschou a , G. Papadakis a , E. Gizeli b,c ,A. Tserepi a, ⇑ a Institute of Nanoscience and Nanotechnology, NCSR ‘‘Demokritos’’, Aghia Paraskevi, Attiki, 15310, Greece b Institute of Molecular Biology & Biotechnology, F.O.R.T.H., 100 N. Plastira str., 70013 Heraklion, Crete, Greece c Department of Biology, University of Crete, Vassilika, Vouton, 71409 Heraklion, Crete, Greece a r t i c l e i n f o  Article history: Received 28 October 2013Received in revised form 1 April 2014Accepted 8 April 2014Available online 18 April 2014 Keywords: Passive micromixerFlexible printed circuitMicrofluidicsLab on a Chip (LoC)DNA digestion a b s t r a c t Apassivemicromixerwithzigzaggeometryisdemonstratedinthisworktoperformsimultaneouslymix-ingofarestrictionenzymewithDNAanddigestionofDNA.Thetotallengthrequiredforcompletemixingis estimated by simulation. The micromixer is fabricated from an imide-based photoimageable dry-filmusing flexibleprintedcircuit technologythat allows easyintegrationof themicrodevice inmorecomplexLab-on-a-Chip (LoC) platforms. When heated at 37  C, the micromixer achieves both complete mixing of the reagents and DNA digestion with restriction enzymes within 2.5min, i.e., a time comparable to theincubation step needed in conventional digestion systems. Thus, it renders further incubation unneces-sary and is proved a valuable component of LoC systems for diagnostic purposes.   2014 Elsevier B.V. All rights reserved. 1. Introduction The basic idea and the vision that leads to the rapid develop-ment of Lab-on-a-Chip (LoC) systems is the integration of several,preferablyall, functions of a (bio)chemical analysis laboratory onachip. Microfluidic devices appropriate for transport, mixing, sepa-ration, and/or reactions, are necessary for LoC applications. Addi-tionally, their operation defines the total performance of LoCsystems; thus, their design is of crucial importance. For example,a well-designed micromixer can reduce the analysis time as wellas the footprint of LoC platforms [1].The application of interest in this work is the enzymatic diges-tion of DNA with restriction enzymes that recognize and cut DNAat specific positions. Restriction enzymes are commonly employedto identify a change (known as single nucleotide polymorphism,SNP) in the genetic sequence that occurs at a site where theseenzymes would normally cut. Effective mixing of the enzyme withDNA sample is required to have a fast digestion process [2]. Thus,the focus of this work is to develop a passive micromixer for theenzymatic digestion of DNA. Passive micromixers do not requireexternal energy (besides the energy required for the pumping of thefluid)asopposedtoactive[3,4]whichusethedisturbancegen-erated by an external field for the mixingprocess. Eventhoughtheactivemicromixersaremoreeffectivethanthepassiveones[5,6],apassive micromixer is chosen in this work, because active micro-mixers entail more complex fabrication processes, and their inte-gration, due to the need of external power source, is moredifficult. In passive micromixers, due to the low Reynolds (Re)number, mixing occurs as a result of diffusion rather than turbu-lence. Hence, mixing is slow; the required length of the microm-ixer, as well as the time for full mixing, are increasedsignificantly compared to flows with high Re.Several designs of passive micromixers have been proposed inthe literature such as Y or T shaped and multi-input channels[7,8] or channels with a – suitably alternating – cross section [9]or channels with zigzag geometry [10]. Other works proposedmicroscopicthree-dimensionalgeometriesinsidethemixingchan-nelsuchasbarriers(posts)[10]orgrooves[11,12].Inthiswork,we combine the benefits of a T shaped channel with the zigzag geom-etry and propose a micromixer which is compatible with printedcircuitboard(PCB)orflexibleprintedcircuit(FPC)technology[13].Inthepast, thefabricationof micromixersrelied onmicro-elec-tromechanical systems (MEMS) technology. Silicon (Si) and glass[14] were the principal substrate materials used for micromixerfabrication. However, Si possesses inadequatemechanical and bio-compatibility properties [15]. In addition, glass is difficult to beprocessed. Recently, theurgentneedforcosteffectiveandbiocom-patible materials led to polymeric fabrication techniques as com-petent candidates. Laser ablation [16], casting soft lithography[17] and hot embossing are some of the readily available   2014 Elsevier B.V. All rights reserved. ⇑ Corresponding authors. E-mail addresses: (G. Kokkoris), (A. Tserepi).Microelectronic Engineering 124 (2014) 42–46 Contents lists available at ScienceDirect Microelectronic Engineering journal homepage:  techniques used in microfabrication of micromixers on materialssuch as Poly(methyl-methacrylate) (PMMA), SU-8, and Poly-dimethylsiloxane (PDMS) [17]. However, there is a need forcheaper,andatthesametime,massproductionamenabletechnol-ogies, such as PCB/FPC compatible ones.In this work, simulation, fabrication, and evaluation of themicromixer are performed. First, the simulation calculates thelengthor thevolumerequiredtoassuremixing. TheNavier–Stokesand continuity equations, together with the mass conservationequationfortheenzymearesolvedwiththecommercialcodeCOM-SOL [18].Secondly,imide-basedphotoimageabledry-filmisimple-mented as substrate material laminated on PCB for the realizationof themicromixer. Thepropertiesof thisfilm, namely, biocompati-bility,aswellaschemicalandthermalstability,arebetterthanthoseexhibited by other polymers, while its photosensitivity allows thewhole fabrication process to be amenable to mass production at alowcost.Simultaneously,thisfilmallowsintegrationofthemicrom-ixerwithothermicrofluidiccomponentsinmorecomplexLoCplat-forms; polyimide has been used as substrate in polymerase chainreaction (PCR) devices [19–21] but the microfluidic chambers orchannelshavebeenformedbylaserablationorplasmaetching,tech-nologies not yet adopted for the mass production of microfluidics.Third, the fabricated micromixer is evaluated for DNA digestion: AfractionofaPCRcontaininganamplified273-basepairs(bp)productwasmixedandreactedwiththerestrictionenzymeat37  Cwithoutfurtherincubationstep. Completedigestionwasachievedinatimecomparabletotheincubationstepoftheconventionalprocess.Only few reports for micromixers used for on-chip enzymaticdigestion exist in the literature. Xie et al. [22] as well as Wangetal.[23]performedenzymaticdigestionsinbatchmodeinastaticmicroreactor incorporated in an integrated microdevice; Fu et al.[2] and Lin et al. [24] developed a continuous flow digestion mic- rodevice which consisted of an active micromixer followed by aseparate serpentinechannel for DNA digestionreaction. Comparedtothepreviousworks,acontinuousflowpassivemicromixerisfab-ricatedinthis work, servingsimultaneouslythemixingof theDNAwiththeenzymeandthedigestionreaction.Besidestheapplicationtargeted, the importanceof this work lies mainlyinthe fabricationtechnologyimplementedforthemicromixerwhichallowstheinte-gration of microfluidic devices with heterogeneous components:electronic circuits, sensors [13], and microheaters [21] that can be utilized for on-chip detection and control in a cost-effective man-ner, following the current trend in LoC technology [25,26].The rest of the work is structured as follows: In Section 2, themathematical model and the measure for the mixing performanceare described. In Section 3, the design of the micromixer and thesimulation results are included. Section 4 describes the fabricationprocess and Section 5 the evaluation of the micromixer. The lastsection includes the conclusions. 2. Mathematical model Evenif bothdiffusionandreactionoccur in the micromixer, themathematicalproblemissimplifiedtoadiffusionproblemofasol-ute (enzyme) into water (solution of DNA); the efficient mixing of the enzyme with the DNA sample is critical for the digestion pro-cess. The model consists of the continuity equation r  u ¼ 0  ð 1 Þ and Navier–Stokes equation q u  r u ¼ r  p þ l r 2 u ;  ð 2 Þ where u isthevectoroffluidvelocityand q , l and  p arethedensity,dynamic viscosity, and pressure of the fluid. The model includesalso the mass conservation equation of the enzyme r ð D r C  Þþ u  r C   ¼ 0  ð 3 Þ where C   and D  aretheconcentrationanddiffusioncoefficient of theenzyme in the DNA solution. No slip condition for the velocity andzero derivative for the concentration are considered at the walls of the micromixer. Fully developed parabolic profiles of flow are con-sidered at the inlets whereas zero derivatives of both velocity andconcentration in the outflow direction are considered at the outlet. The density and the dynamic viscosity of the DNA solution arethose of water at 37  C, i.e. the temperature at which digestiontakes place. The equations are solved in 2d with the commercialcode COMSOL. This assumption has been employed in previousworks [2] and it implies that the dependent variables do not exhi-bit significant gradients in the 3rd dimension. 3d calculationswould have given more accurate results; however, they require agreat amount of memory.The performance of the micromixer is evaluated by the mixingefficiency,  n , [17] at a vertical-to-flow cross section. n ¼ 1   ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 N  X N i ¼ 1 C  i   C   C    2 v uut  ð 4 Þ where   C   is the expected concentration at full mixing and  C  i  is thelocal concentration at point  i  of the cross section.  N   is the numberof points in the cross section. A mixing efficiency between 0.8 and1 is acceptable for several applications [27]. 3. Design of the micromixer and simulation results In Fig. 1, a top-down view of the inlets (Fig. 1a) of the microm- ixer andits zigzaggeometry(Fig. 1b) are shown. Agreater numberof inlets potentially would have increased the mixing efficiency;however, it would have made the operation of the micromixermore complex. The depth of the channel (normal to paper direc-tion) is 60 l m, equal to the thickness of the photopatternableimide-based dry-filmused for thefabrication(Section4). Themin-imum channel width of the design is 100 l m (Fig. 1a), largeenough for reliable photopatterning, and small pressure dropacross the device. Regarding the flowregime, Re in the main chan-nel of the micromixer is from 0.104 to 0.416 for the volumetricflow rates of interest in this work, i.e. from 1.5 to 6 l l/min.The suitable number of zigzags of the micromixer, i.e., thenumber which leads to a high mixing efficiency (see Section 2), Fig. 1.  Design of (a) the inlets and (b) the zigzag geometry of the micromixer. V.E. Papadopoulos et al./Microelectronic Engineering 124 (2014) 42–46   43  is calculated by the simulation. The mixing efficiency vs. the num-berof zigzagsisshowninFig. 2fordifferentvaluesof thediffusioncoefficientoftheenzyme;thevaluesof thediffusioncoefficientsof enzymes in the literature vary from 10  9 to 10  11 m 2 /s [1].Theeffectoftheinletvelocityonthemixingefficiencyisshownin Fig. 3. A smaller number of zigzags are required to achieve thesame mixing efficiency for smaller inlet velocities (flow rates).However, the time for achieving the same mixing efficiency isalmostthesameforallinletvelocities(1.9–2.2min).Thus,theinletvelocity mainly affects the required number of zigzags and as aconsequence the footprint of the micromixer.The simulation recommends a micromixer with ca. 150 zigzags(see Fig. 4), which allows a very efficient mixing if the diffusioncoefficient is 10  10 m 2 /s ( n  =0.98) and an efficient mixing( n  =0.88) if the diffusion coefficient is even smaller(5  10  11 m 2 /s, see Fig. 2). The total volume of the micromixerof  Fig. 4 is 7.5 l l. 4. Fabrication process The process utilized for the fabrication of the micromixer pre-sented herein is schematically shown in Fig. 5. The starting mate-rial used as substrate in this work is commercially available PCB.Commercially available imide-based photosensitive dry film(Dupont PYRALUX  PC1025), 60 l m thick, is laminated (85  C)using a roll laminator directly onto the top of the PCB substrateto constitute the bottom wall of the micromixer. Next, a secondlayer of photosensitive PC1025 is laminated in order to form themicrofluidicnetwork.Aftereachlaminationprocess,standardpho-tolithography and development using 1% w/w aqueous sodium bi-carbonate solution (Na 2 CO 3 ) is performed. During UV exposure of the second PC1025 layer, the micromixer mask is used to formthe microfluidic channel. Next, hard bake is performed at 160  C.The microfluidic component is then sealed using a 70 l m thickcommercial Kapton  polyimide tape with a silicon adhesive on aroll laminator operating at 50  C. A top-down image of the fabri-cated micromixer is shown in Fig. 6.Leakageandflowcontroltestswereperformedonsealedmicro-fuidic devices using a laboratory syringe pump (Chemyx Inc,Fusion 200). The fluidic interfacing was achieved with ahome-made PMMA chip holder (Fig. 7) designed to attach to 501001502002500. D=10 -9  m 2 /sD=5x10 -9  m 2 /sD=10 -10  m 2 /s   m   i  x   i  n  g  e   f   f   i  c   i  e  n  c  y number of zizags D=5x10 -11  m 2 /s 24681012volume of the micromixer ( µ l) Fig. 2.  Mixing efficiency [ n  in Eq. (4)] vs. the number of zigzags (and micromixervolume, see top  x -axis) for different values of the diffusion coefficient of theenzyme. The inlet velocity is 2mm/s, which corresponds to a volumetric flow rateof 3 l l/min. All curves stop at  n  =0.98. A zigzag is defined as a V-shaped (not W-shaped) cell of the channel. 501001502002500.  u =1 mm/s  u =2 mm/s u =4 mm/s   m   i  x   i  n  g  e   f   f   i  c   i  e  n  c  y number of zizags24681012volume of the micromixer ( µ l) Fig. 3.  Mixing efficiency [ n  in Eq. (4)] vs. the number of zigzags (and micromixervolume, top  x -axis) for different values of inlet velocity (1, 2, and 4mm/s whichcorrespond to flow rates of 1.5, 3, and 6 l l/min). The diffusion coefficient of theenzyme is 10  10 m 2 /s. All curves stop at  n  =0.98. A zigzag is defined as a V-shaped(not W-shaped) cell of the channel. Fig. 4.  The design of the proposed micromixer. The circles denote the location of the inlets and outlet of the micromixer. The footprint is 7.98  0.72cm 2 . Fig. 5.  Schematic of the microfabrication process flow.44  V.E. Papadopoulos et al./Microelectronic Engineering 124 (2014) 42–46   commercially available Upchurch nanoport fittings. Fabricateddevices were found to be leak-free when deionized water wasallowed to flow through the microchannels. Furthermore, thebonding strength of the devices was tested under flowing condi-tions and while heating the device up to 45  C on a hot plate. Mostdevices were proved robust under conditions of DNA enzymaticdigestion. 5. Micromixer evaluation for DNA digestion Theevaluationof the micromixerwas based on thedigestionof a PCR product with a restriction endonuclease. A 273-bp DNA wasproduced from human genomic DNA template as described previ-ously [28]. The sequence of the amplified DNA fragment is part of the BRCA1 gene and is associated with breast cancer cases when asingle base insertion occurs at the position 5382 [29]. The 273-bpDNA can be digested with the restriction endonuclease DdeI (NEB,R0175S) into two fragments of 20- and 253-bp, in respect.It should be noted that it was necessary to include, within theDNA and enzyme solutions, 1.5% PEG 8000 and 2mg/ml BSA, topreventsamplelossonthemicrochannelwallsduetothedramaticincrease of the surface-to-volume ratio of the device [30].OurpurposewastoevaluatethecapabilityofthemicromixertoeffectivelymixaPCRproductwitharestrictionendonucleasesolu-tionat roomtemperaturethat wouldleadto completedigestionof theDNAafterincubationat37  C.Additionally,weinvestigatedthepotential of the microdevice to result in rapid DNA digestionwithinthemicrochannelswhenthemixingandreactiontakeplaceat 37  C without further incubation.Initially, a fraction of a PCR reaction containing an amplified273-bp product was mixed with the restriction endonuclease bypipetting gently up and down for several times and was placedin an incubator at 37  C for 5min. Using gel electrophoresis, DNAwas found to be completely digested. We then loaded on themicromixer an equal fraction of the PCR reaction from the firstinlet and the enzyme solution from the second inlet and drovethem through the microchannels using a peristaltic pump. Theflow rate was approximately 3 l l/min (Re=0.208). We placed aneppendorf tube on a heated-block at 37  C and the collected fromthe exit DNA-enzyme mix was incubated for 5 more minutes.Thegel analysis revealedcompletedigestionof the DNAindicatingefficient mixing within the microdevice (Fig. 8a and b). Further-more, we placed the micromixer on a hot plate and adjusted thetemperature of the microdevice at approximately 37  C. Werepeated the previous mixing experiment but we collected theDNA-enzyme mix directly on a tube placed on ice and it wasimmediately loaded on a gel without further incubation. As shownin Fig. 8c, the DNA was completely digested which indicates thatthe efficient mixing taking place within the microchannels alsoleadstorapidDNAdigestion.Theseresultsindicatethatourheated7.5 l l micromixer completes digestion in 2.5min. Although thetime for complete on-chip DNA digestion is not significantlyshorter than that of conventional digestion (5min), the developedmicromixer is advantageous as it renders further incubationunnecessary, while it is indispensable as part of LoC systems fordiagnostic purposes. 6. Conclusion A passive micromixer for enzymatic digestion of DNA is imple-mented by a novel fabrication process. A photoimageable dry filmis utilized under FPC technology processes, which allow easy inte-gration of the micromixer into more complex LoC platforms, using Fig. 6.  Fabricated zigzag micromixer (left) and magnified details (right): inlet junction, part of zig-zag channel. Fig. 7.  Testing of the fabricated micromixer in the home-made PMMA chip holder. Fig. 8.  Gel images of (a) undigested and (b) digested 273-bp fragment. The mixingwas performed at room temperature followed by incubation at 37  C. (c) DNAdigested within the micromixer placed on a hot-plate and without furtherincubation. V.E. Papadopoulos et al./Microelectronic Engineering 124 (2014) 42–46   45  low cost, mass production amenable technologies. Simulation isused for the estimation of the total length of the micromixer. Theproposed passive micromixer achieves both reagent mixing andcompleteDNAdigestionwithintimecomparabletothe incubationstep neededin conventional digestion. This is the first time a dual-purpose micromixer is demonstrated in literature. Previous worksemployed separate microchannels for digestion of DNA after mix-ing. The results presented in this work indicate that our microm-ixer can be employed as part of a LoC diagnostic device wheredigestionofamplifiedDNAcouldbeusedtodetectpointmutationsin genes associated with breast cancer. Future work involves inte-gration of microheaters on the micromixer, evaluation of themicromixer with more time consuming DNA digestion processesas well as integration with a flow-through PCR for performing allDNA amplification, mixing, and enzymatic digestion in one singledevice.  Acknowledgements Thisworkwaspartiallysupportedbytheproject‘‘DoW-DNAonWaves: An integrated diagnostic system’’ (LS7-276, program‘‘Supporting post-doctoral researchers’’, Ministry of Education,Lifelong Learning, and Religious Affairs); the source of funding isthe European Social Fund (ESF) – European Union and NationalResources. References [1] J.M. Ottino, S. Wiggins, Philos. T. R. Soc. A 362 (2004) 923–935.[2] L.M. Fu, C.H. Lin, Biomed. Microdevices 9 (2007) 277–286.[3] Y. Abbas, J. Miwa, R. Zengerle, F. von Stette, Micromachines 4 (2013) 80–89.[4] Y.-K. Lee, C. Shih, P. Tabeling, C.-M. Ho, J. Fluid Mech. 575 (2007) 425.[5] A. Alam, K.Y. Kim, Chem. Eng. J. 181–182 (2012) 708–716.[6] C.Y. Lee, C.L. 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