isolation, digital detection and characterization of ... · exosomes (6.75e + 10 exosomes/ml), the...
TRANSCRIPT
ISOLATION,DIGITALDETECTIONANDCHARACTERIZATIONOFBIOLOGICALNANOPARTICLESFOREXOSOME-BASEDDIAGNOSTICS
MarinaCretich*,LauraSola,FrancescoDaminandMarcellaChiariConsiglioNazionaledelleRicerche,IstitutodiChimicadelRiconoscimentoMolecolare(ICRM),Milano,Italy
Introduction
Silicon CHIPS are functionalized byadsorption of copoly(DMA-NAS-MAPS) ,a ter-copolymer based onN,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS) and 3-(trimethoxysilyl)propyl-methacrylate(MAPS)
a
0
5000
10000
15000
20000
25000
30000
35000
0.1 uM 0.5 uM 1 uM 5 uM 10 uMLabelled amino-modified oligonucleotide concentration
Ave
rage
fluo
resc
ence
Deposited
After washing
b
N
* n
m *
p
O O O O Si(OMe)3O(CH3)2NO O
n=97m=2p=1
a
0
5000
10000
15000
20000
25000
30000
35000
0.1 uM 0.5 uM 1 uM 5 uM 10 uMLabelled amino-modified oligonucleotide concentration
Ave
rage
fluo
resc
ence
Deposited
After washing
b
N
* n
m *
p
O O O O Si(OMe)3O(CH3)2NO O
n=97m=2p=1
Coating procedure is easy and robust: chips areimmersed in a polymer solution (1% w/v in 0.9 Mammonium sulfate) for 30 minutes. The chips arethen rinsed with water, dried under nitrogen andcured under vacuum at 80°C
Exosomes,isolatedfromHEKfibroblastcellsandEVdepletedsupernatant,capturedwithantibodiesagainstCD81,CD63,CD9andIgGnegativecontrolanddetectedbySP-IRIS.
Results
Cellsproduceandreleasemembranevesiclesincellculturesanddifferentbodilyfluids.Aparticulartypeofsuchvesicles,namedexosomes,are30-100nmindiameter.
Exosomesareknowntomediatecommunicationbetweencells,playinganessentialroleininflammation,cellularhomeostasis,survival,transportandregeneration;accordingly,minimally-invasivediagnosisofanumberofdiseasescanbeachievedthroughdetectionofthesevesicles.Inparticular,exosomesareconsideredvaluableforliquidbiopsiesincancerdiagnosissincetheycarrymolecularandproteomiccargofromtheirtumourcelloforigin.Inhumancerebrospinalfluid(hCSF),exosomesarerichreservoirsofbiomarkersforneurologicaldisordersandthereisincreasingevidencethatderegulationofvesiclesecretionplayapathologicalroleinAlzheimer’sdisease.
Thedetection platform Silicon coating
Despite exosomes hold the great promise of revolutionizing the standard ofclinical care, their detection and molecular profiling is still technically challengingdue to their small dimension and low refractive index.In this work we present a method based on Single Particle InterferometricReflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping anddigital counting of various populations of individual exosomes (>50 nm) capturedon a protein microarray-based solid phase chip. We demonstrate these conceptsusing purified exosomes from cell culture and directly from hCSF.Our interferometric imaging method could capture, from a very small hCSF volume(20 uL), nanoparticles that have a size compatible with exosomes, using antibodiesdirected against tetraspanins. With this unprecedented capability, we foreseerevolutionary implications in the clinical field with improvements in diagnosis andstratification of patients affected by different disorders.
(A) SP-IRIS detection principle,monochromatic LED lightilluminates the sensor surface andthe interferometriclly enhancednanoparticle scattering signatureis captured on a CMOS camera.
(B) Demonstrates SP-IRIS signal forpolystyrene nanoparticles with adiameter from 50–200 nm whichcan be used to infer size ofcaptured EVs.
(C) Image of the SP-IRIS chip.(D) Low-magnification
interferometric image showingmicroarray of immobilized captureprobes.
(E) SP-IRIS image of a capture probe.NVDX analysis software recognizecapture spot and detectsnanoparticles captured.
(A,B)Anti-CD81captureprobeimageacquiredbeforeandafterincubationwithpurifiedHEK293cellsderivedexosomes.(C,D)Zoom-boxofparticlesdetectedpre- andpost-incubation.(E–F)Particlecontrasthistogrampre- andpost-incubation.
ExosomespurifiedfromHEKcellline,capturedwithanti- CD81antibodyonsiliconchipanddetectedbySP-IRIS.
(A)andAFM(B,C).(A)SP-IRISImageoftheanti-CD81spotincubatedwithasuspensionofexosomes(6.75E + 10exosomes/mL),theredcircleshighlightthecountablenanoparticles.(B)AFMimageofthesamespotarea:thebluedotsidentifyparticleslargerthan15 nm;theyellowcirclesshowinimage(B)perfectlymatchtheparticlesdetectedbySP-IRISinimage(A).(C)Zoominareaoftheanti-CD81spotshowninthegreenframehighlightstheparticleslargerthan15 nminheightdetectedbyAFMandSP-IRIS.Particlessmallerthan15–10 nmaredetectableonlywithAFMastheyarebelowSP-IRISdetectionlimit
Exosomes,isolatedfromHEKfibroblastcellsandEVdepletedsupernatant,capturedwithantibodiesagainstCD81,CD63,CD9andIgGnegativecontrolanddetectedbySP-IRIS.
INDEX:Integratednanoparticleisolationanddetectionsystemforcompleteon-chipanalysisofexosomesGrantagreementn° 766466
REF:M.Chiari,G.Daaboul,P.Gagni,L.Benussi,P.Bettotti,M.Ciani,M.Cretich,D.Freedman,R.Ghidoni,A.YalcinOzkumur,C.Piotto,D.Prosperi,B.SantiniandM.SelimÜnlü.DigitaldetectionofexosomesbyinterferometricimagingScientificReports(2016)6:3746