Cell -based autonomous biosensing microsystem LiveSense 03/02/12 Nano-Tera.ch  Annual Plenary Meeting
Scope of the project <ul><li>Demonstrate an autonomous cell-based biosensor microsystem for environmental remote monitorin...
The 8 teams in LiveSense project <ul><li>EPFL-LMIS Microsystems Laboratory Philippe Renaud Microfluidics, cell chips, bio-...
Why cell based sensors ? <ul><li>Cell-based biosensors provide a biologically relevant response to toxic compounds and mix...
Mammalian cells : <ul><li>Non-specific toxicity detection </li></ul><ul><li>Strict incubation conditions </li></ul><ul><li...
Genetically modified Bacterial cells: <ul><li>Specificity to chemical compounds </li></ul><ul><li>Easy to incubate </li></...
Bacterial biosensors example  (Jan van der Meer, UNIL) <ul><li>Sample collection </li></ul>03/02/12 nano-tera.ch annual me...
Bacterial biosensors example  (Jan van der Meer, UNIL) <ul><li>Test set-up in village </li></ul>03/02/12 nano-tera.ch annu...
Bacterial biosensors example  (Jan van der Meer, UNIL) <ul><li>Freeze-dried bacteria in closed vials; water sample is adde...
Bacterial biosensors example  (Jan van der Meer, UNIL) <ul><li>Bioluminescence signal produced by the reporter bacteria is...
Emerging contaminants  (Jan van der Meer, UNIL) 03/02/12 nano-tera.ch annual meeting Woutersen et al., 2011
Project highlights <ul><li>Integration of  bacterial biosensors  in microfluidic chips and measurements of  arsenic  with ...
Bacterial biosensors in microfluidic chips <ul><li>Encapsulate the bacteria in agarose beads </li></ul><ul><li>Trapping of...
Bacterial biosensors in microfluidic chips <ul><li>Frozen samples (-20°C) show very good response </li></ul><ul><li>Tested...
Electrochemical measurements with bacterial biosensors <ul><li>LacZ reporter gene for expression of beta-galactosidase </l...
Electrochemical measurements with bacterial biosensors <ul><li>Microfluidic device that allows the trapping of living cell...
Trans Epithelial Electrical Resistance (TEER) <ul><li>Cell model: CaCo-2, human colon carcinoma cell line </li></ul><ul><l...
Trans Epithelial Electrical Resistance (TEER) 03/02/12 nano-tera.ch annual meeting <ul><li>Use a commercially available bi...
Cell contractility toxicology assay 03/02/12 nano-tera.ch annual meeting <ul><li>Many environmental toxins interfere with ...
Cell contractility toxicology assay 03/02/12 nano-tera.ch annual meeting <ul><li>Measurement of pillar displacement by a c...
Liver cell bioreactor 03/02/12 nano-tera.ch annual meeting <ul><li>HepG2 hepatocytes trapped in microfluidic cage </li></u...
Assessment of acetaminophen toxicity 03/02/12 nano-tera.ch annual meeting Lab Chip, DOI: 10.1039/C1LC20212J (2011) <ul><li...
Glucose and lactate sensor 03/02/12 nano-tera.ch annual meeting <ul><li>Sensors for monitoring the cell metabolism. </li><...
Regulating osmolarity of the sample 03/02/12 nano-tera.ch annual meeting <ul><li>Adjust osmolarity of the sample flow thro...
System integration: 03/02/12 nano-tera.ch annual meeting <ul><li>Fluorescence detection with bacterial biosensors </li></u...
First system integration: 03/02/12 nano-tera.ch annual meeting Microfluidic cell incubator Fluorescence sensors µ processo...
First demonstration of the concept: Remote fluorescence detection of bacterial cells 03/02/12 nano-tera.ch annual meeting ...
Summary <ul><li>A set of  cell models , cultivable in microenvironments </li></ul><ul><li>Several  readout  schemes for mo...
03/02/12 nano-tera.ch annual meeting
Secondary sensors for bacterial sensors <ul><li>Fluorescence </li></ul><ul><ul><li>Expression of GPF induced by the report...
Secondary sensors for mammalian cells <ul><li>Trans Epithelial Electrical Resistance (TEER) </li></ul><ul><ul><li>For epit...
Secondary sensors for cell culture <ul><li>Glucose and lactate </li></ul><ul><ul><li>Monitoring of cell metabolism </li></...
Bacterial biosensors example  (Jan van der Meer, UNIL) <ul><li>Arsolux-bioreporter tests for arsenic: </li></ul><ul><ul><l...
Emerging contaminants  (Jan van der Meer, UNIL) <ul><li>Detection limits </li></ul>03/02/12 nano-tera.ch annual meeting Wo...
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LiveSense

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LiveSense

  1. 1. Cell -based autonomous biosensing microsystem LiveSense 03/02/12 Nano-Tera.ch Annual Plenary Meeting
  2. 2. Scope of the project <ul><li>Demonstrate an autonomous cell-based biosensor microsystem for environmental remote monitoring applications </li></ul><ul><li>Scientific objectives: </li></ul><ul><li>Study various cell models for toxicology assays in microbioreactor format </li></ul><ul><li>Develop new physical methods for measuring cell response in situ </li></ul><ul><li>Develop a micro-bioreactor with intergated sensors </li></ul>03/02/12 Nano-Tera.ch Annual Plenary Meeting
  3. 3. The 8 teams in LiveSense project <ul><li>EPFL-LMIS Microsystems Laboratory Philippe Renaud Microfluidics, cell chips, bio-impedance </li></ul><ul><li>UNIL-DMF Department of Fundmental Biology Jan van der Meer Cell biology, gene reporters, bacterial sensors </li></ul><ul><li>HESSO-ISI Industrial Systems Institute Martial Geiser </li></ul><ul><li>Microsystems, electronics, optical sensors </li></ul><ul><li>ETHZ-MAT Biologically Oriented Materials Viola Vogel Biomaterial, cell biology </li></ul><ul><li>CSEM Nanobiotechnology group Martha Liley Surface biochemistry, biomaterials </li></ul><ul><li>EPFL-LEPA Electrochemistry and Analytics Laboratory Hubert Girault Electrochemical sensing, analytical chemistry </li></ul><ul><li>EPFL-IMT Sensors and Actuators Laboratory Nico de Rooij </li></ul><ul><li>Microfluidics, microsensors Peter van der Val </li></ul><ul><li>UNIL-IST Institute of Occupational Health Michael Riedicker </li></ul><ul><li>Health effect of pollution </li></ul>03/02/12 nano-tera.ch annual meeting
  4. 4. Why cell based sensors ? <ul><li>Cell-based biosensors provide a biologically relevant response to toxic compounds and mixtures </li></ul><ul><li>Contrary to analytical chemistry methods, non specific but integrative detection </li></ul><ul><li>Can be extremely sensitive in some cases </li></ul><ul><li>Not only for environmental sensing, but enormous potential in toxicology screening of chemical and pharmacological compounds </li></ul>03/02/12 nano-tera.ch annual meeting
  5. 5. Mammalian cells : <ul><li>Non-specific toxicity detection </li></ul><ul><li>Strict incubation conditions </li></ul><ul><li>Already used for in-vitro toxicology screening in pharma research </li></ul><ul><li>Challenges </li></ul><ul><li>Find best detection methods </li></ul><ul><li>Microbioreactor: long term culture, proliferation </li></ul><ul><li>Sampling environment water while keeping good culture conditions </li></ul><ul><li>Question of the variability and reference measurement </li></ul><ul><li>… . </li></ul>03/02/12 nano-tera.ch annual meeting Epithelial cells on chip, CSEM Hepatocytes on-chip, EPFL_LMIS Fibroblats on nanopillars, ETHZ
  6. 6. Genetically modified Bacterial cells: <ul><li>Specificity to chemical compounds </li></ul><ul><li>Easy to incubate </li></ul><ul><li>Already proven in environmental measurements </li></ul><ul><li>No automated instrument yet </li></ul><ul><li>Challenges </li></ul><ul><li>Storage/conditionning, continuous measurement </li></ul><ul><li>Question of the reference or control measurement </li></ul><ul><li>Design or selection of new bacterial genotypes for new chemical compounds </li></ul><ul><li>… . </li></ul>03/02/12 Bacteria in beads, Unil
  7. 7. Bacterial biosensors example (Jan van der Meer, UNIL) <ul><li>Sample collection </li></ul>03/02/12 nano-tera.ch annual meeting
  8. 8. Bacterial biosensors example (Jan van der Meer, UNIL) <ul><li>Test set-up in village </li></ul>03/02/12 nano-tera.ch annual meeting
  9. 9. Bacterial biosensors example (Jan van der Meer, UNIL) <ul><li>Freeze-dried bacteria in closed vials; water sample is added and mixed </li></ul>03/02/12 nano-tera.ch annual meeting
  10. 10. Bacterial biosensors example (Jan van der Meer, UNIL) <ul><li>Bioluminescence signal produced by the reporter bacteria is read out after 2 h in portable luminometer </li></ul>03/02/12 nano-tera.ch annual meeting
  11. 11. Emerging contaminants (Jan van der Meer, UNIL) 03/02/12 nano-tera.ch annual meeting Woutersen et al., 2011
  12. 12. Project highlights <ul><li>Integration of bacterial biosensors in microfluidic chips and measurements of arsenic with electrochemical microsensors </li></ul><ul><li>Acetaminophen toxicology test on liver cells in microfluidic chips with electrical detection </li></ul><ul><li>TEER chip tested with CaCo-2 epithelial cells </li></ul><ul><li>Microfluidic sensor for online monitoring of cell metabolism and for osmolarity regulation </li></ul><ul><li>Toxicology screening ( ethanol ) based on fibroblast contractility </li></ul><ul><li>Demonstration of a first system integration with microfluidic, pumps, fluorescence and data com </li></ul>03/02/12 nano-tera.ch annual meeting For detailed information, go to the posters
  13. 13. Bacterial biosensors in microfluidic chips <ul><li>Encapsulate the bacteria in agarose beads </li></ul><ul><li>Trapping of the beads on chip for fluorescent and electrochamical detection </li></ul>03/02/12 nano-tera.ch annual meeting
  14. 14. Bacterial biosensors in microfluidic chips <ul><li>Frozen samples (-20°C) show very good response </li></ul><ul><li>Tested with fluorescence micro sensor at HES-SO and with electrochemical sensors developed by LEPA </li></ul>03/02/12 nano-tera.ch annual meeting 0, 10 and 50 µg As/L 60-80 min response time is OK
  15. 15. Electrochemical measurements with bacterial biosensors <ul><li>LacZ reporter gene for expression of beta-galactosidase </li></ul><ul><li>Can be detected by amperometry </li></ul>03/02/12 nano-tera.ch annual meeting LEPA 10 µM As tap
  16. 16. Electrochemical measurements with bacterial biosensors <ul><li>Microfluidic device that allows the trapping of living cells with magnetic beads </li></ul><ul><li>Continuous flow monitoring </li></ul>03/02/12 nano-tera.ch annual meeting LEPA 10 µM As tap
  17. 17. Trans Epithelial Electrical Resistance (TEER) <ul><li>Cell model: CaCo-2, human colon carcinoma cell line </li></ul><ul><li>21 days in culture, Ultra-thin silicon nitride membranes </li></ul>03/02/12 nano-tera.ch annual meeting
  18. 18. Trans Epithelial Electrical Resistance (TEER) 03/02/12 nano-tera.ch annual meeting <ul><li>Use a commercially available bioreactor system for the tests in the lab </li></ul>
  19. 19. Cell contractility toxicology assay 03/02/12 nano-tera.ch annual meeting <ul><li>Many environmental toxins interfere with cell homeostasis and thereby impact cell contractility. </li></ul><ul><li>Probing for changes in cell contractility using a nanopillar array </li></ul>
  20. 20. Cell contractility toxicology assay 03/02/12 nano-tera.ch annual meeting <ul><li>Measurement of pillar displacement by a camera </li></ul>
  21. 21. Liver cell bioreactor 03/02/12 nano-tera.ch annual meeting <ul><li>HepG2 hepatocytes trapped in microfluidic cage </li></ul><ul><li>Electrical impedance measurement </li></ul>LMIS-4
  22. 22. Assessment of acetaminophen toxicity 03/02/12 nano-tera.ch annual meeting Lab Chip, DOI: 10.1039/C1LC20212J (2011) <ul><li>Paracetamol is one of the most common causes of poisoning </li></ul><ul><li>Kinetic measurements in mM/L range </li></ul>LMIS-4
  23. 23. Glucose and lactate sensor 03/02/12 nano-tera.ch annual meeting <ul><li>Sensors for monitoring the cell metabolism. </li></ul>Glucose Measurement Lactate Measurement SAMLAB
  24. 24. Regulating osmolarity of the sample 03/02/12 nano-tera.ch annual meeting <ul><li>Adjust osmolarity of the sample flow through an osmotic membrane with a controlled solution. </li></ul>SAMLAB
  25. 25. System integration: 03/02/12 nano-tera.ch annual meeting <ul><li>Fluorescence detection with bacterial biosensors </li></ul><ul><li>Integration of pump for nutrient perfusion and sample collection </li></ul>50 µg As/L
  26. 26. First system integration: 03/02/12 nano-tera.ch annual meeting Microfluidic cell incubator Fluorescence sensors µ processor + GSM module Power supply Micropumps for perfusion
  27. 27. First demonstration of the concept: Remote fluorescence detection of bacterial cells 03/02/12 nano-tera.ch annual meeting Get SMS with: Reference point Measurement at end point Make florescence measurement Send SMS Send SMS query Start perfusion: Make reference measurement Incubate
  28. 28. Summary <ul><li>A set of cell models , cultivable in microenvironments </li></ul><ul><li>Several readout schemes for monitoring cell response </li></ul><ul><li>Start of system integration </li></ul><ul><li>New opportunities in toxicology screening </li></ul><ul><li>Next steps: </li></ul><ul><ul><li>Validation with toxicants </li></ul></ul><ul><ul><li>Microbioreactor integration </li></ul></ul><ul><ul><li>Environmental sampling </li></ul></ul>03/02/12 nano-tera.ch annual meeting
  29. 29. 03/02/12 nano-tera.ch annual meeting
  30. 30. Secondary sensors for bacterial sensors <ul><li>Fluorescence </li></ul><ul><ul><li>Expression of GPF induced by the reporter gene </li></ul></ul><ul><ul><li>Accumulation of signal with time </li></ul></ul><ul><ul><li>Can be done LED’s and photodiodes </li></ul></ul><ul><li>Electrochemical detection </li></ul><ul><ul><li>Expression of a compound that can react to a substrate to make an electroactive species </li></ul></ul><ul><ul><li>Measurement by amperometry </li></ul></ul><ul><ul><li>Well adapted to microfluidic formats </li></ul></ul>03/02/12 nano-tera.ch annual meeting
  31. 31. Secondary sensors for mammalian cells <ul><li>Trans Epithelial Electrical Resistance (TEER) </li></ul><ul><ul><li>For epithelial layers </li></ul></ul><ul><ul><li>Measures the permabilization of the confluent layer </li></ul></ul><ul><ul><li>Related to damage in junction between cells </li></ul></ul><ul><li>Micro electrode impedance </li></ul><ul><ul><li>For cell suspension or 3D cultures </li></ul></ul><ul><ul><li>Measures the change of cell shape, or membrane and cytosol properties </li></ul></ul><ul><ul><li>Related to overall physiological stress </li></ul></ul><ul><li>Cell contractility </li></ul><ul><ul><li>Many environmental toxins interfere with cell homeostasis and thereby impact cell contractility. </li></ul></ul>03/02/12 nano-tera.ch annual meeting
  32. 32. Secondary sensors for cell culture <ul><li>Glucose and lactate </li></ul><ul><ul><li>Monitoring of cell metabolism </li></ul></ul><ul><ul><li>Enzymatic amperometric sensors </li></ul></ul><ul><ul><li>Integrated in microfluidic format </li></ul></ul><ul><li>Conductivity </li></ul><ul><ul><li>For monitoring osmolarity of the medium </li></ul></ul><ul><ul><li>Same layout as amperometry sensors </li></ul></ul><ul><ul><li>Can be use in conjunction with osmolarity controller </li></ul></ul>03/02/12 nano-tera.ch annual meeting
  33. 33. Bacterial biosensors example (Jan van der Meer, UNIL) <ul><li>Arsolux-bioreporter tests for arsenic: </li></ul><ul><ul><li>Field campaign in Bangladesh performed by UFZ Environmental Research Institute, Leipzig, Germany, in November 2010 </li></ul></ul><ul><ul><li>Used 6000 freeze dried bacterial tests </li></ul></ul><ul><ul><li>Arsenic contamination in household tube wells; > 9 million installed </li></ul></ul><ul><ul><li>Detection limit of bacterial bioreporter system: 1-4 µg As/L </li></ul></ul>03/02/12 nano-tera.ch annual meeting
  34. 34. Emerging contaminants (Jan van der Meer, UNIL) <ul><li>Detection limits </li></ul>03/02/12 nano-tera.ch annual meeting Woutersen et al., 2011 Green = ‘sufficient’ from perspective of international standards * = only very few specific compounds can be targeted: e.g., BTEX, PAHs, phenols, few herbicides, alkanes Compound class Specific reporters ‘ toxicity’ reporters Heavy metals Low µg/L range mg/L range Organic compounds* µg/L - mg/L range mg/L range Mutagens Not detected µg/L range

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