Live Sense, Nano-Tera Presentation 2013

7/28/2019 Live Sense, Nano-Tera Presentation 2013

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30.05.13 nano-tera.channualmeeng 1


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Scope

Demonstrate an autonomous cell-based biosensormicrosystem for environmental remote monitoringapplications

Scientific objectives:

Study various cell models for toxicology assays inmicrobioreactor format

Develop new physical methods for measuring cellresponsein situ

Develop a micro-bioreactor with intergated sensors30.05.13 2Nano-Tera.chAnnualPlenaryMeeng


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Teams

EPFL-LMIS Microsystems Laboratory Philippe RenaudMicrofluidics, cell chips, bio-impedance

UNIL-DMF Department of Fundmental Biology Jan van der MeerCell biology, gene reporters, bacterial sensors

HESSO-ISI Industrial Systems Institute Martial GeiserMicrosystems, electronics, optical sensors

ETHZ-MAT Biologically Oriented Materials Viola VogelBiomaterial, cell biology

CSEM Nanobiotechnology group Martha LileySurface biochemistry, biomaterials

EPFL-LEPA Electrochemistry and Analytics Laboratory Hubert GiraultElectrochemical sensing, analytical chemistry

EPFL-IMT Sensors and Actuators Laboratory Nico de RooijMicrofluidics, microsensors Peter van der Val

UNIL-IST Institute of Occupational Health MichaelRiedicker

Health effect of pollution



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Whycellbasedsensors?

biologically relevant response to toxic compoundsand mixtures non specific but integrative detection, contrary to

analytical chemistry methods,

extremely sensitive in some cases in-vitro toxicology screening of chemical and

pharmacological compounds



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Mammaliancells

already used for in-vitro toxicologyscreening

Challenges

detection method culture stability, control proliferation sampling environment water/air variability reference measurement

30.05.13 nano-tera.channualmeeng5

Epithelial cells on chip, CSEM

Hepatocytes on-chip, EPFL_LMIS

Fibroblats on nanopillars, ETHZ


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GenecallymodifiedBacterialcells

specific to chemical compounds easy to culture already proven in environmental

measurements

Challenges

storage/conditionning continuous measurement control sample design of new bacterial genotypes for new

chemical compounds

30.05.13

Bacteria in beads, Unil


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Signal

processing

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Data

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8

Modulardemonstrators


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Signal

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10

SAMLAB

on-line

adjustment of pH and osmolality

Forward osmosis system


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Signal

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12

Simultaneous measurement of glucose and lactate in a cell-culture mediumusing a single microfluidic cartridge

Pre-treatedSample

Cell-culture

Outlet

Inlet

1 cm

SAMLAB

Glucoseandlactateon-linemicrosensors


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13

CalibraonCurves

GlucoseConcentraon

LactateConcentraon

SAMLAB

Glucoseandlactateon-linemicrosensors


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Oxygen detection principle

LEPA

On-line Oxygen Detection


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Optical images of the tube sensor for on-line conductivity monitoring and

calibration of two conductivity sensors based on Pt and Au electrodes.

LEPA

On-line Conductivity Detection


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16SAMLAB

Metabolitesbasedtoxicology


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Signal

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TEER


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Micro-bioreactorforTEER


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Cell response to a heavyheavy metal was

calculated.

TEER was seen as the mostsensitive and reproducible

method out of the three

-300

-250

-200

-150

-100

-50

0

50

0 2.5 5 10 20 40

DecreaseinResistance

Concentra.onofCopperChloride(PPM)

DecreaseinResistanceinresponsetoCopperChloride

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Posive

Control

Negave

Control

2.5 5 10 20 40

ABsorbance

CopperChloride(PPM)

LactateDehydrogenaseproduc.oninresponsetoCopper

Chloride

0

5000

10000

15000

20000

25000

30000

contr- cont+ 2.5 5 10 20 40

Fluorescence

CopperChlorideConcentra.on(PPM)

Reac.veOxygenSpeciesproduc.oninresponseto

CopperChloride

Copper Chloride toxicity


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Cell response to a acetominophenwas calculated.

TEER was seen to increase at lowerconcentration and then decreaseat higher concentrations

A more sensitive response wasseen compared to other assays

-250

-200

-150

-100

-50

0

50

100

150

0 10 20 30 40 50

ChangeinTEER

()

Concentra.onofAcetominophen(mM)

TEERResponsetoAcetominophen

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

posveNegave 10 20 30 40 50

Absorbance

Acetominophenconcentra.on(mM)

LDHproduc.oninresponsetoAcetominophen

0

5000

10000

15000

20000

25000

Fluorescence

AceotminophenConcentra.on(mM)

Reac.veOxygenSpeciesproduc.oninresponseto

acetominophen

Acetaminophen toxicity


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Mini table-top incubator for epithelial cells includingcontinued TEER measurements over days

Temperature controlled

CO2continued TEERmeasurement

wireless communication


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Healthycell Sickcell Deadcell

Lowfrequencyimpedance

Highfrequencyimpedance

bar=10umgreen: Phalloidin-FITC, blue: DAPI

20mM Acetaminophen, n=4

0 20 40 6040

60

80

10010 kHz (low frequency)

3 MHz (high frequency)

Time (h)

N

ormalized|Z|(%)

Stresssignal

Viabilitysignal

Label-freedisnconof

minorandmajorcelldamage

Meissneretal.,LabChip,11(14),2352-2361,2011

LMIS-4

Impedancespectroscopy


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EkerandMeissneretal.,PLoSONE,8(3),p.e57423.1-12,2013

Lowfrequency

Highfrequency

MCF7DOXcellsdisplaydrugtoxicityat

~150meshigherdrugconcentraons

thanMCF7WTcells

LMIS-4

Cancerdrugresistance


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Signal

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Movie:DICimageofcellspreadingonnanopillar

Laboratory of Applied Mechanobiology Jau-Ye Shiu and Viola Vogel

Howtodetectenvironmentaltoxins

Nothealthcell

Forcebasedtoxicology


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Integrin Activation by Mn2+Long-term Traction Force Monitoring

Mn2+-/-

Mn2+30min

Mn2+-/-

Mn2+30min

Inhibition of cell contractility by a toxin (ML-7)Inhibition of actin polymerization by a toxin Lat BBeforeLatB

AerLatB

Laboratory of Applied Mechanobiology Jau-Ye Shiu and Viola Vogel

Forcebasedtoxicology:on-linemonitoring


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Pillardis

placement(m)

T.Wlchl,V.Pernet,O.Weinmann,JYShiu,A.Guzik-Kornacka,G.Decrey,D.Yksel,H.Schneider,J.Vogel,D.E.Ingber,V.

Vogel,K.Frei,M.E.Schwab,Nogo-Aisanega.veregulatorofCNSangiogenesis,PNAS,2013

The inhibitory effect of Nogo-A Delta 20 on the spreading of brain MVECs can be counterbalanced by blocking the Rho-A-ROCK-myosin pathway

Jau-Ye Shiu and Viola VogelLaboratory of Applied Mechanobiology

EffectofanAngiogenesisInhibitorontheContrac9lity


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Signal

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Bioreporters:

bacterial strains, which

are specifically

designed to produce a

reporter protein in

response to recognition

of a target chemical or

condition.

2m

Bacterialbiosensors


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Bacterial biosensors

Sample collection



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Test set-up in village



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Freeze-dried bacteria in closed vials; water sample isadded and mixed



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Bioluminescence signal produced by the reporterbacteria is read out after 2 h in portable luminometer



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Promoter

Ribosome

binding

site

Regulator

gene

operator

Reporter

gene

Terminator

PARTS (BIOBRICKS)

RULES

Activation

(inducer)

Repression(derepressor)

Buildingthereportergenecircuit


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arsR

AsIII

InabsenceofAsIII:

SystemisOFF:no

reporterismade.

InpresenceofAsIII:

SystemisON:reporterismadeandcellsfluoresce.

Diagramofthegenecircuit

MakinganarsenitesensorinEscherichiacoli


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Images:NinaB

uffi,Siham

Beggah&David

eMerulla

Cellsembeddedinagarose

beads,d=40-70mm

On-chipbacterialbiosensors


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AqueoussampleswithEscherichiacolibioreportercells.

Automacmeasurement Noimageanalysis Repeatabilitybychangingthe

microfluidicchip

Compact portable biosensor for arsenite detection


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Bioreactor

Bacterialcultureon-chip

LMIS-4


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Bacterialcultureon-chip:longtermacvity

LMIS-4


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Conditioning of freeze-dried reporter cells

0 20 40 60 80 100 120

Control

LB+2% glycerol

LB+2%glucose

LB+1% glucose+1% glycerol

LB+2%glucose+2%glycerol

LB+0.7%glucose+0.7%glycerol

+1.7%YE

E.Colisurvival

rateaer

freeze-drying

Freeze-drying

GlucoseandglycerolimprovedsurvivalofE.coliDH5158ArsRgrownfromLBmedium

aerfreeze-drying

Condi.oning

Agaroseencapsulaonprovedunpraccal.Alternaves

weresought,allowingcombinedrehydraon,sample

contacngandfluorescencemeasurement:

Freeze-dryinginHPLCvials

Septums allows injecon of

growth medium and sample.

Fluorescencemeasurement?

Freeze-drying on capillary

walls. Porous plug to retain

cells aer rehydraon, fol-

lowedbysamplecontacng.

Freeze-drying in cell-counng

mini tubes. Cells centrifuged

i nt o boom cap il la ry f or

fluorescencemeasurement

Freeze-drycondionning


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Signal

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Schematic representation of the electrochemical read-out of an arsenic sensitive bacterial

bioreporter and optical photographies of the setup employed for the bioelectrochemical

monitoring of As(III).

Bacterialbiosensor:amperometricdetecon

LEPA

LEPA UNIL


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*AAS = Atomic Absorption Spectroscopy

As Species Concentration /ppbSample No. Bioreporter Reported (AAS)*

1 4.8 0.4 3.3 1.02 7.7 1.6 6.5 0.83 18.7 1.2 17.8 1.14 58.4 1.7 66.0 6.6

Arsenic quantification in Swiss and

Romanian ground water

Method Validation

LEPAUNIL

Bacterialbiosensor:amperometricdetecon

LEPA


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Remotesensing


Get SMS with:Reference point

Measurement at end point

Make florescence measurement

Send SMS

Send SMS query

Start perfusion:Make reference measurement

Incubate


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Summary


A set ofcell models, cultivable in microenvironments Several readout schemes for monitoring cell response Several system integration done Demontrations in toxicology screening

Live Sense, Nano-Tera Presentation 2013

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