Organic electronics at the interface with biology - FUNMAT

Institut Mines-Télécom

Organic electronics at the interface with biology; a biologist’s perspective

Presentation at FunMat

Turku, Finland; 12th Feb. 2015

Róisín M. Owens ([email protected])


“The biologist and the physicist should be friends”

2 Department of Bioelectronics

https://www.youtube.com/watch?v=Vg5cwSBnyQU


Outline

Part I: • Why do we want to interface with biology?

• Why use organic electronic materials for interfacing with biological systems?

• What are the levels of complexity of biological systems? • Examples of biorecognition elements showcasing interface

with organic electronics

Part II: • Organic bioelectronics for tissue monitoring

Future perspectives


Why do we want to interface with biology?


Reasons for interfacing with biology

Fundamentals Diagnostics Treatment

Health Biotechnology The Environment


Why use organic electronic

materials for interfacing with

biological systems?


Traditional applications for organic electronics

Thin film transistors Photovoltaics

DuPont

Someya Lab

Light emitting diodes

Samsung

Astron FIAMM

Heliatek

Department of Bioelectronics – www.bel.emse.fr 7


Organic electronics offer unique opportunities

• Mechanical Properties: Similar to tissue, improved implant stability

• Ideal surfaces/interfaces: high sensitivity, low noise

•Ionic conductivity: electrical interfacing with biological systems

•Processing: low cost fabrication, disposable devices

• Tunability of electronic properties: tailor for specific applications

Organic electronic materials provide a new toolbox for interfacing with biology

Living

Systems +

Biomaterials

Electronic

Elements

electronic input (control)

biological input (monitoring)

cells enzymes DNA

electrodes transistors STM

An enzymatic reaction changes current in a transistor

Application of voltage bias triggers cell growth

10μm


What are the levels of complexity of biological systems?

On what level can we integrate organic

electronics to biological systems?


1. Whole organism

2. Tissue

3. Cells

4. Macromolecular structures

5. Single molecules

Levels of organisation in animal physiology


Tissues as building blocks of organ systems 10 major organ systems

Integration at the level of organs


Improving brain: electrode interface with CPs

Kim et al, Frontiers in Neuroengineering (2007)

Skin probe Surface probe

Depth

probe

Better mechanical matching


Transistor (on surface)

Surface electrode

Depth electrode

ID

VD

Vg

Source Drain

OECT: more sensitive, less invasive

A B

C D

Khodagholy et al, Nature comm; 4, 1575 (2013)

Skin probe Surface probe

Depth

probe

Improving brain: electrode interface with CPs Improved signal transduction


Delivery of neurotransmitters with organic ions pumps

Simon et al, Nat Materials ; 8 (9) (2009)

Spatial control of delivery


Epithelial/Barrier Tissue

Connective Tissue

Muscle Tissue

Nervous Tissue

Extracellular Matrix (ECM): mixture of proteins and sugars underlying all tissue types

Four types of tissue in animals

Integration at the level of tissue


Interfacing with epithelial tissue: OECTs

Caco-2 cells: in vitro model for the GI Tract

Introduction of toxic

compound

Jimison et al, Adv Mater (2012)

Improved signal transduction


Organic electronic materials designed for function

Persson et al, Adv Mater (2011)

OE material provides advantage for tissue engineering

Tunable chemistry


Cellular Diversity

Over 200 different types of animal cells

Integration at the level of cells


Controlling cell migration/proliferation with OE

Lee et al, Biomaterials (2009)

Neurons Muscle cells

Breukers et al, J. Bio. Mat Res Part A (2010)

OE materials combine topographical and electrical stimuli

Tunable chemistry; mixed ionic/electronic signals


Proteins are one of the building blocks of cells and tissues

Polysaccharides

Membranes

DNA/RNA

Proteins

Molecular Biology of the Cell (© Garland Science 2008)

Integration at the level of macromolecules


Electrical control of protein conformation with OE devices

Wan et al, Adv Mat (2011)

PEDOT+: PSS− +M+ + 𝑒− PEDOT0 + PSS−: M+

Salto et al, Langmuir (2008)

Tunable chemistry; mixed ionic/electronic signals


Outline

Part I: • Why do we want to interface with biology?

• Why use organic electronic materials for interfacing with biological systems?

• What are the levels of complexity of biological systems? • Examples of biorecognition elements showcasing interface

with organic electronics

Part II: • Organic bioelectronics for tissue monitoring

Future perspectives


Challenge for my team: Improving diagnostics

Our goal is to develop relevant biological models in vitro with adapted integrated monitoring

We take advantage of advances in device engineering

We develop both devices and models in parallel

We aim to improve the predictive quality of the in vitro model for drug discovery/toxicology



Requirements for in vitro toxicology

Traditionally

New methods

Cell death

Add toxic compound

Mostly optical Low throughput

Time consuming/labor intensive Endpoint assays – not predictive

Morphology IF Western

LDH, apoptosis

Optical: SPR (e.g. biacore), DMR (Epic)

Electrical: Electronic impedance spectroscopy

(e.g. ECIS, CellZscope, xCELLigence)

DNA RNA proteins

Array approach

Imaging Usually dynamic

Medium-high-throughput

But

Ambiguous correlation with cell events Not compatible with imaging (hi-res)

High cost LOW



Epithelial/Barrier Tissue

Connective Tissue

Muscle Tissue

Nervous Tissue

Extracellular Matrix (ECM): mixture of proteins and sugars underlying all tissue types

Four types of tissue in animals

Integration at the level of tissue



Biological target: epithelial/barrier tissue

Barrier Tissue Functions

1. Protection

2. Compartmentalisation

3. Transport & Selective absorption

Skin

Blood Brain Barrier (BBB)

Gastrointestinal tract

Monitoring of barrier tissue function & integrity is an

excellent parameter for predictive toxicology

Kidney tubules



Non-electrogenic mammalian cells

Epithelial cells

Epithelial monolayer

Tissue Species Rpara

(Ω.cm2)

Proximal tubule dog 6–7

Gallbladder rabbit 21

Duodenum rat 98

Jejunum rat 51

Ileum rabbit 100

Distal colon rabbit 385

Urinary bladder rabbit 300000

Electrical resistance of epithelial tissue

varies over several orders of magnitude from the bile duct to the small intestine:



Tight junctions regulate ion conduction in epithelia

Epithelial monolayer



The transducer: organic electrochemical transistor


Strakosas, Bongo et al. Apl Pol Sci (2015)

PEDOT:PSS Electronic and Ionic circuits


Tissue engineering with integrated multi-parameter monitoring (using organic electronic transducers)

Tissue interactions

• Monoculture

• Contact co-

culture

• Non-contact

• Multilayer

system

• 3D culture

Mol. Environment

• Media

composition

• Protein coating

• ECM proteins

Device engineering

• Geometry

• Planar/top-gate

• Topography

• Porous/non-

porous

Exterior conditions

• Temperature

• Humidity

• Gases (O2, CO2)

• Perfusion (flow)

Key challenge:

Improve predictive capability of in vitro model



Integration of OECT with in vitro model of GI tract

Depiction of pathogen infection of epithelium of GI tract from Guttman et al. BBA, 2009

OECT



0 1 2 3 4

5

Epithelium modifies OECT transient response

No cells

Healthy epithelium VG 0.3 V

VD -0.1 V

Jimison, L.H., et al. Adv Mat 24 (44) 2012



Multiplexed OECT for dynamic measurement

Caco-2: model for human

intestinal epithelium

Day 5 Day 21

ZO-1

Nucleus

3-week-old Caco-2 cells

Multiplex OECT integrated with human GI cells on filters

Tria, S. et al Advanced Healthcare Materials, 10.1002/adhm.201300632



OECT integrated with in vitro GI model for toxicology


MOI:1000

MOI:100

MOI:10

MOI:0

WT S. typhimurium Invasion mutant



Disruption of barrier by pathogens in complex matrices

CellZscope OECT

Full-fat, fresh milk




Integration of OECT with in vitro model of kidney tubule

OECT Depiction of kidney tubule



Nephrotox

ELECTRONICS MICROFLUIDICS CELLS



Currently: multi-parameter readout for nephrotoxicity


In situ fluorescence imaging

Fluidic chambers for co-

culture of cells

Media & drug delivery

Cell culture membrane

Live/dead cell viability assay

Efflux sampling for biomarker detection

OECT monitoring of TEER and glucose/lactate

Kim1 western blot

Results from single well; electronics (left) and

brightfield images (right)

Mate-1 and

ZO-1

imaged with

in situ IF


Tissue engineering with integrated multi-parameter monitoring (using organic electronic transducers)

Tissue interactions

• Monoculture

• Contact co-

culture

• Non-contact

• Multilayer

system

• 3D culture

Mol. Environment

• Media

composition

• Protein coating

• ECM proteins

Device engineering

• Geometry

• Planar/top-gate

• Topography

• Porous/non-

porous

Exterior conditions

• Temperature

• Humidity

• Gases (O2, CO2)

• Perfusion (flow)

Key challenge:

Improve predictive capability of in vitro model



Interfacing organic electronic devices with live cells: advantages of planar device

Ionic to electronic conversion: efficient transduction

• Mixed conductivity: signal

transduction

•Processing: disposable devices

• Mechanical Properties:

improved stability, flexible

• Chemistry: application specific

• Ideal interfaces: no oxides,

sensitive

• Optical transparency: high

resolution images

Rivnay, J. et al. Chem. Mater., 26 (1), 2014



Simplification of fabrication process Compatibility with optical

characterization of the cells Potential for correlation of optical and

electrical information

Cells grown directly on the OECT

Planar OECT: combined optical and electronic

𝜏



In situ monitoring of barrier formation of MDCK cells

Optical monitoring Electronic monitoring

Ramuz.,M et al. Adv Mat, 2014



Smaller, faster transistors: Frequency measurements

𝑔𝑚 =𝜕𝐼𝐷𝑆𝜕𝑉𝐺𝑆

coverage Barrier

properties

Department of Bioelectronics 43

Frequency measurement allows differentiation between coverage and barrier functionalities

VGS = 0.01 sin (wt)

Transconductance:


Ion current at low and high frequencies (Z)

LOW

HIGH

Benson et al, Fluids and barriers of the CNS, 2013



Figure 19-3 Molecular Biology of the Cell (© Garland Science 2008)

Cell junctions

The biological target: paracellular ion flow


Frequency response for different cell lines HeLa

Time no cells D2 D4 D6

Freq cut-off

@ -3dB 1560 271 270 460

-3dB

Minimum cut-off

Time no cells D2 D4 D6 D9

Freq cut-off

@ -3dB 1660 230 222 170 300

Minimum cut-off

HEK 293

-3dB



Frequency response for various cell lines


Freq cut-off

@ -3dB 1720 14 14 8 1280

Minimum cut-off


Freq cut-off

@ -3dB 1660 31 23 23 47

Minimum cut-off

Caco-2

MDCK I

-3dB

-3dB



Record baseline for 10 min Add 100mM EGTA at t = 10 min, leave 24 min. Remove EGTA and rinse with calcium containing medium

Calcium switch assay shows full recovery of cells

EGTA specifically disrupts barrier properties resulting in increase in cut off value of transconductance plots

Removal of EGTA results in almost complete recovery



Calcium switch assay demonstrates functionality of cells

Calcium switch assay shows that with this cell type low cut off values indicate barrier function

Remove EGTA Add EGTA



-3dB

PEDOT:PSS channel

Salmonella, green labelled

Salmonella infection of MDCK-I cells

50 µm x 50 µm OECT MDCK-I at day 5

GFP labelled Salmonella typhimurium

Within 2 hours frequency response evolves from barrier properties to cell coverage



Add salmonella

GFP labeled Salmonella infection of MDCK-I cells

Dynamic and simultaneous recording of optical and electrical information After adding salmonella, increase of barrier properties due to sedimentation



Generation of complex impedance traces using OECT


MDCKI

no cells

MDCKI

no cells

a

d

b

c

Figure 1. Harmonic Impedance-based sensing with OECTs. a. Wiring diagram showing operation of OECT, indicating sinusoidal gate input and gate, drain current measurement. The applied VD = -0.6V. b. Example of measured gate input sinusoids at 5 frequencies, applied ?VG=0.1V. Resulting ID and IG are shown in green and blue, respectively. Amplitudes and phase are determined from sinusoidal fits (red). c. Frequency dependence of gm = ? ID/ ?VG, or, the small signal transconductance of an OECT with and without MDCKI cells cultured on the channel of the transistor. d. Frequency dependence of the impedance, |Z|= ?VG/ ? IG for the same samples as (c). The phase shifts with respect to the applied gate modulation is shown at bottom of c,d. Error bars represent the propagation of errors from confidence in the fitted amplitude and phase values from a least squares fit of the sine waves.

Frequency (Hz)

100 101 102 103 104 100 101 102 103 104

Frequency (Hz)

100 101 102 103 104

Frequency (Hz)

MDCK I

MDCK I

OECT

Rcell = 825 Ω cm2 and Ccell = 544 nF/cm2

CellZscope

Rcell = 730 +/-101 Ω cm2 and Ccell = 1µF/cm2*

*literature

Drain current (OECT) provides low-error at low frequency Gate current (traditional impedance) low error at high frequency.


Cellular engineering with integrated monitoring (using organic electronic transducers)

Tissue interactions • Monoculture • Contact co-culture • Non-contact • Multilayer system • 3D culture

Mol. Environment • Media

composition • Protein coating • ECM proteins

Device engineering • Geometry • Planar/top-gate • Topography • Porous/non-

porous

Exterior conditions • Temperature • Humidity • Gases (O2, CO2) • Perfusion (flow)



Cellular engineering with integrated monitoring: improving the biotic/abiotic interface

For metabolite detection (using redox enzymes)

Mimic in vivo environment (improve tissue functionality)



Solution processable CPs: Lithographically fabricated devices

Substrate

Gold

Parylene C

Anti-adhesive

Photoresist

Conducting polymer

Metal lift-off

2× parylene deposition

Lithography and etching

PEDOT:PSS coating

Peel-off

Simple process to define BOTH the conducting and insulating material simultaneously

No wet/ion etching of the polymer conductor

Versatile method: compatible with different polymer materials

Functionalization step



Functionalization of PEDOT:PSS via silanization

X. Strakosas et al., J. Mater. Chem. B (2013).



PEDOT:PSS:PVA OECTs maintain performance

Transconductance:

Time constant τ = 20.6 μs Scale bar: 10 μm

𝑔𝑚 =∆𝐼𝑑

∆𝑉𝑔 = 1.86 mS




Functionalisation of surfaces with PLL




PEDOT:PSS functionalized with GOx for glucose sensing


reactions at the channel reactions at the gate


Change of potential at the gate results in a change in the dedoping of the channel


Optimisation of GOx functionalisation for in vitro


Tuning functionalisation

Goal:

Monitor metabolic activity of cells in

real time (glucose/lactate)

Challenges:

High sensitivity

Stability

Selectivity

Requirements:

Monolithic fabrication

No external mediator


Gate functionalization via a cross linkable hydrogel

Pt-NPs sensitivity Silane stability PEG:PSS selectivity

UV



Successful biofunctionalisation with enzymes

Glucose detection in complex fresh media Lactate detection in complex media

incubated with live cells



In vitro lactate sensing on planar devices



3D spheroids/cysts integrated with OECTs


ZO-1 Actin Nucleus

Huerta et al, APL Materials, in preparation


OECT measurement of cyst resistance



Detection of toxic effects on spheroids with OECT



Future Work

Increase complexity of in vitro models

Increase repertoire of models – towards body on a chip

Monitoring from tissue slices

Use of PEDOT:PSS scaffolds to both host and monitor cells

Modelling of planar OECT data to extract TEER and capacitance

Increase sensitivity of device for monitoring cysts

Continue with metabolite sensing, increase repertoire

Integration of OECT with lipid bilayers +/- ion channels



Identifying new areas for interfacing with biology Devices to stimulate/record electrically active cells

• not just neurons, cardiac cells, muscle cells

• not just CNS, also PNS, neuromuscular junction

Processes involving ion transfer/flow

• Ion channels, signal transduction, oxidative phosphorylation

• Electron transfer chain, redox reactions

• Bio fuel cells

Plants/bacteria/viruses– biotechnology

Materials development Biodegradable/bioerodible/biofunctional – but still highly conducting

Materials designed for specific applications

Conformable, disposable substrates

The future of organic bioelectronics

A!

E! "

E#"

B

C D!


Bibliography

Biochemistry by Jeremy M. Berg ISBN 10 1429276355

Molecular Biology of the Cell 5E by Bruce Alberts ISBN10 0815341067

Principles of Anatomy and Physiology by Gerard J. Tortura ISBN 10 0470233478

Some Light reading:


Acknowledgements

EMSE: Dept of Bioelectronics Ilke Uguz, Nathan Schäfer, Adel Hama, Timothée Roberts, Adam Williamson, Marc Ferro, Jacob Friedlein, Sébastien Sanaur, Seiichi Takamatsu, Pierre Leleux, George Malliaras, Marcel Brandlein, Mary Donahue, Susan Daniel, Mohammed ElMahmoudy, Dimitrios Koutsouras, Xenofon Strakosas, Thomas Lonjaret, Eloise Bihar, Margaret Brennan, Michel Fiocchi, Esma Ismailova, Sahika Inal, Julie Oziat, Yi Zhang, Anna Maria Pappa, Liza Klots, Jonathan Rivnay, Patrick Fournet, Gaëtan Scheiblin

Alumni Leslie Jimison, Scherrine Tria, Manuelle Bongo, Dion Khodagholy, Moshe Gurfinkel, Kaleigh Margita, Marc Ramuz, Yingxin Deng



Tight junction formation in sub-confluent monolayers

Day 1

Day 3

Day 5

ZO-1 E-cadherin Occludin Claudin-1 Brightfield

20 µm

Ramuz.,M et al. Adv Mat, 2014



Equivalent circuits of filter vs planar formats


Rmed

Rjunc

Rsub

Rcell Cmem

ROECT COECT

Rmed

Rjunc Rcell Cmem

ROECT COECT

Rmed


OECT


Integration of OECT with in vitro model of BBB

Cartoon of interactions at blood brain barrier (Abbott)


OECT for monitoring BBB toxicology

hCMEC/D3 + EGTA + trypsin No cells



1 10 100 1000 10000

-50

-40

-30

-20

-10

0

No

rma

lize

d tra

nsco

nd

ucta

nce

(d

B)

Frequency (Hz)

h CMEC/D3

No cell

Comparison with commercial measurement


Organic electronics at the interface with biology - FUNMAT

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