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Introduc)onThistechnicalnotedetailsthecombina0onofSizeExclusion Chromatography (SEC) separa0on andTunable Resis0ve Pulse Sensing (TRPS) analysis ofextracellular vesicles (EVs). This integratedplaCorm for thefirst0meoffers EV researchers asimple, accurate and certain solu0on for theisola0on and characterisa0on of EVs. EVs,membranous vehicles with a range of importantbiological func0ons, are the subject of intenseresearch. Medical diagnos0cs has a compellingneed for the detailed data that EV biomarkersappear to offer. EVs are also being developed astherapeu0cs. Standardised measurementinforma0on is a fundamental requirement for theadop0on of EVs as biomarkers and for EVs astherapeu0cs. The breakthrough created by thisplaCormwill enable the EV field to progress andachieveitspoten0al.

ContextUltra-centrifuga0on and chemical precipita0onmethods are both unsuitable as separa0onmethods fo r severa l reasons inc lud inginconsistency,contamina0onandaggrega0on.

The small physical size and heterogeneity of EVshasalsoexposedthelimita0onsofmostanaly0caltechnologies including confocal microscopy, flowcytometry, dynamic light scaOering (DLS),nanopar0cle tracking analysis (NTA) and Electron-microscopy(EM).EM can resolve EVs but is non-quan0ta0veimprac0cal for rou0ne screening and the vacuumaltersthepar0cles.

SEC + TRPSSECasasepara0onsystempreservesthestructuralintegrity of the EVs1,2,3 and provides a cleanpopula0on of EVs, which makes it possible tomeasurethemconsistently.Itissimple,effec0vetouse and is the only way to provide a standardrepresenta0vesetofEVs.SECcanbeaccessedviathe industry standard qEV columns, aimed at500µL samples or the qEVsingle columns for 100µL samples.

Integrated Platform for EV Analysis: SEC (qEV) + TRPS (qNano)

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TRPSisapowerful,calibratedandcertainapproachtonano-par0clemeasurementwith individualsizeresolu0on of around 1 nm andminimum par0clesize down to~40 nm. TRPS provides par0cle-by-par0cle size distribu0on, accurate numberconcentra0on by size frac0on, and individualpar0cle zeta poten0al. These are achieved byanalyzing individual par0cles as they translocatethrough a single nanopore 4, 5, 6. Each par0cletransi0ng the pore triggers a resis0ve pulse(blockade event), some0mes referred to as theCoulter principle. The size/volume of a par0cle ismeasured by the height of each event, par0clecharge (zetapoten0al) isderivedbyanalysing theshape and width of the pulse and the par0cleconcentra0on is reflected in frequency of theeventsrela0vetoaknowncalibra0onstandard.TheqEVprotocol takes 15minutes, TRPS analysisof a post-qEV sample typically takes 20 minutesincluding calibra0on. Data analysis and the PDFreportgenera0onisrapid.Consequentlythewholeprocedure (purifica0on + analysis) can easily becompleted in less than an hour (Figure 1).Workflowformul0plesamplescanbemanagedtoreducethe0mepermeasurementfurther.DuetotheheterogeneityofEVsandtheneedforstandardiseddata,ithasbecomeveryobviousthatthepar0cleconcentra0onmustbespecifiedwithinagivensizerange(e.g.C50-250nm),whichTRPSdoes.A single number concentra0on is by itselfmeaningless. That appl ies regardless ofmeasurementmethod.

EVsarepresentincomplexbiologicalfluidssuchasserum, plasma, cerebral spinal fluid (CSF), saliva,urine and cell culture media. Contamina0ngproteins and other non-vesicular component (e.g.lipids) can interfere with all methods ofmeasurement and will provide false data ifincluded in biological assays. Consequently thesemust be removed to achieve consistency andaccuracy of the EV specific measurement andanalysis.

SECcolumnshavebeenshowntohavethehighestpurity of EVs with respect to free proteins1. Theyarenowconsideredbymanyresearcherstobetheonlyviablemethodofsepara0onforclinicaluse.This technical note outlines a singleworkflow forEVsisola0onfromcerebralspinalfluidandplasmausingSECbasedqEVcolumnsandanalysisbyTRPS.The whole procedure can be easily applied tootherbiologicalmedia.

Method&ResultsSampleprepara)onExtracellularvesicles(EVs)werepreparedfromovinecerebralspinalfluid andhumanplasma.Allsamplesi

wereclearedof residualcellsanddebrisby threesequen0al10-minutecentrifuga0onsteps (CSF1500g,3000g,10,000g;plasma1500g,3000g,3000g,ineachcasethesupernatantisretained).Pre-equilibratedstandardqEV columnswere loadedwith500µLof sample and500µL frac0onswere collected. TheEVstypicallyeluteinfrac0ons7-9(see2andFigure2)

Sampleobtainedfrominsidetheskullpostmortemi

EVs SEPARATION TRPS ANALYSIS RESULTS

10 0 Time (min)

40 20 30

Mean size: nm

Mode size: nm

C total: Particles / mL

C 50-200nm: Particles / mL

Load 500 µL of

sample Collect EVs

3x 500 µL in PBS Load 35 µL Process Raw

data files

F7 F8 F9

Figure 1: Standardized protocol for EVs separa4on to analysis.GeneralWorkflowforEVsisola4onbySECandanalysisbyTRPS.Theoverallprocedureisusuallyaccomplishedwithin30-40min.

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TRPSSetupAllmeasurementswere conductedusingaqNano(Izon Science Ltd., NZ) and Izon soeware version3.1. The lower fluid cell contained the electrolyte(75μL)andtheupperfluidcellcontained35μLofsample.PriortoTRPSanalysis,allsampleswerepurifiedbyqEV columns as detailed above. Between eachsamplerun,thesystemwaswashedusingPBST(35μL)intotheupperfluidcellseveral0mestoensuretherewasnoresidualpar0clecarryoverbetweensamples.

A detailed descrip0on of such a tunable resis0vepulsesensingdeviceisdescribedelsewhere5,6.Thepore (NP150)was calibratedusing200nm (mean210 nm) CPC polystyrene par0cles of knownconcentra0on(5.0x109/mL)at3pressures(10,8,5cmH2O).Thesampleswerethenanalyzedatthesame voltage and pressures using a consistentbaseline current (e.g. 135 nA ± 2-5% max.) toensurevalidcompara0vedatasetsbetweenruns.

TRPS analysis requires the use of Izon reagents,which includes a coa0ng solu0on for pore pre-treatment,minimisingpar0clebindingtotheporethroughnon-specificinterac0ons.Freshlypreparedreagentswerefiltered (0.22µm)on the day of analysis to avoid contamina0on/par0culatesthatcanoccurover0meandinterferewith TRPS analysis. For TRPS analysis of the qEVfrac0ons, an ini0al dilu0on of 1:5 or 1:10 inelectrolyte was used. This dilu0on was thenop0mised to achieve a transloca0on rate at thehighestopera0ngpressureofapproximately200to1600par0clesperminute. IftheEVconcentra0onis low TRPS analysis takes longer so the samplemay be concentrated using the Merck MilliporeMicrocon-30spinfilters7oranequivalent.

TRPSanalysisofCSFandplasmasamplesThe qEV frac0ons of the CSF and plasma samples were diluted in electrolyte and analyzed by TRPS asoutlinedabove.TheEVsfromplasmaandCSFelutedinqEVfrac0ons7-9withthepeakfrac0onforplasmacontained in F8 and CSF F9 (Figure 2). Some EVs eluted in F10 but this frac0on was typically morecontaminatedwithproteinandusuallydiscarded.Atypicalpar0clesizehistogramandconcentra0ondataisshown in Figure 3. In some instances, where biological samples contain a low level of EVs, addi0onalconcentra0onstepsusingaspinfiltermayberequiredbeforeoraeertheqEVpurifica0on.Thisisfrequentlythecasewithurinesamples,cellculturesupernatantsandfrozenhumanCSFsamples.

Figure2:TypicalqEVsepara4onprofileforplasmaandCSF(500µL).

Figure3:ATRPSmeasurementonapost-qEVsample.

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ConclusionThistechnicalnotedetailsthemethodologyforisola0onandTRPSanalysisofEVs.BiofluidscontainEVsandthesevesiclescanbenowreadilyisolatedbystandardisedqEVsizeexclusionchromatographyandanalyzedbyTRPStoprovidecertaintyandrepeatability.Theprocedureissimple,gentleandcanbecompletedwithin60minutes.WepurifiedEVs fromovineCSFandhumanplasmausingqEVsandperformedTRPSanalysiswithexcellentstabilityat1:10dilu0onofthesamples.TRPSisapowerfulmethodforEVanalysis;itisanon-averaging approach applicable for the analysis of poly-disperse size samples. TRPS par0cle-by-par0clesensing is the only method that provides certain and precise size, concentra0on and zeta poten0almeasurements. The future ofmedical diagnos0cs is expected to include EV profiling. Understanding thebiophysicaldiversityinEVpopula0onsinastandardisedformatisparamountforlinkingtheimpactofEVsproper0es to theirbiological roleand func0on. SEC+TRPS is theonlyway that this canbeachieved forclinicaluse.

References[1] Anita N. Boing; et al. Single-step isola0on of extracellular vesicles by size-exclusion chromatography.JournalofExtracellularVesicles2014,3:23430[2]TheIzontechnicalnote:IzonqEV:anewtoolforrapidEVisola0on[3] Richard J. Lobb; et al. Op0mized exosome isola0on protocol for cell culture supernatant and humanplasma.JournalofExtracellularVesicles2015,4:27031[4]EmmaL.C.Blundell;etal.Emergenceoftunableresis0vepulsesensingasabiosensor.AnalMethods2015,7,7055[5]GeoffreyWillmoO;etal.Useoftunablenanoporeblockaderatestoinves0gatecolloidaldispersions.J.Phys.:Condens.MaOer2010,22,454116[6]RobertVogel;etal.Quan0ta0vesizingofnano/micropar0cleswithatunableelastomericporesensor.Anal.Chem.2011,83,3499−3506[7]MerckMillipore-Microcon-30-Productreference:MRCF0R030