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Introduction

Most conventional cell-based biological analyses are averagedacrosslargegroupsofcells,eventhougheachsinglecellexistsin its own microenvironment, such that population studiescannotcondignlyrepresenttherealconditionsoftheindividualcell. This averaging of diverse cell characteristics in a bulksystem can mislead to overlook the accurate conditions of thesinglecells.Inthisrespectsingle-cellanalysishasbecomethefocus of advanced cell biology in recent decades. Due to thesmallquantitiesof secretions and smallvolumes included in asingle cell, the operation method should be selected prudentlydoastosolvethesedifficultiesduringdetection.Tomeettheseexpectations, interest in microfluidic systems for single-cellanalysis1–8 is increasing,becausethistechniquehasemergedasa pivotal enabling tool for efficient detection. This system iscapable of demonstrating the feasibility of integrating themultiplestepsinvolvedinsingle-cellanalysis,suchasseparating,positioning, chemical stimulations, and detecting. It also has

desirable characteristics, such as the low reagent amountsneeded for analysis, short analysis times and small-spacerequirements. Furthermore this technique offers a precise,rapid,andcost-effectivetoolforsingle-cellanalysis.

Plentyofmicrofluidic-basedsingle-cellanalysissystemshavebeen reported, such as microwell trapping,9–11 physicaltrapping,12,13hydrodynamictrapping,14microfluidictrapping,15,16electricalpositioning,17–19 andmagnetic trapping.20,21 However,these approaches are not suitable for the analysis of secretionfromsingle-adhesivecellsunder flowconditions. Theamountof secretion from a single cell is extremely small, and thesecretiondiffusesintothecell-culturemediuminthemicrofluidicchannel, which makes it difficult to analyze. In a previousreport,22ourdesigneddetectionsystemfornitricoxidesecretedfromstimulatedmacrophagesisdescribedinwhichallprocesses,such as cell culture, chemical stimulation of cells, enzymaticreactionsanddetectionusingathermallensmicroscope(TLM)were integrated intoasinglemicrochip. However, thissystemwasdesignedforabout1000cells inacell-assayplatform,notfor the single-cell level. In order to detect secretion from thesingle-celllevel,asmallsizeofthecell-culturechamberwithachannelinthemicrofluidicsystemisrequired,andthediffusionofsecretionsfromasinglecellshouldbereduced.Inanynew

2011©TheJapanSocietyforAnalyticalChemistry

†Towhomcorrespondenceshouldbeaddressed.E-mail:kitamori@icl.t.u-tokyo.ac.jp

Development of a Microfluidic Platform for Single-cell Secretion Analysis Using a Direct Photoactive Cell-attaching Method

Kihoon JANG,*1 Hong Trang Thi NGO,*1 Yo TANAKA,*1,*2,*3 Yan Xu,*4 Kazuma MAWATARI,*1,*2 and Takehiko KITAMORI*1,*2†

*1DepartmentofAppliedChemistry,GraduateSchoolofEngineering,TheUniversityofTokyo,7-3-1Hongo,Bunkyo,Tokyo113–8656,Japan

*2JapanScienceandTechnologyAgency(JST),7-3-1Sanbacho,Chiyoda,Tokyo102–0075,Japan*3QuantitativeBiologyCenter(QBiC),RIKEN,2-2-3Minatojima-Minamimachi,Chuo,Kobe,

Hyogo650–0047,Japan*4NanoscienceandNanotechnologyResearchCenter,ResearchOrganizationfor21stCentury,

OsakaPrefectureUniversity,1-1Gakuen,Naka,Osaka599–8570,Japan

A precise understanding of individual cellular processes is essential to meet the expectations of most advanced cellbiology.Thereforesingle-cellanalysisisconsideredtobeoneofpossibleapproachtoovercomeanymisleadingofcellcharacteristicsbyaveraginglargegroupsofcellsinbulkconditions.Inthepresentwork,wemodifiedanewlydesignedmicrochipforsingle-cellanalysisandregulatedthecell-adhesiveareainsideacell-chamberofthemicrofluidicsystem.By using surface-modification techniques involving a silanization compound, a photo-labile linker and the2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were covalently bonded on the surface of a microchannel.The MPC polymer was utilized as a non-biofouling compound for inhibiting non-specific binding of the biologicalsamples inside the microchannel, and was selectively removed by a photochemical reaction that controlled the cellattachment.Toachievethedesiredsingle-macrophagepatterningandcultureinthecell-chamberofthemicrochannel,thecelldensityandflowrateoftheculturemediumwereoptimized.Wefoundthatacelldensityof2.0×106cells/mlwastheappropriateconditiontointroduceasinglecellineachcellchamber.Furthermore,themacrophagewasculturedinasmallsizeofthecellchamberinasafewayfor5hataflowrateof0.2μl/minunderthemediumcondition.Thisstrategycanbeapowerfultoolforbroadeningnewpossibilitiesinstudiesofindividualcellularprocessesinadynamicmicrofluidicdevice.

(Received July 6, 2011; Accepted August 18, 2011; Published October 10, 2011)

Original Papers

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design of the microfluidic system, single-cell attachment andculture should be considered for the total system. Single-cellattachmentandculturecanbeachievedbyusingexternalstimulitocontrolthecell-adhesiveareaandthelocationdirectintothecell chamber of the sealed microfluidic system withoutadditional operations. By using ultra violet (UV) lightillumination, non-biofouling reagents can be selectivelyeliminated23insidethecellchamber(Fig.1).

Inthispaper,wedemonstrateasingle-cellarrayandcultureinacellchamberofthemicrofluidicsystem.Wehavepreviouslyshown direct surface modification techniques to achievesingle-cell attachment and culture in a closed microfluidicsystem.23 Thismethodology is basedon the combinationof a2-methacryloyloxyethyl phosphorylcholine (MPC) polymer24andanitrobenzylphotolabilelinker(PL).25MPCpolymer,PL,andasilanecompoundwerechemicallymodifiedonthesurface

of themicrochannel. UVlightenabledustoremovetheMPCpolymer from the surface, and a cell-adhesive surface wasformed.Cell-adhesiveareaswereselectivelycontrolledevenatthe single-cell size level. Microfluidic platforms were alsodesigned to realize a single-cell array and culturing for asingle-cellanalysissystem.Thesmallsizeofthecellchamberwas designed to inhibit the diffusion of secretion from thesingle-cell, and the cell-adhesive area in the cell-chamber wascontrolledbyaphotochemicalreaction.

Experimental

ChipfabricationThe microchip was fabricated by three-step wet etching

(Fig.2a). Briefly, this microchip was composed of two

Fig. 1　Cell-chambercontainedmicrofluidicsystemsinglemacrophagearrayandculture.Preparationofthecell-adhesivesurfacebyUVirradiation.

Fig. 2　(a)Photographsofthefabricatedmicrochip.(b)Microchannel500μmwideand15μmdeep.Cell-chamberwithalinearchannel200μmlong,40μmwide,and5μmdeep.Damstructurewithalinearchannel400μmlong,40μmwide,and2μmdeep.

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borosilicate glass plates (30×70mm), i.e., the cover andbottomplates,withthicknessesof700and200μm,respectively(Fig.2b). The microchannel consisted of four different parts,suchasasidemicrochannel,aflowmicrochannel,acell-chamberandadamstructure;allpartswereetchedatthesametopplate(Fig.2b). Firstly,on the topplate,asidemicrochannelwitha500μm width and 15μm depth was etched by HF afterphotolithography;secondly,aflowmicrochannelwitha40-μmwidth,5μmdepthand3.4cm longandacell-chamberwitha40μmwidth,5μmdepthand200μmlongwereetchedat thesametimeusingthesamemethodasintheformerstep.Finally,the dam structure part with a 40-μm width, 2μm depth and400μmlongwasetchedbyHF(Fig.2b)inthisorder.Finally,four small access holes (diameter=800μm) for the inlet andoutletof thereagentsaswellascellsweremechanicallyboredinto the cover glass plate. After intensive washing andultra-sonicating with deionized water, a patterned cover plateandthenon-patternedbottomplatewasbondedusinganopticalcontact;thatis,theplateswerepolishedtoopticalsmoothness,andthenlaminatedtogetherinanovenat600°C.

PreparationofmacrophagesMacrophages were cultured in 60mm cell culture dishes at

37°C in 5% CO2 in a Dulbecco’s Modified Eagle Medium:Nutrient Mixture F-12 (D-MEM/F-12) 1:1 mixture (Gibco)supplementedwith5%FBS(fetalbovineserum),1%penicillin,and 1% streptomycin (Invitrogen). After the macrophagesbecome confluent, they were washed with 1ml of phosphatebuffered saline (PBS), and detached from the cell culture dishby using 1ml of trypsin (Invitrogen). Detached cells wereadded to5mlofa freshmedium,and thecell suspensionwascentrifuged at 1200rpm for 5min. The supernatant wasdiscarded, followed by resuspension of the machrophages in afresh medium at the required concentration for subsequentexperiments.

MicrochannelmodificationsThe surface-modification method was based on our previous

work.23 The experimental procedures were as follows. Themicrochipwasassembledusingachipholder,andwasconnectedwithcapillaries. Themicrochannelwascleanedusinga0.1MNaOHaqueoussolutionatroomtemperature(RT)for30minat

a flow rate of 5μl/min, followed by rinsing with deionizedwater at a flow rate of 5μl/min for 60min. After dryinginside the channel, the inner surface of the microchannelwas modified by two different methods. Firstly, 3% (v/v)3-aminopropyl-triethoxysilane (APTS) (Sigma-Aldrich) inchloroform was introduced using a syringe at RT for 2h,followedbywashingwithchloroform,ethanol,deionizedwater,and N,N-dimethylformamide (DMF) by using a microsyringepump at 20μl/min for 10 min each. Secondly, vapor-phaseAPTS (0.5ml) was used in oil-bass at 70°C for 1h under avacuumcondition(Fig.3a),followedbywashingwithethanol,deionizedwaterandDMFusingamicro-syringepumpataflowrateof5μl/minfor30min,each.Thesetwodifferentsurfacesof the microchannels were observed under a microscope toconfirmthesurfaceconditions.

To this amino-terminated surface, a nitrobenzyl groupphotolabilelinkerwasmodifiedusinga5mMFmoc-photolabilelinker [4-(4-(1-(9-fluorenylmethoxycarbonylamino) ethyl-2-methoxy-5-nitrophenoxy)-butanoicacid)](AdvancedChemTech),10mM benzotriazol-l-yloxy-tris(dimethylamino) phosphoniumhexafluorophosphate (BOP) (TCI), 1-hydroxy-benzotriazole(HOBt) (TCI), N,N-diisopropylethylamine (DIEA) in DMF atRT for 3h, followed by rinsing with DMF and methylenechloride (MC) at a flow rate of 5μl/min for 20min per each.Then, 30% (v/v) acetic anhydride (Sigma-Aldrich) indichloromethane(DCM)wasusedatRTfor30mintoinactivateallunreactedaminogroups,andwashedwithDCM,DMFataflowrateof5μl/minfor30minpereach.TheFmocprotectinggroupswerethenremovedusing20%(v/v)piperidine(TCI)inDMFataflowrateof5μl/minfor30min,followedbywashingwith DMF and deionized water at a flow rate of 5μl/min for30minpereach.Next,poly[2-methacryloyloxyethylphosphoryl-choline(MPC)-co-methacrylicacid(MA)](Mw=100K,MPC:90mol%, methacrylic acid: 10mol%, synthesized by theconventional radical polymerization technique) was grafted by1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride(EDC) (TCI) coupling for 12h, which created a non-foulingsurface,whichwas then rinsedwithdeionizedwater at a flowrate of 5μl/min for 60min. After drying the microchannelunderanitrogenflow,theMPCpolymer-modifiedcell-chamberwas exposed to UV light (365nm, 1200mW/cm2): the lightpassed throughamicrochipplate from thebottom to topplate

Fig. 3　(a)Directvapor-inducedsurfacemodificationmethodofAPTS. Themicrofluidicchannelwasmodifiedbyvapor.(b)Phase-contrastimagesofthecell-chamberofthemicrochannelaftersurfacemodificationby3%ofAPTSinchloroform.Redcirclesinidicatetheblockedcell-chamberowingtodust and the residue of polymerizedAPTS. (c) Phase-contrast images of the cell-chamber of themicrochannelafteravapor-inducedsurfacemodificationbyAPTS.Scalebar=50μm.

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witha30-μmwidesinglestripephotomask,andwashedunderflowingcondition (5μl/min)usingacombinationofdeionizedwater,ethanol(forsterilization),thendeionizedwateragainfor1heach.Allofthecapillariesandchip-holderswerereplacedwith newly autoclaved ones to guard against contamination.Thefreshmediumwassucked into themicrochannelata flowrate of 5μl/min for 1h, and the microfluidic system wasincubatedat37°Cin5%CO2for2h.

Cellpositioningandcultureinacell-chamberofmicrochannelMicrofluidic chips were prepared as mentioned above. To

accomplishasinglemacrophageinasinglechamberandasafeculture,firstly,thecelldensitywasvariedfrom0.4×106cells/mlto10.0×106cells/mltofindtheoptimalcelldensity.Thecellsuspension was introduced at a flow rate of 5μl/min whileunlocking the bottom side of inlet. After introducing thesingle-cell in a cell-chamber, an optical tweezer(micro-manipulationsystem,MMS-TKS-0912,SIGMAKOKI)wasused tomoveasinglemacrophage to theedgepartof thecell-chamber near the dam structure for cell positioning. Themoved cell was attached to a UV-exposed area in thecell-chamberandat37°Cin5%CO2for1h.Then,thebottomsides of the inlet and the outlet were blocked with a capillarycovertointroducethemediumintothecellchamber.Theflowrate of medium was also varied from 0.1 to 0.6μl/min by amicrosyringe pump during culturing, and the viability of thepositionedsingle-cellwascheckedbytrypanblue.

Results and Discussion

Optimization of the conditions for a single-cell culture in amicrofluidicchannel

Inourpreviousreport,wesucceededinsingle-cellattachmentand culturing inside a microchannel.23 UV light stimulatedremoval of the MPC polymer from the microchannel surface.Forsingle-cellattachmentandarray,UVwasexposedthroughaphotomaskwith a30-μmwide single stripe. Even though themicrochannel modification method was applied to thismicrofluidic system, a problem was found during thesurface-modification step. As shown in a former report,23a silanization step was performed by using 3% APTS in achloroform solution in an I-shapedmicrochannel. In thisnewchip design, a dam structure with a 2-μm depth part wasconsiderably shallow for the modification. Firstly, we triedusing the same condition as described above. However, theconnectingpartof thecellchamberand thedamstructurewasblocked by dust, and the residue of polymerized APTS asFig.3b(redcircle)evenafterfiltrationofAPTSsolution,whichinhibited the next surface-modification step. By contrast,concerning the surface modified by using the vapor-basedmethod, theblockingproblemwassolvedasshowninFig.3c.During washing inside the channel, ethanol, deionized waterandDMFwerewellintroducedwithoutstopping.

Next, for the purpose of safe cell culturing inside acell-chamber condition, the optimum cell culture conditionsshouldbeconfirmed.Theoptimalcelldensityforasingle-cellculture inside a cell-chamber was confirmed in the newlydesignedmicrochannel. The initial step in single-cell analysisinamicrofluidicsystemistointroduceasingle-celltoadesiredplace of the cell-chamber for culturing. The cell density wasvaried from 0.4×106 to 10.0×106cells/ml and the cellsuspensionwasintroducedataflowrateof5μl/min.Ascanbeseen from Fig.4, the trapping ratio of the cell-chambers(10cell-chambersintotal)andsingle-celltrappedcell-chambers

was confirmed according to the cell density. When thecelldensitywas0.4×106,1.0×106,and2.0×106cells/ml,theratiosofthetrappedcell-chamberswere25±7.1,45±7.1and65±7.1% per each, and the ratios of the single-cell trappedcell-chamber were 25±7.1, 30±14.1, and 50±14.1%,respectively.Undertheseconditions,theratioofthesingle-celltrappedcell-chamberstendedtowardincreasedcelldensity. Inthe case of a cell densities of 3.0×106, 5.0×106, and10.0×106cells/ml, the ratios of trapped cell-chambers were60±14.1, 55±21.2, 60±14.1%, per each. The single-cellintroduced cell-chambers were 35±7.1, 20±14.1, and15±7.1%, respectively. The ratio of the single-cell trappedcell-chambers decreased with the cell density over3.0×106cells/ml. Finally, we found that the optimum celldensity for introducing single-cells in each cell-chamber was2.0×106cells/ml.

Single-cellarrayandcultureinamicrofluidicdeviceAfter confirming the optimum cell concentration, a single

macrophage was introduced to the cell-chamber of themicrofluidicdevice,andthecellwasdeliveredtotheedgepartofthecell-chamberusinganopticaltweezertoattachthecellatan UV-irradiated area (Figs.5a and 5b). The structure of themicrofluidicdevicewascomplicated,andtheoptimumflowrateof the medium should be confirmed for safe culturing. Wefoundthatthemacrophagediedafter2hofculturinginsidethecell-chamberofthemicrochannelunderastaticconditionowingto a deficiency of the nutrients and oxygen, which means thatinsidethesmallsizecell-chamberconditionswereverysensitivefor safe cell-culturing. The flow rate was varied from 0.1 to0.6μl/min. When the medium flow rate was from 0.4 to0.6μl/min, a single macrophage was sucked into the damstructure of the channel, which caused cell destruction. Highshear stress in the cell-chamber resulted in sucking thesingle-cell into dam structure. In the caseof a flow rate with0.1μl/min,thecellwasfoundtobedeadafter5hofculturinginsidethecell-chamberbecauseofadeficiencyofnutrientsandoxygen(Fig.5c).Incontrast,ataflowratewith0.2μl/min,thesingle macrophage was placed in a UV exposed area of thecell-chamberwithoutdestruction.After5hofculturing,trypanbluewasintroducedtochecktheviabilityoftheadheredsinglemacrophage in a cell-chamber. As shown in Fig.5d,

Fig. 4　Trapping ratio of cell-chambers and single-cell trappedchambers according to the concentration of macrophage cellsuspension.■,Entirecell-chambers;□,singlecelltrapping.

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the  macrophage was placed in a UV-irradiated area withviability. Thisresult indicated that thesinglemacrophagewascultured in a small size of the cell-chamber inside themicrochannel.

Even though single-cell attachment and culturing wassuccessfully realized by optimization of the culture conditionsinsidethemicrochannel,thedetectionofsecretionfromasinglecell was not carried out. This problem resulted from theexceedinglysmallamountofsecretionfromasinglecell,whichwasnotdeliveredtotheflowchannelfordetection.Toachievethisstrategytosensitiveandhighlyefficientsingle-cellanalysis,theflow-channelpartofthemicrofluidicsystemshouldbemorecarefully considered. Short diffusion distances and the verylarge surface-to-volume ratio in this system could be efficientforanalysis.

Conclusions

A direct, simple surface-regulating technique was used toposition and culture a single macrophage in a cell-chamber ofmicrofluidics. A surface-treatment method including aphotolabile linker and a MPC polymer was introduced forcontrollingthecell-adhesiveareainacell-chamber. TheMPCpolymer was removed selectively by using a photochemicalreaction. We found that cell density of 2.0×106cells/mlwasthe optimum condition to introduce the single-cell in eachcell-chamber. Furthermore,ata flowrateof0.2μl/minof themedium, the macrophage was efficiently attached ontoUV-illuminated area, and was cultured in a small size of thecell-chamber for 5h. These confirmed conditions are theoptimum for analyzing single-cell secretion in a microfluidicdevice.Theproposedmethodoffersnewpossibilitiesinstudiesofsingle-cellanalysisinamicrofluidicdevice.

Acknowledgements

This study was supported by Core Research of EvolutionalScience & Technology (CREST) from Japan Science andTechnologyAgency(JST),theGlobalCenterofExcellenceforMechanicalSystemsInnovation(GMSI)fromtheUniversityof

Tokyo Global COE program, and Grant-in-Aid for YoungScientist (A) (21681019) from the Ministry of Education,Culture,Sports,ScienceandTechnology(MEXT)ofJapan.

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