“Clickable” Organic Electrochemical Transistors

Interfacing the surface of an organic semiconductor with biological elements is a central quest when it comes to the development of efficient organic bioelectronic devices. Here, we present the first example of “clickable” organic electrochemical transistors (OECTs). The synthesis and characterization of an azide-derivatized EDOT monomer (azidomethyl-EDOT, EDOT-N3) are reported, as well as its deposition on Au-interdigitated electrodes through electropolymerization to yield PEDOT-N3-OECTs. The electropolymerization protocol allows for a straightforward and reliable tuning of the characteristics of the OECTs, yielding transistors with lower threshold voltages than PEDOT-based state-of-the-art devices and maximum transconductance voltage values close to 0 V, a key feature for the development of efficient organic bioelectronic devices. Subsequently, the azide moieties are employed to click alkyne-bearing molecules such as redox probes and biorecognition elements. The clicking of an alkyne-modified PEG4-biotin allows for the use of the avidin–biotin interactions to efficiently generate bioconstructs with proteins and enzymes. In addition, a dibenzocyclooctyne-modified thrombin-specific HD22 aptamer is clicked on the PEDOT-N3-OECTs, showing the application of the devices toward the development of organic transistors-based biosensors. Finally, the clicked OECTs preserve their electronic features after the different clicking procedures, demonstrating the stability and robustness of the fabricated transistors.

Au interdigitated microelectrodes (IDE-3, 5/5 m gap/electrode), a Ag/AgCl reference electrode, a Pt counter electrode and a batch cell accessory were obtained from Micrux.

Further electrochemistry measurements
For the organic solvent measurements, a Ag/Ag + electrode was used as RE and a Pt wire as CE. The electrolyte solution was 0.1M TBAF6 in ACN. The electrolyte was thoroughly bubbled with N2 prior to the measurements. For the "electro"clicking procedure, the electrolyte employed was DMF and the same setup was employed (see Click chemistry protocols Section).

Optical Microscope measurements
Optical microscope images were obtained with an Olympus BX51M Microscope.

FO probes fabrication
Fiber optic (FO) probes were fabricated according to a previously reported procedure. 1 In brief, TECSclad step-index multimode fibers (Thorlabs Inc.), with a numerical aperture of 0.39 and a core diameter of 1000 μm were cut to a length of 6.5 cm. At one end, a surface plasmon resonance (SPR)-sensitive zone (1 cm length) was constructed by removing the jacket layer with a stripping tool and the cladding layer by dissolution in acetone. The SPR-zone of the tip was further cleaned with Milli-Q water and isopropanol and blow dried under a flow of N2. As-prepared tips were homogenously sputter-coated with 50 nm of gold and stored under Argon until further usage.

AFM measurements
AFM measurements were performed in tapping mode with a PicoPlus AFM microscope (Molecular Imaging, Agilent Technologies, Germany). PPP-NCHR-50 tips were employed (Nanosensors, Switzerland). The obtained images were processed with the Gwyddion software (gwyddion.net).

ATR-FTIR measurements
Fourier transform infrared spectroscopy in the attenuated total reflection mode (ATR-FTIR) spectra were recorded employing a PerkinElmer Spectrum 100 spectrometer (USA) equipped with a diamond ATR crystal and using a resolution of 2 cm −1 for 48 scans. The spectra were normalized for data processing.

Supplementary Data
NMR spectra of EDOT-N 3 NMR spectra were recorded on an Avance 400 spectrometer ( 1 H NMR: 400 MHz, 13

Optimization of the OECTs obtention protocol Electropolymerization in ACN
The preparation of the devices was performed by electropolymerization employing a 10 mM EDOT-N3 solution in TBAF6 0.1M in ACN, according to a previously reported protocol. 2 The anodic potential limit of the electropolymerization was varied from 1.3 V to 1.5 V in order to study its effect on the polymer film obtained. Data for 1.4 V is shown in Figure S3. Next, the anodic potential limit was increased to 1.5 V (Figure S4). It is observed that thicker PEDOT-N3 films are obtained upon this electropolymerization procedure. Figure S4. Voltammograms for the electropolymerization of PEDOT-N3 on the IDEs (ACN, TBAF6 0.1M, EDOT-N3 10 mM, 40 mV/s, 10 cycles) (A). Output curves for the obtained OECTs (1xPBS) (B). Optical micrographs of the obtained OECTs at different magnifications (C).
When the anodic potential limit employed was 1.3 V (Figure S5), very thin PEDOT-N3 films were obtained. In addition, the transistors were shown to degrade upon the continuous measuring of transfer characteristics, as shown in Figure S5(B). This behavior was not observed in transistors prepared with the other preparation protocols.

Electropolymerization in HClO4
The obtention of the devices was also performed in an acidic solution (HClO4 0.1M, 10 mM EDOT) ( Figure S6) according to a previously reported protocol. 3 It is observed that the films obtained under these conditions are thinner and more homogeneous than those obtained in ACN.

Electropolymerization in HClO4 + SDS
The obtention of the devices was also performed in an acidic microemulsion solution (HClO4 0.1M, 10 mM EDOT, KCl 0.1 M, SDS 0.05M), according to a previously reported protocol. 4 From Figure   S7 it can be observed that the obtained films are more homogeneous than those obtained employing ACN or only HClO4 as electrolyte. Moreover, the effect of the cycle number was studied, and transistors were obtained by performing 5, 10 or 20 electropolymerization cycles. As expected, 5 an increase in the cycle number (and therefore an increase in the thickness) yielded transistors with higher Vth/Vg, gm max.

OECTs parameters computing
With the aim of benchmarking the electropolymerization protocols for the obtention of the PEDOT-N3 OECTs, relevant parameters such as threshold voltage (Vth) and voltage of maximum transconductance (Vg,gm max) were computed. Firstly, Vth was extracted from the transfer characteristics curve plotted in the linear scale, as reported elsewhere. 6,7 The importance of extracting the Vth from the linear regime roots in the uniformity of the charge distribution along the channel observed under these conditions. 8 The maximum transconductance voltage was obtained from the first derivative of the transfer curve, as previously reported. 9 . Figure S8 shows an example for the calculation of both parameters.   In order to study the reproducibility of the chosen protocol, the resistance of 17 different devices was analyzed and shown in a box plot ( Figure S11). It can be observed that the RSD is lower than 15% for the obtained transistors, accounting for a great reproducibility of the obtention method.

PEDOT and PEDOT-N 3 comparison
In order to compare the performance of the PEDOT-N3 transistors with that of PEDOT-based OECTs, PEDOT without azide moieties was electropolymerized on IDEs employing the same protocol as the one chosen for the PEDOT-N3 OECTs (Figure S12(A)). Similar current densities can be observed for the electropolymerization curves of both monomers, indicating comparable thickness of the polymer channel, and allowing the comparison of both transistors. The VG, gm max was calculated and plotted in Figure S12(B). It is observed that PEDOT-N3 OECTs show lower VG, gm max values than PEDOT OECTs. In addition, VG, gm max is very close to 0 V for PEDOT-N3 OECTs which, as it was previously stated, is a highly advantageous feature of these transistors.

PEDOT-N 3 -OECTs characterization Transient measurements
The ON switching of the PEDOT-N3 devices was also evaluated by performing the repeated cycling of the OECTs between 0 V and the ON state while recording the drain-source current at a constant VDS ( Figure S13). Next, the obtained source-drain current response was adjusted to an exponential decay to obtain ton and toff for each set of transistors, as previously described. 6

EIS measurements
Electrochemical Impedance Spectroscopy (EIS) measurements were performed in order to obtain more information about the electrical features of the PEDOT-N3 films deposited onto the IDEs. The spectra were obtained at different potentials and the Nyquist plots are shown in Figure S14(A).
Moreover, the spectra were fitted to the circuit shown in Figure S14(A). The circuit was based in previously reported EIS measurements of PEDOT-based OECTs, 17,18 and consisted of a solution resistance (Rs) connected in series to two parallel branches containing, in one side, a CPE, and in the other branch a series connection of the electronic/charge transfer resistance and a double layer capacitance. The CPE element accounts for the porous interface between the polymer domains and the inner solution interface, showing the typical "capacitance dispersion" and therefore a deviation from the ideal capacitive element. 19 The RE resistance is ascribed to the charge transport process along the conducting polymer domains, while the CDL accounts for the capacitance of the electrode-solution interface of the gold contacts. The results for the fitting are shown in Table S1.
It is worth noting that for the fitting of the spectrum obtained at -0.5 V, a Warburg O element was added, which is suitable for studies involving diffusing species when the DC current is different than zero, i.e., away from the OC potential, where the current would decay in time. 20,21 This is the particular situation of the biased electrode here reported, which at this potential undercomes reduction, as shown in the change of RE in main text, Figure 3. This element has been previously utilized in several works involving the fitting of PEDOT-based electrodes impedance spectra. 20,22 Furthermore, the capacitance was calculated according to = 1/(2 ( ( )) 23 and it is shown in Figure S14(B). The small differences observed in RS can be ascribed to the conducting polymer film resistance in its nonconducting state, which can also contribute to this high frequency resistance, as previously reported. 19 Table S1. Results for the fitting of the EIS spectra.

RE [Ω] Error RE
[Ω]  Figure S14. Nyquist plot of the impedimetric response of the PEDOT-N3 electrode (left) and zoom together with the circuit employed to fit the spectra (right) (A). Capacitance obtained from the EIS spectra (B).

AFM measurements
The morphology of the channel area of the fabricated OECTs was investigated by AFM measurements

ATR-FTIR measurements
To corroborate the presence of the azide moieties after the electropolymerization protocol, ATR-FTIR measurements of the OECTs were performed. From Figure S16 it is observed that the spectrum of the PEDOT-N3 film (obtained from the SDS-HClO4 solution) shows the typical azide band at ca. 2100 cm -1 , in agreement with previously reported studies. 2 Further description of the FTIR spectrum of the fabricated OECTs is presented in Table S2. The spectrum shows the presence of PEDOT-N3 bands as well as SDS ones, corroborating the incorporation of the SDS anions into the polymer film during the polymer electrosynthesis.

Click of redox probes and control experiments
In order to perform a control experiment of the clicking of the ethymyl-ferrocene redox probe on the transistors, PEDOT (without azide moieties) was electropolymerized on IDEs through the same protocol as the one employed for the PEDOT-N3 OECTs. Next, the electrode was exposed to the same clicking process as the PEDOT-N3 one, and the electrochemical and electrical features were obtained ( Figure S17). As described in the main text, no significant differences are observed after the treatment with Et-Fc, accounting for the specificity of the click reaction on the azide-modified OECTs/electrodes. Figure S17. Cyclic voltammetry curves for a PEDOT electrode before and after the click reaction with Et-Fc (50 mV/s, 1xPBS) (A). Threshold voltage and maximum transconductance for a PEDOT OECT before and after the click reaction with Et-Fc (B). Output characteristics for a PEDOT OECT after the click reaction with Et-Fc (C). Figure S18 shows the output characteristics of the PEDOT-N3 OECTs after the click reaction with Et-Fc, proving that the transistors keep their performance after the click functionalization. Figure S18. Output characteristics of the Et-Fc clicked PEDOT-N3 OECTs (1xPBS).
Regarding the clicking of the N-But redox probe, chronoamperometry measurements were employed to perform the clicking of the redox probe. Figure S19 shows an example of the chronoamperometry curves obtained. Moreover, the voltammograms of the PEDOT-N3 electrode before and after the "electro"clicking process are shown in Figure S20, confirming the effective clicking of the redox probe. In addition, it is observed that the transistors conserve their features after the clicking process and the electrochemistry measurements in the organic solvent, proving great stability ( Figure S21).

FO-SPR measurements
The electropolymerization of the PEDOT-N3 film on the FO was performed following the chosen SDS-HClO4-based protocol. To this end, the FO probes were connected to the potentiostat as WE by means of a copper tape, and the electropolymerization area was delimited by a liquid heat shrink tubing, as previously described (Figure S22(A)). 1 In this case, only 5 electropolymerization cycles were employed in order to obtain a thinner polymer film and therefore not overcome the SPR probing depth.
A typical electropolymerization curve is observed in Figure S22(B). From such curves an average PEDOT-N3 film thickness on the fiber of 68 ± 5 nm was derived. It is observed that the current densities values are similar to those observed for the OECTs (when 5 cycles were employed), validating the comparison between the FO-SPR experiments and those employing the transistors as platform. Next, after the electropolymerization of PEDOT-N3 on the FO, a resonance dip can be observed in the reflected spectra measured in air due to the polymer film attached to the fiber surface. (Figure S23(A)).
A picture of the Au-polymer edge is shown in Figure S23(B). water. In addition, and with the aim of taking the polymer back to its conducting state after the clicking procedure and thus recover the optical signal, a few cyclic voltammetry curves were performed in 1xPBS after the click reaction and the FO-PEDOT-N3 was left at its conducting state (Figure S24(A)).
The spectra in air of the PEDOT-N3-FO before and after the acetylene-PEG4-biotin clicking procedure are shown in Figure S24(B). The shifts in the resonance wavelength were analyzed by fitting the to a second-order polynomial, as shown in Figure S25. For the shift obtained for the Biotin-PEG4 clicking.

Equation S1
respectively, and dp corresponds to the polymer/protein layer thickness. The factor / relates the changes in refractive index with the concentration of (bio)molecules bound to the surface and was set to 0.2 mm 3 /mg. 34 Figure S26. Simulated spectra obtained for FO probes coated with a PEDOT-N3 film (dp = 68 nm) and refractive index variation (A). Linear calibration curve for the PEDOT-N3 refractive index (B). Simulated spectra for various refractive indices associated with a NeutrAvidin layer (dp = 5.6 nm) immobilization on FO probes coated with PEDOT-N3 after biotin click reaction (C). Linear calibration curve for the NeutrAvidin refractive index (D).
In order to perform a control experiment for the clicking of the biotin molecules, PEDOT (without azide moieties) was electropolymerized on the FO following the same protocol as for PEDOT-N3.
Next, the PEDOT-FO was subjected to the same clicking protocol employing the acetylene-biotin molecule in DMSO. Figure S27(A) shows the electropolymerization curves of PEDOT on a FO and  Similarly to what was performed for PEDOT-N3-FO, a few cyclic voltammetry curves were performed in 1xPBS after the exposition of the PEDOT-FO to the click reaction conditions, leaving the polymer modified FO at its conducting state (Figure S28(A)). The effect of the potential cycling on the SPR response of the PEDOT-FO is clearly observed in Figure S28(B). These potential-dependent changes of the SPR signal are in agreement with previously reported findings and can be associated to the electrochromic behavior of the PEDOT film, showing significant changes of the resonance wavelength when going from the doped state to the de-doped state of the film. 31 A similar electrochromic behavior was observed for the PEDOT-N3 film as described above.

Clicking of biotin and avidin recognition on OECTs
The same clicking and recognition protocol employed for the FO was used in order to incorporate biotin moieties and NeutrAvidin to the surface of PEDOT-N3 OECTs. From Figure S29 it is observed that the transistors conserve their features after the clicking process in the organic solvent and the NeutrAvidin recognition, showing great stability. On the other hand, biotin-PEDOT-N3 OECTs were also employed in order to anchor streptavidin-HRP conjugate, making use of the biotin-avidin biorecognition. Figure S30 shows the transfer characteristics of the HRP-biotin-PEDOT-N3 OECT together with the calculated transconductance. It is observed that the OECTs present a VG, gm max close to 0 V, endowing the application of 0 V while preserving an optimal gm value.

Thrombin sensing experiments
To check the specificity of the response of the HD22-modified PEDOT-N3 OECTs, the same modification protocol was performed on PEDOT OECTs, without azide moieties, and their response towards thrombin was evaluated ( Figure S31). As described in the main text, the transistors did not show a response towards the cardiac biomarker, probing the specific recognition of thrombin by the HD22 clicked devices. In addition, the same experiment was performed by PEDOT-modified FO setup (main text). Moreover, the PEDOT-N3-HD22 OECTs employed for thrombin sensing showed no alteration of the output characteristics after the modification and recognition process, proving the excellent stability of the developed transistors ( Figure S32). Figure S32. Output characteristics for a DBCO-HD22-modified PEDOT-N3 OECT after thrombin (400 nM) sensing (1xPBS).
A comparison of the developed thrombin biosensing devices with other OECTs-based biosensors for relevant analytes is shown in Table S3.