live sense, nano-tera presentation 2013
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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
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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
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Bacteria in beads, Unil
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Signal
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Modulardemonstrators
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Signal
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SAMLAB
on-line
adjustment of pH and osmolality
Forward osmosis system
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Signal
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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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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
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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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Signal
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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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Bacterial biosensors
Test set-up in village
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Bacterial biosensors
Freeze-dried bacteria in closed vials; water sample isadded and mixed
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Bacterial biosensors
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
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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
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A set ofcell models, cultivable in microenvironments Several readout schemes for monitoring cell response Several system integration done Demontrations in toxicology screening
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