An organic transistor-based system for reference-less electrophysiological monitoring of excitable cells

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SUBJECT AREAS:

BIOMEDICAL
ENGINEERING

ELECTRICAL AND ELECTRONIC

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Received
29 October 2014

Accepted
27 January 2015

Published
6 March 2015

Correspondence and

requests for materials

should be addressed to

[A.B. (annalisa@diee.](mailto:annalisa@diee.unica.it)

[unica.it) or S.M.](mailto:annalisa@diee.unica.it)

[(sergio.martinoia@](mailto:sergio.martinoia@unige.it)

[unige.it)](mailto:sergio.martinoia@unige.it)

## An organic transistor-based system for reference-less electrophysiological monitoring of excitable cells

A. Spanu 1,2, S. Lai 1, P. Cosseddu 1, M. Tedesco 2, S. Martinoia 2 & A. Bonfiglio 1,3

1University of Cagliari – Dept. of Electrical and Electronic Engineering, Piazza d’Armi, 09123 Cagliari, Italy, 2University of Genova

- Dept. of Informatics, Bioengineering, Robotics and System Engineering, Via Opera Pia 13, 16145 Genova, Italy, 3 CNR – Institute
of Nanoscience, Via Campi 213/A, 41125 Modena, Italy.

In the last four decades, substantial advances have been done in the understanding of the electrical behavior
of excitable cells. From the introduction in the early 70’s of the Ion Sensitive Field Effect Transistor (ISFET),
a lot of effort has been put in the development of more and more performing transistor-based devices to
reliably interface electrogenic cells such as, for example, cardiac myocytes and neurons. However, depending
on the type of application, the electronic devices used to this aim face several problems like the intrinsic
rigidity of the materials (associated with foreign body rejection reactions), lack of transparency and the
presence of a reference electrode. Here, an innovative system based on a novel kind of organic thin film
transistor (OTFT), called organic charge modulated FET (OCMFET), is proposed as a flexible, transparent,
reference-less transducer of the electrical activity of electrogenic cells. The exploitation of organic
electronics in interfacing the living matters will open up new perspectives in the electrophysiological field
allowing us to head toward a modern era of flexible, reference-less, and low cost probes with high-spatial and
high-temporal resolution for a new generation of in-vitro and in-vivo monitoring platforms.

he effective connection between cellular tissues and technological platforms is crucial for the success of many
bionic applications as neural prostheses and hybrids for studying information processing in neuronal networks. Moreover, the recent attention for disposable, low-cost and reliable cell-to-chip interface systems for
# T
high-throughput in-vitro toxicity assays and pharmacology is becoming an urgent request because of the new
international regulatory test guidelines (both in Europe and USA). Therefore, engineering the so-called bioelectronic interface is the subject of many technological and basic/applied researches. Specifically related to the
cell (neuronal)-electronic interface, two different kinds of devices have been extensively used over the past thirty
years, namely microelectrodes arrays (MEAs) 1,2 and field effect devices 3–6 . More recently, organic semiconductors
have attracted a considerable interest in this field 7–9 because they have the potential to fulfill many critical
requirements for biomedical and biotechnological applications such as biological compatibility, mechanical
flexibility, and optical transparency. Moreover, devices based on organic semiconductors can be fabricated on
flexible low-cost plastic substrates, with micrometric resolution, over large areas, and using cost-efficient technologies. All these features could allow addressing a wide variety of novel applications ranging from in-vitro to
in-vivo cell biology and addressing unsolved problems of mechanical adaptability (e.g., high density flexible
transducers on catheters) or of multi-parameters analysis on a micro-scale (e.g., disposable and sensorized
smart-petri dish). So far, in addition to passive electrodes made of organic conductive polymers 10, organic
electrochemical transistors (OECTs) have been mainly proposed 11,12 because of their ability to conduct ionic
and electronic charges and to be operated in liquid at very low voltages, which represents a crucial requirement in
presence of living cells or tissues. Organic Thin Film Transistors (OTFTs) have not yet been employed so far to
this aim for two main reasons: 1) they usually need to be operated at relatively high voltages (usually tens of volts);
2) charge carrier mobility in organic semiconductors is orders of magnitude smaller than what generally measured in their standard inorganic counterparts, putting a serious limit on the frequency range of the electrical
signals that might be applied as input for organic amplifiers. A very recent attempt has been done 13 but, as a matter
of fact, the organic transistor employed in the reported experiments is always operated in the off-state and
therefore, this cannot be described as an actual amplifying transducer for the cell activity.

In the following, we will present an optimized device, based on an organic thin film transistor that is able to: i)
be operated at ultra-low voltages (0.5–2 V); ii) amplify signals in the frequency range of cell electrical activity

(10–1000 Hz); iii) work without any external reference electrode.
This important feature is possible thanks to the presence of a second
gate that acts as a reference for the transistors, as further explained
later on in the text. In addition, as the sensing area is separated from
the active channel of the device, this new architecture allows avoiding
several other drawbacks related to the measurement of signals in
liquid environments by means of organic devices, such as, for
instance, the poor stability of organic semiconductors when exposed
to moisture and oxygen.

The basic device constituting the array, named Organic ChargeModulated Field Effect Transistor (OCMFET), is a floating gate
OTFT (Fig. 1a), whose working point is set by means of a control
capacitor (Fig. 1b). A portion of the floating gate, called sensing area,
on which cells are cultured, is exposed, as a direct interface, to the
surrounding bio-electro-chemical environment. In particular, ionic
charge variations occurring in the close proximity of the sensing area
determine a charge separation in the floating gate, leading in turn to a
modulation of the charge carrier density inside the channel of the
transistor (Fig. 1c).

In fact, the potential of the floating gate depends on the voltage
VCG applied to the control gate, on the voltage VDS, on the capacitances present in the structure, and on the charge Q0 entrapped in the
floating gate due to the fabrication process, as described in the following expression:

VFG~ CCF

CTOT

VCGz CDF

CTOT

VDz QSENSEzQ0

CTOT

ð1Þ

Considering all the terms as constants, apart from the charge QSENSE,
the field effect modulation can therefore be described in terms of a
threshold voltage shift of the device:

DVTH~DVFG~{ QSENSE

CTOT

ð2Þ

control capacitor CCF and the parasitic components due to the superposition between floating gate and the metal contacts lying on the
opposite side of the dielectric layer, CSF and CDF). The modulation of
the threshold voltage through the charge QSENSE is the mechanism
that can be employed for sensing all those chemical and biological
reactions that determine a variation of charge onto or in a close
proximity of the sensing area. In particular, a similar device has been
successfully employed for pH sensing 14 and as DNA hybridization
sensor, both in organic 15 and CMOS 16 technology.

In the specific application targeted in this work, i.e. monitoring of
the electrical activity of cells, the charge variation to be sensed by the
device is not a quasi-static charge variation but is related to the rapid
ionic displacement occurring across the cell membrane that faces the
sensing area during an action potential, as previously shown in
Fig. 1c. This small charge variation leads to a charge re-distribution
in the floating gate, thus modulating the density of charge carriers
inside the channel of the transistor. As a result, an output current
variation is obtained and can be straightforward processed by a
dedicated readout circuit. Being the working point of the device set
by means of the control gate, the sensor does not require an additional or external reference electrode, thus overcoming one of the
major drawbacks of MEAs, standard FET-based devices and standard OTFTs. This particular feature, together with the advantages
given by the use of organic materials, makes this device an excellent
candidate for advanced in-vivo bio-electronic interfaces.

A crucial requirement for successfully employing this device with
living cells (especially when targeting in-vivo interfaces) is that the
applied static voltages must be kept as small as possible to reduce
risks of undesired electrochemical processes and enhance sensitivity.
In order to fulfill this constraint and to optimize the cell-transistor
interface for detecting rapidly varying, low amplitude extracellular
electrophysiological signals, we redesigned a structure of ultralowvoltage OTFTs, similarly to what previously described in Ref. 17
(Fig. 2a). In particular, the gate dielectric was fabricated using a
combination of a few nanometers of a native metal oxide layer and
a 40 nm thick Parylene C film 18 . The combination of ultrathin films

being QSENSE the charge coupled to the sensing area, and CTOT 5 CCF
1 CSF 1 CDF the sum of all the capacitances of the structure (i.e. the

Figure 1 | Working principle of the OCMFET. (a), Cross section of an OCMFET device. (b), The working point is set by applying appropriate VGS and
VDS. (c), The charge fluctuation over the sensing area determines a charge re-distribution inside the floating gate, which modulates the charge
carriers density inside the channel of the transistor As a result a variation of the output current is obtained

Figure 2 | Structure and electrical characteristics of the device. (a), Schematic structure of the material layers composing the device. (b), Typical
characteristics of an ultra-low voltage OCMFET right after the fabrication (left) and after 10 days inside an incubator (right). (c), A complete device with 8
OCMFETs and 8 microelectrodes; in the center of the substrate, the sensing pads are surrounded by a glass ring that allows confining the culture medium
with the cells. (d), Top view of the complete experimental setup. The readout electronics in inserted onto a Multichannel Systems ground plate. (e), Gain
vs frequency relationship for an OCMFET device employed for the measurements with cells.

of metal oxide and Parylene C in a similar device structure already
gave very interesting results in terms of charge sensitivity, as demonstrated in the detection of DNA single strands 15 . Another important
requirement is the stability of the organic semiconductor layer. In
Fig. 2b both output and input characteristics are shown for an ultralow-voltage OCMFET right after its fabrication (left) and after 10
days inside an incubator at 37uC and 95% of humidity (right). As it
can be noticed, the current shows a certain degradation (as the active
device area has not been encapsulated), but the transistor behavior is
fully preserved. Eight identical devices, operated with a control gate
voltage of 21.0 V were fabricated on a plastic substrate (Fig. 2c),
having a global size of 50 3 50 mm to ensure compatibility with
standard Multi Channels Systems ground plates (www.
multichannelsystems.com). A glass ring delimited the sensing pads
of the 8 devices, thus allowing to preserve the transistors semiconductor layers from the contact with the culture medium.
Furthermore, a multichannel readout circuit was specifically
designed and realized in order to perform the conditioning of the
signals coming from the sensors. In Fig. 2d, an image of the
OCMFET readout circuit plugged into the Multi Channel Systems
ground plate is shown.

The potential of this device in cell activity monitoring is crucially
correlated to its capability of detecting very small amounts of charge
(less than 1 pC) in a frequency range up to 1 kHz. Therefore, in order
to optimize the frequency response of the OCMFETs, we have
employed a self-aligned structure 18, that allowed obtaining a dramatic reduction of the parasitic capacitances, due to the reduction
of the overlapping area between source and drain electrodes with the

underneath floating gate. The ability of this device to record relatively
high frequency signals is shown in Fig. 2e. The transistor is able to
amplify signals at frequencies up to 1 kHz, a frequency that is higher
than the typical frequency range of signals from electrogenic cells
(both cardiac and neuronal cells).

Since the device has been fabricated with materials that are intrinsically biocompatible, we tested and demonstrated the biocompatibility of the whole structure. As it can be observed in Fig. 3a and 3b,
cardiac cells covering the surface of the sensing electrode appear well
adherent and well differentiated. Furthermore, the healthy condition
of the culture was ultimately proved by the electrophysiological activity recordings made with passive metal microelectrodes (that were
fabricated inside the active area delimited by the glass ring) and by
Calcium imaging. A sustained and repetitive electrical activity at
about 1 Hz frequency was successfully recorded (Fig. 3c) with features comparable to what is routinely obtained with standard MEAs
and silicon FET devices 19,20 . In the Supplementary Information session, short movies are available, showing the intracellular transient of
Ca 21 waves and the correlated cardiomyocytes contractions (Movies
S1 and S2). Furthermore the biocompatibility was demonstrated by
coupling neuronal cultures to our devices (see Supplementary
Information fig. S1). Such networks remained in healthy conditions
for more than 3 weeks, demonstrating the suitability of our system to
long-term in-vitro applications.

In order to prove the ability of the device to record signals generated by living cells, the device was initially tested using primary
cultures of cardiomyocytes from rat embryos (embryonic day 18),
which develop in culture in a few days spontaneous and continuous

Figure 3 | Biocompatibility assessment. (a), Magnification of the sensing
area: a confluent culture of rat cardiomyocytes (8 DIV) is adhering on the
surface. (b), Cardiomyocytes culture fixed after a recording session and
immunostained for the sarcomeric protein Tropomyosin. (c), Few seconds
of spontaneous activity measured with a metallic microelectrode
fabricated near a floating gate pad and recorded with a MEA1060-Inv
Multichannel Systems amplifier.

mechano-electrical activity with a beat rate of 0.4 Hz–4 Hz. The
OCMFET device was able to detect the occurrence of the signals
(similarly to what MEA-based devices do), the beat rate and its rapid
changing related to pharmacological modulations, obtained by applying both positive and negative chronotropic compounds 21,22 . In
Fig. 4a and 4b examples of extracellular action potentials measured,
respectively, with an OCMFET and a metal microelectrode fabricated
on the same chip, are shown. Considering a p-type organic transistor
and a first inverting stage of the readout electronics, the shape of the
single signal shown in Fig. 4a is consistent with the expected current
fluctuation caused by the ionic charge displacement in the early phase
of the cardiac action potential. The IDS fluctuation is driven by the fast
movement of Na 1 ions occurring at the very beginning of the intracellular action potential (Fig. 4c). The fast entrance of Na 1 inside the
cell (phase 0) induce a temporary (0.5–2 ms) displacement of positive
ions rapidly entering in the portion of the cell membrane that faces
the sensing area. Due to the capacitive coupling between the cells and
the sensing area, a displacement charge is induced into the floating
gate area facing the cells; this small and transient amount of charge
induces a negative charge increase in the area underneath the transistor channel and this leads to an increase of the channel current (as
previously shown in Fig. 2c). The subsequent movement of K 1 ions
from the inside to the outside of the cell (phase 1) is responsible for
the temporary (0.5–2 ms) displacement of charge in the floating gate
in the opposite direction, thus causing a decrease of the channel
current. As it can be noticed by comparing Fig. 4a and 4b, the shape
of the extracellular measured signal is similar in the two cases demonstrating the detection capabilities of the reference-less OCMFET
device. Indeed, such inflow and outflow of ions do not influence the
concentration of sodium (and could slightly affect potassium) as the
displacement of ions during an action potential is negligible with
respect to the mM concentration in the extracellular medium.
However, these small charge displacements, thanks to the high capacitive coupling and high sealing resistance 23 determine the detectability of the signals with a mechanism somehow similar as the one
previously reported for silicon based Insulated FET 5

Figure 4 | Comparison between OFET signals, MEA extracellular APs
and intracellular ventricular APs. (a), Single signal measured with an
OCMFET. The fast ionic displacement during the early phase of the
intracellular cardiac AP elicits an increase followed by a decrease of the
current of the OTFT. The shape and the duration are coherent with the
sensing of the events occurring during the upstroke of the intracellular AP.
(b), Rat cardiomyocyte extracellular AP measured with a passive
microelectrode. It is worth noting the similarity of the capacitive part of the
MEA signal and the OFET signal. (c), Cardiac intracellular action
potential; in red the initial part corresponding to the rapid entry of Na 1

ions inside the cell. (c) (inset), A representation of the Na 1 current, the fast
current responsible for the action potential upstroke.

To extensively test our system, we then performed systematic measurements by recording the spontaneous electrophysiological activity
of rat cardiomyocytes (8 Days InVitro - DIV) by means of the
OCMFET device array (Fig. 5a). To further confirm the recording
capabilities of the developed OCMFET based system, we modulated
the cell activity by slowly varying the temperature of the culture’s
medium. We simply changed the control temperature of the thermostat, in contact with the cell plate, from 35uC to 40uC and back. As
expected, and shown in Fig. 5b, the culture’s beating frequency varied
consistently with the temperature variations. Finally, to ultimately
prove that the recorded signals were generated by the cardiomyocyte
cells, after several recordings made in physiological conditions (i.e. at
37uC in a standard culture medium and a controlled atmosphere), the
spontaneous activity of the cardiomyocytes’ culture was pharmacologically manipulated by administering 100 mM of Norepinephrine (a
specific cardio-stimulant that acts on b-adrenergic receptors 21 ), and
then a high-dose (100 mM) of Verapamil (a calcium blocker specific
to the L-type calcium channel that acts as a cardio-relaxant 22 ). As
expected the OCMFET recordings showed modulations of cellular

Figure 5 | Experimental results. (a), Spontaneous activity of a rat cardiomyocytes culture (8 DIV) mantained at 37uC measured with an OCMFET. (b),
Raster plots representing the thermal modulation of the spontaneous activity of the culture. The temperature was incremented from 35uC to 40uC (left
plot) and decreased from 40uC to 35uC (right plot) causing a consistent variation of the beating frequency. (c), Chemical tuning of the culture’s activity.
The spontaneous activity was accelerated by means of the addition of 100 mM of Norepinephrine and then suppressed with 100 mM of Verapamil. (c)
(inset), Beating frequency modulation (statistics on 5 OCMFETs - average and standard deviation): spike-count on 4 min of basal (129 6 4.6), NEmediated (280 6 28.6) and VER-mediated activity (15 6 1.9). (d), Signal propagation reconstruction in a device with multiple recording sites: colored
and black dots represents, respectively, OCMFETs that recorded signals and ‘‘silent’’ OCMFETs (left); raster plot of the spontaneous activity of the
‘‘colored’’ channels indicating a propagation of the signal from site 14 to site 41 (right). (e), Action potentials of striatal cells from rat embryo (21 DIV)
mantained at 37uC measured with an OCMFET. (e) (inset), Particular of a neuronal extracellular action potential.

activity as reported in Fig. 5c. All the measurements relative to the
pharmacological modulation of the electrical activity were performed
while maintaining the culture at constant temperature (37uC) and the
same reliable results were obtained in 5 recording devices simultaneously. With this array configuration, it was possible to estimate the
propagation speed of the electrical signal (around 0.4 m/s, in agreement with values measured by means of other techniques 24 ).
Figure 5d shows the raster plot of the propagation of the action
potential among the cell monolayer, as recorded by different
OCMFET devices fabricated onto the same substrate.

Measurements performed with several OCMFETs showed IDS variations ranging from hundreds of pA to few nA. Considering an
average IDS variation of 1 nA and representative values of the electronic parameters of the device (a trans-conductance gm of 300 pA/
mV and the sum of the capacitances CTOT of 100 pF, see
Supplementary Information, Table S1), we were able to estimate,
using the OCMFET’s equations, a corresponding charge variation
of,0.3 pC. By assuming that this variation is entirely due to the
ionic charge crossing the cell membrane during the upstroke of an
action potential and by considering a typical membrane capacitance
of 1 mF/cm 2, we may compute a value of about 300 mm 2 for the
effective area where this charge variation occurs. This value is consistent with the typical adhesion area of the cardiomyocyte soma,
thus indicating the validity of the transduction principle. To test the
suitability of our system for future applications with neuronal cells,
the device was also tested with rat striatal cells. Figure 5e shows a
preliminary recording of the electrical activity of a culture of rat
striatal neurons maintained in vitro for 21 days (21 DIV). A picture
of the neurons cultured on the same plastic platform together with
the comparison with a signal recorded by a standard MEA is also
available in the Supplementary Information Session. Interestingly,
calculation of the maximal amplitude of the signal-to-noise ratio 26

provided a value up to 3.2 (similarly to what is routinely obtained
with standard MEAs)

In summary, the reliable detection of action potentials (both in
physiological conditions and upon stimulation obtained by heating/
cooling steps and by pharmacological manipulations) from cardiac
cells, and the consistency of the beating frequency with that recorded
with standard microelectrodes fabricated onto the same substrates,
demonstrates the capability of the reference-less OTFT-based device
to efficiently transduce electrophysiological signals from electrogenic
cells. The suitability of the proposed system for future applications
with neurons was also preliminary successfully tested with rat striatal
neurons.

Moreover, a simple working mechanism based on the capacitive
coupling between the floating gate of the transistor and the cellular
membrane crossed by the ionic charge during the action potential,
justifies the observed signal shape and amplitude.

The obtained results support the use of this device for the detection
of the electrical activity of any kind of excitable and spontaneously
active cells and suggest new applications in the neuro-electronic
interface domain. The features of the presented platform could be
further exploited for realizing smart, disposable substrates for cell
cultures at low cost, thus opening an innovative perspective for functional cell monitoring both in-vitro and in-vivo.

Methods
Sensor fabrication. All devices were fabricated on a 175 mm thick polyethylene
terephthalate (PET) substrate (Goodfellow). At first, a metallic layer (Al or Ti,
Goodfellow) was thermally evaporated onto the substrate and patterned by means of
a standard photolithographic process. This patterned metallic film act as the floating
gates in the final sensor architecture. In particular, the terminal sensing pads of the
floating gates have a diameter ranging from 50 to 150 mm. The microelectrodes were
patterned during this step of the fabrication process. An UV-Ozone treatment was
subsequently performed in order to enhance the growth of the superficial native oxide
layer. After that, 30–50 nm of Parylene C (Specialty Coating Systems) were deposited
onto the entire substrate. A gold layer was then evaporated and patterned using a selfalignment process as described in Ref 18 to obtain the drain source and control gate

contacts. The obtained self-aligned low-voltage OTFTs have a W/L (channel Width/
channels Length) ratio of about 650. A thick layer of photoresist was finally deposited
and photo-lithographed in order to expose only a portion of the pads and protect the
rest of the surface. Afterwards, the Parylene C layer was etched from both the floating
gates pads and the microelectrodes by means of a plasma oxygen exposure 27 . After the
photoresist removal, 2 ml of a solution of 6,13-Bis(triisopropylsilylethynyl)pentacene
(TIPS Pentacene, Sigma Aldrich) in toluene (0.5 wt %) were drop casted directly over
the channel of the transistors. Finally, a glass ring (1.5 cm in diameter) was glued onto
the substrate with a thin rim of polydimethylsiloxane (PDMS) in order to delimit the
cell culture region. A careful cleaning of the chip surface with ethanol, acetone and
deionized water preceded every step of the process.

Experimental setup. The experimental setup consisted of a multichannel (16
channels) dedicated readout and conditioning electronics. Each channel consists of
three main blocks: a first inverting I/V converter with a 1 MV feedback resistor, a 2 nd

order high pass Butterworth filter with a cut-off frequency of 150 Hz, a 3 rd order low
pass Butterworth filter with a cut-off frequency of 1.3 kHz; all channels share an
adjustable biasing circuit for the OCMFETs polarization (VDS 5 VGS 5 21 V for all
the reported measurements). The total voltage gain of the circuit is 110. The realized
custom circuit is connected to a Multichannel Systems acquisition board for A/D
conversion, acquisition and storage (www.multichannelsystems.com). The
measurements with the passive microelectrodes were performed by simply
substituting the OCMFET readout electronics with a MEA1060-inv amplifier (www.
multichannelsystems.com) provided with the necessary external reference electrode.
All the measurement sessions were carried out inside a Faraday cage in order to
minimize the electrical environmental noise on the system.

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Acknowledgments
The research leading to these results has received funding from the Italian Ministry of
University and Research (MIUR) under the project PRIN2011-MIND. S.L. gratefully
acknowledges Sardinia Regional Government for the financial support of his PhD
scholarship (P.O.R. Sardegna F.S.E. Operational Programme of the Autonomous Region of
Sardinia, European Social Fund 2007–2013-Axis IV Human Resources, Objective l.3, Line
of Activity l.3.1.). The authors thank Giorgio Carlini for the constant support during the
design and the realization of the multichannel readout circuit; they also would like to thank
Dr. Enrico Defranchi for helpful discussions and his scientific support. The authors also
thank Prof. Fabrizio Barberis and Dr. Alberto Lagazzo for the support with plasma oxygen
treatments.

Author contributions

A.S. made the entire experimental work, from the design and fabrication of the devices to
the implementation of the measurement setup for the electrophysiological measurements.
He also contributed to write the paper. S.L. contributed to the design and discussions about
the working models of the device. He also contributed to write the paper. P.C. contributed to
the fabrication procedure and to the discussion of the results. He also contributed to write
the paper. M.T. took entirely care of the preparation of cell cultures and contributed to the
experiments with living cells. She also contributed to write the paper. S.M. coordinated the
work, focusing in particular on the design of the measurement platform and on the
electrophysiological experiments. He also contributed to write the paper. A.B. coordinated
the work, focusing in particular on the design and fabrication of the device and working
platform. She coordinated the writing of the paper.

Additional information
[Supplementary information accompanies this paper at http://www.nature.com/](http://www.nature.com/scientificreports)
[scientificreports](http://www.nature.com/scientificreports)

Competing financial interests: The authors declare no competing financial interests.

How to cite this article: Spanu, A. et al. An organic transistor-based system for
reference-less electrophysiological monitoring of excitable cells. Sci. Rep. 5, 8807;
DOI:10.1038/srep08807 (2015).

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