Electrosynthesis of Biocompatible Free-Standing PEDOT Thin Films at a Polarised Liquid|Liquid Interface

The versatility of conducting polymers (CPs) facilitates their use in energy conversion and storage, sensor, and biomedical technologies, once processed into thin films. Hydrophobic CPs, like poly(3,4-ethylenedioxythiophene) (PEDOT), typically require the use of surfactant additives, such as poly(styrenesulfonate) (PSS), to aid their aqueous processability as thin films. However, excess PSS diminishes CP electrochemical performance, biocompatibility, and device stability. Here, we report the electrosynthesis of PEDOT thin films at a polarised liquid|liquid interface, a method non-reliant on conductive solid substrates that produces free-standing, additive-free, biocompatible, easily transferrable, and scalable 2D PEDOT thin films of any shape or size in a single-step at ambient conditions. We demonstrate the PEDOT thin film’s superior biocompatibility as scaffolds for cellular growth, opening immediate applications in organic electrochemical transistor (OECT) devices for monitoring cell behaviour over extended time periods, bio-scaffolds and medical devices, without the requirement for physiologically unstable and poorly biocompatible PSS.

In this work, we report a major advance in the use of interfacial electrosynthesis at an ITIES to prepare free-standing, additive-free, reproducible, easily transferrable, scalable, and biocompatible PEDOT thin films in a single step at optimised ambient conditions. The external electrochemical driving force, the interfacial Galvani potential difference (∆ o w ), is applied using double potential step chronoamperometry (DPSCA). The IET reaction between an aqueous Ce 4+ oxidant and organic EDOT monomer, and subsequent oligomer deposition at the L|L interface, can be assisted or hindered by manipulation of ∆ o w . We demonstrate an emergent enhanced biocompatibility of free-standing PEDOT thin films prepared at the ITIES, compared with films prepared by drop-casting commercial PEDOT:PSS surfactant-4 free ink, using normal human retina pigment epithelium (hTERT RPE-1) cells. The latter are a physiologically pertinent cell line given the application of PEDOT films in maculopathies. 21,22 We functionally modified the PEDOT and PEDOT:PSS films through the incorporation of bioactive proteins and monitored the consequential effects on cell behaviour in each case. These findings foreshadow potential applications as suitable 2D conductive substrates for RPE and electrically active photoreceptor cells and the development of organic electrochemical cell transistors (OECTs) capable of electrochemically monitoring cell growth over >24 hr periods.

The mechanism of PEDOT interfacial electrosynthesis
As illustrated in Fig. 1a(i)-(v), PEDOT thin film interfacial electrosynthesis progresses along five distinct stages with time. Initially, IET occurs between the aqueous Ce 4+ oxidant and EDOT organic monomer forming monomeric radical cations (EDOT •+ ) in the diffusion layer on the organic side of the ITIES (Fig. 1a(i)). For IET to proceed with appreciable kinetics, the ITIES must be polarised positively (as discussed vide infra) with Δ o w set to a value near the positive extreme of the Galvani polarisable potential window. EDOT •+ species are stabilised by the weakly coordinating organic anion, tetrakis(pentafluorophenyl)borate (TB -), 23 and further couple with each other or EDOT monomers to form dimers in the diffusion layer.
Continuous EDOT •+ generation by IET and ensuing radical coupling steps ultimately lead to the formation of cationic PEDOT oligomers that also coordinate with TBto maintain electroneutrality ( Fig. 1a(i)). These coupling steps result in the release of protons on the organic side of the ITIES (Supplementary Fig. 1) that will be stabilised by the PEDOT thin film itself or by water present inside the film. 24 5 Fig. 1 Interfacial adsorption involving ion-pairing between the cationic PEDOT oligomers and aqueous electrolyte anions (herein SO4 2-) takes place once the oligomers reach a critical size after an induction period ( Fig. 1a(ii)). SO4 2anions displace the weakly coordinating organic TBanions during interfacial adsorption, ultimately becoming the sole dopant anion in the PEDOT thin film. This deposition process is driven by the energetically favourable reduction of the interfacial tension ( ) between the two repulsive phases upon oligomer adsorption. 25  In the next step, nucleation and growth of adsorbed PEDOT oligomers takes place at the interface. The PEDOT oligomers act as floating interfacial bipolar electrodes, providing abundant catalytic sites as electrical short-cuts to catalyse IET between the Ce 4+ and EDOT species (Fig. 1a(iii)). [26][27][28] Due to this autocatalytic effect, IET proceeds at a much lower overpotential than at a bare ITIES, with a higher kinetic rate. Thus, the PEDOT islands show rapid 2D growth, parallel to the L|L interface. The gaps between individual rapidly growing islands of PEDOT disappear and a highly compact 2D PEDOT thin film coalesces at the ITIES that is flat on both sides, with a thickness of ~50 nm ( Fig. 1a(iv)). At this point, a physical barrier now exists between the Ce 4+ and EDOT species at the ITIES. However, IET continues through the conductive PEDOT thin film and is subject to the influence of the diffusion of SO4 2counter-anions through the film to maintain electroneutrality locally, i.e., "p-doping". Continued IET initiates a secondary 3D growth process into the organic phase as the thickness of the PEDOT thin film increases. This controllable secondary growth process leads to the formation of a highly porous 3D structure, up to ~ 850 nm thick ( Fig. 1a(v)).

Electrochemically initiating and controlling PEDOT thin film interfacial electrosynthesis
IET between Ce 4+ and EDOT leading to a 2D PEDOT thin film is not a spontaneous process at an aqueous|α, α, α-trifluorotoluene (TFT) interface ( Supplementary Fig. 3). To initiate CP thin film formation, the aqueous|TFT interface must be polarised using a potentiostat in conjunction with a four-electrode electrochemical cell ( Fig. 1b and Supplementary Fig. 4).
The resulting free-standing 2D PEDOT thin film could be recovered from the L|L interface, thin film formation at the ITIES after 300 DPSCA cycles ( Supplementary Fig. 11). The Tyndall effect was used to explore the partition of small PEDOT oligomers from the organic to aqueous phase during interfacial electrosynthesis ( Supplementary Fig. 12). 11 Scanning electron microscopy (SEM) of a 2D PEDOT thin film prepared by DPSCA (150 cycles) revealed an asymmetric "Janus" morphology ( Fig. 3a and Supplementary Fig. 13).

Microscopic analysis
One side is flat and featureless at the nanoscale, while the other shows a rough, porous 3D structure. The PEDOT thin films adhere to any solid substrate, with the thin film closely  Fig. 16). Initially, after 50 DPSCA cycles, the thin film shows 2D growth parallel to the ITIES, a highly compact structure that is flat on both sides, and a thickness of 40 -60 nm ( Fig. 3e(i)). With continued interfacial electrosynthesis (up to 150 DPSCA cycles), secondary 3D growth begins to extend into the organic phase as the thickness of the PEDOT thin film increases ( Fig. 3e(ii)). This controllable secondary growth process leads to the formation of a very porous 3D structure with a thickness of up to ~ 850 nm after prolonged (> 300 DPSCA cycles) interfacial electrosynthesis ( Fig. 3e(iii)).
Transmission electron microscopy (TEM) studies revealed that the PEDOT thin film is exceptionally stable under the TEM beam (80 kV), signalling a high thermal conductivity 14 and providing an opportunity to further investigate the PEDOT thin film's nanostructure.
Both bright-field ( Fig. 3f(i)) and dark-field ( Fig. 3f(ii)-(iii)) mode TEM images show that the flat aqueous side consists of a compact layer of PEDOT nanofibers that run parallel to the ITIES. The diameter of the PEDOT nanofibers varies from < 5 nm to above 50 nm. We propose that the nanofibers with a diameter of < 5 nm are first to be deposited at the ITIES during interfacial electrosynthesis, forming an initial compact layer. Subsequently, the nanofiber diameter increases as the thin film grows down into the organic phase.

Spectroscopic, conductivity and electrochemical analysis
Ex situ spectroscopic analysis was performed on PEDOT thin films transferred to suitable solid substrates (Supplementary section 6). A bipolaron band was observed by UV/vis absorbance, signifying that the PEDOT thin film is in an oxidised state 29 and p-doped ( Supplementary Fig. 17). Raman spectroscopy also confirmed that the PEDOT thin film is pdoped, with high π-π conjugation and a benzenoid (coiled) configuration to the polymer chain ( Supplementary Fig. 18), the more stable form when PEDOT is highly doped. 30 Following 48 hours of cell growth on each film, marked differences were observed in hTERT RPE-1 cell growth dynamics (Fig. 4). Overall, in the presence of PEDOT thin films, cells exhibited a greater degree of proliferation and showed a stretched morphology, associated with actin bundle stress fibre formation, alluding to the more biocompatible nature of PEDOT versus PEDOT:PSS ( Fig. 4a-b). These data re-enforce the challenges of using PSS at bio-interfaces and differentiate our investigations from many evaluating PEDOT:PSS in cell culture, where lack of obvious cytotoxicity is incorrectly construed as high biocompatibility. Next, each film was evaluated for suitability of active biomolecule incorporation. Collagen was selected as an active biomolecule due to its known influence on cell proliferation and adhesion, which could potentially mitigate the poor cellular growth seen on PEDOT:PSS samples. While only marginal effects were observed in cells grown on PEDOT:PSS films, functionalisation of PEDOT thin films resulted in a marked amplification in cell proliferation and cell spreading, indicative of robust adhesion receptor engagement and bioactivation of the cell cycle programme (Fig 4a).

Discussion
Interfacial electrosynthesis at an ITIES bridges the fields of purely homogeneous (chemical) electron transfer reactions between redox couples, which are difficult to control, and finely controlled (electrochemical) heterogeneous electron transfer at conventional solid electrodeelectrolyte interfaces. This work demonstrates that interfacial electrosynthesis directly produces PEDOT thin films of any shape or size in a single step, with distinctive molecular architectures inaccessible in bulk solution or at solid electrode-electrolyte interfaces, and emergent properties that facilitate technological advances. Electrochemical control of thinfilm nucleation and growth at the L|L interface allows fine control over the morphology, transitioning from 2D films (flat on both sides with a thickness <50 nm) to "Janus" 3D films (with a flat and rough side, each showing distinct physical properties, and thickness >850 nm) by simply tuning the number of DPSCA cycles implemented. The PEDOT thin films were highly p-doped (approaching the theoretical limit), showed high π-π conjugation, and were processed directly as thin films without insulating PSS. As a result, these films were highly conductive without post-processing, with their ex situ conductivity determined as 554 In situ UV/vis absorbance measurements were performed using the parallel beam configuration that is illustrated in Supplementary Fig. 10 Fig. 24). ITO electrodes were used to 29 investigate the PEDOT films scan rate dependence ( Supplementary Fig. 25

Data availability
All data is available from the corresponding author M.D.S on request.