You are on page 1of 46

30.05.

13  

nano-­‐tera.ch  annual  mee3ng  

1  

Scope  
•  Demonstrate an autonomous cell-based biosensor microsystem for environmental remote monitoring applications

Scientific objectives: •  Study various cell models for toxicology assays in microbioreactor format •  Develop new physical methods for measuring cell response in situ •  Develop a micro-bioreactor with intergated sensors
30.05.13   Nano-­‐Tera.ch          Annual  Plenary  Mee3ng   2  

Teams  
•  •  EPFL-LMIS Microsystems Laboratory Microfluidics, cell chips, bio-impedance UNIL-DMF Department of Fundmental Biology Cell biology, gene reporters, bacterial sensors Industrial Systems Institute Microsystems, electronics, optical sensors Philippe Renaud Jan van der Meer

•  •  •  •  •  • 

HESSO-ISI

Martial Geiser Viola Vogel Martha Liley Hubert Girault Nico de Rooij Peter van der Val Michael

ETHZ-MAT Biologically Oriented Materials Biomaterial, cell biology CSEM Nanobiotechnology group Surface biochemistry, biomaterials

EPFL-LEPA Electrochemistry and Analytics Laboratory Electrochemical sensing, analytical chemistry EPFL-IMT UNIL-IST Riedicker Sensors and Actuators Laboratory Microfluidics, microsensors Institute of Occupational Health Health effect of pollution

30.05.13  

nano-­‐tera.ch  annual  mee3ng  

3  

Why  cell  based  sensors  ?  
•  biologically relevant response to toxic compounds and 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

30.05.13  

nano-­‐tera.ch  annual  mee3ng  

4  

Mammalian  cells  
•  already used for in-vitro toxicology screening

Epithelial cells on chip, CSEM

Challenges •  •  •  •  •  detection method culture stability, control proliferation sampling environment water/air variability reference measurement
30.05.13   nano-­‐tera.ch  annual  mee3ng  

Hepatocytes on-chip, EPFL_LMIS

Fibroblats on nanopillars, ETHZ 5  

Gene3cally  modified  Bacterial  cells  
•  specific to chemical compounds •  easy to culture •  already proven in environmental measurements Challenges
Bacteria in beads, Unil

•  •  •  • 

storage/conditionning continuous measurement control sample design of new bacterial genotypes for new chemical compounds
30.05.13  

General  concept  
TER   >   >   osm  
Nutrients    +   Temp.   control  

Z(f)  

<   fluo   v   glu   pH   I   F  

Data   comm  

Signal   processing     control  

30.05.13  

Nano-­‐Tera.ch          Annual  Plenary  Mee3ng  

7  

Modular  demonstrators  

8

Osmolarity  and  pH  control  
TER   >   >   osm  
Nutrients    +   Temp.   control  

Z(f)  

<   fluo   v v     glu   glu   pH   pH   I   F  

Data   comm  

Signal   processing     control  

30.05.13  

Nano-­‐Tera.ch          Annual  Plenary  Mee3ng  

9  

Forward osmosis system  
on-line adjustment of pH and osmolality

SAMLAB

10

Metabolites  sensing  
TER   >   >   osm  
Nutrients    +   Temp.   control  

Z(f)  

<   fluo   v v     glu   glu   pH   pH   I   F  

Data   comm  

Signal   processing     control  

30.05.13  

Nano-­‐Tera.ch          Annual  Plenary  Mee3ng  

11  

Glucose  and  lactate  on-­‐line  microsensors  
Simultaneous measurement of glucose and lactate in a cell-culture medium using a single microfluidic cartridge

Cell-culture

Inlet

Pre-treated Sample

1 cm

Outlet

SAMLAB

12

Glucose  and  lactate  on-­‐line  microsensors  
Calibra3on  Curves  

Lactate  Concentra3on   Glucose  Concentra3on  
13

SAMLAB

On-line Oxygen Detection

Oxygen detection principle

LEPA

On-line Conductivity Detection

Optical images of the tube sensor for on-line conductivity monitoring and calibration of two conductivity sensors based on Pt and Au electrodes.
LEPA

Metabolites  based  toxicology  

SAMLAB

16

TEER  based  toxicology  
TEER   TER   >   >   osm  
Nutrients    +   Temp.   control  

Z(f)  

<   fluo   v   glu   pH   I   F  

Data   comm  

Signal   processing     control  

30.05.13  

Nano-­‐Tera.ch          Annual  Plenary  Mee3ng  

17  

Micro-­‐bioreactor  for  TEER  

Copper Chloride toxicity  
50   Decrease  in  Resistance   0   -­‐50   -­‐100   -­‐150   -­‐200   -­‐250   -­‐300   Concentra.on  of  Copper  Chloride  (PPM)   0   2.5   5   10   20   40   A B s o r b a n c e   Decrease  in  Resistance  in  response  to  Copper  Chloride   0.9   0.7   0.6   0.5   0.4   0.3   0.2   0.1   0   Posi3ve   Nega3ve   Control   Control   2.5   5   10   20   40   0.8   **   Lactate  Dehydrogenase  produc.on  in  response  to  Copper   Chloride  

Copper  Chloride  (PPM)  

30000   25000   Fluorescence   20000   15000   10000   5000   0  

Reac.ve  Oxygen  Species  produc.on  in  response  to   Copper  Chloride  

•  Cell response to a heavy heavy metal was calculated. •  TEER was seen as the most sensitive and reproducible method out of the three

contr  -­‐   cont  +   2.5  

5  

10  

20  

40  

Copper  Chloride  Concentra.on  (PPM)  

Acetaminophen toxicity  
150   100   Change  in  TEER  (Ω)   Absorbance   50   0   -­‐50   -­‐100   -­‐150   -­‐200   -­‐250   Concentra.on  of  Acetominophen  (mM)   0   10   20   30   40   50  

TEER  Response  to  Acetominophen  

0.7   0.6   0.5   0.4   0.3   0.2   0.1   0  

LDH  produc.on  in  response  to  Acetominophen  

pos3ve  Nega3ve  

10  

20  

30  

40  

50  

Acetominophen  concentra.on  (mM)  

25000   Fluorescence   20000   15000   10000   5000   0  

Reac.ve  Oxygen  Species  produc.on  in  response  to   acetominophen  

•  Cell response to a acetominophen was calculated. •  TEER was seen to increase at lower concentration and then decrease at higher concentrations

Aceotminophen  Concentra.on  (mM)  

•  A more sensitive response was seen compared to other assays

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

• Temperature controlled •  CO2 •  continued TEER

measurement •  wireless communication

21

Impedance  based  toxicology  
TER   >   >   osm  
Nutrients    +   Temp.   control  

Z(f)  

<   fluo   v   glu   pH   I   F  

Data   comm  

Signal   processing     control  

30.05.13  

Nano-­‐Tera.ch          Annual  Plenary  Mee3ng  

22  

Impedance  spectroscopy    
Low  frequency  impedance    
20mM Acetaminophen, n=4
Normalized |Z| (%)
100

10 kHz (low frequency) 3 MHz (high frequency)

80

Viability  signal   Stress  signal  
0 20 40 60

Healthy  cell  

«Sick»  cell  

Dead  cell  

60

40

High  frequency  impedance    

Time (h)

à  Label-­‐free  dis3nc3on  of   minor  and  major  cell  damage    
green: Phalloidin-FITC, blue: DAPI Meissner  et  al.,  Lab  Chip,  11  (14),  2352  -­‐  2361,  2011   LMIS-4
bar=10um  

Cancer  drug  resistance  

Low  frequency   High  frequency  

MCF7  DOX  cells  display  drug  toxicity  at   ~150  3mes  higher    drug  concentra3ons   than  MCF7  WT  cells  

LMIS-4

Eker  and  Meissner  et  al.,  PLoS  ONE,  8  (3),  p.  e57423.1-­‐12,  2013  

Cell-­‐based  toxicology:  force  measurement  
TER   >   >   osm  
Nutrients    +   Temp.   control  

Z(f)  

<   fluo   v   glu   pH   I   Z(f)   F  

Data   comm  

Signal   processing     control  

30.05.13  

Nano-­‐Tera.ch          Annual  Plenary  Mee3ng  

25  

Force  based  toxicology  
Not  health  cell  

How  to  detect  environmental  toxins  

Movie:  DIC  image  of  cell  spreading  on  nanopillar  

Laboratory of Applied Mechanobiology

Jau-Ye Shiu and Viola Vogel

Force   based  toxicology  :  on-­‐line   monitoring   Integrin Activation by Mn Long-term Traction Force Monitoring
2+
Mn2+-­‐/-­‐   Mn2+-­‐/-­‐  

Mn2+  30min  

Mn2+  30min  

Inhibition of actin polymerization by a toxin Lat B
Before  Lat  B  

Inhibition of cell contractility by a toxin (ML-7)

Amer  Lat  B  

Laboratory of Applied Mechanobiology

Jau-Ye Shiu and Viola Vogel

Effect  of  an  Angiogenesis  Inhibitor  on  the  Contrac9lity    

The inhibitory effect of Nogo-A Delta 20 on the spreading of brain MVECs can be counterbalanced by blocking the Rho-A-ROCKmyosin pathway
Pillar  displacement  (μm)   T.  Wälchl,  V.  Pernet,  O.  Weinmann,  JY  Shiu,  A.  Guzik-­‐Kornacka,  G.  Decrey,  D.  Yüksel,  H.  Schneider,  J.  Vogel,  D.  E.  Ingber,  V.   Vogel,  K.  Frei,  M.  E.  Schwab,  Nogo-­‐A  is  a  nega.ve  regulator  of  CNS  angiogenesis,  PNAS,  2013.  

Laboratory of Applied Mechanobiology

Jau-Ye Shiu and Viola Vogel

Bacterial  biosensor  
TER   >   >   osm  
Nutrients    +   Temp.   control   Nutrients  

Z(f)  

<   fluo   fluo   v   glu   pH   I   F  

Data   comm  

Signal   processing     control  

30.05.13  

Nano-­‐Tera.ch          Annual  Plenary  Mee3ng  

29  

Bacterial  biosensors   Bioreporters:
bacterial strains, which are specifically designed to produce a reporter protein in response to recognition of a target chemical or condition.
2  µm  

Bacterial biosensors
•  Sample collection

30.05.13  

nano-­‐tera.ch  annual  mee3ng  

31  

Bacterial biosensors
•  Test set-up in village

30.05.13  

nano-­‐tera.ch  annual  mee3ng  

32  

Bacterial biosensors
•  Freeze-dried bacteria in closed vials; water sample is added and mixed

30.05.13  

nano-­‐tera.ch  annual  mee3ng  

33  

Bacterial biosensors
•  Bioluminescence signal produced by the reporter bacteria is read out after 2 h in portable luminometer

30.05.13  

nano-­‐tera.ch  annual  mee3ng  

34  

Building  the  reporter  gene  circuit

Terminator Ribosome binding site operator

RULES Repression (derepressor)
Reporter gene

Promoter

PARTS (”BIOBRICKS”)

Regulator gene

Activation (inducer)

Making  an  arsenite  sensor  in  Escherichia  coli  
Diagram  of  the  gene  circuit  

Native (feedback)
AsIII

In  absence  of  AsIII:     System  is  “OFF”:  no   reporter  is  made.  

arsR

In  presence  of  AsIII:     System  is  “ON”:  reporter  is   made  and  cells  fluoresce.  

On-­‐chip  bacterial  biosensors  
Cells  embedded  in  agarose   beads,  d=40-­‐70  mm  

Images: Nina Buffi , Siham Beggah & Davide Merulla

Compact portable biosensor for arsenite detection  
•  Aqueous  samples  with   Escherichia  coli  bioreporter   cells.   •  Automa3c  measurement   •  No  image  analysis   •  Repeatability  by  changing  the   microfluidic  chip  

38

Bacterial  culture  on-­‐chip  

Bioreactor  

LMIS-4

Bacterial  culture  on-­‐chip  :  long  term  ac3vity  

LMIS-4

Freeze-­‐dry  condi3onning  
Freeze-­‐drying   Glucose  and  glycerol  improved  survival  of  E.   coli  DH5α  1598  ArsR  grown  from  LB  medium   amer  freeze-­‐drying  

Conditioning of freeze-dried reporter cells
LB+0.7%glucose+0.7%glycerol +1.7%YE LB+2%glucose+2%glycerol LB+1% glucose+1% glycerol LB+2%glucose LB+2% glycerol Control 0 20 40 60 80 100 120

E.  Coli  survival   rate  amer   freeze-­‐drying  

Condi.oning     Agarose  encapsula3on  proved    unprac3cal.  Alterna3ves   were  sought,  allowing  combined  rehydra3on,  sample   contac3ng  and  fluorescence  measurement:   Freeze-­‐drying  in  HPLC  vials   Septums   allows   injec3on   of   growth   medium   and   sample.   Fluorescence  measurement?  

Freeze-­‐drying   on   capillary   walls.   Porous   plug   to   retain   cells   amer   rehydra3on,   fol-­‐ lowed  by  sample  contac3ng.  

Freeze-­‐drying   in   cell-­‐coun3ng   mini   tubes.   Cells   centrifuged   into   boxom   capillary   for   fluorescence  measurement  

Bacterial  biosensor:  amperometric  detec3on  
TER   >   >   osm  
Nutrients    +   Temp.   control   Nutrients  

Z(f)  

<   fluo   v v     glu   pH   I   I   F  

Data   comm  

Signal   processing     control  

30.05.13  

Nano-­‐Tera.ch          Annual  Plenary  Mee3ng  

42  

Bacterial  biosensor:  amperometric  detec3on  

LEPA

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).

LEPA–UNIL

Bacterial  biosensor:  amperometric  detec3on  
Method Validation
Arsenic quantification in Swiss and Romanian ground water
As Species Concentration /ppb
Sample No.
1
2
3
4

*AAS

Bioreporter
4.8 ± 0.4
7.7 ± 1.6
18.7 ± 1.2
58.4 ± 1.7

Reported (AAS)*
3.3 ± 1.0
6.5 ± 0.8
17.8 ± 1.1
66.0 ± 6.6

= Atomic Absorption Spectroscopy

LEPA

Remote  sensing  
Start perfusion: Send SMS query
Make reference measurement Incubate

Get SMS with:
Reference point Measurement at end point

Make florescence measurement

Send SMS

30.05.13  

nano-­‐tera.ch  annual  mee3ng  

45  

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

30.05.13  

nano-­‐tera.ch  annual  mee3ng  

46