Portable and Autonomous PEDOT-Modified Flexible Paper-Based Methanol Fuel Cell Sensing Platform Applied to L1CAM Recombinant Protein Detection

This work describes first a 5-stack direct methanol fuel cell (DMFC) based on poly(3,4-ethylenedioxythiophene)-modified paper (PEDOT/PB-DMFC), which acts as an energy source and biosensor, coupled to an electrochromic cell (EC). It is autonomous and monitors the biosensor response by color change, as appropriate for point-of-care (POC) applications. In detail, DMFC strips were developed from square Whatman paper, and the EC was made on baking paper treated with polydimethylsiloxane (PDMS). The PEDOT/PB-DMFCs operate in a passive mode with a few microliters of diluted methanol. The biosensor layer was obtained on the anode ink (a composite of EDOT, oxidized multiwalled carbon nanotubes, and carbon black with platinum and ruthenium) by electropolymerizing 3,4-ethylenedioxythiophene (EDOT), in situ, in the presence of L1CAM. Each PEDOT/PB-DMFC single cell generates a voltage in the range of 0.3–0.35 V depending on the cell, and a five-cell stack delivers a 1.5–1.6 V voltage range when fed with 0.5 M methanol. The fabricated PEDOT/PB-DMFC/biosensor was calibrated against L1CAM, showing linear responses from 1.0 × 10–12 to 1.0 × 10–8 M with a detection limit of 1.17 × 10–13 M (single cell mode). When the EC was connected to the PEDOT/PB-DMFC device, a color gradient was observed. Overall, this work opens horizons to the use of biosensors even in places with energy scarcity and offers an alternative to reducing the current energy demand.


Supplementary Tables
• Table S1.Electrochemical data and normalized power calculation of the results presented in Figure 3 A-D.
• Table S2.Electrochemical data and power calculation of the results presented in Figure 5-A/B, D/E.
• Table S3.RGB coordinates determined at three different locations with the blue coordinate, and calculation of the average values and standard deviation.

Reagents and solutions.
All the reagents were of analytical grade and the water was an ultrapure Milli-Q.A solution of Nafion ® [20 wt%, alcohol based (propanol/ethanol) Quintech] was applied to a ≈ 2.2 cm 2 square paper strip (Whatman grade 40 filter papers) and covered on both sides with carbon based inks consisting of: Carbon Black with PtRu (40:20 wt%) catalyst (C/PtRu) on Vulcan XC-72R (Quintech) mixed with oxidized Multi Walled Carbon Nanotubes (MWCNT-COOH) (Sigma Aldrich) for the anode side, and Carbon Black with Pt (10 wt%) catalyst (C/Pt) on Vulcan XC-72R (Quintech) for the cathode side.The loading of the Pt catalyst in the anode layer was set as 2 mg/cm 2 and 0.5 mg/cm 2 for the cathode electrode.The inks were prepared using 2-propanol (IPA) (99.8%,Panreac) as solvent and Nafion ® (20 wt%.) as binder using a microtip ultrasonic system.A solution of 0.01 M EDOT (97%, Alfa Aesar) in IPA/H2O (4/1) was added to the anode ink and sonicated in ultrasonic bath for 60 s.Titanium (IV) oxide (anatase, Sigma Aldrich) was also added to the formulation for evaluating TiO2 applicability as conductive component in ink.Different amounts of MWCNT-COOH were also tested in the ink formulation.
A paraffin solution (Sigma Aldrich) was diluted in 2-propanol (50/50) and applied around the active area of the PB-DMFC assembly (on both sides of the paper).It hydrophobized and strengthens the paper substrate.
Conductive silver ink (RS Pro) was applied to the top edges for electrical connection (each side with an independent terminal, insulated with tape on the non-electrical side to avoid short circuits).Two buffer media were used in this work: 10 mM MES buffer (pH 6.0) obtained by dissolving 2-morpholinoethanesulfonic acid buffer (AppliChem) and PBS buffer with (pH 7.2) obtained by dissolving phosphate-buffered saline (Amresco).A recombinant human L1CAM protein (His-tag) (MyBiosource) stock solution was prepared by diluting the commercial reagent in buffer; less concentrated standards were prepared by accurately diluting the stock solution in the range of 1.0 × 10 -12 -1.0 × 10 -8 M.
The polymerization solution consisted of 0.01 M EDOT (97%, Alfa Aesar) prepared in PBS (25 mL solution for each electrode), and L1CAM protein was used as a template during polymerization.Template removal was performed electrochemically in 0.5 M H2SO4 (Sigma Aldrich).Electrochemical measurements were performed using an aqueous methanol solution (Honeywell) of 0.5 M for calibrations and 1.0 M for initial stabilization of the fuel cell.A pretreated human serum (Cormay ® HN, PZ Cormay S.A) was used for the selectivity studies.The pretreatment consisted of adding silver nitrate (AgNO3, 0.1 M) to the pure serum, to reduce the effect of chloride ions (0.1 M Cl -).In this procedure, 10 mg of AgNO3 was added to 1 mL of pure serum and left in a stir plate for 30 min.The mixture was then centrifugated at 10 000 rpm for 10 min, and a solid white precipitate (AgCl) was deposited at the bottom of the flask (Figure S1).This treated serum was 100x diluted in MES buffer and spiked with selected target concentrations.This medium was spiked with the same biomarker concentrations that were prepared in the buffer.
For the selectivity tests, L1CAM standard solutions were prepared in the presence of interfering species.
The paper supported EC was developed on top of baking paper, modified with polydimethylsiloxane (PDMS commercial kit SYLGARD TM ).After curing the PDMS, a promotor for gold adhesion, 3-Mercaptopropylmethyldimethoxysilane, MPTMS (Sigma Aldrich), was spin-coated on the surface of the paper/PDMS.A commercial dispersion of poly(2,3-dihydrothieno-1,4-dioxin)-poly(styrenesulfonate) (PEDOT:PSS) (Sigma Aldrich) was diluted in dimethyl sulfoxide (DMSO, 95/5(%)) and spread in the middle ring of the EC.0.1 M solution of lithium perchlorate (Sigma Aldrich ® ) was used as the electrolyte in the EC paper device.

Equipment and apparatus
Electrochemical measurements were performed at lab-ambient conditions (21±2 o C) using a Methrom Autolab potentiostat/galvanostat with a PGSTAT302N, controlled by Nova software (NOVA 2.1).The certified current resolution of the PGSTAT302N is 30•10 -15 A; fairly enough for reliable tracking of the output current and power of the produced devices ranging in several  and W, respectively.The electrochemical a Hitachi (TG/DTA7200) instrument.The morphology of the PEDOT/PB-DMFCs was studied using a scanning electron microscope (SEM) with cross-section and microstructure analysis.

Electrochemical and electrochromic assays
The electrochemical performance of the PEDOT/PB-DMFCs was evaluated using the typical polarization curves used to characterize fuel cell systems (obtained by Sampled DC technique).Calibrations of the PEDOT/PB-DMFCs/biosensor and the PEDOT/PB-DMFCs/NIP were performed by incubating L1CAM standard solutions in the range of 1.0 × 10 -12 -1.0 × 10 -8 M in MES buffer.Prior to this, blank stabilization was performed by incubating the MES buffer on the modified anode until electrical signal stabilization was reached.During calibration, each solution (100 µL) was incubated on the anode surface for 20 minutes, starting with the lowest concentration.The system was covered with a glass square to efficiently distribute the solution over the entire active area and avoid evaporation.After each incubation and electrochemical measurement, the anode sensor area was carefully washed with ultrapure water and dried with nitrogen.This procedure was repeated for increasing L1CAM concentration standards prepared in MES buffer and diluted Cormay ® serum (100 ×) to evaluate the ability of the PEDOT/PB-DMFC/biosensor to discriminate L1CAM.
For the selectivity assays, 1.0 × 10 -10 M L1CAM concentration was selected and incubated directly in the PEDOT/PB-DMFCs/MIP and the signal was measured.The same L1CAM concentration was doped with the different selected interferents and later incubated in the sensor area using different PEDOT/PB-DMFC assemblies (L1CAM standard + interferent for PEDOT/PB-DMFC/biosensor unit).
Calibrations with the hybrid PEDOT/PB-DMFC/biosensor stack connected to the EC were performed in buffer after the hybrid stack stabilized to the maximum voltage (≈1.5 V).The anode region of the different PEDOT/PB-DMFCs electrically connected to each other was incubated with the same concentration standards of L1CAM used in the single-cell calibrations.After washing the sensing anode with ultrapure water, methanol was added to the fuel cells anodes.After 5 minutes of stabilization, a 48 Mp smartphone camera with a resolution of 8000 x 6000 pixels (Samsung Galaxy A51) was used to take a photo of the EC S-9 center ring and the color coordinates of these images were analyzed using ImageJ software (blue coordinate selection).A typical smartphone, with a similar medium resolution can be used to reproduce the data.
. Pre-treatment steps of the human serum (Cormay®) for L1CAM standards preparation: (1)add AgNO3 to the pure Cormay ® serum; (2)-stir in a stirring plate for 30 minutes until a yellow/white turve solution is formed; (3)centrifugation at 10000 rpm during 10 minutes until the solution appears light yellow with a white precipitate in the bottom of the flask, indicating the removing of the chloride ions present in the initial serum; (0.1 M of Clpresent in the initial serum) (4) -Cormay ® serum pre-treated is compatible for incubation in the anode side of the PEDOT/PB-DMFC with a 100x MES buffer dilution; (5)-example of a stabilization protocol using the diluted pre-treated serum in a PEDOT/PB-DMFC assembly containing the MIP sensor, evidencing a stable system after 4/5 incubations.• Figure S2.Steps in the PEDOT/PB-DMFC development.(1)the Nafion ® electrolyte incorporation in the Whatman paper, (2) ink deposition (anode/cathode) in both sides of the electrolyte, (3) paraffin impermeabilization in the surrounding area of the electrodes (50/50 paraffin/IPA), and (4) electrical connector's integration on the top edges with silver ink.; (5) this final assembly is isolated with glue tape to avoid leaking of the methanol solution and avoid short circuit; (6) the final PEDOT/PB-DMFC operates with a solution of methanol (0.5 M -1 M) which is adsorbed in a square of adsorbent paper and with the cathode exposed to air; (7) a typical polarization and power curves after signal activation and stabilization; PEDOT/PB-DMFC contains a MIP sensor for L1CAM detection.• Figure S3.Example of the polarization curve calculations to obtain the normalized current density and power, highlighting the maximum power of each system, used for comparison of all the developed systems.• Figure S4.Steady-state cyclic voltammograms obtained in a 3-electrode system in a PBS buffer solution (the 5 th cycle is presented, −0.3 V to 1.2 V, 50 mV/s), for the assembly of the PEDOT sensing polymer on the anode layer of PEDOT/PB-DMFC-MIP in comparison with the control PEDOT/PB-DMFC-NIP and corresponding removal step in sulfuric acid solution.• Figure S5.SEM analysis of a PEDOT/PB-DMFC/MIP and PEDOT/PB-DMFC/NIP electrodes, before and after activation and stabilization, evidencing some roughness modifications.• Figure S6.Power curves were obtained in consecutive electrochemical readings using independent PEDOT/PB-DMFCs assemblies after stabilization and prior incubation with MES buffer (A), pre-treated diluted (100x) Cormay ® serum (B) using 0.5 M MeOH solution for the readings.• Figure S7.Photographs of the PEDOT/PB-DMFC-stack; the electrical set-up (A), the anode sensing side(B), and connected to the cathodes (C).

Figure S1 -
Figure S1 -Pre-treatment steps of the human serum (Cormay®) for L1CAM standards preparation: (1)add AgNO3 to the pure Cormay ® serum; (2)-stir in a stirring plate for 30 minutes until a yellow/white turve solution

Figure S3 - 13 Figure S4 - 14 Figure S5 -Figure S6 - 16 Figure S7 -
Figure S3 -Example of the polarization curve calculations to obtain the normalized current density and

Table S3
RGB coordinates determined at three different locations with the blue coordinate, and calculation of the average values and standard deviation.