A hybrid living/organic electrochemical transistor based on the Physarum polycephalum cell endowed with both sensing and memristive properties

A hybrid bio-organic electrochemical transistor based on the Physarum polycephalum cell, showing a multifunctional operation (transistor and memristive-like response), has been demonstrated.

One of the main interests in bioelectronics, besides the relevant expected impacts in bio-medicine and prosthetics, is driven by the aim to emulate abilities that, essentially present in living beings, can scarcely be reproduced with artificial man-made devices. Even the simplest living organisms, for example, learn from and adapt themselves to stimuli from the surrounding environment. A challenging perspective is the integration of these adaptive/learning behaviours into artificial systems, possibly interfacing them with existing devices and technologies. Even though the great efforts of the scientific community in this direction have brought enormous progress in interfacing living beings with electronic devices, currently artificial models can barely mimic the basic properties of the simplest living organism in an oversimplified way.1 Remarkable state of the art work also aims at demonstrating the feasibility of biodevices and bio-inspired systems.2,3

In this framework, quite a relevant evolution has been determined by the concept of a memristor, introduced theoretically4 and hence realized experimentally.5 The basic concept underlying a memristor involves a device inherently endowed with memory, the resistance of which can switch from an insulating to a conductive state, depending on the sequence of electrical signals experienced. Hence, a memristor is particularly well suited for mimicking the learning behaviour of biosystems, opening novel perspectives in information processing.6

In bioelectronics, a strongly evolving novel strategy is based on organic electronics and in particular on organic electrochemical transistors (OECTs).7–9 OECTs are very promising as biocompatible sensing devices,10–12 as electrodes interfacing neurons13 and nervous systems, as well as active elements for bioelectronics,14 and for the recently proposed iontronics (ion-based signal handling and processing including bio-actuation).15,16 Organic bioelectronic devices, based on organic semiconductors, are increasingly attracting the scientific community since they operate with electrolytes in the liquid phase and at low bias voltages (<1 V), and are fully biocompatible.7,8

An OECT consists essentially of a semiconducting polymer channel in contact with an electrolyte, properly confined by a PDMS-well. The gate electrode is immersed into the electrolyte and the overlapping area between the organic polymer and the electrolyte defines the channel of the OECT, where the ionic interchanges can take place.8 At present, the most popular conducting polymer is poly (3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate), PEDOT:PSS. The operating mechanism of the OECT is based on the reversible doping/de-doping of the channel: upon application of a drain–source voltage V ds, holes drift within the transistor channel, generating a drain–source current I ds (the on state); when a positive voltage V gs is applied, cations M+ from the electrolyte penetrate into the PEDOT:PSS channel and de-dope it according to a redox reaction (the off state).17,18 Even though the electrolyte is often a simple physiological solution, such as NaCl or phosphate buffered saline (PBS),19 OECTs based on PEDOT:PSS work efficiently even with more complex solutions, such as cell culture media,19 solid-gels20 and micellar electrolytes,21 hence becoming a suitable playground for addressing very relevant questions concerning cell functioning,11,12 signalling and stress, drug delivery systems and processes,10 neuronal and brain functions and working principles (including synaptic and post-synaptic processes).13,22

We report here on a novel hybrid bioelectronic organic electrochemical device based on a living being – the Physarum polycephalum cell (PPC), a multinuclear single-cell mass of protoplasm belonging to the family of myxomycetes, in the past defined as fungi, nowadays simply slime moulds. PPC lives in humid and dark environments. The studied form, in particular, is the plasmodium, PPC vegetative form; it looks like an amorphous yellow mass with networks of protoplasmic tubes branching towards nutrients. Its foraging behaviour can be seen as a computation: data are represented by spatial configurations of attractants and repellents, and results by the structure of protoplasmic networks.23 Therefore, PPC is widely studied for unconventional computing24 applications as it has demonstrated the capability of resolving optimization problems.

We demonstrate that this hybrid “living” device operates reproducibly both as a transistor and as a memristive device, and its peculiar features pave the way to novel strategies based on the integration of organic bioelectronics with memristive approaches. The choice of PPC was based on its unique, recognized properties of “intelligence”, “creativity” and “capacity of learning” that are being increasingly investigated.25 For example, during its life cycle, and especially when it seeks food, PPC is able to remember already trodden paths, in order not to retrace them. For this reason, PPC has recently been exploited as the main material in non-conventional computing, robot-Physarum and PPC-based network circuits.26–28 In addition, it is rather easy to keep the PPC alive: it requires room temperature, dark conditions and humidity. The PPC colony needs to be fed with oat flakes and periodically replanted to fresh substrates. Therefore, given a little care and constant attention it is possible to grow PPC in its yellow plasmodial stage.

The OECT channel was made of PEDOT:PSS, a p-type semiconductive polymer widely used in bioelectronics because of its demonstrated properties of stability and biocompatibility.29,30 Before patterning, the solution was doped with diethylene glycol 20% (Sigma) with 0.05% by vol. of dodecyl benzene sulfonic acid (DBSA) surfactant (Sigma Aldrich), in order to enhance its electrical conductivity and film-forming properties, respectively.31,32 The OECT channel, with a final width of 2 mm, was patterned on a square glass slide of 2 × 2 cm and the PEDOT:PSS was spun onto the substrate using a first ramp of 6 s (at 450 rpm) followed by a 30 s plateau at 1500 rpm. The final film thickness was d ∼ 100 nm, as measured using a profilometer. Devices were finally baked on a hotplate at 120 °C for 120 min. A PDMS well of about 500 μL in volume was used to confine the mould onto the channel, defined by the overlapping area of the mould with the PEDOT:PSS stripe.

Plasmodium of Physarum polycephalum was cultivated in a glass box, kept in a dark, humidifying atmosphere with a water bath, on wet towels and fed with oat flakes. Cultures were periodically replanted to a fresh substrate. Fresh PPC was placed into the PDMS well for each electrical measurement. Therefore, after setting up the OECT, a 150 μl blob of fresh PPC was picked from the growing box and manually inserted into the PDMS well. The operation was carried out with the help of a small spatula taking care that the mould made contact with the underlying PEDOT:PSS channel. Subsequently, the gate electrode was inserted into the PPC's body without touching the polymeric channel. All the measurements were performed under dark conditions in order to preserve the PPC. More details on the culture growth can be found in literature.33

Electrical measurements were carried out using a 2 channel source/measure precision unit (Agilent B2902A), controlled by home-made LabView software. Before the experiments, the OECT channel was immersed in DI water for 1 hour in order to properly hydrate the PEDOT:PSS layer, while the gate electrodes were cleaned to remove any residue. Two types of measurements were carried out: in the first set, a 3-electrode device was used under a transistor-mode configuration (see Fig. 1A), recording the output and transfer characteristics. In the second set of measurements, the source electrode was interdicted, and a 2-terminal device was exploited to carry out I–V cyclic measurements, with the polymeric film used as the reference electrode and the metal wire as the working electrode. The PPC-OECT device was tested acquiring the typical output and transfer characteristics, as well as the kinetic curves.

Hereafter, we define the “kinetic curve” as the measurement of source–drain current (I ds) vs. time recorded under a constant drain voltage (V ds = –0.4 V) and by varying the gate voltage V gs according to a step-like scan mode, that is by applying voltage steps in the range 0–2 V with step heights increasing progressively by 0.2 V. Kinetic curves are generally acquired by operating OECTs in sensor mode in order to extract the modulation ratio ΔI/I 0 = (I – I 0)/I 0, where I 0 is the current value for V gs = 0 V, and I is the current value for V gs > 0 V. The modulation ratio is the typical parameter for quantifying the performance of OECTs used in sensor mode.34,35 In addition, the transfer curves show the channel current I ds flowing between the source and drain electrodes as a function of the gate voltage (V gs) under a constant drain voltage V ds = –0.4 V. On the other hand, output characteristics consist of the channel current I ds recorded as a function of the drain voltage V ds under a constant (variable) gate voltage V gs, resulting thus in a set of I ds vs. V ds curves parameterized by V gs. The gate current was simultaneously acquired during all the measurements. In order to obtain a steady-state curve, each point of the channel current was acquired with a delay of 30 s after the voltage application. Finally, a 2-terminal device was used to investigate the electrochemical response of the different electrodes inserted into the PP cell. This set of measurements was performed applying a bias between the PEDOT:PSS film and the mould, contacting the electrode inserted into it. The measurements were carried out via a series of voltage scans with steps of 0.2 V separated by a 10 s delay: the first range is between 0 and 2 V, followed by a scan between +2 to –2 V, and finally from –2 V to 0 V.

Control experiments have shown that the interaction of the gate electrode with the mould does not affect the viability of the PPC because, after insertion, the membrane of the PPC rearranges itself in a new state of equilibrium.

Our extensive study of the electrochemical transistor (hereafter referred as PPC-OECT) has been carried out by systematically changing the gate electrode material, that is platinum (Pt), silver (Ag) and gold (Au). In fact it has been recognized that the nature of the gate electrode can affect the device response in quite a relevant way, due to the different electrochemical reactivity between the metallic electrode and electrochemically active species.17 On the basis of the results reported in ref. 17, we have tried to highlight the connection between the electrical response of the device and intracellular mechanisms driven by electrochemical processes in the body of the PPC.

We first made a full standard electrical characterization by performing electrical measurements in a transistor-mode configuration (output and transfer characteristics). This characterization demonstrated the OECT-like operation. Then, we performed cyclic current–voltage measurements (I–V), by using the device in a 2-terminal configuration, where the OECT organic semiconducting layer works as the reference electrode and the metal gate plays the role of the working electrode.

A major result of our work is that the PPC-OECT shows operation as a multifunctional device, consisting of both a transistor-like and a memristor response, and is fully satisfactory for proper implementation as a memristive element. The switching between the OECT mode and the memristor mode is realized by interdicting the drain electrode. Such a hybrid device, based on interfacing organic electronics with living systems, is ideally suitable for building artificial bio-inspired systems. At the same time, the hybrid living/organic interface is an ideal electrochemically-active microenvironment suitable for studying in situ and in real-time both intracellular bioprocesses (whether induced or not by interaction with the environment) as well as collective properties of living organisms, including self-replicating systems. We use a living organism as an active device element, so that our PPC-OECT represents a prototypal test architecture aimed at showing the possibility to ideally scale down OECTs towards microscopic structures and, at the same time, a tool suitable to study membrane effects, eventually related, for example, to specific pathologies. Moreover, the memristive device counterpart can be used in a multifunctional device view for the recording/storing of specific cellular activities.

Fig. 1A shows a schematic of the hybrid PPC-OECT structure, where the yellow area represents the slime mould, while the black stripe between the source and drain electrodes represents the PEDOT:PSS film and the gate electrode is placed inside the PPC.

Fig. 1B shows the typical kinetic curves, I ds(t), measured by switching the gate voltage in the range 0–1.6 V with steps of 0.2 V. The device response upon application of gate voltage steps is defined by the modulation ratio ΔI/I 0 = (I – I 0)/I 0. The comparison among the typical current modulations observed for the three different gate electrodes is reported in Fig. 1C. As already indicated above, generally, ΔI/I 0 vs. V gs curves are used for expressing the sensing capability of OECTs in the presence of different analytes in an aqueous suspension, and/or different concentrations for a given analyte.

In our case, the comparison in Fig. 1C is used in order to show in a clear way how the mould reacts electrochemically in the presence of different gate electrode materials. In this respect, it is known that an OECT can work in two operating regimes, namely Faradaic and non-Faradaic (capacitive).17,36 In the Faradaic mode, a redox reaction occurs at the gate surface, generating a current in the gate–source circuit that decreases the potential drop at the gate/electrolyte interface, therefore increasing the effective gate voltage (Veffg) acting on the transistor channel.

Consequently, an increasing amount of ions is injected into the polymer film, de-doping it and hence inducing a significant decrease of the source–drain current, which, in turn, results in an increased current modulation. When the capacitive mode is dominant, an electrical double layer is formed at the gate/electrolyte interface. In this case, a significant potential drop arises at this interface, which reduces the Veffg acting on the polymeric film underneath, therefore limiting its de-doping.

As previously observed, the electrode material determines the electrochemical regime under which the PPC-OECT operates. As expected,17,37 the Ag gate, being redox-active, generates the highest current modulation over the whole voltage range investigated. On the other hand, Pt and Au, both without any significant redox reactivity, show lower current modulations. For instance, ΔI/I 0 at V gs = 0.8 V is 0.41 for the Au-gate, 0.65 for the Pt-gate and 0.95 for the Ag-gate. In the PPC-OECT a non-Faradaic regime is hence expected when a Pt or an Au gate are used. Fig. 1C shows the progressive gate voltage shift towards lower gate voltages of the transfer curves depending on the material of the gate electrode (Au < Pt < Ag), indicating that the Veffg increases progressively. In particular, focusing on the modulation value ΔI/I 0 of 0.5, the voltage gate shift between the Ag and Au gates is ∼0.5 V, whereas that between Pt and Au is ∼0.2. This is the effect induced by the specific reactivity of the gate material with the saline environment inside the cell, confirming what we expected from the abovementioned electrochemical considerations. These findings already give a first strong indication that the PPC-OECT provides valuable and direct “in situ” information about the electrochemical state of the mould cell itself and hence, possibly, of its interaction with the environment.

Fig. 2A–C compare the typical output characteristics (I ds vs. V ds at different V gs) for the three different gate electrodes investigated. In all cases, the output curves show that the devices work properly as transistors, operating in depletion mode in a very similar way to more standard electrolyte-gated transistors (even though the saturation regime occurring at voltages higher than 1 V was not investigated in order to avoid water electrolysis). We have observed an excellent biocompatibility of PEDOT:PSS with respect to the slime mould cell, confirming evidences reported in literature for this polymer when interfaced with several kinds of bio-systems, e.g. proteins,38 cells,19 and bacteria.39

Fig. 2D–F report the typical PPC-OECT transfer characteristics (I ds vs. V gs, V ds = –0.4 V) comparing once again the performance of Ag, Pt and Au gate electrodes. The hysteresis loops were investigated by recording the transfer characteristics in a cyclic mode, i.e. using a scan sweep with increasing gate voltage followed by a backward sweep. In particular, V gs was varied between –2 to 2 V, starting from 0 V, with step voltages of 0.2 V, at different bias step durations, e.g. 2, 5 and 10 s. In the positive range (0 < V gs < 2 V) the action of V gs induces an incorporation of cations into the PEDOT:PSS, causing its de-doping. Hence we observe a decrease of the channel current up to a saturation of the curves that depends on the gate material. In the negative range of the gate bias (–2 < V gs < 0 V) we observe in all cases that a higher gate voltage is needed to establish the original doping level of the PEDOT:PSS channel, i.e. for the cations to desorb from the polymer backbone towards the PPC. In particular, in the case of an Ag-gate, the change of the backward current is quite small in the negative V gs range, while it saturates at –1.5 V for the Pt-gate electrode and at about –2 V for the Au-gate electrode. Such behaviour suggests that chemical processes occur at the gate/PPC interface. The I ds current flowing in the polymeric channel at V gs = –2 V corresponds to the polymer intrinsic current. This current value is hundreds of μA higher than at zero-gate voltage, therefore, in the negative branch of the hysteresis curves, the ionic diffusion from the PEDOT:PSS towards the PPC is assisted by V gs. The high negative V gs required to (re-)dope the polymer could be understood by taking into account the fact that the mould cell can be thought of as a highly viscous electrolyte, and that ions should pass through the cell membrane to control/modify the polymer state.

The overall behaviour resembles that of a gel phase, and this would indicate that PPC works as a “quasi” solid electrolyte.40,41 This is an oversimplified picture since it does not consider that PPC is a cell and, consequently, for the actual mechanisms involved in the release/incorporation of ions, one should take into account more complex membrane mechanisms. Such biological mechanisms require further investigation and are currently an object of study.

A complementary observation concerns the area of the hysteresis curves, which have been found to be dependent on the gate material. In particular, the lowest area was found for the Ag-gate and the largest one for the Pt-gate electrode. In general, hysteresis arises from the competition between the dynamics of cation adsorption/desorption and the timescale with which the doping/de-doping occurs.10,42 The increase of the hysteresis area is hence related to the different operating regimes under which the OECT works according to the material of the gate electrode. More specifically, as already mentioned, the Ag-OECT operating regime (with a saline electrolyte) is almost fully Faradaic, and is characterized by a negligible potential drop at the gate/electrolyte interface,35,36 while with Au and Pt gates, OECTs are expected to work mostly in a capacitive operation mode (non-Faradaic mode), above all at lower gate voltages. It is worth noting that for Pt and Au (inert electrode materials) the channel current I ds (in the range of mA) is much higher than the gate current I gs (in the range of μA). Instead, when Ag is the gate electrode, I gs is considerably higher (on average, by a factor of 5), this being the fingerprint of a Faradaic regime.17

A similar consideration holds for the I ds steady-state level (saturation) in the positive range of V gs. Fig. 2–F shows that the Ag-gate gives a full-saturation level already at V gs < +1 V, a value that is achieved at higher voltages with Au (about 1.8 V), while a real onset of saturation is not observed for Pt in the range studied. This behaviour further indicates possible electrochemical reactions taking place at the PPC/Ag-gate electrode (as already indicated by the curves in Fig. 1B). In fact, we expect that a Faradaic reaction at the Ag-gate electrode results in a higher modulation and a fast response of the device, that is in faster channel current decrease (de-doping phase) and enhancement (desorption of cations from the channel). These are exactly the trends observed in the data of Fig. 1B.

The main question here is on how the PPC affects the channel current of the device, that is the conductive state of the PEDOT:PSS thin film. Specifically, the de-doping mechanism induced by the injection of cations from the electrolyte upon gate biasing should be completely reversible, so that the subsequent doping process (as observed in Fig. 2D–F) can actually take place after a de-doping process. Since the hole density in the PEDOT:PSS film is influenced by the dopant density, that is the ionic concentration of the electrolyte,43 the transfer characteristics of Fig. 2D–F indicate that the PPC acts as a reservoir of cations which can be exchanged with the PEDOT:PSS film upon suitable gate biasing. We envisage that since the cell membrane (5 nm thick) is in contact with the PEDOT:PSS film, the cations contained in the intracellular matrix can cross the cell membrane, possibly through the ion channels under proper polarization, both towards and from the PEDOT:PSS, and this cationic motion allows to de-dope/dope the polymer, respectively. Cations that cross the cell membrane towards the PEDOT:PSS are driven by the applied V gs > 0 V, so their motion will be faster than that of cations diffusing back from PEDOT:PSS to the cell membrane when V gs = 0 V.

This is a good indication that the PPC-OECT response could be related to the transmembrane mobility of ions contained in the cell. In order to assess the specificity of the response induced by the cell, and hence the ability of our system to study such effects, we made comparative measurements with standard physiological solutions.

Fig. 2G–I show the results related to the transfer characteristics measured with 0.15 M NaCl as the electrolyte, and using the same procedures adopted for the corresponding curves recorded in the case of the PPC-OECT (Fig. 2D–F). Of particular interest are the differences observed in the transfer characteristics, where the hysteresis loops are clearly different from those obtained with PPC and reported in Fig. 2D–F. In particular, the following differences can be observed in the hysteresis loops:

(i) Shape: the transfer curves for both NaCl and PPC-based devices clearly show a specific shape dependence on the gate material; in particular, in the case of PPC-OECT, a widening of the hysteresis loop is observed. The maximum widening is centred at different V gs values, according to the gate material (–0.5, 0, –1.2 V for Au, Pt, and Ag, respectively); this effect could be ascribable to the specific reaction of the intracellular matrix of the PPC with the gate material, resulting in a fingerprint of the electrochemical state of the PPC interior.

(ii) The transfer curves measured with NaCl show a progressive current decay upon measurement cycles, due to an over-oxidation effect induced by the largest bias reached in each cycle (+2 V);44 on the other hand, the PPC-OECT shows highly reproducible transfer curves with no loss of conductivity of PEDOT:PSS in the bias range investigated, resulting in a peculiar feature of the metallic gate/PPC/PEDOT:PSS system.