Monolithic integration of a smart temperature sensor on a modular silicon-based organ-on-a-chip device

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Delft University of Technology

Monolithic integration of a smart temperature sensor on a modular silicon-based

organ-on-a-chip device

Martins Da Ponte, Rolando; Gaio, Nikolas; van Zeijl, Henk; Vollebregt, Sten; Dijkstra, Paul; Dekker, Ronald;

Serdijn, Wouter; Giagka, Vasiliki

DOI

10.1016/j.sna.2020.112439

Publication date

2021

Document Version

Final published version

Published in

Sensors and Actuators A: Physical: an international journal devoted to research and development of

physical and chemical transducers

Citation (APA)

Martins Da Ponte, R., Gaio, N., van Zeijl, H., Vollebregt, S., Dijkstra, P., Dekker, R., Serdijn, W., & Giagka,

V. (2021). Monolithic integration of a smart temperature sensor on a modular silicon-based organ-on-a-chip

device. Sensors and Actuators A: Physical: an international journal devoted to research and development of

physical and chemical transducers, 317, 1 - 7. [112439]. https://doi.org/10.1016/j.sna.2020.112439

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ContentslistsavailableatScienceDirect

Sensors

and

Actuators

A:

Physical

j o u r n a l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s n a

Monolithic

integration

of

a

smart

temperature

sensor

on

a

modular

silicon-based

organ-on-a-chip

device

Ronaldo

Martins

da

Ponte

a,∗

_,

_Nikolas

_Gaio

b

_,

_Henk

_van

_Zeijl

a

_,

_Sten

_Vollebregt

a

_,

Paul

Dijkstra

c

_,

_Ronald

_Dekker

a,d

_,

_Wouter

_A.

_Serdijn

a

_,

_Vasiliki

_Giagka

a,e

a_Faculty_of_Electrical_Engineering,_Mathematics_and_Computer_Science,_Department_of_{Microelectronics,}_TU_Delft,_Mekelweg_4,₂₆₂₈_CD,_The_Netherlands

b_BIOND_Solutions_B.V.,_TU_Delft,_Mekelweg_4,₂₆₂₈_CD,_The_Netherlands

c_Philips_Innovation_Services,_MMD,_Eindhoven₅₆₁₆_LZ,_The_Netherlands

d_Philips_Research,_Eindhoven₅₆₅₆_AE,_The_Netherlands

e_Department_of_System_Integration_and_{Interconnection}_{Technologies,}_Fraunhofer_Institute_for_Reliability_and_{Microintegration,}_IZM_{Gustav-Meyer-Allee}

25,13355Berlin,Germany

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23July2020

Receivedinrevisedform

18September2020

Accepted13November2020

Availableonline21November2020

Keywords: Organs-on-a-chip

Smarttemperaturesensor

Time-modedomainsignalprocessing

MEMS

CMOSmonolithicintegration

MEMS-electronicsco-fabrication

a

b

s

t

r

a

c

t

Oneofthemanyapplicationsoforgan-on-a-chip(OOC)technologyisthestudyofbiologicalprocessesin humaninducedpluripotentstemcells(iPSCs)duringpharmacologicaldrugscreening.Itisofparamount importancetoconstructOOCsequippedwithhighlycompactinsitusensorsthatcanaccuratelymonitor, inrealtime,theextracellularﬂuidenvironmentandanticipateanyvitalphysiologicalchangesofthe culture.Inthispaper,wereporttheco-fabricationofaCMOSsmartsensoronthesamesubstrateas oursilicon-basedOOCforreal-timeinsitutemperaturemeasurementofthecellculture.Theproposed CMOScircuitisdevelopedtoprovidetheﬁrstmonolithicallyintegratedinsitusmarttemperature-sensing systemonamicromachinedsilicon-basedOOCdevice.Measurementresultsonwaferrevealaresolution oflessthan±0.2◦_C_and_a_nonlinearity_error_of_less_than_0.05%_across_a_temperature_range_from₃₀_to

40◦C.Thesensor’stimeresponseismorethan10timesfasterthanthetimeconstantofthe convection-coolingmechanismfoundforamediumcontaining0.4mlofPBSsolution.Allinall,thisworkisthe ﬁrststeptowardsrealizingOOCswithseamlessintegratedCMOS-basedsensorscapabletomeasure,in realtime,multiplephysicalquantitiesfoundincellcultureexperiments.Itisexpectedthattheuseof commercialfoundryCMOSprocessesmayenableOOCswithverylargescaleofmulti-sensingintegration andactuationinaclosed-loopsystemmanner.

1. Introduction

Organ-on-a-chip(OOC)isanemergenttechnologyinwhicha microﬂuidicperfusionplatformforculturinghumaniPSCsisused tomimicaminituarizedversionofanexplicitorgananatomyand physiology.Thistechnologyhasbeendevelopedtosubstitute tradi-tionalinvitroandanimalmodelsthatareofteninaccuratetopredict thehumanphysiology[1,2].StudiesindicatethatOOCscanplaya transformativeroleinthedrugdevelopmentcyclebybridgingthe gapbetweenpreclinicalstudiesandhumantrials,whilereducing thepharmaceuticalR&Dcoststo10–26%[3].

∗ Correspondingauthor.

E-mailaddress:r.martinsdaponte@tudelft.nl(R.M.daPonte).

URL:http://www.bioelectronics.tudelft.nl(R.M.daPonte).

For the construction of these OOC systems, various micro-and nano-fabrication technologies have been used, including soft lithography on elastomeric materials. The simplicity, fast turnaroundtime,andrelativelylowcostofthistechniqueaffords quickexperimentationofnewdesigns.Examplesofsuchdesigns includeOOCsfortheheart[4],theliver[5],thekidney[6],the lung[7],andtumours[8,29].Ontheotherhand,shortcomingsof suchmethodsincludelimiteddevicethroughputwhichisacrucial featureforhigh-volumemanufacturing.

To overcome this, MEMS (Microelectromechanical Systems) technologybasedonsiliconwaferlevelprocessingprovestobea veryefﬁcientoptionformicromachininghigh-aspectratio struc-tureswithsubmicrometerresolution, andover a widerange of materials[9].Althoughdependentonhighlyspecializedand expen-siveequipment,thehighinitialcostsarecounteractedwhenlarge productionvolumeisanticipated.Additionally,MEMSprocesses often are compatible with CMOS(complementary metal oxide

https://doi.org/10.1016/j.sna.2020.112439

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R.M.daPonte,N.Gaio,H.vanZeijletal. SensorsandActuatorsA317(2021)112439

semiconductor) technologywhichallows monolithicintegration of dedicated interface electronics for thermal, optical, pH,and label-freesensingnecessarytodesigncompactcellculturesystems

[10].Furthermore,theinsitureal-timeanalysisofferedbythese microelectronicsystemscouldrevealnewinsightsintointra-and inter-cellularsignallingpathways.

Currently, theaforementionedanalysisassumesthatthepH, temperature (∼37◦_C), _humidity ₍_∼95% _RH) _and _gaseous

atmo-sphere (CO2/O2 levels) around the cell culture medium are

regulatedbyincubators.Theseparametersshouldbekeptconstant sincetheyplayapivotalroleintheoptimalgrowthandmaximum productivityofthecellculture[11].However,variationsdooccur, whichcausestressinthecellsthatcanrespondinvariousways rangingfromtheactivationofsurvivalpathwaystotheinitiation ofsenescence.

Especially,recurrenttemperaturevariationsinthecellculture should be carefully monitoredas they may severelyaffect the experiments.Causesforsuchvariationscanbeduetodeviations betweentheincubator’stemperaturesetpointandthe tempera-tureofthecells,thefrequencyanddurationoftheincubator’sdoor opening,andthedurationthecellsareoutsidetheincubatorfor inspection[12].Aboveall,timespentoutsidetheincubator rep-resentsalarger,morevariable,factorthatislikelytoimpactcell health. Adropoftheculturetemperaturetoroomtemperature resultsinaconsiderabledecreaseincellgrowthalong withthe accumulationofcellsintheG1phase[13,14].Infact,therateat which thetemperatureofthecellculturedecreasesoutsidethe incubatorisunknowntolifescienceresearchers.Thishas moti-vated us toconstruct a real-time CMOStemperature sensor to monitortheinsitutemperatureoftheculturethroughoutthe cell-divisioncycle.

Avarietyofelectricalcellculturetemperaturesensing meth-odshasbeenpresentedinpreviousliterature:commercialT-type thermocouples madeof copperand constantan wires[15],NTC (negativetemperaturecoefﬁcient)thermistors[16]orcommercial PT-100RTDs(resistancethermometerdetectors)[17].Acommon drawbackoftheaforementionedsolutionsmanifestsitselfwhen interfacingthesensor’soutputwithreadoutsystemsthatare out-sidetheculturingenvironment.Inthisscenario,withthesensor remotefromreadoutelectronics,varioussourcesoferrors(noise, interference,distortion,crosstalk,etc.)maybeintroducedoverthe channelandimpairthemeasurement.Moreover,suchcommercial sensorsarenotverycompactforOOCapplications.

Non-electrical temperature sensing methods have also been reported, such asliquid crystal displays[18], ﬂuorescent poly-mericthermometers[19],andoptoacousticmethods[20].These solutions,however,arenotverycompactbecausetheydependon expensiveandbulkyinstrumentationlaboratoryequipment[21]

toopticallymapthetemperatureoftheculture.Moreover,these systemshavepoorresolution(∼1◦_C),_do_not_easily_allow_the

inte-grationofclosed-loopsystemsanddonotofferthehighthroughput ofsilicon-basedmicrosystems.

Totackletheseshortcomings,wehavefabricatedasmart tem-perature sensoronthesamesiliconsubstrateusedtoconstruct ourcustommicromachinedorgan-on-a-chipdevice.Asa conse-quence,thesystemismademorecompactandtherobustnessto varioussourcesoferrorsisenhanced.Toaccomplishthis,weused asimple,robust,andcustomin-houseintegratedcircuit(IC) tech-nology[22].We havepreviouslyusedthistechnologytodesign andcharacterizeasuitabletemperaturesensor[23].Inthispaper, wearepresentingacomplete,seamlesslyintegratedinsitusmart temperature-sensingsystemonanOOC.Tothebestoftheauthors’ knowledgethisistheﬁrsttimesuchintegrationisperformedusing acustom-designedsensingandconditioningcircuitfabricatedon thesamesiliconsubstrateasthatoftheOOC.

Fig.1. ArtisticimpressionoftheCytostrechsystem.

Asopposed toaSystem-in-Package(SiP) approach,in which anoutsourcedASIC(Application-speciﬁcIntegratedCircuit)is het-erogenously integrated ontheOOC device,our solutionavoids theuseofchipmountingtechnology(e.g.wireanddiebonding) thatusuallyrequiresextrapackagingprotectionoftheassembled components.Incontrast,ourseamlessintegrationminimizesthe extraprocessingstepsandprecludespotentialmechanicalstresses causedbymismatchesinthethermalexpansioncoefﬁcientofthe variousdissimilarcomponentsandmaterialstobeusedinaSiP sce-nario.Finally,theholisticCMOS-MEMSco-designapproachoffers thepossibilitytoconformandbetteraccomodatetheinclusionof CMOSelectronicsovervariousMEMStopologies.

2. Materialsandmethods

Our OOC platform, presented here as Cytostretch [24], is modular, customizable, silicon-based and microfabricated with cleanroom-compatibleprocesses.Themaincomponentsofthe sys-temaredepictedinFig.1,wheretheCytostretchchipsarebonded toa PCBthatincludesa mouldedmulti-wellplatefor culturing thecells.Thechipsincludeapneumatically-activated freestand-ingdog-bone-shapedPDMSmembranetoaccommodatethecell culture(module1)whiledeliveringmechanicalstimulitothecells invariousin-vitrostudies,additionalfeatures,suchas through-membranemicro-poresforbiologicalsignalexchange(module2), on-membranegroovesforcellalignment(module3),in-membrane titaniumnitride(TiN)microelectrodesformonitoringactivityfrom electricallyactivecells(module4),andtitanium(Ti)straingauges tomeasurethedeformationofthePDMSmembraneduring inﬂa-tion(module5).More detailsonthesespeciﬁcmodulescanbe foundin[25].Thesmarttemperaturesensorpresentedinthispaper isthesixthmoduleofthisOOCplatform.Thismodulewas accom-plishedthankstothemonolithicintegrationofthissmartsensor onthebacksideofourOOCdevice.Thetemperatureofthecellsis sensedasaresultoftheheattransferredfromtheculturemedium tothecrystallinesiliconthroughthermalconductionmechanisms associatedwithelasticvibrationsofthelattice(i.e.phonons trans-port).

2.1. Thesmarttemperaturesensingmodule

Thesmartsensingmodulewasdesignedtodetecttheinsitu temperatureofthecultureandconvertitintoaperiodicdigital elec-tronicsignal,whichcarriesthetemperatureinformationencoded inthetimedomain.Torealizethis,thesystemconsistsoftwomain blocks:aproportionaltoabsolutetemperature(PTAT)current gen-erator(employingNPNbipolartransistorstosensethetemperature information)andarelaxationoscillator(Fig.2).

Thecircuitoperationcanbeunderstoodfromthesystem dia-gramof Fig.2.During start-up,theoutputofthecomparatoris settoalogic“0”whichturnstheCMOSswitches( ¯)onforthe comparisonphase.Asaresult,aPTATcurrentisintegratedinthe capacitoranditsvoltage(Vc)isrampedup.WhenVcequalsvoltage

VH,theoutputofthecomparatortogglesandturnstheotherpairof

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Fig.2.MainblocksofthesmartCMOStemperaturesensor:aPTATgeneratorandarelaxationoscillator.

CMOSswitches()on.ThevoltageVcisnowrampeddownviathe

PTATcurrentsinkuntilitreachesVLendingthedischargingcycle.

Oncestarted,thisprocesscontinuesindeﬁnetelytoyieldasignalin whichtheperiodisPTATaccordingtotheequationT=CV/IPTAT,

whereVisthedifferencebetweenthethresholdvoltagesVHand

VL,CisthecapacitanceandIPTATisthePTATcurrentgenerated.

2.1.1. PTATgenerator

ThePTATcurrentgeneratoryieldsacurrentthatisproportional tothevoltagedrop(VBE)acrosstheresistorR.Sincethisvoltageis

thedifferenceoftwobase-emittervoltages,thecurrentproduced, I=VBE/R,isPTAT.Currentmirrorswitha m:1ratioconveythe

copyofthisPTATcurrenttothecapacitor.Thecollectorvoltages areforcedtobeequal,regardlessofvariationsinthepowersupply orinthetemperature,viathenegativefeedbackloopthatincludes theopamp(OperationalAmpliﬁer),thebipolardevices(Q1andQ2)

andtheresistor.TheexpressionofthecurrentacrossresistorRis: IPTAT =

UT

R ln(mn), (1)

whereUTisthethermalvoltage(≈26mVatroomtemperature),m

isthecurrentmirrorratioandnisthebipolaremitterarearatio. Hence,theresponsivityofthisblockismostlydeterminedbythe currentmirrorandemitterarearatios.Inthedesign,valuesof5 and4formandn,respectively,werechosen.Inaddition,a start-upcircuitforthisPTATcellwasimplementedtoensureitscorrect operatingpoint.

2.1.2. Relaxationoscillator

Therelaxationoscillatorisrealizedbyafeedbackcontrol per-formed by the comparator, the PTAT current source and the hysteresiscircuit.Hysteresis isa functionalrequirementforthe relaxationoscillatortoworkproperly.Thehysteresiscircuitwas implementedwithninestackeddiode-connectedbipolardevices biasedwithacopyofthePTATcurrentsoastoproducethevoltages VHandVL,at37◦C,of6and3.5V,respectively.Thehysteresiswas

madeinverselyproportionaltotheabsolutetemperature(CTAT)to increasethecircuitresponsivitywithrespecttotheperiod. 2.2. Microfabricationonsiliconsubstrate

The cofabrication of MEMS and CMOS on a single silicon substrateistypicallyadverseintermsofcosts.Thisproblem exac-erbatesinmoreadvancedCMOStechnologiesduetotheincreased number of masks and processing steps required. For instance, adding high-performance verticalbipolar devices toa standard

0.18␮mCMOStechnologycanaddupto10–20extra photolitho-graphicmasksandincreasethecostsby20–30%[26].

UsingasimplerCMOSprocesswithfewerphotomasksand pro-cessstepsismoreattractive.Ourin-houseCMOSprocesscomprises seven photomasks which yields a more cost-effective solution (Fig.3a–h)whileofferinga holisticCMOS-MEMS co-design.The cofabricationprocedureusesaMEMS-lastprocessingstrategy[27]. Inthisapproach,theMEMSstructuresarefabricatedby deposit-ingandmicromachiningCMOS-compatiblematerialsontopofthe fabricatedCMOSelectronics.

2.3. CMOSfabrication

Adouble-polishedp-typesiliconwaferwith₁₀₀of crystal-lographicorientationand5cmofresistivityisusedtostartthe alignmentlayer(zerolayer)deﬁnition.A2-␮mthicknessofp-type epitaxiallayerwasgrown onthetopof thesilicon waferwith 1.0e16ions/cm3 _of_boron_doping_in_order_to_obtain_a _precise

p-dopantconcentrationrequiredtoincludeNPNbipolartransistors intheCMOSprocess.

A20-nmbarrierwasformedusingwetoxidationtoscreen co-implantedparticles.Then-wellandthecollectorareaoftheNPN bipolartransistorarepatternedusingtheﬁrstphotomask(Fig.3a) witha3.1␮mofphotoresistthickness.A5e12-doseofphosporus implantationat150keVofenergyisfollowedby415minof anneal-ingfordopingredistribution.A230-nmofoxideiscreatedasa result.Anoxidestrippingisperformedusingabuffered hydroﬂu-oricacid(BHF)solutionwith1:7ofselectivity.Anotherdirtbarrier oxidelayerisgrowntoprocessthesubsequentsteps.

Thesecondphotomaskisusedtodeﬁnethen-typediffusion areasfortheCMOStransistorsandtheemitterareaforthe bipo-lartransistors(Fig.3b)using5.0e15ions/cm3 _dose_of_arsenic_at

40.0keVofenergy.Thethirdphotomaskdeﬁnesthep-type diffu-sionareasfortheCMOSaswellasthebaseareaofthebipolars (Fig.3c)using4.0e14ions/cm3_of_boron_at_20.0_keV_of_energy._An

optimaldoseimplantationshouldbeinvestigatedhereduetothe trade-offbetweentheintrinsic currentgain of theNPNbipolar devicesandthecurrentdrivecapacityofthePMOSdevices.

Next,athresholdvoltageadjustmentiscarriedoutusinganet doseof20e11ions/cm3_at_25.0_keV_in_the_fourth_quadrant_of_the

wafer.Subsequently,thedirtbarrieroxideisremovedusinga1:7 BHFsolution,followedby9minofwetoxidationfordopant acti-vationandtocreatethe100-nmofgateoxide.Afterthisstep,the thresholdvoltagesforthenMOSandpMOStransistors,inthefourth quadrant,aresettoabout2.0Vand−2.5V,respectively.

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R.M.daPonte,N.Gaio,H.vanZeijletal. SensorsandActuatorsA317(2021)112439

Fig.3.FabricationstepsinacustomCMOS-MEMStechnologyforthesmartsensordeviceontheCytostretchplatform.Nottoscale.

Inthenextstep(fourthmask),thecontactopeningsare pat-terned and wet-etched with a 1:7 BHF solution (Fig. 3d). The interconnects and the gate material are created by sputtering 200nm of AlSi (1%) and patterning with the ﬁfth photomask (Fig.3e).The1%ofsiliconcomposition inthealuminum avoids spikes in the shallow metal-diffusion interfaces. The process followswithadepositionof2-␮mofSiO2usingPECVD

(plasma-enhancedchemicalvapourdeposition)atthefrontsideofthewafer tocreateatthesametimetheMIM(metal-insulator-metal) capaci-tordielectricsandthestoppingmaskfortheDRIE(deepreactiveion etching)stepthatdeﬁnesthePDMSmembranearea.Tosharethis processstep,acleartrade-offistobemadeintheco-designphase. Ideally,thethinnerthedielectricoftheMIMcapacitorthebigger thecapacitance,thus,thehigherthetotalcapacitanceperarea.This resultsinamorearea-savingsolution.Ontheotherhand,enough oxidethicknessheadroomshouldbeprovidedintheDRIEstepto ensureareliablehardmasklanding.Asaconsequence,duringthe co-designphase,abiggersiliconareahasbeengrantedtotheMIM capacitorgivenaminimumof2-␮mthicknessofSiO2tobeused.

Theviasareopenedusingplasmaetchingafterpatterningthe sixthphotomask(Fig.3f)withphotoresist.Thesecond metalliza-tion(Fig.3g)usesa3.1-␮mthicknessofsputteredAlSi(1%).This stepsimultaneouslypatternsthesecondlevelofinterconnectsof thesmartsensortogetherwiththecontactpadsandthe electri-cal interconnects outside themembrane areaoftheCytostrech (Fig.3h).

2.4. MEMSbulkmicromaching

Followingthe laststep of thesmart sensormicrofabrication (Fig.3h),a5-␮mPECVDSiO2isdepositedonthebacksideofthe

wafertopreparethesubstratefortheDRIEstep.TheCytostretch membraneareaisthenpatternedonthesamebacksidebydry etch-ing(Fig.3j).Subsequently,a15-␮m-thickPDMSlayerisspunonto thefrontofthewaferat3500rpmfor30sandcuredfor1hat90◦C (Fig.3j).Next,300nmofAlSi(1%)issputteredatroomtemperature ontopofthePDMSfilmcreated.TheAlismaskedwith4␮mof pho-toresist(PR)(AZECI3027)anddry-etched(Fig.3k).Thelithography and etchingprocesses usedare optimized tocircumventissues causedbythedifferencebetweentheexpansioncoefficientsofthe PDMSandthePR.Besidesservingasahardmasktolaterexpose theelectricalcontacts,theAllayerreducestheeffectsofthe differ-encesinexpansioncoefficientsbyactingasabufferlayerbetween thePDMSandthePR.

Subsequently, the membrane is released removing the Si and the SiO2 layers from underneath the membrane using

DRIE and BHF,respectively (Fig.3l).Finally, anetchmixture of phosporic/acetic/nitricacid(PES77-19-04)isusedtoremovethe aluminiumonthetopofPDMSandmakethemembranefully trans-parent.ThisdoesnotremovetheSifromtheAlSi(1%),though.

Fig.4. SmarttemperaturesensormonolithicallyintegratedontheCytostretchchip.

3. Experimental

ThemicrofabricateddeviceisshowninFig.4.Thetotalchipsize is7×7mm2_and_less_than_15%_of_this_area_was_used_for_the_smart

sensor.

Staticanddynamicresponsemeasurementswerecarriedoutto characterizethesensor’sperformance.Theresolutionandlinearity wereextractedbymeansofaseriesofstaticresponse measure-mentsin whichthetemperaturewasmaintainedconstantwith respecttotime.Thedynamicresponsemeasurementwasusedto characterizethesensor’sresponsespeedbyapplyingabrief tem-peraturepulsebymeansofapre-heatedPBSsolution.

3.1. Staticresponse:drymeasurements

The static response measurements were carried out with a microprobestationwhichincludesathermalchucktosweepthe temperatureofthewaferoverthedesiredrange.Acommercial PT-100temperaturesensorwasattachedtothethermalchucktoseta well-calibratedreferenceandthe4-pointprobesmethodwasused tomeasureitsresistancechanges.

Atemperaturesweepfrom25to100◦Cwith5◦Cincrementwas carriedoutinordertomeasuretheresponsivityandthe nonlinear-ityofthetemperature-to-timeconversion.Theresponsivity and themaximumnonlinearityerrorobtainedfromthismeasurement was57.1ns/◦Cand0.26%,respectively(Fig.5).

Atemperature measurement over a shorter range (30–40◦C with1◦Cincrement)wasalsoperformedinordertocharacterize

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Fig.5. Temperature-to-timeconversionofthesmarttemperaturesensor.The

least-squarespolynomialregressionrevealsa57.1ns/◦Cofresponsivity.

Fig.6.CytostrechchipsmountedonaPCBcontainingwellsforcellculture

experi-ments.

thesensor’slinearitywithinatemperaturespanthatisclosertothe cellcultureapplication.Asimplelinearregressionofthedataover thistemperaturerangerevealsa99.988%ﬁtwiththelinearmodel. Hence,a0.05%ofmaximumnonlinearityerrorwasfoundoverthis range.

Thejitterisameasureofthedeviationoftheperiodicsignalfrom itstrueperiodicityanditaffectstheresolutionthatthesensorcan achieve.Thetotalrootmeansquare(rms)jitterwasmeasuredat 37◦Cformorethan50,000samplesand isequalto2.78ns.The sensor’sresolution(R)iscalculatedastheratioofthejitterfor3 andtheresponsivity,andequals:

R=3_57.1×2.78_ns/_◦ns_C =0.15◦C(3), (2) 3.2. Dynamicresponse:wetmeasurements

To measurethesensor’sdynamic response,thewafers were dicedandfourdifferentdicewereassembledonasemi-ﬂexible PCBcontainingfourdifferentwellsintendedforcellculture exper-imentation(Fig.6).

The measurementswereinitiatedwiththeambient temper-ature at 28◦C (±0.5◦_C) _and _after _{approximately} ₁₅_s _the _wells

wereﬁlledwith0.4mlofPBS(Phosphate-bufferedsaline)solution pre-heated toatemperatureof32◦C.Here,three differenttime constantsareinvolvedintheheattransferexchange(Fig.7):(1) thetimeconstantassociatedwiththeconvection-cooling mecha-nismsbetweenthemediumandtheambienttemperature(1),(2)

associatedwiththethermalconductionhappeningattheinterface betweenthemediumbulkandthesiliconcrystallattice(2),(3)

andassociatedwiththeintrinsicdelaybetweenthesiliconlattice andthesensoritself(3).

Fig.7.Different timeconstants involvedontheheat transferof thesystem.

Timeconstant1happensbetweenthemediumandtheambienttemperature,2

betweenthemediumandthesiliconlatticeand,3,betweenthesiliconandthe

sensor.

Fig.8. Dynamicresponseofthesensortoabrieftemperaturepulse.

TheresultofthismeasurementisplottedinFig.8forten dif-ferentmeasurements(darkgreenlines)takenatdifferenttimes withinaday.Thepinklineonthiscurveindicatesthemeanvalue calculatedforthese10samples,whereastheshadedlightgreen regionsencompassesthe±3conﬁdencelevelaroundthemean.

Anexponentialcurveisalsoﬁttedonthedatatoindicatethe tendencyofthesamples.Fromthiscurve,thetimeconstant associ-atedwiththeheatlossbetweenthemediumandtheambient(1)

hasbeenextractedanditisroughly50seconds.Thecumulative timeconstant(2+3)associatedwiththermalconductioninthe

siliconandthesensor’sresponsehasalsobeenderivedfromthe slopeinthecurvearoundt=15s,anditisonaverage1.5s.

Theratioofthethermalresistancesduetoconductionbetween themediumandthesiliconsurfacecomparedtothethermal resis-tanceduetotheheatlossmechanismsgiveanindicationoftheBiot number.Thisdimensionlessquantitywascalculatedtobeabout 0.03anditimpliesthattheheatconductioninsidethebodyismuch fasterthantheheatconvectionawayfromitssurface,and temper-aturegradientsarenegligibleinsideofit.HavingaBiotnumber smallerthan0.1labelsasubstanceas“thermallythin”,and tem-peraturecanbeassumedtobeconstantthroughoutthematerial’s volume.

4. Resultsanddiscussion

The results presented in the previous section demonstrate thecapabilityofmonolithicallyintegratingCMOSfunctionalityin silicon-basedOOCdevicesforreal-timeinsitutemperature mea-surementsofthecellculture.Ourin-houseCMOStechnologyhas beenusedasaresearchtoolfortheproofofconcept.Thishasbeen theﬁrststeptorealize OOCswithintegrated CMOS

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functional-R.M.daPonte,N.Gaio,H.vanZeijletal. SensorsandActuatorsA317(2021)112439

ityformulti-sensingmanyotherdifferentphysicalquantities(pH, glucose,glutamate,growthfactors,etc.)inthecellculture.Asthe numberofsensorsintheplatformincreases,morecorrelationscan beperformedforstoichiometricoptimizationsoftheculture.

Previousliteraturehasextensivelyusedcommercialsensorsfor thispurpose,whicharenotcompact,notscalableanddonotallow forhighdensitymulti-sensingintegration.

We expectthattheuseofmoreadvanced CMOS technologi-calnodescanenableverylargescaleofmulti-sensingintegration and actuationina closed-loopsystem.Tothat end,thesensors shallyieldthenecessaryaccuracyandrespondfastenoughforthe application.

Forinstance,theresultsobtainedwiththedynamicresponse measurementsindicatethatthesensor’sresponse(≈1.5s)ismuch fasterthanthetimeittakestoelapseonetimeconstant(≈50s)of theconvection-coolingmechanism.Inthiscase,thesensorcould beincorporatedinaclosed-loopconﬁgurationwithlocalheaters tokeeptheinsitutemperatureascloseaspossibletoitsreference forthemaximumtimewhenoutsidetheincubator.

In addition,thecurveobtainedfromthecoolingmechanism indicatesthattherateofheatlossinthemediumwherethecells areculturedtendstofollowanexponentialdecayanditis pro-portionaltothetemperaturedifferencebetweenthemediumand itssurroundings.Suchresultisalsoarelevantinformationtothe biologistsduringthetimethecultureisoutsidetheincubatorfor inspection.

Withrespecttotheresultsfoundinthestaticmeasurements, thesensor’sresolution(0.15◦Candlinearitysuggestthatthesensor canpotentiallymonitortemperatureincrementsoftheculturethat couldgiveanindicationofthemetabolicgrowthrateasaresultof theheatdissipatedduetoenthalpychanges[28].

It is important to notice that the sensor’s circuit design is not optimizedforbestperformance. Therefore,circuit improve-mentscanbemadetoachievebetterresolutionorresponsitivity, ifneeded. Regardingthemicrofabricationchallenges,care must be takenwhen processing materialssuch asPDMS. After etch-ing,residuesmaystill remainatthesurface whichcanhamper furthersystem-levelintegrationprocessessuchaswirebonding. Non-selectiveover-etching,ontheotherhand,mightpartiallyor completely remove thematerialsbeneath. Hence,process opti-mizationduringtheetchingphasesisofparamountimportance forthereliabilityandreproducibilityoftheendproduct.

5. Conclusions

Cellculturesaremaintainedatanappropriatetemperatureand gasmixtureinsideacellincubator.Becausetheinsituculture condi-tionsmayvary,especiallywhenthecultureisoutsidetheincubator, itisadvantageoustoconstructOOCsthatareequippedwith sen-sorsthatcanaccuratelymeasureinreal-timetheinsituconditions ofthecells.

In thiswork,weinvestigatedthemonolithic integrationofa CMOSsmarttemperaturesensorinourMEMSOOCdevice.By com-biningCMOSandMEMStechnologymonolithically,itispossible to create OOCsto accommodatethecells over differentMEMS structureswhileintegratinghigh-densityCMOSelectronicsforvery compactsystemsthatcanmeasureinsituphysicalquantitiesinthe culturemedium.

TheCMOS-MEMScofabricationmethodusedhereyielded,for theﬁrsttime,anOOCdevicewithintegratedCMOSsensing func-tionalityforareal-timeinsitutemperaturemeasurementofthecell culture.Ourin-houseCMOStechnologyhasbeenusedasaresearch toolfortheproofofconcept.

Moreover,asaresultofsharingcommonprocessstepsandby minimizingthenumberofCMOSmasksused,amorecost-effective

andscalablesolutionisobtained.Inordertomeetspeciﬁc require-mentsofboth technologies,aholisticco-designphase hasbeen followedsothetrade-offsbetweencircuitperformanceand micro-machingreliabilityaretakenintoconsideration.

Measurementresultsofoursmarttemperaturesensorindicate thattemperatureincrementsof0.2◦Ccanbeaccuratelymonitored. Thiscouldpotentiallybeusedtogiveanindicationofthemetabolic growthratewhenthecultureisinsidetheincubator.Thesensor’s timeresponsefoundwasapproximately1.5swhichismuchfaster thanthetimeittakesforthetemperatureofa0.4mlmediumto dropby1◦C.

ThisworkistheﬁrststeptowardsconstructingOOCswith inte-gratedCMOSelectronicsformulti-sensingrelevantinformationin thecellculture(O2,CO2,pH,glucose,glutamate,growfactors,etc.).

Itis expectedthattheuseofmore advancedCMOSnodesmay enablepowerfulOOCswithaverylargedegreeofmulti-sensing integrationandactuationinaclosed-loopsystemmanner.

Authors’contribution

RonaldoMartinsdaPonte:maininvestigator,writing–original draftpreparation,conceptualization,methodology,measurements, formalanalysis,datacuration,visualization,fundingacquisition. NikolasGaio:conceptualization,resources,methodologysupport, reviewing original draft.Henk vanZeijl: methodology support, resources, reviewing original draft. Sten Vollebregt: methodol-ogysupport,resources,measurementsupport,reviewingoriginal draft.PaulDijkstra:resources.RonaldDekker:conceptualization, resources, project administration. Wouter A. Serdjin: concep-tualization, supervision, resources, funding acquisition, project administration,reviewingoriginaldraft.VasilikiGiagka: concep-tualization, supervision, resources, funding acquisition, project administration,reviewingoriginaldraft.

Conﬂictsofinterest

Therearenoconﬂictstodeclare.

Acknowledgments

ThisworkwassupportedbytheNationalCouncilforScientiﬁc andTechnologicalDevelopment(CNPq),Brazil.Partofthework thatisdiscussedinthisarticlewasalsoconductedunderthe ECSEL-02-2014programInForMed,grantnumber:2014-2-662155.The authorswouldliketothankMassimoMastrangelifromTUDelft andVirgilioValente,nowinRyersonUniversity,forthesupportand fruitfuldiscussionsaboutOOCs.NielsRijkersofPhilips,Eindhoven, forhishelponthemicrosystemintegrationoftheCytostretch sam-ples.LoekSteenwegofEKLoptimizedtheparametersfordicingthe wafers.WilliamQuirós-SolanoandBrunoMoranafromTUDelft suppliedtheOOCwellsforintegrationandthefour-terminal sens-inggearformeasurement,respectively.LukaszPakulahelpedwith thesetupconﬁgurationforautomaticmeasurements.

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Biographies

RonaldoMartinsdaPontereceivedhisM.Sc.degreefromtheFederalUniversity

ofSantaCatarina,Florianópolis,Brazil,in2015.Heiscurrentlyworkingtoward

hisPh.D.degreeattheSectionBioelectronics,DelftUniversityofTechnology,The

Netherlands.HisresearchinterestsincludetheMEMS-CMOSintegrationfor

opto-geneticbraininterfacesandlab-on-CMOSdevices.HeisaStudentMemberofthe

IEEE.

NikolasGaioreceivedtheM.Sc.(cumlaude)inBiomedicalEngineeringin2015and

thePh.D.degree,in2019fromDelftUniversityofTechnology,Netherlands.Hisareas

ofinterestsincludeOrgan-on-Chip,stretchableelectronicsandmicroﬂuidicdevices.

HereceivedtheBestStudentPaperAwardatthe2015IEEESensors,thepublicPoster

AwardattheICTOpenConferencein2016,andtheLushPrize(YoungResearcher)

in2018.HeisthefounderandCTOofBIONDSolutionsB.V.

Dr.Ing.H.W.vanZeijl(1958)studiedphysicsattheTechnicalCollegeinRijswijk

wherehereceivedtheengineersdiplomain1980.InthatyearhejoinedtheDelft

UniversityofTechnology(TUD)wherehecooperatedindifferentresearchproject

relatedtoradiationphysics,Lithography,IC-technology,MEMs,packagingand3D

integration.In2005hereceivedaPh.D.degreefromTUD.Hedevelopedseveral

technologycoursesandlectures.CurrentlyheisscientiﬁcdirectoroftheChip

Inte-grationTechnologyCentre(CITC)inNijmegen(theNetherlands)andemployedat

TUD.Heisa(co-)authorofmorethan80papersandseveralpatents.

StenVollebregtreceivedhisM.Sc.in’09(cumlaude)andPh.D.in’14fromDelft

UniversityofTechnology.HecurrentlyisanassistantprofessorintheLaboratoryof

ElectronicComponents,TechnologyandMaterialsintheMicroelectronics

Depart-mentoftheDelftUniversityofTechnology.Hisfocusisontheintegrationofnovel

materials,likecarbonnanotubes,grapheneandwide-bandgapsemiconductor,in

microelectronicsandmicrosystemsforsensorapplications.HeisaseniorIEEE

mem-berandhasco-authoredover30journalpublications,4bookchaptersandholds3

patents.

PaulDijkstrareceivedtheM.S.degreeinChemicalengineeringfromEindhoven

Uni-versityofTechnology,Eindhoven,TheNetherlandsin1992.Currentlyheisworking

asPrincipalArchitectMicroAssemblyatPhilipsInnovationServicesinthe

depart-mentMEMS&MicroDevices.Hehas25yearsofexperienceinassemblytechnologies

intheﬁeldofelectronicpackageinnovationandmicrodeviceassembly.Mainfocus

atthismomentisonthedevelopmentofnewemergingapplicationsinareaslike

photonics,inandonbodydevices,MicroﬂuidicsandMEMSpackages.

RonaldDekkerreceivedhisM.Sc.inElectricalEngineeringfromtheTechnical

Uni-versityofEindhovenandhisPh.D.fromtheTechnicalUniversityofDelft.Hejoined

PhilipsResearchin1988whereheworkedonthedevelopmentofRFtechnologies

formobilecommunication.Since2000hisfocusshiftedtotheintegrationofcomplex

electronicsensorfunctionalityonthetipofthesmallestminimalinvasive

instru-ments.In2007hewasappointedparttimeprofessorattheTechnicalUniversityof

DelftwithafocusonOrgan-on-Chipandbioelectronicsmedicines.Hepublishedin

leadingjournalsandconferencesandholdsinexcessof70patents.

WouterA.SerdijnisafullprofessorinbioelectronicsatDelftUniversityof

Tech-nology,whereheheadstheSectionBioelectronics,andaMedical-Deltahonorary

professoratbothDelftUniversityofTechnologyandtheErasmusMedical

Cen-ter,Rotterdam.Hisresearchinterestsincludeintegratedbiomedicalcircuitsand

systemsforwearable,injectableandimplantablemedicaldevices,suchascardiac

pacemakers,cochlearimplants,neurostimulators,bioelectronicmedicineand

elec-troceuticals.WouterA.SerdijnisanIEEEFellow,anIEEEDistinguishedLecturerand

amentoroftheIEEE.In2016,hereceivedtheIEEECircuitsandSystemsMeritorious

ServiceAward.Moreinformationcanbefoundat:http://bioelectronics.tudelft.nl/

∼wout.

VasilikiGiagkareceivedtheM.Eng.degreeinelectronicandcomputerengineering

fromtheAristotleUniversityofThessaloniki,Greece,in2009,andthePh.D.degreein

AnalogueandBiomedicalElectronicsandImplantedDevicesfromUniversityCollege

London,U.K.in2014.SheiscurrentlyAssistantProfessorintheBioelectronicsGroup,

DelftUniversityofTechnology,NetherlandsandHeadoftheTechnologiesfor

Bio-electronicsresearchgroup,atFraunhoferIZMInstitute,Berlin,Germany.Heresearch

focusesonthedesignandfabricationofactiveneuralinterfaces.Sheservesonthe

IEEECircuitsandSystemsSocietyBioCASTechnicalCommittee,andasAssociate