patlisci2, nano-tera 2016

8/17/2019 PATLiSci2, Nano-Tera 2016

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PATLiSci II – Probe Array Technology for Life Sciences /Rapid Sensing of Cancer

cells

Indentation-typeAFM

targetadsorption(sensing)

orce spectroscopy mappingllows to identify breast cancerells among healthy cells inisse by their characteristicistogram of elasticity modleales"

Label free# ampli$cation free and tisse sample basedetection of biomar%ers sing the cantile!ers insensing mode in the same FL&' AF( head com)plements information abot stats of a tmor"Case stdies* melanoma# breast cancer"

Implementation of cantile!er arrays for parallel ac+isitionof force !s" distance cr!es in a commercial FL&' AF(head ,-A-.SRF A0 Liestal1 increases throghpt of thetechni+e"

Partners* ni!ersity of 2asel# ni!ersity 3ospital 2asel#&PFL# CS&(

-A-.SRF A0PATLiSci


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The clinical !ale of newdiagnostic tools

Based on the current diagnostics,

patients are undertreated or more oftenovertreated

Overtreatment includes chemotherapy (and all its side eects) ormore etensive surgery (and its additional ris!s and more di"cult

recovery)#


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TLiSci 4 – Probe Array Technologiese Sciences – Rapid Sensing of Canc

Partners*ni!ersity of 2asel# ni!ersity3ospital 2asel# &PFL# CS&(#

$!in and %reast cancer are among the most comcancers of the &orld

'he faster cancer can %e detected, the %etter e need rapid diagnostic tools#

It has %een found that cancerous %reast cells havdierent elasticity than healthy ones#

PATLiSci


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Atomic Force (icroscopy ,AF(1

Palpation Indentation

(acrometer scale -anometer scale

'he tip touches andfeels the

moleclarstrctres

'he e5aminer touches andfeels the

patient6s body

3ow can we measre this7

Cell and Cell ) &C( interactions occr at the nano s

How physical properties of tissues

contribute

to cancer development and

progression?


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Partners*ni!ersity of 2asel# ni!ersity3ospital 2asel# &PFL# CS&(#

*antilevers can pro%e the elasticity of surface loca%y force spectroscopy#

PATLiSci


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le!er arrays ha!e been microfabric&()&PFL

PATLiSci


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e (apping Spectroscopy on 2iopsincer cell elasticity is pro%ed %y force spectroscopyrce-distance curves) in a grid on the %iopsy surfac

ousands of force +distance curves are evaluateding a stiness histogram#

e shape of the histogram clearly allo&s to distingualthy tissue from cancerous cells

e results from classical histology, uorescent in-sit%ridiation and force spectroscopy maps are comp

omar!er detection %y cantilever sensing#

PATLiSci


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2reast Tisse Composition

www.proteinatlas.org

histology

cell sie. /-0 1m


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mary tmor pro$le and the ad8acent tisse re!eal a cphenotype detected in the lymph node metastases

Field eect evaluation

2rognostic32redictive Mar!er

405 specimensmeasured &ithforcespectroscopy.


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Actin367AlamininActin367A

*ancer cells BM Fi%ro%lasts 8*M

*ollagen I

Co)cltre of cancer cells and$broblasts on nati!e 2(

cancer cells

BM

9%ro%lasts

:;


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Cancer cells Re;ection CAFs

2(

Cancer cells

=*'44/ cells > *AFs, 4? FB$, start after : days co-culture, imaging time /5h

CAFs

Cancer cells prepared on basalmembrane for calibration prposes

@; 1m


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*om%ination of force and opticalmicroscopy

for live cell and tissue imaging


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e Spectroscopy 3istogram Analysi

resence of cancer cells in a %iopsy is detectedn the histogram

isadvantage. It ta!es several hours

mprovement &ithin 2A'i$ci .sing a cantilever array speeds up diagnosisaster data acDuisition applied using dedicated

ard&are of a 7ano$urf FleAFM head

PATLiSci


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• Cse cantilever array

• Cse E*$8 (vertical-cavity

surface emitting laser) array

&ith 4 E*$8 aligned to

each cantilever

• $&itch E*$8s on (and o)

fast

• $ynchronise readout of

position sensitive detector tomeasure the deection of

each cantilever

• Increase speed of data

acDuisition VCSEL array

Photodetector

Cantilever array

*opyright ;4/ *$8M Mi 2age 4@


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2roof of principle.• E*$8 array. @;µm pitch, λ G

0@;nm

• pac!aged

• dedicated driver electronics

created

• Hay-tracing simulations to optimise

optical design

• Optimisation of readout of position

sensitive detector

• Alignment. many degrees of

freedom *opyright ;4/ *$8M Mi 2age 4/


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• Bench-top set up

2roof of principle.

2$6

*antilever array

Beamsplitters

nd lens

E*$8array > 4stlens

$ample

*opyright ;4/ *$8M Mi 2age 45


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Integration in a commercialAFM.

• Modi9ed FleAFM• Modi9ed optics + tilted

• Addition of alignment

system for E*$8 array

(,y,,θ)

$tandard 7anosurf FleAFM

Modi9ed FleAFM &ith tilted optics and E*$8 alignment

E*$8 alignment

'ilted optics



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Optics.•

Optics is tilted#• $tandard FleAFM optics not

&ide enough for cantilever

arrays &ithout modi9cation#

• Hay tracing predicts the

eect of a%erration on the

dierent cantilever spots



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E*$8s.•

7e& E*$8 electronics.• Faster s&itching speed (J;;!=)

for %etter precision &hen

determining the spot position on

the 2$6

• Modulation of the driver signal at

J::M= allo&s us to avoid

oscillations in the E*$8 output

• *an %e controlled from FleAFM

soft&are

*opyright ;4/ *$8M Mi 2age ;


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Alignment.•

7eed to reduce the manydegrees of freedom in the

%enchtop system to %e

compati%le &ith the FleAFM

• J degrees of freedom retained

in the E*$8 positioning. ,y,

and Θ

• eight restrictions on

alignment system*A6 dra&ings of alignment

system for E*$8s



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o support force spectroscopy 9ndings cantilere also operated in sensing mode to detectiomar!ers for cancer#

peci9c %inding of molecules to pro%es on thantilevers leads to %ending due to surface

tress change

PATLiSci


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lanoma tmor progression

PATLiSci

Radial growthphase :erticalgrowth phase

M# Eol!enandt. Maligne Melanome. In.Dermatologie und Venerologie.

O# Braun-Falco u# a# (8d#), Eerlag$pringer, ;;@, $# 4:4:+4:J

Melanoma on a patientLs s!in(source . 7ational *ancerInstitute)#

6ierent gro&th phases.Hadial, vertical, circulating tumor cells

Metastatic malignant melanoma at the heart

i!iMedia *ommons

1960 20060.000

0.002

0.004

0.006

0.008

0.010

r a t e

o f i n c i d e n c e

year

development of lifetime risk for

melanoma in the last 50 years


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e


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rocedre

totalRNA

evice !et"p

functionalization

injectionisolation

c# Hybridization

a)

b)

PATLiSci


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PATLiSci


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PATLiSci


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psies al&ays contain healthy ,wildtype1 BHAF#

&e &ill al&ays see a BHAF &ildtype signal (green cr

elanoma tmor cells are present, the cantileverctionalied &ith 2RAF :=>>& mar!er &ill detect it#

en &e &ill see a BHAF E/;;8 signal (red cr!e

PATLiSci


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o% for the red cr!e?

ple and rapid diagnostics of melanoma (BHAF E/;;

(elanoma tmor present -o s%in cancer

PATLiSci


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s on melanoma %iopsies. all samples correctly ide

positi!es -o false

PATLiSci


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gram representation* all biopsies correctly id

e positi!es -o false negati!esPATLiSci


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imating the detection limit

s of cell line H7A etracts containing BHAF E/;;8d type to assess the detection limit. %etter than

@ ? mutant in &ild type#PATLiSci

Institut fr 2athologie, Basel;4:N;:N40


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:J

-o labelling# no PCR ampli$cation#

no se+encing


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In 4; ? of melanoma patients a dierent mutatioBHAF E/;; instead of BHAF E/;;8 + is responsi%lfor s!in cancer

By choosing appropriate 67A pro%es on thecantilevers, &e can detect %oth BHAF E/;;8 and

BHAF E/;;

cting di@erent mtations sing cantile! sensors

PATLiSci


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PATLiSci


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9etect 2RAF :=>>& 9etect 2RAF :=>>

PATLiSci

@;? of melanomapatients

4;? of melanomapatients


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se 4* 2reast Cancer

ast cancer is detected %y evaluation of =8H leveman epidermal gro&th factor )

ay, certain aggressive mammary carcinomas can

ated &ith therapeutic anti%odies (trastuuma%)

tection of =8H overepression allo&s to identifyients &ho &ill respond to such a therapyPATLiSci


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perimental procedre

ollecting

%iopsy

'issuesample

3 i s t o

l o g y

& 5 t r a c t R - A

Cantile!ersensing

Florescentin sit hybridi)

Bation ,3&R41

PATLiSci


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PATLiSci


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3&R4Positi!e nmsignal

3&R4 -egati!e)D nm signal

PATLiSci

C il 4 C il


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-mbe

rType of Tmor

R-A

yield"

,Eg1

R-A

conc"

,ng/El1

Cantile!

er

response

s

3&R4

stats

Cantile!

er

sensing

reslt

<

Invasive ductal

carcinoma4;# ::

-@ nm

negati

!e

negati!

e

4Invasive ductal

carcinoma;#@@0 40#/

-; nm

negati

!e

negati!

e

6uctal carcinoma

in situ4#/ /@#J

>4; nm

positi!

e

positi!e

G

=ighly

dierentiated

lo%ular carcinoma

9&0RA9&9 R-A

SA(PL&

4# /J#4J

>@ nm

negati

!e

positi!e

DInvasive ductal

carcinoma#04 :#@4

-@ nm

negati

!e

negati!

e

=

Invasive ductal

carcinoma

9&0RA9&9 R-A

SA(PL&

4#/4 @:#0

>J; nm

negati

!e

positi!e

=8H

Bdetec

t

ion

results

correctly identi$ed" 4 false positi!es de to degraded biopsy R

PATLiSci


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-anoSrf Fle5AF( head

.ptics andpositioning stage:CS&L board ,CS&(1

gration of Cantile!er Array Sensinhnology into the Fle5AF( head

PATLiSci


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LEDs represent

the eight VCSELs

SDA SCL GND

$ontrol electronics for %$!&'s via 4()it *2$(+"s

PATLiSci

l t ti f i h


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6-levels valve device

Kent Scientific Sringe

!"#p$nverted #icr%sc%pe&le'A&(

Valves indicat%r

!C f%r #%nit%ring the cantilever arra

and the ill"#inati%n %f the VCSELs

lementation of peripheryted microscope# !al!e# syringe pmp

PATLiSci

t d l t ,C>>> A li ti


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>>"Applicatio

PATLiSci

eado t test


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eadot test

PATLiSci

l i


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onclsions

setup for detection of %reast cancer andelanoma %ased on a 7ano$urf FleAFM hea

as %een developed#

reast cancer diagnosis is %ased on %oth forcpectroscopy and cantilever array sensingiomar!er detection

PATLiSci

t t


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emonstrators

Mem%rane $urface$tress$ensors for an8lectronic 7ose

PATLiSci

7ano$urfFleAFMfor Force$pectros-copy using a

canti-lever array

word abot biopsy sample +ality*


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word abot biopsy sample +ality*-A +ality control ,electrophoresis

tal R-A ,


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parison of measrements with total Ri@erent +ality ,sable !s" degraded1

0ood sample*

patlisci2, nano-tera 2016

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