Engineering5(2019)173–180Contents lists available at ScienceDirectEngineeringResearch
SyntheticBiology—Article
AnAdditiveManufacturingApproachthatEnablestheFieldDeploymentofSyntheticBiosensors
DanielWoloznya,JohnR.Lakeb,PaulG.Movizzob,ZhichengLongb,?,WarrenC.Ruderb,?abDepartmentofBiologicalSystemsEngineering,VirginiaPolytechnicInstituteandStateUniversity,Blacksburg,VA24061,USADepartmentofBioengineering,UniversityofPittsburgh,Pittsburgh,PA15219,USAarticleinfoabstract
The tools of synthetic biology can be used to engineer living biosensors that report the presence of analytes. Although these engineered cellular biosensors have many potential applications for deployment outside of the lab, they are genetically modi?ed organisms (GMOs) and are often considered dangerous. Mitigating the risk of releasing GMOs into the environment while enabling their use outside a laboratory is critical. Here, we describe the development of a biosensing system consisting of a synthetic biological circuit, which is engineered in Escherichia coli that are contained within a unique 3D-printed device housing. These GMOs detect the chemical quorum signal of Pseudomonas aeruginosa, an opportunistic pathogen. Using this device, the living biosensor makes contact with a specimen of interest without ever being exposed to the environment. Cells can be visually analyzed in the ?eld within culture tubes, or returned to the lab for further analysis. Many biosensors lack the versatility required for deployment in the ?eld, where many diseases can go undiagnosed due to a lack of resources and equipment. Our bioassay device utilizes 3D printing to create a portable, modular, and inexpensive device for the ?eld deployment of living biosensors.
ó 2019 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and Higher Education Press Limited Company. This is an open access article under the CC BY-NC-ND license
Articlehistory:Received13May2018Revised25September2018Accepted17December2018Availableonline22December2018Keywords:SyntheticbiologyAdditivemanufacturingBiosensors1.IntroductionSyntheticbiologyisacombinationofbiologyandengineeringthatattemptstogainnewunderstandingandutilizationofthenaturalworldbyapplyingengineeringprinciplestodesigngeneticelementsforcellularcontrol.Commonexamplesofsyntheticbiologyincludememoryswitch[1]andoscillator[2]circuitsthatenablecellstoswitchbetweendistinctstatesbasedonchemicalorotherinput.Thisabilitytosenseaninputfromtheenvironmentenablescellstoserveasabiosensor.Asyntheticallyengineeredbiosensor[3–6]takesadvantageoftheversatilityoflivingcellstodetectandprocessinputsintoanalyzableoutputs.Livingcells,especiallybacteria,cansurviveundermanyconditions[7,8]andcanbeengineeredtodetectanddiagnoseinfectiousbiomarkersinclinicalsettings.Geneticallymodi?edorganisms(GMOs)areregulatedbymanylawsandgovernmentalbodies[9–13],makingthemdif?culttodeployoutsideoflaboratorysettings.Toallowengineered?Correspondingauthors.E-mailaddresses:(W.C.Ruder).longzhicheng@pitt.edu(Z.Long),warrenr@pitt.edubiosensorstobedeployedfordiseasediagnosisinthe?eld,theseregulationsmustbesatis?ed.Additivemanufacturing,intheformofthree-dimensional(3D)printing,canserveasasolutiontoiso-lateGMOsfromtheenvironmentwhenusedinthe?eld.Criticaladditivemanufacturingapproacheswere?rstcommer-cializedthreedecadesago[14].Sincethen,additivemanufacturingtechnologyhasevolvedrapidly,making3Dprinterswidelyavail-able.The3Dprintersutilizedforthisworkfunctionbysuccessivelydepositinglayersofextruded?lamentstobuilda3Dstructure[15].Thesetypesof3Dprintershavealreadybeenproventosuc-cessfullydesign,print,andtestmedicallyrelevantdevices[16,17].Inthiswork,wechosetoemployarobustapproachfordetect-ingthequorumsignalofPseudomonasaeruginosa(P.aeruginosa)thatisfrequentlyusedinsyntheticbiology(Fig.1(a)).ThelivingbiosensorconsistsofengineeredEscherichiacoli(E.coli)thataredesignedtodetectthepresenceofthisquorumsignal,N-3-oxododecanoylhomoserinelactone(3O-C12).ThelivingbiosensorwasconstructedbyintroducingcomponentsofP.aeruginosa’snative3O-C12signalingpathwaytoE.coli.Here,atranscriptionfactor,LasR,actsasasignaltransducerbybinding3O-C12anddimerizing.Thiscomplexthenbindstoatranscriptionactivationdomainfoundwithinitscognatepromoter,PLas[6,18,19],which174D.Woloznyetal./Engineering5(2019)173–180Fig.1.Fielddetectionof3O-C12throughtheuseofaGMOanda3D-printedenclosure.(a)The3O-C12biosensorwasengineeredtoconstitutivelyexpressaprotein,LasR,whichisabletobinda3O-C12molecule.Onceboundto3O-C12,theLasRproteindimerizesandbindstoaDNAregioncontainedinthePLaspromoter,whichpromotestheexpressionofmCherry.(b)Thebiosensorisplacedina3D-printedenclosure.Abiologicalsamplecanthenbeextractedandplacedinasampletubethatisconnectedtothe3D-printeddevice.Thedevicecoveristhenpressed,causingthebiosensortofallintothesample.(c)Thebiosensorturnsredtoreportexposureto3O-C12inthesample;thissignalcanthenbequanti?edusing?owcytometry.Histogramsrepresentdatacollectedfor10000eventsforboththeun-inducedandthe100nmoláLà13O-C12samples(threesamples).isalsotransplantedintoE.coli.Inourdesign,oncebound,LasRpro-motestheexpressionofthegeneformCherry,ared?uorescentprotein.Wespeci?callychose3O-C12asthechemicaltobedetectedduetoitsrelevanceinmedicalsettings[20–25].Asanexampleoftheclinicalrelevanceofthisapproach,inruralandless-developedareas,lunginfectionsfromtheorganismP.aeruginosacanbeacquiredinlocalclinics.UponlunginfectionbyP.aeruginosa(Fig.1(b)),theorganismmaybefoundinlungspu-tumsamples.Thissputumcanbecollectedandplacedwithincom-merciallyavailablesampletubes.Asaresult,anapproachenablingthesesamplestobeimmediatelyanalyzedinthe?eld,usingthetoolsofsyntheticbiology,wouldbeidealforearlydiagnosis.Inthesolutionpresentedhere,thesampletubeismatedwithauniquecapcontainingthesyntheticbiologicalsensor(Fig.1(b)).Thisunique3D-printeddevicecompletelyencasesthelivingbiosensor,allowingitstransportfromthelaboratory,whilealsoallowingthesensortobeintroduceddirectlytoasample.Whenthesputumsampleisreadytobeanalyzed,forceisappliedtothedevicebytheuser’s?nger,causingthebacterialbiosensortobreakoutofthehousingandfallintothesample.Detectionof3O-C12,whichwillbeproducedbyP.aeruginosainthesampleorisalreadypresentinthesample,causesthelivingbiosensortoproducemCherry(Fig.1(c))andturnsthesamplered;thiscolorcanbedetectedthrough?uorescenceanalysisorvisuallywiththehumaneye.Thus,acolorimetricchangeinthesamplecanindi-catethepresenceofP.aeruginosainthelungs.P.aeruginosafunctionsasanopportunisticpathogeninpatientswithcystic?brosis,causingrecurringinfectionsofthelungsandrespiratorytract.Currentdiagnostictechnologytypicallytakestheformofenzyme-linkedimmunosorbentassays(ELISAs)[26]andreal-timepolymerasechainreaction(PCR)assays[27],whichareeitherexpensiveorrequireextensivetrainingtoadminister.ThesensingapproachdescribedherecouldserveasasimpleandinexpensivecomplementtocurrentdiagnosistechniquesfordetectingP.aeruginosa.Sincethecellularbiosensorcanbegrownwithinasecure,3D-printeddeviceforcontainment,thisdevicecanbeusedasaplatformforthedeploymentofsyntheticallyengi-neeredbiosensorsinthe?eld.2.Materialandmethods2.1.CellcultureandmolecularcloningTheE.colistrainsusedinthisstudywerederivedfromtheE.coliK12parentstrainandarelistedinTable1[28].E.colicellsweregrowninbatchcultureat37°Cinlysogenybroth(LB,ThermoFisherScienti?cInc.,USA)media[29].Resistanttransformantswereselectedusing100lgámLà1kanamycin(ThermoFisherScien-ti?cInc.,USA).Forcloning,NEBòTurboCompetentE.coli(NewEnglandBiolabsòInc.,USA)wasusedasthehosttopropagatetheplasmids.Cellsweregrownat37°CinLBmediasupplementedwithkanamycin.Forgrowthonplates,LBmediawassupple-mentedwith2%agar(w/v)andkanamycin.TheplasmidsusedinthisstudyarelistedinTable1andwereconstructedthroughstandardmolecularcloningapproaches[29,30].Thesensorplasmidwasconstructedby?rstamplifyingan843bpPLtetO-1–lasRDNAregionfromaplasmid,pKDT17,whichwasorderedfromAddgene.org,andoriginallycamefromthelaboratoryofDr.PeterGreenberg.A711bpmCherrygenewasampli?edtoproduceanRBS–mCherry–t0fragment,whichwasinsertedintopKDT17toobtaintheLasRsensorplasmid,pDW065(Fig.2).2.2.Dose–responsecharacterizationTostudythisgenenetwork’sdoseresponse,starterculturesofE.coliwithpDW065weregrownovernightinLBmediasupple-mentedwith50lgámLà1kanamycin.TheculturesweredilutedTable1
Listofplasmids,strains,relevantcharacteristics,andorigins.NameMutationsOriginNEBTurboòE.coliK12wildtypeNewEnglandBiolabsòInc.MG1655WTE.coliK12wildtypeE.ColiGeneticStockCenterpWR011PLtetO-1–mCherryRuderLabpKDT17lasR,lasB::lacZPearsonetal.[28]pDW065aPLtetO-1–lasR,PLtetO-1–mCherryThisstudypDW065PLtetO-1–lasR,PLas–mCherryThisstudyD.Woloznyetal./Engineering5(2019)173–180175Fig.2.3O-C12sensorplasmidmap.The3O-C12sensor,alsoknownaspDW065,iscomposedofthreemainelements:theplasmidbackbone,thelasRcassette,andthemCherrycassette.Thebackboneconsistsofanampicillin-resistancecassetteandapUCoriginofreplication.ThelasRgeneisplacedunderthecontrolofaPLtetO-1promoter,whilethemCherrygeneisunderthecontrolofthePLaspromoter,whichcontainsabindingsitefortheLasRprotein.to0.2OD600(i.e.,anopticaldensitymeasuredat600nm)andallowedtogrowuntilreaching0.6OD600.Thecultureswerethendilutedagain,supplementedwith0–100lmoláLà13O-C12(Sigma-AldrichCorporation,USA),andmaintainedattheexponen-tialphaseat37°Cuntilfullyinduced(about8h).Culturesat0.6OD600weredilutedto1:100in?lteredphosphate-bufferedsaline(PBS,ThermoFisherScienti?cInc.,USA)and?uorescencewasmeasuredusinganAccuriTMC6?owcytometer(Becton,DickinsonandCompany,USA).2.3.DesignandfabricationofacrylonitrilebutadienestyrenedevicesThe3D-printeddevicewasdesignedusingAutodeskInventorò(AutodeskInc.,USA),whereitwasrenderedintoa3Dmodel.ThedesignedmodelwasthenexportedasanSTL?le,whichisa?leformatthatcanbeusedbyZ-suiteò(ZortraxCompany,Poland),thesoftwarethatwasusedtotranslatethe3Dobjectintoasetofprinterinstructions.TheZ-suiteòsoftwarewasthenusedtoexportthe?leasaz-code?le,whichwasusedinaZortraxM200(ZortraxCompany,Poland)toprintthe3Ddesignusinganacrylonitrilebutadienestyrene(ABS)variant,ABSULTRAT.ThisproprietaryABSvariantiscomposedofABS(90%–100%),stabilizer(0%–5%),lubricants(0%–2%),mineraloil(0%–4%),tallow(0%–4%),wax(0%–4%),polycarbonate(PC,0%–3%)andanti-oxidant(<2%).2.4.DevicesurfacepolishingInordertosmooththesurfaceroughnessofthedevicecomponentspost-printing,the3D-printedcomponentswere?rstplacedonasterilesurface.Papertowelswereattachedtotheinnersurfaceofa1000mLbeakerandsaturatedwithacetone.Thebeakerwasinvertedoverthedevicecomponents,andcarewastakentoavoiddirectcontactbetweentheliquidacetoneandthecomponents.After1h,thebeakerwasremovedandthedeviceswereexposedtoambientairfor2h.2.5.DeviceuseandoperationToencasethe3O-C12livingbiosensorintothe3D-printeddevice,a40lLagarplugwasaddedtothebacterialwellinthehousing;theengineeredE.colicellswerethenplatedontotheagarsurface.Afterovernightculture,theplungerwaspressedintothehousingandapress-?tsealwasformedtoisolatethebacterialcellsfromtheenvironment.Thecapcontainingthelivingbiosensorcouldthenbesafelytransferredtothe?eldandstoredinarefrigeratorforupto30dbeforeuse.Whenreadyforuse,thesampletobeexaminedwasplacedintoa14mLculturetube.Theculturetubecapwasremovedandtheassembleddevicecontainingthelivingbiosensorwasthensnappedintoplaceontotheculturetube.Toactivatethebiosensing,thecoverwas?rmlypressedwithalateralpinchgripuntilthebacterialwellfellintotheculturetube.Thecellculturewasthenincubatedat37°Cfor8–9handsubsequentlydetectedwith?uorescencespectroscopy,orsimplywiththehumaneye.Forthe?owcytometryanalysisusedinthisstudy,sampleswerecollectedatanOD600of0.6anddilutedto1:100indeionizedwater.FluorescencemeasurementswereacquiredusinganAccuriTMC6?owcytometer.Followingthecompletionofexperiments,thesystemcanbesterilizedinanautoclave;or,ifanautoclaveisnotavailable,thecomponentscanbedisassembledandimmersedinsodiumhypochlorite(i.e.,bleach).2.6.GeneregulatorynetworkmodelingWeengineeredanE.colibiosensorcapableofexpressingmCherrywhenexposedto3O-C12.Theactivator,LasR,wasconstitutivelyproducedinaninactivestate.When3O-C12isintroducedtothecell,itbindstoLasR.Thiscomplexactivatesageneregulatorynetwork.Aswedidnotengineeranypost-transcriptionalregulation,thegeneralbehavioroftheregulatorygenenetworkscanbemodeledbythedynamicsofthetranscriptionalresponsesite.Tocapturetheunderlyingdynamicsofoursystem,wedecidedtoemploytheMichaelis–Mentenformalismforthemodelingtranscription.Asweusedahighcopycountplasmid,wecanassumethatstochasticeffectsarenegligibleforspeci?cgeneticcomponents.Thisallowsustosimplifyoursimulationwithasetofordinarydifferentialequations.Byassumingthattheinducer,3O-C12,iswellmixedwithinourreactionchamberandthattransportof3O-C12intothecytosolisconsistentamongindividualcells,wecanmodelthetranscriptionalresponseto3O-C12asshowninEq.(1).Notethatthisequationassumesthatthemajorityofpost-transcriptionalevents(translationandproteinmaturation)occurconsistentlyandlinearlyinresponsetomRNAproduction.Thisisafairassumption,ifwealsoassumethattheribosomebindingsitestrengthsareconstantandthatthereisanabundanceofresourcesandribosomeswithinthecell.d?mCherryi???3O-C12??ndt?V1maxKnntV1leakàd1?mCherryi??e1T1t?3O-C12??Eq.(1)relatesthetimerateofchangeofmCherrytoanumberofinputsandparametersthatdrivetranscriptionalevents.InherentinthismodelistheassumptionthatLasRconcentrationswithinthecellsremainrelativelyconstant.The?rsttermontherightsideofEq.(1)isaHillfunction[31]relatingtheintracellularconcentrationof3O-C12,?3O-C12??,totherateofmCherryproduction.Thistermisafunctionoftheconcentrationofarabinosebutitincludesparametersforthemaximumtranscriptionrate,V1max,andthekineticcoef?cient,K1.Inaddition,theHillcoef?cient,n,relatesthedegreeofcooperativenessoftheRNApolymerasebindingtothepromotersite.176D.Woloznyetal./Engineering5(2019)173–180ThesecondtermontherightsideofEq.(1),V1leak,describesthe‘‘leak”ofthepromotersite.Inphysicalterms,thisistherateofmCherryproducedintheabsenceof3O-C12.The?naltermontherightsidedescribestherateofdegradationofmCherrywithinthecell.Thistermrelatesadecayconstant,d1,totheconcentrationofmCherry,?mCherryi??.Thisequationmaybe?ttoexperimentaldatainordertobetterpredictandmodelunderlyingdynamics.2.7.Modelderivation:doseresponsecurve?tting,simulation,andplottingExperimentaldatawereanalyzedand?taccordingtoEq.(1)usingtheMATLABò(MathWorksInc.,USA)optimizationtoolbox.Onceparametervalueswereidenti?ed,?tswerecon?rmedwiththeMATLABcurve-?ttingtoolbox.Thegoodnessof?tforthesimulatedequationswasevaluatedusingtheR2value.SimulationswerecodedinPythonandnumericallyintegratedusingLSODE[32]intheFORTRANlibrary.Simulations,withexperimentaldata,wereplottedinPythonusingtheMatplotliblibrary.2.8.Modelderivation:?niteelementanalysisofbiosensorFiniteelementanalysiswasperformedusingCOMSOL(COMSOLMultiphysicsòv5.2,COMSOLInc.,Sweden).AllsimulationresultsrepresentastationarystudyperformedusingtheSolidMechanicsmodule.AutodeskInventorsolidmodel?leswereimportedintoCOMSOLtode?nethegeometryforthesesimulations.MaterialpropertieswerethenenteredintoCOMSOLtode?nethepropertiesofourABSmaterialforanalysis.Boundaryconstraintsandappliedloadswerethende?ned.AllsimulationplotsshownhereweregeneratedusingCOMSOL’sintegratedgraphics.3.ResultsThe3D-printeddevicewasdesignedtoattachtoacommonlaboratorysampletubeformfactor(Fig.3(a))toensureeaseofuse.Thedeviceconsistsoftwocomponents(Fig.3(b)),referredtohereinasthe‘‘housing”and‘‘cover.”Thecoverservestoisolatethecontentsenclosedinthehousinginteriorfromthesurroundingenvironment.Inaddition,thetopofthecoverisdesignedtobeergonomicforpressingwithathumborfore?nger.Onceassem-bled,thedevicecanbeenclosedwithinarectangularprismwithdimensionsofapproximately0.026m?0.026m?0.023m.Across-sectionalschematicoftheassembleddevice(Fig.3(c))showsthehousingandcoverheldtogetherbyapress-?t.Thehousingholdsabacterialwell,whichcontainsrichagarwithasmalllawnofbacteria;thisservestosustainandcontainthebacterialbiosen-sorwithinthedevice.Next,weaddedsmallmechanicalfeaturestothedevice,thedesignofwhichwasbasedonmodelingwitha?niteelementmethodsoftwaresuite[33],COMSOLMultiphysicsò.ItisimportanttonotethatourmodelswerebasedonconstructionwithABS,athermoplastic.Thermoplasticpolymersbecomepliableafterheating,hardenuponcooling,andarefrequentlyusedin3Dprinting.ABSisoftenusedbecauseitsglasstransitiontemperatureof105°Cmakesitwell-suitedfor3Dprinting,astheextrudertemperatureinmany3Dprintersissetat230°C[34].Thishighprintingtemperaturealsoenhancesthesterilityoftheprinteddevice.Inadditiontothecover,housing,andbacterialwellfeaturesdiscussedabove,wedesignedasmallmechanicalfeaturethatenablesanaxialforce(i.e.,aforceapplieddirectlydownwardonthecover,alongthemajoraxisofthesampletube)tocausethebottomofthehousingtoquicklyseparatefromthehousing,thusreleasingthebiosensorintothesampleduringa(designed)catastrophicfailure.Inordertocausethisfastyielding,wecreatedtwodepressedannulifeaturesinthehousingbottom(onboththeinteriorandexterior,showninFigs.4and5withadditionaldetail).Herein,werefertotheseannulias‘‘stress-focusingcutouts.”Thesecutoutfeaturesservetoconcentratethestressesthatdevelopfromappliedaxialforceonthecover,thusallowingsimpleactivationofthedevicebymeansofauser’sthumbusingapinchgrip.FiniteelementanalysiswasusedtonumericallyevaluatethestressesthatdevelopedwithinthedevicewhenforcewasappliedFig.3.Devicedetailsandschematic.(a)The3D-printeddevicecanbeattachedtoatesttube,whereitformsaseal.Thisdeviceisseparatedintotwoparts:thecoverandthehousing.(b)Thedevicecoverissealedontothehousingusingapress-?t.(c)Thedevicehousingcontainsabacterialculturewell,whichservesasachamberinwhichculturenutrients,agar,andlivingbiosensorscanbedeposited.Thehousingisdesignedwithastress-focusingcutout,whichservestoconcentratethepressingforcetransmittedfromthecovertoseparatethebacterialwellfromthehousing.(d)Thestressesconcentratedbythestress-focusingcutoutweremodeledusingCOMSOLtoensurethatthedeviceisusablewithaone-handedlateralpinchgrip.Max:maximum;min:minimum.D.Woloznyetal./Engineering5(2019)173–180177Fig.4.Deviceboundaryconstraints,loads,andmeshelementsforstresssimulation.(a)Boundaryconstraints.Thepurplehighlightedboundaryofthebiosensorhousingisthesole?xedboundaryconstraintusedforallsimulations.Allotherboundariesarefree.(b)Boundaryloads.Thepurplehighlightedboundaryshowsthesurfacewherethesensor-activatinglidappliesadistributedpressureloadduringuse.Allsimulationsusedatotalloadof100Ndistributedacrossthishighlightedarea.(c)Meshelements.COMSOLprede?ned‘‘normal”meshingwasusedforallsimulations.Fig.5.Cross-sectionsofthreedifferentdeviceiterationswithvonMisesstressdistribution.(a)Finaldesigncon?gurationandstressdistributionofthestress-focusingcutoutportionofthebiosensorhousing;(b)analternativedesignwitharcscomingtoapoint;(c)analternativedesignwithpeaks.Thelargeststressandbestdistributionofhighstress,asshownhere,resultedinthe?naldesignin(a)beingchosen.Allsimulationshadthefollowingmaterialproperties:amaterialdensityof1150kgámà3,Young’smodulusof2GPa,andPoisson’sratioof0.35.Allsimulationsusedatotalloadof100Ndistributedacrosstheboundaryloadarea.COMSOLprede?ned‘‘normal”meshingwasusedforallsimulations.tobeconstructedfromarelativelyhigh-strengthmaterial,whilestillrequiringarelativelysmallforcetoactivatethesensorbybreakingthecutout.Thedevicewasalsodesignedtobesimpletoassembleanduse,asshowninFig.6.A40lLagarplugwasaddedtothebacterialwellandthe3O-C12livingbiosensorwasplatedonitssurface.Thedevicecoverwasthen?tontothehousingandattachedtoaculturetube,whichisolatesthebiologicalsamplefromtheenvi-ronment.Theuser’sthumbisthenusedtoactivatethedevice.Thisappliedforceonthecoverdevelopsstressinthestress-focusingcutoutwithinthehousing.Thebacterialwellthenbreaksawayfromthehousingandfallsintothesample.Afteranalysis,thecovercanbepushedintothehousingtocreatethesecondstageofthepress-?tseal,thuspreventingthesamplewithinthetesttubefromescapingduringaleak,intheeventthatthedeviceisinvertedoragitated.Thissealalsoallowstheusertotransferthebiohazardouswastebacktoabiosafetylabforproperdisposal.Uponimmersioninthesample,thebiosensorwasgrownfor8h.The3O-C12-activatedbiosensorthenproducedmCherryprotein.Asshowninthe?owcytometryanalysis(Fig.7(a)),theinducedbiosensorshowedashiftin?uorescenceemittedat590nm,whichwasdistinctlyseparatefromtheun-inducedbiosensorcontrol?uorescentpeak.Inourdose–responseexperiment(Fig.7(b)),0.1lmoláLà1of3O-C12wassuf?cienttoinducethelivingbiosensortoproducevisible?uorescenceinthetubeafter8hgrowth.Whenquanti?edwitha?owcytometer,thedynamicrangeofthesensorwasobservedtobeintherangeof0.01–1lmoláLà13O-C12.4.DiscussionAlthoughsyntheticbiology-basedbiosensorscanbeversatileinthedetectionofchemicalinputs,manyarecurrentlycon?nedtoalaboratorysettingduetoregulationsthatrestricttheuseandtransportofGMOs.Itisworthnotingthatapaper-basedsyntheticbiosensorwasrecentlydevelopedbyPardeeetal.[35].Thispaper-basedbiosensorwasdevelopedbyfreeze-dryingcell-freesyntheticgenenetworksonpapertocreatematerialsthathadthefundamentaltranscriptionandtranslationpropertiesofacellyetremainedabiotic.Thepaper-basedapproachoffersfasterdetectionthancell-basedlivingbiosensorsandeliminatestheuseoflivingbacterialcellsfordetection,whichmakesitsuitableforthesafedeploymentofengineeredgenecircuitsbeyondthelab.However,thepaper-basedbiosensorrequiresdifferentsampleprocessingandadifferentsetofspecializedmaterials.Thus,thesystemwedescribehereallowsthecomplementaryduringitsuse.Thestresspro?lethroughthestress-focusingcutoutwasmodeledusingCOMSOLfordifferentdesignsinordertoarriveatthe?nalversion,whichispresentedinthispaper[33].Anevenlydistributedpressureloadwasappliedtothesurfaceofthehousingbythecovertogeneratestressinthecutout.Threedifferentdesignsofthestress-focusingcutoutweremodeledinordertoidentifythestressmagnitudeandconcentrationinthecutoutforagivenappliedforce.Relativetootherpreliminarydesigns,the?naldesignpresentedhere(Fig.3(d))hasboththehighestmaxi-mumstressdevelopedwithinthecutoutforagivenappliedforceandthebestspatialdistributionofthehighstressconcentrationthroughthecutout.Thisdesignofthecutoutallowsourdevice
一种可实现合成生物传感器现场部署的增材制造方法 - 图文
