livesense

Cell-based autonomous biosensing microsystem

LiveSense

12/04/23 1Nano-Tera.ch Annual Plenary Meeting

Scope of the projectScope of the project

• 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

12/04/23 2Nano-Tera.ch Annual Plenary Meeting

The 8 teams in LiveSense projectThe 8 teams in LiveSense project

• EPFL-LMISMicrosystems 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-MATBiologically Oriented Materials Viola VogelBiomaterial, cell biology

• CSEM Nanobiotechnology group Martha LileySurface biochemistry, biomaterials

• EPFL-LEPAElectrochemistry 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 Michael RiedickerHealth effect of pollution

12/04/23 nano-tera.ch annual meeting 3

Why cell based sensors ?Why cell based sensors ?

• Cell-based biosensors provide a biologically relevant response to toxic compounds and mixtures

• Contrary to analytical chemistry methods, non specific but integrative detection

• Can be extremely sensitive in some cases

• Not only for environmental sensing, but enormous potential in toxicology screening of chemical and pharmacological compounds


Mammalian cells :Mammalian cells :

• Non-specific toxicity detection• Strict incubation conditions• Already used for in-vitro toxicology screening

in pharma research

Challenges• Find best detection methods• Microbioreactor: long term culture,

proliferation• Sampling environment water while keeping

good culture conditions• Question of the variability and reference

measurement• ….

12/04/23 nano-tera.ch annual meeting5

Epithelial cells on chip, CSEM

Hepatocytes on-chip, EPFL_LMIS

Fibroblats on nanopillars, ETHZ

Genetically modified Bacterial cells:Genetically modified Bacterial cells:

• Specificity to chemical compounds• Easy to incubate• Already proven in environmental

measurements• No automated instrument yet

Challenges• Storage/conditionning, continuous

measurement• Question of the reference or control

measurement• Design or selection of new bacterial genotypes

for new chemical compounds• ….

12/04/23

Bacteria in beads, Unil

Bacterial biosensors example (Jan van der Meer, UNIL)Bacterial biosensors example (Jan van der Meer, UNIL)

• Sample collection



• Test set-up in village



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



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


Emerging contaminants (Jan van der Meer, UNIL)Emerging contaminants (Jan van der Meer, UNIL)


Woutersen et al., 2011

Project highlightsProject highlights

• Integration of bacterial biosensors in microfluidic chips and measurements of arsenic with electrochemical microsensors

• Acetaminophen toxicology test on liver cells in microfluidic chips with electrical detection

• TEER chip tested with CaCo-2 epithelial cells

• Microfluidic sensor for online monitoring of cell metabolism and for osmolarity regulation

• Toxicology screening (ethanol) based on fibroblast contractility

• Demonstration of a first system integration with microfluidic, pumps, fluorescence and data com


For detailed information, go to the posters

Bacterial biosensors in microfluidic chipsBacterial biosensors in microfluidic chips

• Encapsulate the bacteria in agarose beads• Trapping of the beads on chip for fluorescent and

electrochamical detection


Bacterial biosensors in microfluidic chipsBacterial biosensors in microfluidic chips

• Frozen samples (-20°C) show very good response• Tested with fluorescence micro sensor at HES-SO and with

electrochemical sensors developed by LEPA


0, 10 and 50 µg As/L

60-80 min response time is OK

Electrochemical measurements with bacterial biosensorsElectrochemical measurements with bacterial biosensors

• LacZ reporter gene for expression of beta-galactosidase• Can be detected by amperometry

12/04/23 nano-tera.ch annual meeting 15LEPA

10 µM As

tap

Electrochemical measurements with bacterial biosensorsElectrochemical measurements with bacterial biosensors

• Microfluidic device that allows the trapping of living cells with magnetic beads

• Continuous flow monitoring

12/04/23 nano-tera.ch annual meeting 16LEPA

10 µM As

tap

Trans Epithelial Electrical Resistance (TEER)Trans Epithelial Electrical Resistance (TEER)

• Cell model: CaCo-2, human colon carcinoma cell line• 21 days in culture, Ultra-thin silicon nitride membranes


Trans Epithelial Electrical Resistance (TEER)Trans Epithelial Electrical Resistance (TEER)


• Use a commercially available bioreactor system for the tests in the lab

Cell contractility toxicology assayCell contractility toxicology assay


• Many environmental toxins interfere with cell homeostasis and thereby impact cell contractility.

• Probing for changes in cell contractility using a nanopillar array

Cell contractility toxicology assayCell contractility toxicology assay


• Measurement of pillar displacement by a camera

Liver cell bioreactorLiver cell bioreactor

12/04/23 nano-tera.ch annual meeting 21LMIS-4

• HepG2 hepatocytes trapped in microfluidic cage• Electrical impedance measurement

Assessment of acetaminophen toxicityAssessment of acetaminophen toxicity


Lab Chip, DOI: 10.1039/C1LC20212J (2011)

LMIS-4

• Paracetamol is one of the most common causes of poisoning

• Kinetic measurements in mM/L range

Glucose and lactate sensorGlucose and lactate sensor


• Sensors for monitoring the cell metabolism.

SAMLAB

Glucose Measurement Lactate Measurement

Regulating osmolarity of the sampleRegulating osmolarity of the sample

12/04/23 nano-tera.ch annual meeting 24SAMLAB

• Adjust osmolarity of the sample flow through an osmotic membrane with a controlled solution.

System integration:System integration:


• Fluorescence detection with bacterial biosensors• Integration of pump for nutrient perfusion and sample

collection

50 µg As/L

First system integration:First system integration:


Microfluidic cell incubator

Fluorescence sensors

µ processor + GSM module

Power supply

Micropumps for perfusion

First demonstration of the concept:Remote fluorescence detection of bacterial cellsFirst demonstration of the concept:Remote fluorescence detection of bacterial cells


Get SMS with:Reference pointMeasurement at end

point

Make florescence measurement

Send SMSMake florescence measurement

Send SMS

Send SMS query

Start perfusion:Make reference measurementIncubate

SummarySummary


• A set of cell models, cultivable in microenvironments

• Several readout schemes for monitoring cell response

• Start of system integration• New opportunities in toxicology screening

• Next steps:– Validation with toxicants– Microbioreactor integration– Environmental sampling

Secondary sensors for bacterial sensorsSecondary sensors for bacterial sensors

• Fluorescence– Expression of GPF induced by the reporter gene– Accumulation of signal with time– Can be done LED’s and photodiodes

• Electrochemical detection– Expression of a compound that can react to a substrate to make an

electroactive species– Measurement by amperometry– Well adapted to microfluidic formats


Secondary sensors for mammalian cellsSecondary sensors for mammalian cells

• Trans Epithelial Electrical Resistance (TEER)– For epithelial layers– Measures the permabilization of the confluent layer– Related to damage in junction between cells

• Micro electrode impedance– For cell suspension or 3D cultures– Measures the change of cell shape, or membrane and cytosol properties– Related to overall physiological stress

• Cell contractility– Many environmental toxins interfere with cell homeostasis and thereby

impact cell contractility.


Secondary sensors for cell cultureSecondary sensors for cell culture

• Glucose and lactate– Monitoring of cell metabolism– Enzymatic amperometric sensors– Integrated in microfluidic format

• Conductivity– For monitoring osmolarity of the medium– Same layout as amperometry sensors– Can be use in conjunction with osmolarity controller


Bacterial biosensors example (Jan van der Meer,

UNIL)

• Arsolux-bioreporter tests for arsenic:

– Field campaign in Bangladesh performed by UFZ Environmental Research Institute, Leipzig, Germany, in November 2010

– Used 6000 freeze dried bacterial tests– Arsenic contamination in household tube wells; > 9 million

installed– Detection limit of bacterial bioreporter system: 1-4 µg As/L


Emerging contaminants (Jan van der Meer, UNIL)

• Detection limits


Woutersen et al., 2011

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

Green = ‘sufficient’ from perspective of international standards

* = only very few specific compounds can be targeted: e.g., BTEX, PAHs, phenols, few herbicides, alkanes

livesense

Technology

annual meeting lepa

annual meeting woutersen

cellbased biosensors

cell chips

meer cell biology

channual plenary meeting

modified bacterial cells

electrochemical sensors